# Large scale identification of pellicle and cell-free liquid phase associated proteins in Bacillus amyloliquefaciens L-17

Tassadit Ouidir, Julie Hardouin, Claire-Emmanuelle Marcato-Romain, Elisabeth Girbal-Neuhauser, Yassine Nait Chabane

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Tassadit Ouidir, Julie Hardouin, Claire-Emmanuelle Marcato-Romain, Elisabeth Girbal-Neuhauser, Yassine Nait Chabane. Large scale identification of pellicle and cell-free liquid phase associated proteins in Bacillus amyloliquefaciens L-17. Current Research in Microbial Sciences, 2025, 8, 10.1016/j.crmicr.2025.100387. hal-05368531

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# Large scale identification of pellicle and cell-free liquid phase associated proteins in *Bacillus amyloliquefaciens* L-17

Tassadit Ouidir<sup>a,b</sup>, Julie Hardouin<sup>c,d</sup>, Claire-Emmanuelle Marcato-Romain<sup>a</sup>, Elisabeth Girbal-Neuhauser<sup>a</sup>, Yassine Nait Chabane<sup>a,*</sup>

## ARTICLE INFO

Keywords:

Pellicle proteins

Cell-free liquid phase

EPS

Proteomic

Antimicrobial peptide

Enzymes

## ABSTRACT

*Bacillus amyloliquefaciens* is a soil-associated and plant growth-promoting bacterium. It is the focus of numerous studies due to its ability to sporulate, form biofilms, produce antimicrobial peptides and commercial enzymes. The ability of *B. amyloliquefaciens* L-17 to form floating biofilm at the air-liquid interface “pellicle” was previously demonstrated. This pellicle exhibits a highly structured architecture which is provided by loosely and tightly matrix bound polysaccharides and proteins. In this study, a first large scale proteomic investigation of both the pellicle and the cell-free liquid phase of L-17 strain was performed. An approach based on physical and chemical extraction of the pellicular matrix combined with protein analysis by mass spectrometry identified 87 weakly matrix-bound proteins and 62 tightly bound proteins. A total of 131 pellicle-associated proteins were identified, including (i) the conserved proteins TasA and TapA, involved in biofilm formation and cohesion (ii) BslA, important for biofilm hydrophobicity (iii) several enzymes that make nutrients available and protect the biofilm from competitors (iv) flagellin and (v) proteins involved in the sporulation process. Proteomic characterization of the cell-free liquid phase underlying the analyzed pellicle allowed the identification of 423 proteins including 118 proteins yet identified in the matrix of the pellicle. The proteins identified specifically in the liquid phase include enzymes involved in the biosynthesis process of non-ribosomal peptides and a variety of commercial enzymes such as proteases, lipases, aminotransferases, peroxidases and phytases. This provides valuable clues to promote the industrial and agricultural application of the cell-free liquid phase of *B. amyloliquefaciens* L-17.

## 1. Introduction

Most bacteria privilege the biofilm lifestyle where they are held together by self-produced extra polymeric substances (EPSs) commonly comprised of polysaccharides, proteins, lipids and DNA (Devi et al., 2023; Flemming and Wuertz, 2019; Karygianni et al., 2020). Biofilm is a highly orchestrated social life strategy where inhabitants engage in co-ordinated living to fight against the environmental fluctuations (Costerton et al., 1995; Parrilli et al., 2022; Tallawi et al., 2017). The diversity of microorganisms forming biofilms and EPSs composition heterogeneity, provide biofilm different properties which can negatively or positively impact various aspects of our daily life (Devi et al., 2023; Moore-Ott et al., 2022; Ouidir et al., 2022; Pradeep and Arratia, 2022).

The beneficial biofilm research field is fast moving owing to the bio-logical relevance in natural systems and human applications including bioremediation, water treatment, food field and agricultural settings (Morikawa, 2006; Mukhi and Vishwanathan, 2022; Philipp et al., 2024).

The non-pathogenic bacterium, *Bacillus subtilis*, has been the major model organism for the study of Gram-positive bacteria for many decades, due to its ease of genetic manipulation and its extensive industrial and agricultural application (Arbour et al., 2023; Arnaouteli et al., 2021; Vlamakis et al., 2013). These studies commonly used the ancestral strain, *B. subtilis* NCIB 3610, in three experimental systems including (i) pellicle, a floating biofilm on air-liquid surfaces (ii) rough colony formed on semi-solid agar surfaces, and (iii) biofilm formation on plant roots (Cairns et al., 2014). In all these models, *B. subtilis* leads cooperative life

<footnote><sup>a</sup> Laboratoire de Biotechnologies Agroalimentaire et Environnementale (LBAE) URU 4565, Université de Toulouse, IUT de Toulouse Auch Castres, IUT A Paul Sabatier, 24 rue d'Embaquès, Auch 32000, France</footnote>

<footnote><sup>b</sup> Beaulieu-Lavacant General and Technological Agricultural Education High School, Route de Tarbes, Auch 32020 CEDEX 9, France</footnote>

<footnote><sup>c</sup> Université de Rouen Normandie, INSA Rouen Normandie, CNRS, Normandie Universite, PBS UMR 6270, Rouen, France</footnote>

<footnote><sup>d</sup> University of Rouen Normandy, INSERM US 51, CNRS UAR 2026, HeRacLeS PISSARO, Rouen, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: yassine.nait-chabane@iut-tlse3.fr (Y. Nait Chabane).</footnote>
through cell differentiation, labor repartition and EPSs production (López et al., 2009). The EPSs of *B. subtilis* biofilm are majorly composed of polysaccharides, TasA, TapA, and BsIA crucial proteins. Together, they create a complex interaction network that gives the biofilm its architecture and biochemical and physical characteristics (Branda et al., 2006, 2001; Milton and Cavanagh, 2023; Romero et al., 2010). Despite their omnipresence in the natural environments, it is however necessary to note that pellicles are far less studied compared to solid surface attached biofilms (Vaccari et al., 2017). This lifestyle is also found in organisms belonging to the *B. subtilis* species complex, such as members of the *Bacillus amyloliquefaciens* (OGBa) operational group (Ngalimat et al., 2021; Zaidi-Ait Salem et al., 2021b).

*B. amyloliquefaciens* has long been the center of research interest. This ubiquitous species is known for its excellent ability to plant colonization and protection from pathogens (Jiao et al., 2021; Wang et al., 2017). It is considered as plant growth-promoting (PGPR) bacteria due to its abilities to fix nitrogen, solubilize phosphate, and produce siderophores and phytohormones, as well as antimicrobial compounds and a wide range of commercial enzymes (Ngalimat et al., 2021). However, biofilm formed by this species remain very little studied compared to those of *B. subtilis* (Kimani et al., 2016; Zaidi-Ait Salem et al., 2021b; Zhao et al., 2016). Interestingly, Deka et al. (2019) has shown that the EPSs produced by *B. amyloliquefaciens* p16 is able to confer acid tolerance to the bacterium and improve the structural stability of the soil by increasing its aggregation when inoculated. On the other hand, Qiu et al. (2014) identified key proteins involved in *situ* root colonization and biofilm formation in *B. amyloliquefaciens* SQR9 by comparing the proteomic profiles of planktonic and root-colonized SQR9 cells.

During the biofilm or pellicle formation in *Bacillus* species, two populations, sessile and planktonic, coexist and can exchange. Cells from the biofilm can migrate to the planktonic population while cells from the planktonic population can enter the biofilm (El-Khoury et al., 2021). Thus, the cell-free liquid phase (CFLP) seems interesting to explore. Cell-free supernatant of *Bacillus* species, including *B. amyloliquefaciens*, planktonic culture is increasingly attracting researchers because of its richness in bacterial bioactive compounds (Latorre et al., 2016; Naamala et al., 2022; Park et al., 2003; Xu et al., 2019). Several studies have shown its effectiveness for fight against biofilms formed by clinical (Islam et al., 2022; Pedretti et al., 2024) or foodborne pathogens (Hamza et al., 2018; Park et al., 2023), but also against pathogenic fungus attacking plant crops (Gao et al., 2017; Schmiedeknecht et al., 1998; Zhao et al., 2022). This cell-free supernatant is a promising alternative to traditional antimicrobial or anti-biofilm molecules having potential issues related to environmental pollution, microbial resistance and human health. In fact, it is filled with antimicrobial arsenal non-ribosomal peptides (i.e. iturin, surfactin, fengycin) (Puan et al., 2023; Ranjan et al., 2023; Tran et al., 2022). Moreover, the *B. amyloliquefaciens* extracellular medium contains various enzymes including α-amylase, proteases, lipases, cellulases, xylanases, pectinases, aminotransferases, barnases, peroxidases, and laccases (Farhat-Khemakhem et al., 2018; Ngalimat et al., 2021).

Results from the whole genome sequence of *B. amyloliquefaciens* L-17 revealed that it could be a potential strain for study of biofilm formation, and for investigation in biocontrol agents against plant pathogens and enzymes production (Zaidi-Ait Salem et al., 2021a). Zaidi-Ait Salem et al. (2021b) underlined the ability of L-17 strain to survive on air-liquid interfaces forming thick floating biofilms. They highlighted that the mature pellicle exhibits rough hydrophobic surface on the air interface and a smooth hydrophilic texture on the surface in contact with the liquid. The same authors reported that the pellicle matrix is composed of loosely and tightly bound polysaccharides and proteins providing the pellicle cohesion and architecture (Zaidi-Ait Salem et al., 2021b). Continuing this work, we have firstly investigated the proteome of the pellicle matrix of *B. amyloliquefaciens* L-17 strain. Therefore, we combined the physical and chemical protocol matrix extraction and the high accuracy tandem mass spectrometry analysis to have the first board view

of matrix proteins of L-1-7 strain. As far as we know, no proteomic study has been previously carried out on the matrix of mature biofilm or pellicles. In the second part of this present study, we performed, for the first time, a deep proteomic characterization of the CFLP to establish an initial repertoire of interest proteins in *B. amyloliquefaciens* L-17 liquid phase environment.

## 2. Materials and methods

### 2.1. Bacterial strain and growth conditions

*Bacillus amyloliquefaciens* L-17 was provided by the Culture Collection of the Laboratoire de Biotechnologies Agroalimentaire et Environnementale (culture collection WDCM 1016, LBAE-UPS, Auch, France). An overnight culture in rich trypticase soya broth (TSB) medium was performed at 30 °C and shaking at 150 rpm. This pre-culture was used to inoculate a glucose, fructose minimal medium (MMGF) (glucose 10 g·L⁻¹, fructose 10 g·L⁻¹, MgSO₄ 0.05 g·L⁻¹, C₆H₁₁FeNO₇(Ferric ammonium citrate) 0.025 g·L⁻¹, H₃BO₃ 3 × 10⁻⁵ g·L⁻¹, CoCl₂·6H₂O 21 × 10⁻⁵ g·L⁻¹, ZnSO₄·7H₂O 11 × 10⁻⁵ g·L⁻¹, MnCl₂·4H₂O 4 × 10⁻⁶ g·L⁻¹, CuSO₄·5H₂O 2 × 10⁻⁶ g·L⁻¹, NiCl₂·6H₂O 1 × 10⁻⁶ g·L⁻¹, Na₂MO₄·2H₂O 3 × 10⁻⁶ g·L⁻¹, Na₂HPO₄ 4.33 g·L⁻¹, K₂HPO₄ 2.65 g·L⁻¹, CaCl₂ 0.0625 g·L⁻¹, NH₄Cl 6 g·L⁻¹, pH = 7) at final concentration of 10⁷ CFU·mL⁻¹. Pellicles in triplicate were formed in 1 L Erlenmeyer flasks containing 400 mL of culture medium for 72 h at 30 °C under static conditions.

### 2.2. EPSs extraction by physical and chemical methods

The extraction of pellicle EPSs is based on the combination of sequenced physical and chemical extractions. As previously described, the pellicle obtained at the air-liquid interface was recovered by forceps and was washed three times with PBS. The underlying liquid phase was used for a parallel proteomic analysis. The pellicle was then suspended in 20 mL of PBS (2 g VSS mL⁻¹) and ground with an ULTRA-TURRAX homogenizer (3 × 15 s and 8000 rpm), aiming to release the loosely bound EPSs (LB-EPSs). After centrifugation of the suspension (20,000 g, 15 min and 4 °C), the supernatant containing the LB-EPSs proteins was recovered and the pellet was resuspended in 20 mL NaOH (0.1 M) to extract tightly bound EPSs (TB-EPSs). After incubation (60 min, 30 °C), the supernatant was separated from the remaining insoluble materials by centrifugation (20,000 g, 15 min and 4 °C) and the obtained soluble extract (TB-EPSs) was recovered. In each LB-EPSs and TB-EPSs fraction, protein concentration was evaluated using the Bradford assay. Then, the LB-EPSs and TB-EPSs were stored at -20 °C for further analysis.

### 2.3. Cell-free liquid phase proteins (CFLP) precipitation

The liquid phase underlying the pellicle was centrifuged (10,000 x g, 20 min, 4 °C) to remove planktonic bacteria. The proteins of this cell-free liquid phase (CFLP) were precipitated with trichloroacetic acid (TCA). One volume of TCA 100 % (w/v) was added to 4 vol of bacteria-free growth medium. After incubation for 15 min at 4 °C, the protein pellet was collected by centrifugation (10,000 x g, 20 min, 4 °C), and washed with ice-cold acetone. Proteins were solubilized in buffer containing Tris-HCl 20 mM, NaCl 150 mM. Protein concentration was evaluated using the Bradford assay, then the sample was stored at -20 °C.

### 2.4. In gel protein digestion

Twenty-five µg of proteins from either the LB-EPSs, TB-EPSs and CFLP fractions were mixed with SDS loading buffer (63 mM Tris-HCl, pH 6.8, 10 mM DTT, 2 % SDS, 0.02 % bromophenol blue, 10 % glycerol), then loaded onto an SDS-PAGE stacking gel (7 %). A short electrophoresis was performed (10 mA, 2 h). After migration, the gels were stained with Coomassie blue and destained with a solution containing 50 %
ethanol, 10 % acetic acid and 40 % deionized water. The revealed protein band from each fraction was excised, washed with water, and then immersed in reductive medium (5 mM DTT). Cysteines were irreversibly alkylated with 25 mM iodoacetamide in the dark. Following washing steps in water, gel bands were submitted to protein digestion with trypsin (2 µg per each band). The digestion was achieved after 3 h at 37 °C. Several steps of peptide extraction were performed in H₂O / CH₂CN/trifluoroacetic acid (TFA) mixtures (49.5 / 49.5 / 1). The peptide mixtures were dried and stored at -20 °C until MS analysis.

## 2.5. Tandem mass spectrometry

All experiments were done on an LTQ-Orbitrap Elite coupled with an Easy nLC II system (both from Thermo Scientific) and were carried out in three biological replicates. The samples (0.2 µg) were injected onto an enrichment column (C18 PepMap100, Thermo Scientific). The separation was achieved with an analytical column needle (NTCC-360/internal diameter: 100 µm; particle size: 5 µm; length: 153 mm, NikkyoTechnos, Tokyo, Japan). The mobile phase consisted of H₂O/0.1 % FA (buffer A) and ACN/FA 0.1 % (buffer B). Tryptic peptides were eluted at a flow rate of 300 nL/min using a three-step linear gradient: from 2 to 40 % B over 75 min, from 40 to 80 % B in 4 min and 11 min at 80 % B. The mass spectrometer was operated in positive ionization mode with a capillary voltage and a source temperature set at 1.5 kV and 275 °C, respectively. The samples were analyzed using the CID (collision induced dissociation) method. The first scan (MS spectra) was recorded in the Orbitrap analyzer (R = 60,000) with the mass range *m/z* 400–1800. Then, the 20 most intense ions were selected for MS² experiments. Singly charged species were excluded for MS² analysis. Dynamic exclusion of already fragmented precursor ions was applied for 30 s, with a repeat count of 1, a repeat duration of 30 s and an exclusion mass width of ± 10 ppm. Fragmentation occurred in the linear ion trap analyzer with collision energy of 35. All measurements in the Orbitrap analyzer were performed with on-the-fly internal recalibration (lock mass) at *m/z* 445.12002 (polydimethylcyclosiloxane).

## 2.6. Database searches

Raw data files were processed using Proteome Discoverer 1.4 software (Thermo Scientific). Peak lists were searched using the MASCOT search engine (Matrix Science) against *B. amyloliquefaciens* L-17 National Center for Biotechnology Information (NCBI) database (with 4001 entries). Database searches were performed with the following parameters: 2 missed trypsin cleavage sites allowed; variable modifications: carbamidomethylation of cystein, oxidation of methionine. The parent ion and daughter ion tolerances were 5 ppm and 0.35 Da, respectively. For the identification, a false discovery rate below 1 % was selected.

## 2.7. Label-free quantitative and statistical analyses

Label-free quantitative analysis was achieved with spectral counting method in which the abundance of peptide is measured as the number of Peptide Spectral Matching (PSM) events. A protein abundance is measured by the total number of MS/MS spectra associated with it from all its supporting peptides (Lundgren et al., 2010; Ning et al., 2012). Thus, protein abundance was analyzed for the 10 proteins having the highest PSM in each fraction (TB, LB and CFLP). The PSMs were normalized and ANOVA test with significant differences were considered at *p* < 0.05 (Prism 5.0, GraphPad Software, Inc.). The PSMs corresponding to each protein in the different replicates of TB, LB and CFLP samples are provided in the Supplementary information (Table SI).

## 2.8. Subcellular localization, biological function and protein-protein interaction network

Subcellular localization, molecular function and biological process

data were acquired after BLAST sequence similarity search for the identified proteins, and from the UniProtKB database (www.uniprot.org). The functional categories of the identified proteins were achieved by Clusters of Orthologous Genes (COGs) database (http://www.ncbi.nlm.nih.gov/COG/)(Galperin et al., 2021) The protein-protein interaction network, including both physical interactions and functional associations (Szkarczyk et al., 2023), was analyzed using STRING (v 12.0) database (https://string-db.org/) with high confidence (0.7) parameter.

# 3. Results and discussion

## 3.1. Identification of pellicle associated proteins

*B. amyloliquefaciens* L-17 bacteria colonize the air liquid interfaces and coats themselves with exopolymeric substances (EPSs) of their own to form a floating biofilm. Zaidi-Ait Salem et al. (2021b) highlighted the importance of proteins in the aggregation and cohesion of this matrix yet rich in polysaccharide.

In this study, we implemented a protocol combining both an ULTRATURRAX grinding aiming to release the loosely bound (LB) EPSs and NaOH treatment targeting tightly bound (TB) EPSs. In fact, in biofilms, the LB-EPSs form a loose dispersible slime layer without a distinct boundary. The inner TB-EPSs layer remains tightly packed surrounding the cells (Mahto et al., 2022). Here, we carried out, for the first time, an in-depth proteomic analysis to obtain a global overview of the matrix proteins of this strain. The proteomic strategy is represented in Fig. 1a. After database research, 87 and 62 proteins were identified in LB and TB fractions respectively (Table 1). This result reveals that proteins are distributed in the LB and TB EPS layers and fulfil various functions such as structuring, defense, enzymatic metabolism, etc.

The SipW-dependent-type signal peptide (WP_015388008.1), homologous to *B. subtilis* biofilm matrix protein TasA (84 % sequence identity), is over-presented in both LB and TB fractions (Fig. 2a and b). This result is not surprising since this protein has already been characterized as the major protein component of the *B. subtilis* biofilm matrix, where it has been reported to form functional amyloid fibers contributing to biofilm structure and stability (Böhning et al., 2022). Among the major proteins identified in LB and TB fractions, we also found the DUF1016 domain-containing protein (WP_003152178.1) having 74 % sequence identity with *B. subtilis* 168 biofilm surface layer hydrophobin BsIA (Fig. 2a and b). This protein is required for hydrophobicity and architecture of *B. subtilis* biofilms (Arnaouteli et al., 2016). Interestingly, the flagellin (WP_015387631.1), *hag* gene product, is among the major proteins in LB-fraction (Fig. 2a). This can be explained by the fact that some bacteria regain their motility to enter a planktonic lifestyle in the liquid phase. In *B. thuringiensis* 407, Houry et al. (2012), discovered that a subpopulation consisting of 0.1–1 % of cells within a biofilm, referred to as stealth swimmers, could remain motile thanks to their flagella creating transient tunnels highlighting the reversible elastic properties of the matrix. The over-presence of flagellin in the LB-fraction might suggest the presence of stealth swimmers in *B. amyloliquefaciens* L-17 pellicle. This hypothesis could be confirmed using time-lapse confocal laser microscopy at regular intervals to visualize and highlight the involvement of flagella in biofilm structuring or dispersion.

Of the proteins identified, 18 (13 %) were found in both LB and TB fractions (Table 1) and Forty-four proteins (34 %) were specifically identified in the TB-fraction and 69 (53 %) in the LB-fraction (Fig. 2c). This may suggest that the pellicle of *B. amyloliquefaciens* L-17 is more provided in loosely bound proteins that were detached by mechanical disruption of the pellicle. As far as we know, no work using the EPSs sequential extraction to characterize other monospace biofilm models has been reported in the literature. This strategy is often used to study multispecies biofilms such as granular activated sludge (Bou-Sarkis et al., 2024; Caudan et al., 2012) and membrane bioreactors (Lin et al., 2014; Teng et al., 2020). For example, in a membrane bioreactor, Teng
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Schematic representation of experimental workflow for analysis of (a) pellicle and (b) liquid phase associated proteins in *B. amyloliquefaciens* L-17. LB Loosely Bound, TB: Tightly Bound, EPSs: Extra-Polymeric Substances, CFLP: Cell Free Liquid Phase.</figcaption></figure>

et al. (2020) showed that the LB-EPSs had a higher ratio of proteins to polysaccharides than TB-EPSs. Yu et al. (2010) found that LB-EPSs fraction contained much higher proteins than TB-EPSs fraction in aerobic granules. It is widely accepted that sample preparation is an essential stage affecting the overall efficiency of proteomic studies. Prefractionation method allows to visualize a deeper picture of proteome and promoted identification of the less abundant proteins which are buried in the complex mixture (Duong and Lee, 2023). Our results support this, since 131 unique proteins were identified in the pellicle matrix of *B. amyloliquefaciens* L-17 (Table 1) thanks to combining physical and chemical EPSs extraction steps. An additional step extraction, based on a robust urea treatment, would be necessary to access to highly aggregated proteins of amyloid and/or hydrophobic type (Zaidi-Ait Salem et al., 2021b).

We compared our results with the only proteomic study that highlights proteins involved in the first phase of biofilm formation or in *situ* roots colonization in *B. amyloliquefaciens* SQR9 (Qiu et al., 2014). The discussed proteins related to SQR9 strain biofilm formation are not identified in the L-17 strain. This might be explained by the difference in experimental conditions, model biofilms, bacterial strains, and proteomic strategies used in the two studies. We have therefore extended our comparison to other species close to *B. amyloliquefaciens*, such as *B. Subtilis*.

## 3.2. Description of pellicle associated proteins

Subcellular localization, molecular function and biological process data of the identified proteins were obtained from the UniProtKB database (www.uniprot.org) (Table 1). However, we were faced with the lack of directly available data for the proteins of the *B. amyloliquefaciens* L-17 strain. We did BLAST sequence similarity searches for each of the 131 identified proteins via Uniprot database. We noticed that most of these proteins (77 %) have a strong homology (up to 70 % sequence identity) with those of *B. subtilis* 168 strain for which the databases are more informed (Table 1). It is known that these two species are genetically very close even though physiologically and

biochemically they are clearly distinct (Ngalimat et al., 2021; Welker and Campbell, 1967; Zhang et al., 2016). We therefore focused on this homology to collect data on the cellular location, molecular function, biological process, and protein-protein interaction of identified proteins. For the 29 proteins having low homology (<70 % sequence identity) with that of *B. subtilis* 168, we considered that this information is unknown (Table 1).

### 3.2.1. Subcellular localization of pellicle associated proteins

Among the identified matrix pellicle proteins, 13 % were predicted with an extracellular region localization (Fig. 3a). These include, among others, the major proteins TasA, BslA and flagellin (hag gene product). Some proteins (8 %) were predicted with dual localization. For example, the glycoside hydrolase family 3 protein (WP_015388768.1), homologous to the Beta-hexosaminidase NagZ of *B. subtilis* 168 (75 % sequence identity), is predicted with both extracellular and cytoplasmic subcellular locations. This protein is associated with peptidoglycan recycling, for which a crucial role in modulating biofilm accumulation has been demonstrated in *Neisseria gonorrhoeae* (Bhoopalan et al., 2016). Otherwise, we found that the pellicle matrix contains a large fraction of cytoplasmic (23 %) and membrane proteins (10 %) including plasma membrane, periplasmic space and cell-surface bounded proteins. These results implicate the involvement of cell lysis in modulating biofilm proteome composition (Fong and Yildiz, 2015). Several studies reported that *B. subtilis* can sporulate within the biofilm matrix (Azulay et al., 2021; Faille et al., 2014; Srinivasan et al., 2018). The spores are found enriched at the tips of the so-called fruiting bodies which are small visible aerial projections that rise from the surface of a biofilm (Branda et al., 2001). Interestingly, we emphasize that few proteins (5 %) are associated with the coat or wall spore, suggesting that *B. amyloliquefaciens* L-17 could also sporulate inside the pellicle matrix. Microscopic analysis seems to be necessary to confirm this and provide more information on the distribution of spores within the pellicle. We also note that the location of several proteins (41 %) remained unknown.
<table><caption>**Table 1**

List of identified proteins in pellicle matrix of *B. amyloliquefaciens* L-17 showing protein distribution in LB and TB fractions and their cellular localization and functions.</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with <i>B. subtilis</i> 168 proteins</th><th>COG symbol<sup>h</sup></th><th>LB-fraction<sup>i</sup></th><th>TB-fraction<sup>j</sup></th></tr></thead><tbody><tr><td colspan="10"><strong>Amino acid transport and metabolism</strong></td></tr><tr><td>WP_003154560.1</td><td>aminopeptidase</td><td>ampS</td><td>aminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>87</td><td>E</td><td>x</td><td></td></tr><tr><td>WP_003154169.1</td><td>aspartate-semialdehyde dehydrogenase</td><td>comER</td><td>pyrroline-5-carboxylate reductase activity</td><td>L-proline biosynthetic process</td><td>unknown</td><td>79</td><td>E</td><td>x</td><td></td></tr><tr><td>WP_014305170.1</td><td>gamma-glutamyltransferase</td><td>ggt</td><td>proteolysis/glutathione hydrolase activity</td><td>leukotriene C4 gamma-glutamyl transferase activity</td><td>extracellular region</td><td>98</td><td>E</td><td>x</td><td>x</td></tr><tr><td>WP_003154048.1</td><td>type I glutamate-ammonia ligase</td><td>glnA</td><td>ligase (glu, ATP, nucluotid, Mg, metal binding)</td><td>nitrogen catabolite repression of transcription/ negative regulation of DNA-templated transcription/ cellular response to nitrogen levels</td><td>unknown</td><td>97</td><td>E</td><td></td><td>x</td></tr><tr><td>WP_015388449.1</td><td>peptide ABC transporter substrate-binding protein</td><td>oppA</td><td>peptide transmembrane transporter activity</td><td>protein transport/ establishment of competence for transformation/ sporulation</td><td>cell membrane</td><td>88</td><td>E</td><td></td><td>x</td></tr><tr><td>WP_003151992.1</td><td>leucyl aminopeptidase</td><td>pepA</td><td>metalloaminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>76</td><td>E</td><td>x</td><td></td></tr><tr><td>WP_015388480.1</td><td>3-phosphoserine/ phosphohydroxythreonine transaminase</td><td>serC</td><td>aminotransferase/O-phospho-L-serine:2-oxoglutarate aminotransferase activity/pyridoxal phosphate binding</td><td>amino-acid biosynthesis</td><td>cytoplasm</td><td>85</td><td>EH</td><td></td><td>x</td></tr><tr><td colspan="10"><strong>Carbohydrate transport and metabolism</strong></td></tr><tr><td>WP_015388729.1</td><td>starch-binding protein</td><td>amyE</td><td>alpha-amylase activity/glycosidase/ hydrolase</td><td>carbohydrate metabolism</td><td>extracellular region</td><td>86</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_003151102.1</td><td>fructose-bisphosphate aldolase</td><td>fbaA</td><td>fructose-bisphosphate aldolase activity/tagatose-bisphosphate aldolase activity</td><td>lyase/glycolysis/sporulation</td><td>cytoplasm</td><td>99</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_003151611.1</td><td>type I glyceraldehyde-3-phosphate dehydrogenase</td><td>gapA</td><td>Oxidoreductase/NADP binding</td><td>glycolysis</td><td>unknown</td><td>97</td><td>G</td><td>x</td><td></td></tr><tr><td>WP_015388768.1</td><td>glycoside hydrolase family 3 protein</td><td>nagZ</td><td>beta-N-acetylhexosaminidase activity</td><td>peptidoglycan biosynthetic process</td><td>extracellular region/plasma membrane</td><td>75</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_015387727.1</td><td>oxalate decarboxylase family bicupin</td><td>oxdC</td><td>oxalate decarboxylase activity</td><td>oxalate metabolic process</td><td>periplasmic space/ cytoplasm</td><td>87</td><td>G</td><td>x</td><td>x</td></tr><tr><td>WP_015388558.1</td><td>pectate lyase</td><td>pel</td><td>pectate lyase activity/metal ion binding</td><td>pectin catabolic process</td><td>extracellular region</td><td>76</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_014306184.1</td><td>glycoside hydrolase family 68 protein</td><td>sacB</td><td>unknown</td><td>carbohydrate metabolism</td><td>unknown</td><td>90</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_003151103.1</td><td>fructose-6-phosphate aldolase</td><td>tal</td><td>aldehyde-lyase activity</td><td>carbohydrate metabolic process</td><td>cytoplasm</td><td>90</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_015388167.1</td><td>carbohydrate-binding protein</td><td>xynD</td><td>glycosidase/hydrolase</td><td>carbohydrate metabolism/ polysaccharide degradation/ xylan degradation</td><td>extracellular region</td><td>90</td><td>G</td><td></td><td>x</td></tr><tr><td>WP_003150769.1</td><td>glycoside hydrolase family 16 protein</td><td>ydhD</td><td>glycosidase/hydrolase/chitin binding</td><td>carbohydrate metabolism/ polysaccharide degradation/ sporulation</td><td>extracellular region/spore wall</td><td>72</td><td>GE</td><td></td><td>x</td></tr></tbody></table>

Cell cycle control, cell division, chromosome partitioning, Inorganic ion transport and

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<table><caption>Table 1 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>LB-fraction<sup>i</sup></th><th>TB-fraction<sup>j</sup></th></tr></thead><tbody><tr><td colspan="10"><strong>metabolism, Cell wall/ membrane/envelope biogenesis</strong></td></tr><tr><td>WP_003155789.1</td><td>multicopper oxidase family protein</td><td>cotA</td><td>oxidoreductase</td><td>sporulation</td><td>periplasmic space</td><td>77</td><td>D</td><td>x</td><td></td></tr><tr><td>WP_015387430.1</td><td>spore coat protein</td><td>cotF</td><td>unknown</td><td>sporulation</td><td>spore coat</td><td>71</td><td>D</td><td>x</td><td>x</td></tr><tr><td>WP_015388201.1</td><td>N-acetylmuramoyl-L-alanine amidase</td><td>cwlC</td><td>N-acetylmuramoyl-L-alanine amidase activity/peptidoglycan binding/ hydrolase</td><td>peptidoglycan catabolic process/sporulation</td><td>extracellular region/spore wall</td><td>77</td><td>D</td><td>x</td><td>x</td></tr><tr><td>WP_003153438.1</td><td>stage IV sporulation protein A</td><td>spoIVA</td><td>ATP hydrolysis activity</td><td>sporulation</td><td>spore wall/ endospore cortex</td><td>96</td><td>D</td><td>x</td><td></td></tr><tr><td>WP_003155477.1</td><td>gamma-type small acid-soluble spore protein</td><td>sspE</td><td>unknown</td><td>sporulation</td><td>unknown</td><td>84</td><td>D</td><td>x</td><td></td></tr><tr><td>WP_003155170.1</td><td>hypothetical protein</td><td>yrpD</td><td>unknown</td><td>unknown</td><td>unknown</td><td>88</td><td>D</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Cell wall/membrane/envelope biogenesis</strong></td></tr><tr><td>WP_015388018.1</td><td>alanine racemase</td><td>alr2</td><td>catalyzes the interconversion of L-alanine and D-alanine. pyridoxal phosphate binding</td><td>D-alanine biosynthetic process/ peptidoglycan biosynthetic process</td><td>cytoplasm</td><td>84</td><td>M</td><td>x</td><td></td></tr><tr><td>WP_015387749.1</td><td>chitosanase</td><td>csn</td><td>glycosidase/hydrolase</td><td>carbohydrate metabolic process</td><td>extracellular region</td><td>88</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_003154166.1</td><td>4-hydroxy-tetrahydrodipicolinate synthase</td><td>dapA</td><td>4-hydroxy-tetrahydrodipicolinate synthase activity</td><td>mino-acid biosynthesis</td><td>cytoplasm</td><td>94</td><td>ME</td><td>x</td><td></td></tr><tr><td>WP_003155677.1</td><td>LTA synthase family protein</td><td>ltaS1</td><td>metal ion binding</td><td>cell wall organization</td><td>extracellular region/plasma membrane</td><td>85</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_032865795.1</td><td>glycoside hydrolase family 18 protein</td><td>sleL</td><td>glycosidase Hydrolase</td><td>sporulation</td><td>spore coat</td><td>78</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_015388671.1</td><td>hypothetical protein</td><td>Unknown</td><td>unknown</td><td>unknown</td><td>extracellular region</td><td>74</td><td>M</td><td>x</td><td></td></tr><tr><td>WP_003151206.1</td><td>endo-1,4-beta-xylanase XynA</td><td>xynA</td><td>glycosidase/Hydrolase</td><td>carbohydrate metabolism/ polysaccharide degradation/ xylan degradation</td><td>unknown</td><td>95</td><td>MG</td><td>x</td><td></td></tr><tr><td>WP_015388168.1</td><td>glucuronoxylanase xynC</td><td>xynC</td><td>glycosidase/Hydrolase</td><td>carbohydrate metabolism/ xylan degradation/ polysaccharide degradation/ sphingolipid metabolic process</td><td>extracellular region</td><td>98</td><td>M</td><td>x</td><td></td></tr><tr><td>WP_015388752.1</td><td>DUF1906 domain-containing protein</td><td>ybfG</td><td>unknown</td><td>unknown</td><td>cell membrane</td><td>84</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_003154742.1</td><td>glycoside hydrolase family 18 protein</td><td>ydhD</td><td>chitin binding chitin activity</td><td>chitin catabolic process/ carbohydrate metabolism/ sporulation</td><td>extracellular region</td><td>72</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_003150840.1</td><td>choloylglycine hydrolase family protein</td><td>yxeI</td><td>hydrolase</td><td>antibiotic resistance</td><td>unknown</td><td>76</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_003150773.1</td><td>DUF3298 and DUF4163 domain-containing protein</td><td>yxiT</td><td>unknown</td><td>unknown</td><td>unknown</td><td>84</td><td>M</td><td></td><td>x</td></tr><tr><td>WP_088005523.1</td><td>PQQ-binding-like beta-propeller repeat protein</td><td>yxaL</td><td>increases the processivity of the PcrA helicase but does not bind to DNA</td><td>extracellular region</td><td>unknown</td><td>76</td><td>M</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Defence mechanisms</strong></td></tr><tr><td>WP_004393188.1</td><td>peroxiredoxin</td><td>ahpC</td><td>antioxydant/oxidoreductase/ peroxidase/stress response</td><td>hydrogen peroxide catabolic process/response to oxidative stress</td><td>cytoplasm</td><td>98</td><td>V</td><td>x</td><td>x</td></tr></tbody></table>

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<table><caption>Table 1 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>LB-fraction<sup>i</sup></th><th>TB-fraction<sup>j</sup></th></tr></thead><tbody><tr><td>WP_007614902.1</td><td>alkyl hydroperoxide reductase subunit F</td><td>ahpF</td><td>oxidoreductase/translocase</td><td>electron transport</td><td>cell membrane</td><td>95</td><td>V</td><td></td><td>x</td></tr><tr><td>WP_015388172.1</td><td>cellulase family glycosylhydrolase</td><td>ahpF</td><td>oxidoreductase/translocase</td><td>electron transport</td><td>cell membrane</td><td>95</td><td>V</td><td></td><td>x</td></tr><tr><td>WP_015387507.1</td><td>catalase</td><td>katE</td><td>catalase activity/heme binding</td><td>hydrogen peroxide catabolic process/response to oxidative stress</td><td>cytoplasm</td><td>84</td><td>V</td><td></td><td>x</td></tr><tr><td>WP_139,886,654.1</td><td>class A beta-lactamase</td><td>penP</td><td>hydrolase</td><td>antibiotic resistance</td><td>unknown</td><td>91</td><td>V</td><td></td><td>x</td></tr><tr><td>WP_152,514,787.1</td><td>multifunctional 2',3'-cyclic-nucleotide 2'-phosphodiesterase/3'-nucleotidase/5'-nucleotidase</td><td>yfkN</td><td>metal ion binding/nucleotide binding</td><td>nucleotide catabolic process</td><td>extracellular region/periplasmic space</td><td>74</td><td>VF</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Energy production and conversion</strong></td></tr><tr><td>WP_003151173.1</td><td>F0F1 ATP synthase subunit beta</td><td>atpD</td><td>unknown</td><td>unknown</td><td>cell membrane</td><td>96</td><td>C</td><td>x</td><td></td></tr><tr><td>WP_003151167.1</td><td>F0F1 ATP synthase subunit C</td><td>atpE</td><td>proton-transporting ATP synthase activity, rotational mechanism/lipid binding</td><td>ATP synthesis/Hydrogen /ion transport</td><td>plasma membrane/multi-pass membrane protein</td><td>100</td><td>C</td><td></td><td>x</td></tr><tr><td>WP_003152455.1</td><td>malate dehydrogenase</td><td>mdh</td><td>oxidoreductase</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>96</td><td>C</td><td></td><td>x</td></tr><tr><td>WP_015388516.1</td><td>FAD-binding oxidoreductase</td><td>ygaK</td><td>oxidoreductase activity/FAD binding</td><td>unknown</td><td>Unknown</td><td>85</td><td>C</td><td>x</td><td></td></tr><tr><td>WP_003156110.1</td><td>FAD-binding oxidoreductase</td><td>yvdP</td><td>oxidoreductase</td><td>sporulation</td><td>spore coat</td><td>98</td><td>C</td><td>x</td><td></td></tr><tr><td>WP_003151173.1</td><td>F0F1 ATP synthase subunit beta</td><td>atpD</td><td>unknown</td><td>unknown</td><td>cell membrane</td><td>96</td><td>C</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Inorganic ion transport and metabolism</strong></td></tr><tr><td>WP_003155700.1</td><td>spore coat protein CotJC</td><td>cotJC</td><td>spore coat composition</td><td>sporulation</td><td>Unknown</td><td>96</td><td>P</td><td>x</td><td></td></tr><tr><td>WP_003156778.1</td><td>ABC transporter substrate-binding protein</td><td>feuA</td><td>part of the ABC transporter complex FeuABC/YusV involved in import of the catecholate siderophores bacillibactin and enterobactin.</td><td>iron ion transport</td><td>Cytoplasm/cell membrane</td><td>83</td><td>P</td><td>x</td><td></td></tr><tr><td>WP_003155420.1</td><td>catalase KatA</td><td>katA</td><td>oxidoreductase/peroxidase</td><td>hydrogen peroxide catabolic process</td><td>cytoplasm</td><td>94</td><td>P</td><td>x</td><td>x</td></tr><tr><td>WP_012118382.1</td><td>methionine ABC transporter substrate-binding lipoprotein MetQ</td><td>metQ</td><td>unknown</td><td>Amino-acid transport</td><td>cell membrane</td><td>87</td><td>P</td><td>x</td><td>x</td></tr><tr><td>WP_003153772.1</td><td>superoxide dismutase</td><td>sodF</td><td>superoxide dismutase activity</td><td>unknown</td><td>cytoplasm</td><td>80</td><td>P</td><td></td><td>x</td></tr><tr><td>WP_014305921.1</td><td>right-handed parallel beta-helix repeat-containing protein</td><td>ywoF</td><td>unknown</td><td>unknown</td><td>extracellular region</td><td>74</td><td>P</td><td></td><td>x</td></tr><tr><td>WP_003156656.1</td><td>metal ABC transporter substrate-binding protein</td><td>znuA</td><td>metal ion binding</td><td>zinc ion transport/cell adhesion</td><td>plasma membrane</td><td>83</td><td>P</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Lipid transport and metabolism</strong></td></tr><tr><td>WP_003154310.1</td><td>acyl carrier protein</td><td>acpA</td><td>growing fatty acid chain in fatty acid biosynthesis</td><td>fatty acid metabolism</td><td>cytoplasm</td><td>100</td><td>I</td><td>x</td><td></td></tr><tr><td>WP_003156672.1</td><td>triacylglycerol lipase</td><td>estA</td><td>triglyceride lipase activity</td><td>lipid catabolic process</td><td>extracellular region</td><td>76</td><td>I</td><td>x</td><td></td></tr><tr><td>WP_003156315.1</td><td>glucose 1-dehydrogenase</td><td>gdh</td><td>oxidoreductase/glucose 1-dehydrogenase [NAD(P)] activity</td><td>sporulation</td><td>unknown</td><td>90</td><td>I</td><td>x</td><td></td></tr><tr><td>WP_003155182.1</td><td>glucose 1-dehydrogenase</td><td>yhxC</td><td>oxidoreductase</td><td>unknown</td><td>cytoplasm</td><td>92</td><td>I</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Nucleotide transport and metabolism/Inorganic ion transport and metabolism</strong></td></tr><tr><td>WP_003150717.1</td><td>IMP dehydrogenase</td><td>guaB</td><td>oxidoreductase</td><td>GMP biosynthesis/purine biosynthesis</td><td>cytoplasm</td><td>96</td><td>F</td><td>x</td><td></td></tr><tr><td>WP_015387718.1</td><td>assimilatory sulfite reductase (NADPH) flavoprotein subunit</td><td>cysJ</td><td>flavin adenine dinucleotide binding/ FMN binding</td><td>cysteine biosynthetic process/amino-acid biosynthesis</td><td>cytoplasm</td><td>78</td><td>FP</td><td>x</td><td></td></tr></tbody></table>

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<table><caption>Table 1 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>LB-fraction<sup>i</sup></th><th>TB-fraction<sup>j</sup></th></tr></thead><tbody><tr><td colspan="10"><strong>Post translational modification, protein turnover, chaperones</strong></td></tr><tr><td>WP_003155195.1</td><td>S8 family serine peptidase</td><td>aprE</td><td>serine protease</td><td>fibrinolysis/proteolysis/ sporulation</td><td>extracellular region</td><td>85</td><td>O</td><td>x</td><td></td></tr><tr><td>WP_003154079.1</td><td>S8 family peptidase</td><td>aprX</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region/cytoplasm</td><td>83</td><td>O</td><td></td><td>x</td></tr><tr><td>WP_015388265.1</td><td>S8 family serine peptidase</td><td>bpr</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>70</td><td>O</td><td>x</td><td>x</td></tr><tr><td>WP_014305002.1</td><td>HslU-HslV peptidase ATPase subunit</td><td>clpY</td><td>ATP hydrolysis activity</td><td>proteolysis</td><td>unknown</td><td>93</td><td>O</td><td>x</td><td></td></tr><tr><td>WP_004264608.1</td><td>ATP-dependent zinc metalloprotease FtsH</td><td>ftsH</td><td>ATP-dependent peptidase activity</td><td>protein catabolic process/cell cycle/sporulation</td><td>plasma membrane</td><td>96</td><td>O</td><td>x</td><td>x</td></tr><tr><td>WP_014304883.1</td><td>S8 family peptidase</td><td>isp</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>93</td><td>O</td><td>x</td><td></td></tr><tr><td>WP_003155497.1</td><td>serine protease</td><td>mpr</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>74</td><td>O</td><td></td><td>x</td></tr><tr><td>WP_003151600.1</td><td>hypothetical protein</td><td>nprB</td><td>metal ion binding</td><td>proteolysis</td><td>extracellular region</td><td>83</td><td>O</td><td></td><td>x</td></tr><tr><td>WP_015388283.1</td><td>M4 family metallopeptidase</td><td>nprE</td><td>metalloendopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>83</td><td>O</td><td></td><td>x</td></tr><tr><td>WP_003155249.1</td><td>peptidylprolyl isomerase</td><td>prsA</td><td>protein folding AND TRANSPORT</td><td>peptidyl-prolyl cis-trans isomerase activity</td><td>plasma membrane</td><td>85</td><td>O</td><td>x</td><td></td></tr><tr><td>WP_014306056.1</td><td>S8 family serine peptidase</td><td>vpr</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>80</td><td>O</td><td>x</td><td>x</td></tr><tr><td>WP_007410388.1</td><td>ATP-dependent protease ATP-binding subunit ClpC</td><td>clpC</td><td>ATP hydrolysis activity/chaperone/repressor</td><td>chaperone/establishment of competence for transformation</td><td>cytoplasm</td><td>98</td><td>O</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Replication, recombination and repair</strong></td></tr><tr><td>WP_003153447.1</td><td>non-specific DNA-binding protein Hbs</td><td>hbs</td><td>DNA binding</td><td>chromosome condensation</td><td>cytoplasm</td><td>100</td><td>L</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Secondary metabolites biosynthesis, transport and catabolism</strong></td></tr><tr><td>WP_003151745.1</td><td>aldo/keto reductase</td><td>yvgN</td><td>aldo-keto reductase (NADP) activity</td><td>small molecule metabolic process</td><td>unknown</td><td>90</td><td>Q</td><td></td><td>x</td></tr><tr><td colspan="10"><strong>Signal transduction mechanisms</strong></td></tr><tr><td>WP_003153224.1</td><td>transporter substrate-binding domain-containing protein</td><td>artP</td><td>ligand-gated monoatomic ion channel activity</td><td>amino acid transport</td><td>plasma membrane</td><td>82</td><td>TE</td><td></td><td>x</td></tr><tr><td>WP_003154064.1</td><td>RNA chaperone Hfq</td><td>hfq</td><td>RNA binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>T</td><td>x</td><td>x</td></tr><tr><td>WP_015388734.1</td><td>TerD family protein</td><td>yceC</td><td>stress response</td><td>Stress response</td><td>cytoplasm</td><td>82</td><td>T</td><td></td><td>x</td></tr><tr><td>WP_003156648.1</td><td>TerD family protein</td><td>yceD</td><td>stress response</td><td>Unknown</td><td>unknown</td><td>93</td><td>T</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Transcription</strong></td></tr><tr><td>WP_003156143.1</td><td>cold shock protein CspC</td><td>cspC</td><td>nucleic acid binding</td><td>transcription/transcription regulation</td><td>cytoplasm</td><td>98</td><td>K</td><td></td><td>x</td></tr><tr><td colspan="10"><strong>Translation, ribosomal structure and biogenesis</strong></td></tr><tr><td>WP_004264654.1</td><td>50S ribosomal protein L25/ general stress protein Ctc</td><td>ctc</td><td>5S rRNA binding</td><td>translation</td><td>cytoplasm</td><td>71</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_007410399.1</td><td>elongation factor G</td><td>fusA</td><td>translation elongation factor activity</td><td>protein biosynthesis</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_003155605.1</td><td>type I methionyl aminopeptidase</td><td>mapB</td><td>initiator methionyl aminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>94</td><td>J</td><td></td><td>x</td></tr><tr><td>WP_004393063.1</td><td>50S ribosomal protein L9</td><td>rplI</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>91</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_003156433.1</td><td>50S ribosomal protein L10</td><td>rplJ</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 1 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>LB-fraction<sup>i</sup></th><th>TB-fraction<sup>j</sup></th></tr></thead><tbody><tr><td>WP_003156436.1</td><td>50S ribosomal protein L7/L12</td><td>rplL</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_003154288.1</td><td>50S ribosomal protein L19</td><td>rplS</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>91</td><td>J</td><td>x</td><td>x</td></tr><tr><td>WP_003152495.1</td><td>50S ribosomal protein L20</td><td>rplT</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_003152662.1</td><td>50S ribosomal protein L27</td><td>rpmA</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td></td><td>x</td></tr><tr><td>WP_003156464.1</td><td>30S ribosomal protein S10</td><td>rpsJ</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_003154296.1</td><td>30S ribosomal protein S16</td><td>RPSP</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td></td><td>x</td></tr><tr><td>WP_003156451.1</td><td>elongation factor Tu</td><td>tuf</td><td>translation elongation factor activity</td><td>translation</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td></tr><tr><td>WP_015387848.1</td><td>SGNH/GDSL hydrolase family protein</td><td>rpmE2</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>91</td><td>J</td><td></td><td>x</td></tr><tr><td>WP_003156431.1</td><td>50S ribosomal protein L1</td><td>rplA</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>91</td><td>J</td><td>x</td><td></td></tr><tr><td colspan="10"><strong>Unknown</strong></td></tr><tr><td>WP_003154023.1</td><td>lytic polysaccharide monooxygenase</td><td>ctc</td><td>Unknown/Ribonucleoprotein</td><td>unknown/translation</td><td>unknown/ cytoplasm</td><td>50</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003155052.1</td><td>peptide-binding protein</td><td>appA</td><td>peptide transmembrane transporter activity</td><td>peptide transport/ sporulation</td><td>periplasmic space</td><td>73</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003152178.1</td><td>DUF1016 domain-containing protein/biofilm surface layer hydrophobin BsIA</td><td>bslA/ yuaB</td><td>biofilm formation</td><td>unknown/Biofilm formation</td><td>extracellular region</td><td>74</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_003150988.1</td><td>hypothetical protein</td><td>bslB</td><td>surface hydrophobicity/minor role in biofilm architecture</td><td>Unknown/Biofilm formation</td><td>extracellular region</td><td>79</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_015388185.1</td><td>hypothetical protein</td><td>cotC</td><td>Unknown</td><td>Unknown/sporulation</td><td>unknown</td><td>45</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_003154111.1</td><td>outer spore coat protein CotE</td><td>cotE</td><td>identical protein binding</td><td>sporulation</td><td>spore coat</td><td>96</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_021734090.1</td><td>hypothetical protein</td><td>cotG</td><td>unknown</td><td>sporulation</td><td>Unknown</td><td>81</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_015388659.1</td><td>sporulation hydrolase CotR</td><td>cotR</td><td>hydrolase activity</td><td>lipid degradation/ sporulation</td><td>spore coat</td><td>96</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015388113.1</td><td>peptoglycan endopeptase</td><td>cwlS</td><td>lytic endotransglycosylase activity</td><td>cell wall biogenesis/ degradation/proteolysis</td><td>cell surface</td><td>68</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015388651.1</td><td>hypothetical protein</td><td>floT</td><td>unknown</td><td>unknown/regulation of cell shape</td><td>unknown</td><td>28</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_015387631.1</td><td>flagellin</td><td>hag</td><td>unknown/structural molecule activity</td><td>bacterial-type flagellum</td><td>unknown/ extracellular region</td><td>56</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003151908.1</td><td>hypothetical protein</td><td>mpr</td><td>unknown/serine-type endopeptidase activity</td><td>unknown/proteolysis</td><td>unknown/ extracellular region</td><td>31</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_015387670.1</td><td>carboxylesterase/lipase family protein</td><td>pnbA</td><td>Unknown/cholinesterase activity</td><td>unknown</td><td>unknown</td><td>63</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_003154135.1</td><td>stage V sporulation protein SpoVS</td><td>spoVS</td><td>nucleic acid binding</td><td>sporulation /cell division</td><td>unknown</td><td>100</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003155280.1</td><td>alpha/beta-type small acid-soluble spore protein</td><td>sspB</td><td>double-stranded DNA binding</td><td>DNA topological change/ sporulation</td><td>unknown</td><td>89</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_003154036.1</td><td>hypothetical protein</td><td>ssuD</td><td>unknown/alkanesulfonate monooxygenase activity</td><td>unknown/alkanesulfonate catabolic process</td><td>unknown</td><td>30</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_015388007.1</td><td>amyloid fiber anchoring/assembly protein TapA</td><td>tapA</td><td>unknown/required for the proper anchoring and polymerization of TasA amyloid fibers at the cell surface</td><td>extracellular region/bacterial biofilm matrix</td><td>unknown</td><td>47</td><td>Unknown</td><td></td><td>x</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 1 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>LB-fraction<sup>i</sup></th><th>TB-fraction<sup>j</sup></th></tr></thead><tbody><tr><td>WP_015388008.1</td><td>SipW-dependent-type signal peptide-containing protein/biofilm matrix protein TasA</td><td>tasA</td><td>major protein component of the biofilm extracellular matrix /amyloid fibers that bind cells together in the biofilm/antibacterial/spore coat assembly</td><td>sporulation</td><td>extracellular region/Forespore intermembrane space</td><td>84</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_015388356.1</td><td>phage tail sheath family protein</td><td>xkdK</td><td>unknown</td><td>unknown</td><td>unknown</td><td>86</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015388720.1</td><td>GTP-binding protein</td><td>yciC</td><td>GTP binding/hydrolase activity</td><td>unknown</td><td>unknown</td><td>84</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_015387422.1</td><td>DUF1259 domain-containing protein</td><td>ycxD</td><td>unknown/transaminase activity</td><td>unknown/alpha-amino acid metabolic process</td><td>unknown</td><td>27</td><td>Unknown</td><td>x</td><td>x</td></tr><tr><td>WP_014305702.1</td><td>hydrolase</td><td>yddQ</td><td>unknown/hydrolase activity</td><td>unknown</td><td>unknown</td><td>31</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_024084955.1</td><td>YdhK family protein</td><td>ydhK</td><td>unknown</td><td>unknown</td><td>unknown</td><td>61</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015388281.1</td><td>hypothetical protein</td><td>ylaE</td><td>unknown</td><td>unknown</td><td>unknown</td><td>52</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_014305020.1</td><td>insulinase family protein</td><td>ymfH</td><td>metallopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>86</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015388142.1</td><td>IseA DL-endopeptidase inhibitor family protein</td><td>yoeB</td><td></td><td></td><td>unknown</td><td>77</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_014304250.1</td><td>DUF4879 domain-containing protein</td><td>yolA</td><td></td><td></td><td>unknown</td><td>27</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003153543.1</td><td>hypothetical protein</td><td>yqgA</td><td>unknown</td><td>unknown</td><td>unknown/ extracellular region</td><td>52</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003152143.1</td><td>type 1 glutamine amidotransferase</td><td>yraA</td><td>unknown/peptidase activity</td><td>unknown/proteolysis</td><td>unknown</td><td>38</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015387776.1</td><td>YukJ family protein</td><td>yukJ</td><td>unknown</td><td>unknown</td><td>unknown</td><td>81</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_015387655.1</td><td>ribonuclease</td><td>yvaE</td><td>unknown/amino-acid betaine transmembrane transporter activity</td><td>unknown/transport</td><td>unknown/plasma membrane</td><td>43</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_015387689.1</td><td>M28 family peptidase</td><td>ywaD</td><td>unknown/peptidase activity</td><td>unknown/proteolysis</td><td>unknown/ extracellular region</td><td>48</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_003151188.1</td><td>VWA domain-containing protein</td><td>ywmC</td><td>unknown</td><td>unknown</td><td>unknown</td><td>66</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_187,650,984.1</td><td>hypothetical protein</td><td>ywmD</td><td>unknown</td><td>unknown</td><td>unknown</td><td>21</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_152,514,778.1</td><td>hypothetical protein</td><td>YycH</td><td>unknown/metal ion binding</td><td>unknown</td><td>unknown/ plasma membrane</td><td>25</td><td>Unknown</td><td></td><td>x</td></tr><tr><td>WP_003151740.1</td><td>hypothetical protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>81</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_003155828.1</td><td>DUF1906 domain-containing protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>49</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>WP_004393267.1</td><td>hypothetical protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>66</td><td>Unknown</td><td>x</td><td></td></tr><tr><td>Total</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>72</td></tr></tbody></table>

<footnote>a Accession number</footnote>

<footnote>b Protein name in the NCBI database for *B. amyloliquefaciens* L-17.</footnote>

<footnote>c Gene designation.</footnote>

<footnote>d Subcellular localization.</footnote>

<footnote>e molecular function and.</footnote>

<footnote>f biological process data obtained from the UniProKB database after BLAST sequence similarity searches.</footnote>

<footnote>g Identity (%) in UniProt Protein BLAST: percentage of identical amino acids between the *B. amyloliquefaciens* L-17 proteins sequence and that of *B. subtilis* 168.</footnote>

<footnote>h COG category symbol: functional categories according Clusters of Orthologous Genes (COG) database.</footnote>

<footnote>i LB-fraction: proteins loosely bound to the matrix that were detached by mechanical disruption of the pellicle.</footnote>

<footnote>j TB-fraction: proteins tightly bound to the matrix that were chemically extracted of the pellicle.</footnote>

<footnote>In green, the identified proteins in L-17 strain having low % identity (<70 %) with that of *B. subtilis* 168.</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Protein abundance assessment in the (a) loosely bound (LB) and (b) tightly bound (TB) fractions in *B. amyloliquefaciens* L-17 pellicle matrix. Analysis was performed by the spectral counting method and focused on the ten proteins with the higher PSM (Peptide Spectral Match) parameter in each fraction. (c) Venn Diagram showing the distribution of identified proteins in LB and TB fractions. LB fraction contains proteins detached by mechanical disruption of the pellicle. TB fraction includes proteins chemically extracted from the pellicle.</figcaption></figure>

### 3.2.2. Biological function of pellicle associated proteins

The 131 identified pellicle associated proteins were classified in clusters of orthologous genes (COGs) categories to have a clearer vision of their physiological role (Table 1). The distribution in various functional categories is summarized in Fig. 3b The protein-protein interaction network mapped by STRING database highlighted three main protein groups which will be discussed, in more depth, in the following sections (Fig. 3c).

### 3.2.3. Biofilm structuring proteins

As described in Section 3.1, TasA is the most prevalent protein in the analyzed pellicle of *B. amyloliquefaciens* L-17. In *B. subtilis* NCBI 3610, Romero et al. (2010) reported that TasA forms amyloid fibers which would provide structure to the extracellular matrix pellicle and, in conjunction with other EPSs components, hold the cells together. In the same strain, Steinberg et al. (2020) demonstrated that TasA acted as a signal stimulating a subset of biofilm cells to revert to a motile phenotype. Deletion of tasA has other effects, including the downregulation of

genes related to sporulation and an increase in expression of EPSs matrix and antimicrobial secondary metabolite-related genes (Cámara-Almirón et al., 2020; Khambhati et al., 2021). A similar regulation may be expected in *B. amyloliquefaciens* L-17 since flagellin and several sporulation-related proteins were identified in the pellicle matrix with the absence of gene products of the *epsA*-O operon, which oversees the production of EPSs matrix carbohydrates (Branda et al., 2001). However, the absence of the *epsA*-O operon products could also be linked to their loss during the analysis. Indeed, proteomics provides a global but non-exhaustive analysis of the proteins present in specific environments and time.

According to protein-protein interaction (STRING) parameters, TasA would interact with TapA protein (Fig. 3c). In this study, TapA (WP_015388007.1), the amyloid fiber anchoring/assembly protein TapA, was identified in the TB-fraction (Table 1). This protein forms a minor component of the TasA fibers and is required for their assembly in *B. subtilis* (Cairns et al., 2014). It has been reported that the *tapA* deletion strain is defective for pellicle formation and in the absence of *tapA*, TasA
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Distribution of proteins identified in *B. amyloliquefaciens* L-17 in the pellicle matrix according to their (a) cellular localization and (b) their functional categories. The cell membrane localization includes plasma membrane, periplasmic space and cell-surface bounded proteins. The category "Others" groups together functional categories with <3 proteins (i.e. transcription; replication, recombination and repair; nucleoid transport and metabolism; secondary metabolites biosynthesis, transport and catabolism). (c) Protein-protein interaction map of the identified pellicle associated proteins in the L-17 strain visualized by STRING program with high confidence parameter. The analysis was achieved using gene names, the corresponding protein names are reported in Table 1.</figcaption></figure>
<table><caption>**Table 2**
Proteins identified in the cell free liquid phase (CFLP) of *B. amyloliquefaciens* L-17 and their cellular localization and biological functions. Proteins identified in both CFLP and pellicle (LB and TB fractions) are highlighted with a cross.</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td colspan="11"><strong>Amino acid transport and metabolism</strong></td></tr><tr><td>WP_014305170.1</td><td>gamma-glutamyltransferase</td><td>ggt</td><td>glutathione hydrolase activity/ leukotriene C4 gamma-glutamyl transferase activity</td><td>proteolysis/ glutathione catabolic process</td><td>extracellular region</td><td>98</td><td>E</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003151173.1</td><td>F0F1 ATP synthase subunit beta</td><td>pepA</td><td>metalloaminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>76</td><td>E</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_015388449.1</td><td>peptide ABC transporter substrate-binding protein</td><td>oppA</td><td>peptide transmembrane transporter activity</td><td>protein transport/ establishment of competence for transformation/ sporulation</td><td>cell membrane</td><td>88</td><td>E</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154048.1</td><td>type I glutamate-ammonia ligase</td><td>glnA</td><td>Ligase (glu, ATP, nucluotid, Mg) metal binding</td><td>nitrogen catabolite repression of transcription/negative regulation of DNA-templated transcription/cellular response to nitrogen levels</td><td>unknown</td><td>97</td><td>E</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_015388480.1</td><td>3-phosphoserine/phosphohydroxythreonine transaminase</td><td>serC</td><td>Aminotransferase/O-phospho-L-serine:2-oxoglutarate aminotransferase activity/pyridoxal phosphate binding</td><td>amino-acid biosynthesis</td><td>cytoplasm</td><td>85</td><td>EH</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151992.1</td><td>leucyl aminopeptidase</td><td>pepA</td><td>metalloaminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>76</td><td>E</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154169.1</td><td>aspartate-semialdehyde dehydrogenase</td><td>comER</td><td>pyrroline-5-carboxylate reductase activity</td><td>L-proline biosynthetic process</td><td>unknown</td><td>79</td><td>E</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154560.1</td><td>aminopeptidase</td><td>ampS</td><td>aminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>87</td><td>E</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003150810.1</td><td>peptidase T</td><td>pepT</td><td>tripeptide aminopeptidase activity</td><td>proteolysis</td><td>cytoplasm</td><td>78</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388345.1</td><td>peptide ABC transporter substrate-binding protein</td><td>dppE</td><td>peptide transmembrane transporter activity</td><td>protein transport/ sporulation</td><td>cell membrane/ periplasmic space</td><td>79</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388451.1</td><td>ornithine carbamoyltransferase</td><td>argF</td><td>ornithine carbamoyltransferase activity</td><td>arginine biosynthetic process via ornithine</td><td>cytoplasm</td><td>82</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388348.1</td><td>M55 family metallopeptidase</td><td>dppA</td><td>aminopeptidase activity</td><td>proteolysis/ Sporulation</td><td>unknown</td><td>83</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153463.1</td><td>chorismate mutase</td><td>aroH</td><td>chorismate mutase activity</td><td>aromatic amino acid biosynthesis</td><td>cytoplasm</td><td>84</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156637.1</td><td>glycine/betaine ABC transporter</td><td>opuAC</td><td>transmembrane transporter activity</td><td>amino acid transport</td><td>cell membrane/ membrane raft</td><td>85</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387510.1</td><td>5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase</td><td>yxjG</td><td>5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase activity</td><td>methionine biosynthetic process</td><td>unknown</td><td>86</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_014305305.1</td><td>tryptophan synthase subunit beta</td><td>trpB</td><td>tryptophan synthase activity</td><td>tryptophan biosynthetic process</td><td>cytoplasm</td><td>86</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388136.1</td><td>glutamate synthase large subunit</td><td>gltA</td><td>glutamate synthase activity</td><td>amino-acid biosynthesis</td><td>unknown</td><td>88</td><td>E</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_007611083.1</td><td>aminotransferase A</td><td>dapX</td><td>transaminase activity</td><td>lysine biosynthetic process via diaminopimelate</td><td>cytoplasm</td><td>88</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388703.1</td><td>4-aminobutyrate-2-oxoglutarate transaminase</td><td>gabT</td><td>4-aminobutyrate transaminase activity</td><td>gamma-aminobutyric acid catabolic process</td><td>cytoplasm</td><td>88</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153679.1</td><td>lysine 2,3-aminomutase</td><td>kamA</td><td>lysine 2,3-aminomutase activity</td><td>L-lysine catabolic process to acetate</td><td>unknown</td><td>89</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387777.1</td><td>alanine dehydrogenase</td><td>ald</td><td>alanine dehydrogenase activity</td><td>L-alanine catabolic process/sporulation</td><td>cytoplasm// plasma membrane</td><td>89</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156640.1</td><td>glycine/proline betaine ABC transporter ATP-binding protein OpuAA</td><td>opuAA</td><td>ABC-type quaternary ammonium compound transporting activity</td><td>amino acid transport</td><td>cell membrane</td><td>90</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_007408146.1</td><td>3-isopropylmalate dehydratase small subunit</td><td>leuD</td><td>3-isopropylmalate dehydratase activity</td><td>amino acid biosynthetic process</td><td>cytoplasm</td><td>92</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155800.1</td><td>2,3-butanediol dehydrogenase</td><td>bdhA</td><td>(R,R)-butanediol dehydrogenase activity</td><td>butanediol biosynthetic process</td><td>extracellular region/ cytoplasm</td><td>93</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387765.1</td><td>threonine synthase</td><td>thrC</td><td>threonine synthase activity</td><td>amino-acid biosynthesis</td><td>cytoplasm</td><td>94</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151052.1</td><td>spermidine synthase</td><td>speE</td><td>spermidine synthase activity</td><td>spermidine biosynthetic process</td><td>cytoplasm</td><td>95</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151153.1</td><td>serine hydroxymethyltransferase</td><td>glyA</td><td>glycine hydroxymethyltransferase activity</td><td>amino-acid biosynthesis</td><td>cytoplasm</td><td>95</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152598.1</td><td>acetolactate synthase small subunit</td><td>ilvH</td><td>acetolactate synthase regulator activity</td><td>amino-acid biosynthesis</td><td>cytoplasm</td><td>98</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387606.1</td><td>acetolactate synthase AlsS</td><td>alsS</td><td>acetolactate synthase activity/flavin adenine dinucleotide binding</td><td>cytoplasm</td><td>cytoplasm</td><td>84</td><td>EH</td><td>x</td><td></td><td></td></tr><tr><td>WP_014304782.1</td><td>ABC transporter ATP-binding protein</td><td>appD</td><td>ATP hydrolysis activity</td><td>protein transport/ establishment of competence for transformation/ sporulation</td><td>plasma membrane</td><td>83</td><td>EP</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151199.1</td><td>urease subunit beta</td><td>ureB</td><td>urease activity</td><td>urea catabolic process</td><td>cytoplasm</td><td>76</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152866.1</td><td>late competence protein ComER</td><td>comER</td><td>pyrroline-5-carboxylate reductase activity</td><td>L-proline biosynthetic process</td><td>unknown</td><td>79</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387836.1</td><td>dipeptidase PepV</td><td></td><td>acetylornithine deacetylase activity</td><td>arginine biosynthetic process</td><td>unknown</td><td>80</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154692.1</td><td>cupin domain-containing protein</td><td>mtnD</td><td>acireductone dioxygenase [iron(II)- requiring] activity</td><td>methionine metabolic process</td><td>unknown</td><td>83</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152778.1</td><td>bifunctional cystathionine gamma-lyase/ homocysteine desulfhydrase</td><td>mccB</td><td>cystathionine gamma-synthase activity</td><td>cysteine biosynthetic process via cystathionine/ homocysteine catabolic process</td><td>cytoplasm</td><td>84</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388331.1</td><td>5- methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase</td><td>metE</td><td>5- methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase activity</td><td>amino-acid biosynthesis/ Methylation</td><td>extracellular region/ cytoplasm</td><td>89</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_014306158.1</td><td>arginase</td><td>rocF</td><td>arginase activity/manganese ion binding</td><td>arginine metabolic process/urea cycle</td><td>cytoplasm</td><td>91</td><td>E</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003151938.1</td><td>homoserine dehydrogenase</td><td>hom</td><td>homoserine dehydrogenase activity</td><td>amino-acid biosynthesis</td><td>cytoplasm/ extracellular region</td><td>93</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153209.1</td><td>branched-chain amino acid dehydrogenase</td><td>yqiT</td><td>glutamate dehydrogenase [NAD(P)+] activity/leucine dehydrogenase activity</td><td>leucine catabolic process</td><td>unknown</td><td>94</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387842.1</td><td>bifunctional 3-deoxy-7-phosphoheptulonate synthase/chorismate mutase</td><td>aroA</td><td>3-deoxy-7-phosphoheptulonate synthase activity</td><td>aromatic amino acid family biosynthetic process</td><td>unknown</td><td>95</td><td>E</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388257.1</td><td>carbamoyl-phosphate synthase (glutamine-hydrolyzing) large subunit</td><td>pyrAB</td><td>aspartate carbamoyltransferase activity/ ATP binding</td><td>amino-acid biosynthesis/ Pyrimidine biosynthesis</td><td>cytoplasm</td><td>92</td><td>EF</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388490.1</td><td>D-amino-acid transaminase</td><td>dat</td><td>D-alanine:2-oxoglutarate aminotransferase activity</td><td>D-amino acid biosynthetic process</td><td>cytoplasm</td><td>74</td><td>EH</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152600.1</td><td>ketol-acid reductoisomerase</td><td>ilvC</td><td>ketol-acid reductoisomerase activity/ magnesium ion binding/NADP binding</td><td>amino-acid biosynthesis</td><td>cytoplasm</td><td>96</td><td>EH</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Carbohydrate transport and metabolism</strong></td></tr><tr><td>WP_015387727.1</td><td>oxalate decarboxylase family bicupin</td><td>oxdC</td><td>oxalate decarboxylase activity</td><td>oxalate metabolic process</td><td>periplasmic space/ cytoplasm</td><td>87</td><td>G</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_021734196.1</td><td>M42 family metallopeptidase</td><td>yhfE</td><td>aminopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>83</td><td>GE</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003150769.1</td><td>glycoside hydrolase family 16 protein</td><td>yhdfD</td><td>glycosidase/Hydrolase/chitin binding</td><td>carbohydrate metabolism/ polysaccharide degradation/ sporulation</td><td>extracellular region/spore wall</td><td>72</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388558.1</td><td>pectate lyase</td><td>pel</td><td>pectate lyase activity/metal ion binding</td><td>pectin catabolic process</td><td>extracellular region</td><td>76</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388729.1</td><td>starch-binding protein</td><td>amyE</td><td>alpha-amylase activity/ glycosidase/ hydrolase</td><td>Carbohydrate metabolism</td><td>extracellular region</td><td>86</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388167.1</td><td>carbohydrate-binding protein</td><td>xynD</td><td>glycosidase/hydrolase</td><td>carbohydrate metabolism/ polysaccharide degradation/xylan degradation</td><td>extracellular region</td><td>90</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151102.1</td><td>fructose-bisphosphate aldolase</td><td>fbaA</td><td>fructose-bisphosphate aldolase activity/tagatose-bisphosphate aldolase activity</td><td>lyase/glycolysis/ sporulation</td><td>cytoplasm</td><td>99</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388172.1</td><td>cellulase family glycosylhydrolase</td><td>eglS</td><td>beta-glucosidase activity/cellulase activity</td><td>cellulose catabolic process</td><td>extracellular region/Cell surface</td><td>94</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151103.1</td><td>fructose-6-phosphate aldolase</td><td>tal</td><td>aldehyde-lyase activity</td><td>carbohydrate metabolic process</td><td>cytoplasm</td><td>90</td><td>G</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151611.1</td><td>type I glyceraldehyde-3-phosphate dehydrogenase</td><td>gapA</td><td>oxidoreductase/NADP binding</td><td>glycolysis</td><td>unknown</td><td>97</td><td>G</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_015387871.1</td><td>glycoside hydrolase family 43 protein</td><td>abnA</td><td>arabinan endo-1,5-alpha-1- arabinosidase activity</td><td>arabinan catabolic process</td><td>extracellular region</td><td>84</td><td>G</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_021734251.1</td><td>carbohydrate kinase</td><td></td><td>kinase activity</td><td>phosphorylation</td><td>unknown</td><td>88</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388180.1</td><td>transketolase</td><td>tkt</td><td>transketolase activity</td><td>pentose-phosphate shunt</td><td>cytoplasm</td><td>90</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_007408647.1</td><td>glucose-6-phosphate isomerase</td><td>pgi</td><td>glucose-6-phosphate isomerase activity</td><td>glycolytic process</td><td>cytoplasm</td><td>94</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156759.1</td><td>phosphoglucosamine mutase</td><td>glmM</td><td>phosphoglucosamine mutase activity</td><td>peptidoglycan biosynthetic process</td><td>cytoplasm</td><td>95</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_007408096.1</td><td>M42 family metallopeptidase</td><td>ysdC</td><td>aminopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>90</td><td>GE</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152432.1</td><td>metal-dependent hydrolase</td><td>ytkL</td><td>hydrolase activity</td><td>unknown</td><td>unknown</td><td>92</td><td>GE</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388184.1</td><td>peptidase G2</td><td>yobO</td><td></td><td>unknown</td><td>unknown</td><td>81</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154656.1</td><td>glucose-specific PTS transporter subunit IIBC</td><td>ptsG</td><td>D-glucosamine PTS permease activity/glucose transmembrane transporter activity/kinase activity</td><td>phosphoenolpyruvate-dependent sugar phosphotransferase system</td><td>plasma membrane</td><td>93</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151620.1</td><td>2,3-bisphosphoglycerate-independent phosphoglycerate mutase</td><td>gpml</td><td>2,3-bisphosphoglycerate-independent phosphoglycerate mutase activity</td><td>sporulation/glycolysis</td><td>cytoplasm</td><td>96</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151616.1</td><td>phosphoglycerate kinase</td><td>pgk</td><td>phosphoglycerate kinase activity</td><td>glycolysis/phosphorylation</td><td>cytoplasm</td><td>98</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151618.1</td><td>triose-phosphate isomerase</td><td>tpiA</td><td>triose-phosphate isomerase activity</td><td>gluconeogenesis/glycerol catabolic process</td><td>cytoplasm</td><td>98</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153241.1</td><td>NADP-dependent phosphogluconate dehydrogenase</td><td>gndA</td><td>phosphogluconate 2-dehydrogenase activity/NADP binding</td><td>pentose-phosphate shunt, oxidative branch</td><td>unknown</td><td>99</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_007409956.1</td><td>phosphopyruvate hydratase</td><td>eno</td><td>phosphopyruvate hydratase complex</td><td>sporulation/Glycolysis</td><td>extracellular region/cytoplasm</td><td>99</td><td>G</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152370.1</td><td>M42 family metallopeptidase</td><td>ytoP</td><td>metallopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>88</td><td>GE</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Cell cycle control, cell division, chromosome partitioning, Inorganic ion transport and metabolism, Cell wall/ membrane/ envelope biogenesis</strong></td></tr><tr><td>WP_015387430.1</td><td>spore coat protein</td><td>cotF</td><td>Unknown</td><td>sporulation</td><td>spore coat</td><td>71</td><td>D</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388201.1</td><td>N-acetylmuramoyl-L-alanine amidase</td><td>cwlC</td><td>N-acetylmuramoyl-L-alanine amidase activity/peptidoglycan binding/hydrolase</td><td>peptidoglycan catabolic process/sporulation</td><td>extracellular region/spore wall</td><td>77</td><td>D</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003155789.1</td><td>multicopper oxidase family protein</td><td>cotA</td><td>oxidoreductase</td><td>sporulation</td><td>periplasmic space</td><td>77</td><td>D</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003155477.1</td><td>gamma-type small acid-soluble spore protein</td><td>sspE</td><td>unknown</td><td>sporulation</td><td>unknown</td><td>84</td><td>D</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003153438.1</td><td>stage IV sporulation protein A</td><td>spoIVA</td><td>ATP hydrolysis activity</td><td>sporulation</td><td>spore wall/ endospore cortex</td><td>96</td><td>D</td><td>x</td><td></td><td>x</td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003155170.1</td><td>hypothetical protein</td><td>spoIVA</td><td>ATP hydrolysis activity/</td><td>sporulation</td><td>spore wall/ endospore cortex plasma membrane</td><td>96</td><td>D</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388222.1</td><td>DUF4115 domain-containing protein</td><td>ymfM</td><td>DNA binding</td><td>unknown</td><td>Unknown plasma membrane</td><td>72</td><td>D</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387885.1</td><td>GerMN domain-containing protein</td><td>gerM</td><td>Unknown</td><td>sporulation</td><td>Unknown plasma membrane</td><td>82</td><td>D</td><td>x</td><td></td><td></td></tr><tr><td>WP_014304355.1</td><td>penicillin-binding transpeptidase domain-containing protein</td><td>pbpC</td><td>peptidoglycan L,D-transpeptidase activity/penicillin binding</td><td>cell wall organization/ regulation of cell shape</td><td>Unknown plasma membrane</td><td>76</td><td>DM</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152647.1</td><td>cell shape-determining protein MreB</td><td>mreB</td><td>ATP binding</td><td>cell morphogenesis</td><td>cytoplasm</td><td>90</td><td>D</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154424.1</td><td>cell division protein FtsZ</td><td>ftsZ</td><td>GTPase activity</td><td>cell division</td><td>cytoplasm</td><td>95</td><td>D</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Cell motility</strong></td></tr><tr><td>WP_003154244.1</td><td>flagellar basal body rod protein FlgG</td><td>flgG</td><td>bacterial-type flagellum assembly</td><td>bacterial-type flagellum-dependent swarming motility</td><td>bacterial flagellum</td><td>72</td><td>N</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Cell wall/ membrane/ envelope biogenesis</strong></td></tr><tr><td>WP_015388671.1</td><td>hypothetical protein</td><td>yxaL</td><td>unknown</td><td>unknown</td><td>extracellular region</td><td>74</td><td>M</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003155677.1</td><td>LTA synthase family protein</td><td>ltaS1</td><td>metal ion binding</td><td>cell wall organization</td><td>extracellular region/plasma membrane</td><td>85</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154166.1</td><td>4-hydroxy-tetrahydrodipicolinate synthase</td><td>dapA</td><td>4-hydroxy-tetrahydrodipicolinate synthase activity</td><td>mino-acid biosynthesis</td><td>cytoplasm</td><td>94</td><td>ME</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003150773.1</td><td>DUF3298 and DUF4163 domain-containing protein</td><td>yciT</td><td>unknown</td><td>unknown</td><td>unknown</td><td>84</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154742.1</td><td>glycoside hydrolase family 18 protein</td><td>ydhD</td><td>chitin binding chitin activity</td><td>chitin catabolic process/Carbohydrate metabolism/ sporulation</td><td>extracellular region</td><td>72</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_032865795.1</td><td>glycoside hydrolase family 18 protein</td><td>sleL</td><td>glycosidase Hydrolase</td><td>sporulation</td><td>spore coat</td><td>78</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388752.1</td><td>DUF1906 domain-containing protein</td><td>ybfG</td><td>unknown</td><td>unknown</td><td>cell membrane</td><td>84</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387749.1</td><td>chitosanase</td><td>csn</td><td>glycosidase/Hydrolase</td><td>carbohydrate metabolic process</td><td>extracellular region</td><td>88</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388168.1</td><td>glucuronoxylanase xynC</td><td>xynC</td><td>glycosidase/Hydrolase</td><td>carbohydrate metabolism/xylan degradation/ polysaccharide degradation/ sphingolipid metabolic process</td><td>extracellular region</td><td>98</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_088005523.1</td><td>PQQ-binding-like beta-propeller repeat protein</td><td>yxaL</td><td>Increases the processivity of the PcrA helicase, but does not bind to DNA.</td><td>extracellular region</td><td>extracellular region</td><td>76</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388018.1</td><td>alanine racemase</td><td>alr2</td><td>catalyzes the interconversion of L-alanine and D-alanine, pyridoxal phosphate binding</td><td>cytoplasm</td><td>cytoplasm</td><td>84</td><td>M</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151206.1</td><td>endo-1,4-beta-xylanase XynA</td><td>xynA</td><td>glycosidase/Hydrolase</td><td>D-alanine biosynthetic process/ peptidoglycan biosynthetic process</td><td>carbohydrate metabolism/</td><td>95</td><td>MG</td><td>x</td><td></td><td>x</td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_015388080.1</td><td>PBP1A family penicillin-binding protein</td><td>ponA</td><td>penicillin binding/peptidoglycan glycosyltransferase activity</td><td>Polysaccharide degradation/Xylan degradation</td><td>unknown</td><td>74</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151892.1</td><td>SIS domain-containing protein</td><td>frlB</td><td>glutamine-fructose-6-phosphate transaminase (isomerizing) activity</td><td>peptidoglycan biosynthetic process/ response to antibiotic</td><td>unknown</td><td>86</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388267.1</td><td>UDP-N-acetylmuramate dehydrogenase</td><td>murB</td><td>UDP-N-acetylmuramate dehydrogenase activity/flavin adenine dinucleotide binding</td><td>carbohydrate metabolism</td><td>cytoplasm</td><td>87</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154428.1</td><td>undecaprenyldiphospho-muramoylpentapeptide beta-N-acetylglucosaminyltransferase</td><td>murG</td><td>glycosyltransferase activity</td><td>cell cycle/cell division/cell shape/ peptidoglycan synthesis</td><td>cytoplasm</td><td>90</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152281.1</td><td>ABC transporter ATP-binding protein</td><td>ytrE</td><td>transmembrane transporter activity</td><td>transport</td><td>cell membrane/ peripheral membrane protein</td><td>92</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154436.1</td><td>UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase</td><td>murE</td><td>UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase activity</td><td>cell cycle/cell division/cell shape/ peptidoglycan synthesis</td><td>cytoplasm</td><td>78</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_014304858.1</td><td>N-acetylmuramoyl-L-alanine amidase</td><td>xlyA</td><td>N-acetylmuramoyl-L-alanine amidase activity</td><td>peptidoglycan catabolic process/ sporulation</td><td>extracellular region</td><td>80</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_003150715.1</td><td>D-alanyl-D-alanine carboxypeptidase</td><td>dacA</td><td>serine-type D-Ala-D-Ala carboxypeptidase activity</td><td>cell wall organization/ peptidoglycan biosynthetic process</td><td>extracellular region/plasma membrane plasma membrane</td><td>88</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156191.1</td><td>outer membrane lipoprotein carrier protein LolA</td><td>ydcC</td><td>unknown</td><td>sporulation</td><td>cytoplasm</td><td>90</td><td>M</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387544.1</td><td>dTDP-4-dehydrorhamnose 3,5-epimerase family protein</td><td>spsL</td><td>dTDP-4-dehydrorhamnose 3,5-epimerase activity</td><td>extracellular polysaccharide biosynthetic process/ dTDP-rhamnose biosynthetic process</td><td>cytoplasm</td><td>100</td><td>ME</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154355.1</td><td>extracellular matrix/biofilm regulator RemA</td><td>remA</td><td>DNA-binding</td><td>Transcription</td><td>unknown</td><td>77</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387611.1</td><td>Cof-type HAD-IIB family hydrolase</td><td>YwtE</td><td>magnesium ion binding/phosphatase activity</td><td>riboflavin biosynthetic process</td><td>cytoplasm</td><td>82</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387992.1</td><td>Nif3-like dinuclear metal center hexameric protein</td><td>yqfO</td><td>metal ion binding</td><td>unknown</td><td>cytoplasm</td><td>84</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155224.1</td><td>heme-degrading oxygenase HmoB</td><td>hmoB</td><td>heme oxygenase (decyclizing) activity</td><td>metal ion binding</td><td>cytoplasm</td><td>84</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388476.1</td><td>ferrochelatase</td><td>cpfC</td><td>ferrochelatase activity</td><td>heme biosynthetic process</td><td>cytoplasm</td><td>84</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_003150671.1</td><td>dihydroneopterin aldolase</td><td>folB</td><td>dihydroneopterin aldolase activity</td><td>tetrahydrofolate biosynthetic process</td><td>cytoplasm</td><td>89</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153510.1</td><td>aspartate 1-decarboxylase</td><td>panD</td><td>aspartate 1-decarboxylase activity</td><td>pantothenate biosynthetic process</td><td>cytoplasm</td><td>92</td><td>H</td><td>x</td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_015388486.1</td><td>Cof-type HAD-IIB family hydrolase</td><td>yhaX</td><td>magnesium ion binding/phosphatase activity</td><td>stress response</td><td>cytoplasm</td><td>87</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388517.1</td><td>phosphomethylpyrimidine synthase ThiC</td><td>thiC</td><td>4 iron, 4 sulfur cluster binding</td><td>thiamine biosynthetic process</td><td>cytoplasm</td><td>91</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153372.1</td><td>6,7-dimethyl-8-ribityllumazine synthase</td><td>ribH</td><td>6,7-dimethyl-8-ribityllumazine synthase activity</td><td>riboflavin biosynthetic process</td><td>cytoplasm</td><td>93</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153401.1</td><td>phosphoglycerate dehydrogenase</td><td>serA</td><td>formate dehydrogenase (NAD+) activity/phosphoglycerate dehydrogenase activity</td><td>L-serine biosynthetic process</td><td>cytoplasm</td><td>94</td><td>H</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152427.1</td><td>molybdenum cofactor biosynthesis protein MoaB</td><td>moaB</td><td>unknown</td><td>molybdenum cofactor biosynthesis</td><td>cytoplasm</td><td>82</td><td>HP</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151011.1</td><td>heme-dependent peroxidase</td><td>chdC</td><td>oxidoreductase/metal ion binding/peroxidase activity</td><td>heme biosynthetic process</td><td>unknown</td><td>94</td><td>HP</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152221.1</td><td>1,4-dihydroxy-2-naphthoyl-CoA synthase</td><td>menB</td><td>1,4-dihydroxy-2-naphthoyl-CoA synthase activity</td><td>menaquinone biosynthetic process</td><td>cytoplasm</td><td>96</td><td>HP</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152253.1</td><td>methionine adenosyltransferase</td><td>metK</td><td>methionine adenosyltransferase activity/magnesium ion binding</td><td>S-adenosylmethionine biosynthetic process</td><td>cytoplasm</td><td>98</td><td>HP</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Defense mechanisms</strong></td></tr><tr><td>WP_004393188.1</td><td>peroxiredoxin</td><td>ahpC</td><td>antioxydant/Oxidoreductase/ Peroxidase/Stress response</td><td>hydrogen peroxide catabolic process/ response to oxidative stress</td><td>cytoplasm</td><td>98</td><td>V</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015387507.1</td><td>catalase</td><td>katE</td><td>catalase activity/heme binding</td><td>hydrogen peroxide catabolic process/ response to oxidative stress</td><td>cytoplasm</td><td>84</td><td>V</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_139,886,654.1</td><td>class A beta-lactamase</td><td>penP</td><td>hydrolase</td><td>antibiotic resistance</td><td>unknown</td><td>91</td><td>V</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_007614902.1</td><td>alkyl hydroperoxide reductase subunit F</td><td>ahpF</td><td>oxidoreductase/Translocase</td><td>electron transport</td><td>cell membrane</td><td>95</td><td>V</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_152,514,787.1</td><td>multifunctional 2',3'-cyclic-nucleotide 2'-phosphodiesterase/3'-nucleotidase/5'-nucleotidase</td><td>yfkN</td><td>metal ion binding/nucleotide binding</td><td>nucleotide catabolic process</td><td>extracellular region/ periplasmic space</td><td>74</td><td>VF</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_014305062.1</td><td>dUTP diphosphatase</td><td>yncF</td><td>dUTP diphosphatase activity/ magnesium ion binding</td><td>nucleotide metabolism</td><td>unknown</td><td>87</td><td>VF</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388087.1</td><td>glutathione peroxidase</td><td>bsaA</td><td>phospholipid-hydroperoxide glutathione peroxidase activity</td><td>cellular response to oxidative stress</td><td>cytoplasm</td><td>84</td><td>VI</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151815.1</td><td>DNA starvation/stationary phase protection protein</td><td>mrgA</td><td>DNA-binding/oxidoreductase activity, acting on metal ions</td><td>stress response</td><td>unknown</td><td>75</td><td>VP</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152242.1</td><td>DNA starvation/stationary phase protection protein</td><td>dps</td><td>oxidoreductase activity, acting on metal ions</td><td>stress response</td><td>unknown</td><td>91</td><td>VP</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154758.1</td><td>organic hydroperoxide resistance protein</td><td>ohrB</td><td>unknown</td><td>stress response</td><td>unknown</td><td>81</td><td>V</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152408.1</td><td>GAF domain-containing protein</td><td>ytsP</td><td>unknown</td><td>stress response</td><td>cytoplasm</td><td>87</td><td>VT</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Energy production and conversion</strong></td></tr><tr><td>WP_003152455.1</td><td>malate dehydrogenase</td><td>mdh</td><td>oxidoreductase</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>96</td><td>C</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151167.1</td><td>F0F1 ATP synthase subunit C</td><td>atpE</td><td>proton-transporting ATP synthase activity, rotational mechanism/ lipid binding</td><td>ATP synthesis/ Hydrogen /ion transporton transport</td><td>plasma membrane</td><td>100</td><td>C</td><td>x</td><td>x</td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_015388516.1</td><td>FAD-binding oxidoreductase</td><td>ygaK</td><td>oxidoreductase activity/ FAD binding</td><td>unknown</td><td>unknown</td><td>85</td><td>C</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003156110.1</td><td>FAD-binding oxidoreductase</td><td>yvdP</td><td>oxidoreductase</td><td>sporulation</td><td>spore coat</td><td>98</td><td>C</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_015388129.1</td><td>molybdopterin oxidoreductase family protein</td><td>yoaE</td><td>iron-sulfur cluster binding</td><td>unknown</td><td>unknown</td><td>80</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154530.1</td><td>pyruvate dehydrogenase (acetyl-transferring) E1 component subunit alpha</td><td>pdhA</td><td>pyruvate dehydrogenase (acetyl-transferring) activity</td><td>acetyl-CoA</td><td>acetyl-CoA</td><td>86</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156323.1</td><td>antibiotic biosynthesis monooxygenase</td><td>ycnE</td><td>monooxygenase activity</td><td>biosynthetic process from pyruvate</td><td>cytoplasm</td><td>88</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153791.1</td><td>FMN-dependent NADH-azoreductase</td><td>azoR1</td><td>oxidoreductase activity, acting on NAD(P)H, NAD(P) as acceptor</td><td>aromatic compound catabolic process/ response to toxic substance</td><td>unknown</td><td>90</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153484.1</td><td>ubiquinol-cytochrome c reductase iron-sulfur subunit</td><td>qcrA</td><td>2 iron, 2 sulfur cluster binding</td><td>aromatic hydrocarbons catabolism/response to toxic substance</td><td>plasma membrane</td><td>91</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154490.1</td><td>pyruvate carboxylase</td><td>pyc</td><td>pyruvate carboxylase activity</td><td>oxidoreductase activity</td><td>cytoplasm</td><td>91</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_014305704.1</td><td>iron-containing alcohol dehydrogenase</td><td>yugJ</td><td>alcohol dehydrogenase (NADP<sup>+</sup>) activity</td><td>pyruvate metabolic process</td><td>cytoplasm</td><td>92</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388115.1</td><td>2-oxoglutarate dehydrogenase E1 component</td><td>odhA</td><td>oxoglutarate dehydrogenase complex</td><td>methylglyoxal reductase (NADPH-dependent, acetol producing)</td><td>cytoplasm</td><td>92</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388116.1</td><td>2-oxoglutarate dehydrogenase complex</td><td>odhB</td><td>dihydrolipoyllysine-residue</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>92</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151719.1</td><td>FMN-dependent NADH-azoreductase AzoRB</td><td>azoR2</td><td>succinyltransferase activity</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>94</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155386.1</td><td>NAD(P)H-dependent oxidoreductase</td><td>yhcB</td><td>unknown</td><td>response to toxic substance/aromatic compound catabolic process</td><td>cell membrane</td><td>94</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152575.1</td><td>succinate dehydrogenase iron-sulfur subunit</td><td>sdhB</td><td>electron transfer activity</td><td>NAD(P)H dehydrogenase (quinone) activity</td><td>plasma membrane</td><td>94</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154907.1</td><td>NAD(P)/FAD-dependent oxidoreductase</td><td>yjlD</td><td>NAD(P)H dehydrogenase (quinone) activity</td><td>tricarboxylic acid cycle/Electron transport</td><td>plasma membrane</td><td>95</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152573.1</td><td>succinate dehydrogenase flavoprotein subunit</td><td>sdhA</td><td>electron transfer activity</td><td>aerobic electron transport chain</td><td>plasma membrane</td><td>96</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151170.1</td><td>FOF1 ATP synthase subunit alpha</td><td>atpA</td><td>ATP binding</td><td>tricarboxylic acid cycle/Electron transport</td><td>plasma membrane</td><td>98</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_014304945.1</td><td>dihydrolipoyl dehydrogenase</td><td>pdhD</td><td>dihydrolipoyl dehydrogenase activity</td><td>proton motive force-driven ATP synthesis</td><td>plasma membrane</td><td>98</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387882.1</td><td>electron transfer flavoprotein subunit alpha/FixB family protein</td><td>etfA</td><td>specific electron acceptor for other dehydrogenases</td><td>tricarboxylic acid cycle/Electron transport</td><td>cytoplasm</td><td>81</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388029.1</td><td>dihydrolipoyl dehydrogenase</td><td>bfmBC</td><td>dihydrolipoyl dehydrogenase activity/flavin adenine dinucleotide binding</td><td>proton motive force-driven ATP synthesis</td><td>cytoplasm</td><td>83</td><td>C</td><td>x</td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_015388104.1</td><td>nitroreductase family protein</td><td>yodC</td><td>oxidoreductase activity</td><td>aromatic compound catabolic process/ response to toxic substance</td><td>cytoplasm</td><td>84</td><td>c</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151012.1</td><td>phosphate acetyltransferase</td><td>pta</td><td>phosphate acetyltransferase activity</td><td>acetyl-CoA biosynthetic process</td><td>cytoplasm</td><td>93</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_014305071.1</td><td>aconitate hydratase AcnA</td><td>citB</td><td>2-methylisocitrate dehydratase activity/4 iron, 4 sulfur cluster binding/aconitate hydratase activity/ iron-responsive element binding/ mRNA 3'-UTR binding</td><td>citrate metabolic process/propionate metabolic process, methylcitrate cycle/ regulation of sporulation/ tricarboxylic acid cycle</td><td>cytoplasm</td><td>93</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387863.1</td><td>NADP-dependent isocitrate dehydrogenase</td><td>icd</td><td>isocitrate dehydrogenase (NADP+) activity/ magnesium ion binding</td><td>glyoxylate bypass/ Tricarboxylic acid cycle</td><td>unknown</td><td>94</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_014305566.1</td><td>citrate synthase</td><td>citZ</td><td>citrate (Si)-synthase activity</td><td>carbohydrate metabolic process/ tricarboxylic acid cycle</td><td>cytoplasm</td><td>94</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154526.1</td><td>pyruvate dehydrogenase complex</td><td>pdhC</td><td>dihydrolipoyllysine-residue succinyltransferase activity</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>96</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154281.1</td><td>succinate-CoA ligase subunit alpha</td><td>sucD</td><td>succinate-CoA ligase (GDP-forming) activity</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>97</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154283.1</td><td>ADP-forming succinate-CoA ligase subunit beta</td><td>sucC</td><td>succinate-CoA ligase (GDP-forming) activity</td><td>tricarboxylic acid cycle</td><td>cytoplasm</td><td>98</td><td>C</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Inorganic ion transport and metabolism</strong></td></tr><tr><td>WP_012118382.1</td><td>methionine ABC transporter substrate-binding lipoprotein MetQ</td><td>metQ</td><td>unknown</td><td>amino-acid transport</td><td>cell membrane</td><td>87</td><td>P</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003155420.1</td><td>catalase KatA</td><td>katA</td><td>oxidoreductase/Peroxidase</td><td>hydrogen peroxide catabolic process</td><td>cytoplasm</td><td>94</td><td>P</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_014305921.1</td><td>right-handed parallel beta-helix repeat-containing protein</td><td>ywoF</td><td>unknown</td><td>unknown</td><td>extracellular region</td><td>74</td><td>P</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003156656.1</td><td>metal ABC transporter substrate-binding protein</td><td>znuA</td><td>metal ion binding</td><td>zinc ion transport, cell adhesion</td><td>plasma membrane</td><td>83</td><td>P</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003156778.1</td><td>ABC transporter substrate-binding protein</td><td>feuA</td><td>part of the ABC transporter complex FeuABC/YusV involved in import of the catecholate siderophores bacillibactin and enterobactin.</td><td>iron ion transport</td><td>cytoplasm/cell membrane</td><td>83</td><td>P</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003155700.1</td><td>spore coat protein CotJC</td><td>cotJC</td><td>spore coat composition</td><td>sporulation</td><td>unknown</td><td>96</td><td>P</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_004393259.1</td><td>iron-hydroxamate ABC transporter substrate-binding protein</td><td>yxeB</td><td>unknown</td><td>iron ion transport</td><td>plasma membrane/ periplasmic space</td><td>72</td><td>P</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153772.1</td><td>superoxide dismutase</td><td>sodF</td><td>superoxide dismutase activity</td><td>unknown</td><td>unknown</td><td>80</td><td>P</td><td>x</td><td></td><td></td></tr><tr><td>WP_007408225.1</td><td>formate/nitrite transporter family protein</td><td>yrhG</td><td>formate transmembrane transporter activity</td><td>formate transport/ nitrite transport</td><td>plasma membrane</td><td>84</td><td>P</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388253.1</td><td>sulfate adenylyltransferase</td><td>sat</td><td>sulfate adenylyltransferase (ATP) activity</td><td>sulfate assimilation</td><td>unknown</td><td>87</td><td>P</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_015387719.1</td><td>assimilatory sulfite reductase (NADPH) hemoprotein subunit</td><td>cysI</td><td>sulfite reductase complex (NADPH)</td><td>cysteine biosynthetic process/amino-acid biosynthesis</td><td>cytoplasm</td><td>90</td><td>P</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153025.1</td><td>superoxide dismutase SodA</td><td>sodA</td><td>superoxide dismutase activity/metal ion binding</td><td>removal of superoxide radicals</td><td>cytoplasm</td><td>92</td><td>P</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Intracellular trafficking, secretion, and vesicular transport</strong></td></tr><tr><td>WP_003151414.1</td><td>preprotein translocase subunit SecA</td><td>secA</td><td>protein-exporting ATPase activity</td><td>intracellular protein transmembrane transport</td><td>cell membrane/cell envelope Sec protein transport complex</td><td>94</td><td>U</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Lipid transport and metabolism</strong></td></tr><tr><td>WP_003156672.1</td><td>triacylglycerol lipase</td><td>estA</td><td>triglyceride lipase activity</td><td>lipid catabolic process</td><td>extracellular region</td><td>76</td><td>I</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003156315.1</td><td>glucose 1-dehydrogenase</td><td>gdh</td><td>oxidoreductase/glucose 1-dehydrogenase [NAD(P)] activity</td><td>sporulation</td><td>unknown</td><td>90</td><td>I</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003155182.1</td><td>glucose 1-dehydrogenase</td><td>yhxC</td><td>oxidoreductase</td><td>unknown</td><td>unknown</td><td>92</td><td>I</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003154310.1</td><td>acyl carrier protein</td><td>acpa</td><td>carrier of the growing fatty acid chain in fatty acid biosynthesis.</td><td>fatty acid metabolism</td><td>cytoplasm</td><td>100</td><td>I</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003152442.1</td><td>acetyl-CoA carboxylase carboxyl transferase subunit alpha</td><td>accA</td><td>carboxyl- or carbamoyltransferase activity</td><td>fatty acid biosynthetic process/response to antibiotic</td><td>cytoplasm</td><td>88</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153776.1</td><td>aldehyde dehydrogenase family protein</td><td>dhaS</td><td>phenylacetaldehyde dehydrogenase activity</td><td>cellular aldehyde metabolic process</td><td>unknown</td><td>90</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152391.1</td><td>acetate-CoA ligase</td><td>acsa</td><td>acetate-CoA ligase activity</td><td>acetyl-CoA biosynthetic process</td><td>cytoplasm/cell membrane</td><td>90</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153973.1</td><td>YbgC/FadM family acyl-CoA thioesterase</td><td>yneP</td><td>fatty acyl-CoA hydrolase activity</td><td>unknown</td><td>unknown</td><td>92</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156602.1</td><td>L-glutamate gamma-semialdehyde dehydrogenase</td><td>putC</td><td>1-pyrroline-5-carboxylate dehydrogenase activity</td><td>proline catabolic process to glutamate</td><td>plasma membrane</td><td>97</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155056.1</td><td>beta-ketoacyl-ACP synthase II</td><td>fabF</td><td>3-oxoacyl-[acyl-carrier-protein] synthase activity</td><td>fatty acid biosynthetic process</td><td>cytoplasm</td><td>92</td><td>IQ</td><td>x</td><td></td><td></td></tr><tr><td>WP_014305231.1</td><td>3-phytase</td><td>phy</td><td>3-phytase activity</td><td>unknown</td><td>extracellular region</td><td>72</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388475.1</td><td>beta-ketoacyl-ACP synthase III FabHB</td><td>fabHB</td><td>3-oxoacyl-[acyl-carrier-protein] synthase activity</td><td>fatty acid biosynthetic process/secondary metabolite biosynthetic process</td><td>cytoplasm</td><td>80</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155001.1</td><td>enoyl-ACP reductase FabI</td><td>fabI</td><td>enoyl-[acyl-carrier-protein] reductase (NADH) activity</td><td>cellular response to cold/fatty acid elongation</td><td>unknown</td><td>95</td><td>I</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156407.1</td><td>2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase</td><td>ispF</td><td>2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase activity</td><td>isopentenyl diphosphate biosynthetic process, methylerthritol 4-phosphate pathway</td><td>unknown</td><td>95</td><td>I</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td colspan="11"><strong>Mobilome: prophages, transposons</strong></td></tr><tr><td>WP_007407274.1</td><td>phage major capsid protein</td><td>xkdG</td><td>Possibly a prophage capsid protein.</td><td>unknown</td><td>extracellular region</td><td>91</td><td>X</td><td>X</td><td></td><td></td></tr><tr><td colspan="11"><strong>Nucleotide transport and metabolism</strong></td></tr><tr><td>WP_003150717.1</td><td>IMP dehydrogenase</td><td>guaB</td><td>oxydoreductase</td><td>GMP biosynthesis/ Purine biosynthesis</td><td>cytoplasm</td><td>96</td><td>F</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387718.1</td><td>assimilatory sulfite reductase (NADPH) flavoprotein subunit</td><td>cysJ</td><td>flavin adenine dinucleotide binding/ FMN binding</td><td>cysteine biosynthetic process/amino-acid biosynthesis</td><td>cytoplasm</td><td>78</td><td>FP</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003155232.1</td><td>HIT family protein</td><td>hit</td><td>catalytic activity</td><td>nucleotide metabolic process</td><td>unknown</td><td>85</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153455.1</td><td>nucleoside-diphosphate kinase</td><td>ndk</td><td>nucleoside diphosphate kinase activity</td><td>nucleotide metabolism</td><td>cytoplasm</td><td>90</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153486.1</td><td>menaquinol-cytochrome c reductase cytochrome b/c subunit</td><td>ndk</td><td>nucleoside diphosphate kinase activity</td><td>nucleotide metabolism</td><td>cytoplasm</td><td>90</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388096.1</td><td>purine-nucleoside phosphorylase</td><td>deoD</td><td>purine-nucleoside phosphorylase activity</td><td>purine nucleoside catabolic process</td><td>cytoplasm</td><td>90</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387764.1</td><td>TIGR01457 family HAD-type hydrolase</td><td>yutF</td><td>alkaline phosphatase activity</td><td>unknown</td><td>cytoplasm</td><td>91</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154392.1</td><td>bifunctional pyrimidine operon transcriptional regulator/uracil phosphoribosyltransferase</td><td>pyrR</td><td>uracil phosphoribosyltransferase activity</td><td>DNA-templated transcription termination</td><td>plasma membrane</td><td>94</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_004393102.1</td><td>adenylosuccinate synthase</td><td>purA</td><td>adenylosuccinate synthase activity</td><td>IMP metabolic process</td><td>cytoplasm</td><td>94</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155784.1</td><td>glutamine-hydrolyzing GMP synthase</td><td>guaA</td><td>GMP synthase activity</td><td>GMP biosynthetic process</td><td>cytoplasm</td><td>97</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_004264656.1</td><td>ribose-phosphate diphosphokinase</td><td>prs</td><td>kinase activity</td><td>nucleotide biosynthesis</td><td>cytoplasm</td><td>95</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td>WP_007410407.1</td><td>adenylate kinase</td><td>adk</td><td>adenylate kinase activity</td><td>nucleoside diphosphate metabolic process</td><td>cytoplasm</td><td>95</td><td>F</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Posttranslation modification, protein turnover, chaperones</strong></td></tr><tr><td>WP_004264608.1</td><td>ATP-dependent zinc metalloprotease FtsH</td><td>ftsH</td><td>ATP-dependent peptidase activity</td><td>protein catabolic process/cell cycle/ sporulation</td><td>plasma membrane</td><td>96</td><td>O</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388265.1</td><td>S8 family serine peptidase</td><td>bpr</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>70</td><td>O</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003155497.1</td><td>serine protease</td><td>mpr</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>74</td><td>O</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388283.1</td><td>M4 family metallopeptidase</td><td>nprE</td><td>metalloendopeptidase activity</td><td>proteolysis</td><td>extracellular region</td><td>83</td><td>O</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154079.1</td><td>S8 family peptidase</td><td>aprX</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>extracellular region/ cytoplasm</td><td>83</td><td>O</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_014304883.1</td><td>S8 family peptidase</td><td>isp</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>93</td><td>O</td><td>x</td><td>x</td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_014305002.1</td><td>HslU-HslV peptidase ATPase subunit</td><td>clpY</td><td>ATP hydrolysis activity</td><td>proteolysis</td><td>unknown</td><td>93</td><td>O</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003155249.1</td><td>peptidylprolyl isomerase</td><td>prsA</td><td>protein folding and transport</td><td>peptidyl-prolyl cis-trans isomerase activity</td><td>plasma membrane</td><td>85</td><td>O</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003155195.1</td><td>S8 family serine peptidase</td><td>aprE</td><td>serine protease</td><td>fibrinolysis/proteolysis/sporulation</td><td>extracellular region</td><td>85</td><td>O</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_007410388.1</td><td>ATP-dependent protease ATP-binding subunit ClpC</td><td>clpC</td><td>ATP hydrolysis activity/Chaperone/Repressor</td><td>chaperone/establishment of competence for transformation</td><td>cytoplasm</td><td>98</td><td>O</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_015387737.1</td><td>trypsin-like peptidase domain-containing protein</td><td>htrB</td><td>serine-type endopeptidase activity</td><td>proteolysis</td><td>plasma membrane</td><td>76</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153552.1</td><td>carboxypeptidase M32</td><td>ypwA</td><td>metallocarboxypeptidase activity</td><td>proteolysis</td><td>unknown</td><td>83</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153641.1</td><td>peptide-methionine (S)-S-oxide reductase MsrA</td><td>msrA</td><td>peptide-methionine (S)-S-oxide reductase activity</td><td>protein modification process</td><td>unknown</td><td>87</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156398.1</td><td>protein arginine kinase</td><td>mcsB</td><td>kinase activity</td><td>phosphorylation</td><td>extracellular region/cytoplasm</td><td>91</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387840.1</td><td>thioredoxin family protein</td><td>typp</td><td>unknown</td><td>unknown</td><td>unknown</td><td>92</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_007408050.1</td><td>thiol peroxidase</td><td>tpx</td><td>thioredoxin peroxidase activity</td><td>unknown</td><td>unknown</td><td>94</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151513.1</td><td>ATP-dependent Clp endopeptidase proteolytic subunit ClpP</td><td>clpP</td><td>ATP-dependent peptidase activity</td><td>protein quality control for misfolded or incompletely synthesized proteins/Stress response</td><td>cytoplasm</td><td>96</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152893.1</td><td>molecular chaperone DnaK</td><td>dnaK</td><td>ATP-dependent protein folding chaperone</td><td>protein refolding/stress response</td><td>cytoplasm</td><td>97</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388098.1</td><td>S41 family peptidase</td><td>ctpA</td><td>serine-type endopeptidase activity</td><td>proteolysis/signal transduction</td><td>periplasmic space</td><td>81</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387889.1</td><td>ATP-dependent protease ATP-binding subunit ClpX</td><td>clpX</td><td>ATP hydrolysis activity/ATP-dependent protein folding chaperone</td><td>protein catabolic process/cell division</td><td>unknown</td><td>96</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152618.1</td><td>trigger factor</td><td>tig</td><td>protein folding chaperone/peptidyl-prolyl cis-trans isomerase activity</td><td>cell division/Cell cycle</td><td>cytoplasm</td><td>96</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155941.1</td><td>chaperonin GroEL</td><td>groEL</td><td>GroEL-GroES complex/ATP-dependent protein folding chaperone</td><td>protein refolding/response to heat</td><td>extracellular region/Cytoplasme</td><td>98</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152560.1</td><td>thioredoxin</td><td>trxA</td><td>protein-disulfide reductase activity</td><td>cell redox homeostasis</td><td>cytoplasm</td><td>99</td><td>O</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153447.1</td><td>non-specific DNA-binding protein Hbs</td><td>hbs</td><td>DNA binding</td><td>chromosome condensation</td><td>cytoplasm</td><td>100</td><td>L</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_004392898.1</td><td>DNA gyrase subunit A</td><td>gyrA</td><td>DNA topoisomerase type II (double strand cut, ATP-hydrolyzing) complex</td><td>DNA topological change</td><td>cytoplasm</td><td>93</td><td>L</td><td>x</td><td></td><td></td></tr><tr><td>WP_004392911.1</td><td>DNA polymerase III subunit beta</td><td>dnaN</td><td>3'-5' exonuclease activity</td><td>DNA strand elongation involved in DNA replication</td><td>cytoplasm</td><td>94</td><td>L</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154145.1</td><td>recombinase RecA</td><td>RecA</td><td>ATP-dependent DNA damage sensor activity/damaged DNA binding</td><td>unknown</td><td>unknown</td><td>96</td><td>L</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003151902.1</td><td>ribonuclease</td><td>bsn</td><td>nuclease activity</td><td>unknown</td><td>extracellular region</td><td>78</td><td>L</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388679.1</td><td>ATP-dependent RNA helicase CshA</td><td>cshA</td><td>ATP hydrolysis activity/RNA helicase activity</td><td>bibosome biogenesis/Stress response</td><td>cytoplasm/plasma membrane</td><td>97</td><td>L</td><td>x</td><td></td><td></td></tr><tr><td>WP_004392979.1</td><td>single-stranded DNA-binding protein SsbA</td><td>ssbA</td><td>single-stranded DNA binding</td><td>DNA recombination/DNA repair</td><td>cytoplasm</td><td>99</td><td>L</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Secondary metabolites biosynthesis, transport and catabolism</strong></td></tr><tr><td>WP_003151745.1</td><td>aldo/keto reductase</td><td>yvgN</td><td>aldo-keto reductase (NADP) activity</td><td>small molecule metabolic process</td><td>unknown</td><td>90</td><td>Q</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003152184.1</td><td>DinB family protein</td><td>yuaE</td><td>unknown</td><td>unknown</td><td>unknown</td><td>70</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151318.1</td><td>acetolactate decarboxylase</td><td>alsD</td><td>acetolactate decarboxylase activity</td><td>acetoin biosynthetic process</td><td>unknown</td><td>82</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td>WP_004393101.1</td><td>VOC family protein</td><td>yycE</td><td>unknown</td><td>unknown</td><td>unknown</td><td>72</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td>WP_197,923,070.1</td><td>amino acid adenylation domain-containing protein</td><td>srfAB</td><td>amino acid activation for nonribosomal peptide biosynthetic process/phosphopantetheine binding/</td><td>secondary metabolite biosynthetic process</td><td>cytoplasm</td><td>72</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td>WP_003150879.1</td><td>D-alanine-poly(phosphoribitol) ligase subunit DltA</td><td>dltA</td><td>ATP binding/D-alanine [D-alanyl carrier protein] ligase activity</td><td>lipoteichoic acid biosynthetic process</td><td>cytoplasm</td><td>78</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388560.1</td><td>NADP-dependent oxidoreductase</td><td>yfmJ</td><td>15-oxoprostaglandin 13-oxidase activity</td><td>Aromatic hydrocarbons catabolism/ Detoxification</td><td>unknown</td><td>87</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388106.1</td><td>tautomerase family protein</td><td>yolI</td><td>isomerase activity</td><td>unknown</td><td>unknown</td><td>74</td><td>Q</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Signal transduction mechanisms</strong></td></tr><tr><td>WP_003154064.1</td><td>RNA chaperone Hfq</td><td>hfq</td><td>RNA binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>T</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388734.1</td><td>TerD family protein</td><td>yceC</td><td>unknown</td><td>stress response</td><td>cytoplasm</td><td>82</td><td>T</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003156648.1</td><td>TerD family protein</td><td>yceD</td><td>Stress response</td><td>Unknown</td><td>Unknown</td><td>93</td><td>T</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151456.1</td><td>HPr(Ser) kinase/phosphatase</td><td>hprK</td><td>protein serine/threonine kinase activity</td><td>regulation of carbohydrate metabolic process</td><td>cytoplasm</td><td>96</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_004264734.1</td><td>cyclic di-AMP receptor DarA</td><td>darA</td><td>unknown</td><td>unknown</td><td>cytoplasm</td><td>97</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388712.1</td><td>cystine ABC transporter substrate-binding lipoprotein TcyA</td><td>tcyA</td><td>unknown</td><td>amino acid transport</td><td>plasma membrane</td><td>84</td><td>TE</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155112.1</td><td>YajQ family cyclic di-GMP-binding protein</td><td>yitK</td><td>nucleotide binding</td><td>unknown</td><td>cytoplasm</td><td>88</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153343.1</td><td>anti-sigma F factor antagonist</td><td>spoIIAA</td><td>anti-sigma factor antagonist activity</td><td>sporulation</td><td>Unknown</td><td>92</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152237.1</td><td>S-ribosylhomocysteine lyase</td><td>luxS</td><td>S-ribosylhomocysteine lyase activity</td><td>quorum sensing</td><td>Unknown</td><td>93</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156645.1</td><td>TerD family protein</td><td>yceE</td><td>unknown</td><td>unknown</td><td>Unknown</td><td>95</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155391.1</td><td>serine/threonine protein kinase PrkA</td><td>prkA</td><td>protein kinase activity</td><td>unknown</td><td>Unknown</td><td>97</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154502.1</td><td>translational GTPase TypA</td><td>bipA</td><td>GTPase activity</td><td>ribosome biogenesis</td><td>cytoplasm</td><td>97</td><td>T</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Transcription</strong></td></tr><tr><td>WP_015387570.1</td><td>DNA-directed RNA polymerase subunit delta</td><td>rpoE</td><td>Nucleotidyltransferase</td><td>transcription</td><td>cytoplasm</td><td>84</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152998.1</td><td>RNA polymerase sigma factor RpoD</td><td>sigA</td><td>sigma factor activity/DNA binding</td><td>transcription regulation</td><td>cytoplasm</td><td>98</td><td>K</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003156441.1</td><td>DNA-directed RNA polymerase subunit beta'</td><td>rpoC</td><td>DNA-directed 5'-3' RNA polymerase activity</td><td>DNA-templated transcription/response to antibiotic cell cycle/cell division/cell shape/ peptidoglycan synthesis</td><td>cytoplasm</td><td>98</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_004264660.1</td><td>septation regulator SpoVG</td><td>spoVG</td><td>unknown</td><td>DNA-templated transcription elongation</td><td>unknown</td><td>96</td><td>KD</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152766.1</td><td>transcription elongation factor GreA</td><td>greA</td><td>RNA polymerase binding/DNA binding</td><td>DNA-templated transcription elongation</td><td>cytoplasm</td><td>95</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154195.1</td><td>transcription termination factor NusA</td><td>nusA</td><td>DNA-binding transcription factor activity</td><td>DNA-templated transcription termination</td><td>cytoplasm</td><td>95</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156440.1</td><td>DNA-directed RNA polymerase subunit beta</td><td>rpoB</td><td>DNA-directed 5'-3' RNA polymerase activity</td><td>DNA-templated transcription/response to antibiotic regulation of DNA-templated transcription</td><td>cytoplasm</td><td>98</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154263.1</td><td>GTP-sensing pleiotropic transcriptional regulator CodY</td><td>codY</td><td>DNA-binding transcription repressor activity</td><td>DNA-templated transcription</td><td>cytoplasm</td><td>99</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156549.1</td><td>DNA-directed RNA polymerase subunit alpha</td><td>rpoA</td><td>DNA-directed 5'-3' RNA polymerase activity</td><td>DNA-templated transcription</td><td>cytoplasm</td><td>99</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151236.1</td><td>sporulation transcriptional regulator SpoIID</td><td>spoIID</td><td>DNA-binding transcription factor activity</td><td>sporulation/ transcription regulation</td><td>unknown</td><td>100</td><td>K</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153177.1</td><td>sporulation transcription factor SpoOA</td><td>spoOA</td><td>phosphorelay response regulator activity/ transcription cis-regulatory region binding/calcium ion binding</td><td>single-species surface biofilm formation/ positive regulation of sporulation resulting in formation of a cellular spore/positive regulation of DNA-templated transcription/ Sporulation</td><td>cytoplasm</td><td>96</td><td>KT</td><td>x</td><td></td><td></td></tr><tr><td>WP_003219701.1</td><td>two-component system response regulator DegU</td><td>Stat</td><td>DNA-binding</td><td>phosphorelay signal transduction system/ positive regulation of DNA-templated transcription</td><td>cytoplasm</td><td>100</td><td>KT</td><td>x</td><td></td><td></td></tr><tr><td>WP_169,510,469.1</td><td>transition state genes transcriptional regulator AbrB</td><td>abrB</td><td>DNA-binding</td><td>sporulation/negative regulation of DNA-templated transcription</td><td>unknown</td><td>97</td><td>KV</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Translation, ribosomal structure and biogenesis</strong></td></tr><tr><td>WP_003156451.1</td><td>elongation factor Tu</td><td>tuf</td><td>GTPase activity</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_014304716.1</td><td>YheC/YheD family protein</td><td>yheC</td><td>ATP binding</td><td>sporulation</td><td>peripheral membrane</td><td>74</td><td>JEHQ</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003154288.1</td><td>50S ribosomal protein L19</td><td>rplS</td><td>structural constituent of ribosome</td><td>translation</td><td>protein /spore coat</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>WP_004264654.1</td><td>50S ribosomal protein L25/general stress protein Ctc</td><td>ctc</td><td>5S rRNA binding</td><td>translation</td><td>cytoplasm</td><td>91</td><td>J</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003156433.1</td><td>50S ribosomal protein L10</td><td>rplJ</td><td>structural constituent of ribosome</td><td>translation</td><td>cytoplasm</td><td>71</td><td>J</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003154296.1</td><td>30S ribosomal protein S16</td><td>RPSP</td><td>structural constituent of ribosome</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_004393063.1</td><td>50S ribosomal protein L9</td><td>rplI</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_007410399.1</td><td>elongation factor G</td><td>fusA</td><td>translation elongation factor activit</td><td>protein biosynthesis</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152495.1</td><td>50S ribosomal protein L20</td><td>rplT</td><td>Binds directly to 23S ribosomal RNA and is necessary for the in vitro assembly process of the 50S ribosomal subunit. It is not involved in the protein synthesizing functions of that subunit (By similarity).</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003156436.1</td><td>50S ribosomal protein L7/L12</td><td>rplL</td><td>Forms part of the ribosomal stalk which helps the ribosome interact with GTP-bound translation factors. Is thus essential for accurate translation. large ribosomal subunit rRNA binding</td><td>Translation</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_003156464.1</td><td>30S ribosomal protein S10</td><td>rpsJ</td><td></td><td>translation</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_015387878.1</td><td>phenylalanine-tRNA ligase subunit beta</td><td>pheT</td><td>Aminoacyl-tRNA synthetase</td><td>phenylalanyl-tRNA aminoacylation</td><td>cytoplasm</td><td>88</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387901.1</td><td>aspartate-tRNA ligase</td><td>aspS</td><td>aspartate-tRNA ligase activity</td><td>protein biosynthesis</td><td>cytoplasm</td><td>89</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152480.1</td><td>threonine-tRNA ligase</td><td>thrS</td><td>threonine-tRNA ligase activity</td><td>threonyl-tRNA aminoacylation</td><td>cytoplasm</td><td>93</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388055.1</td><td>23S rRNA pseudouridine(2605) synthase RluB</td><td>rluB</td><td>23S rRNA pseudouridine(2605) synthase activity</td><td>maturation of LSU-rRNA from tetracistronic rRNA transcript (SSU-rRNA, LSU-rRNA, 4.5S-rRNA, 5S-rRNA)</td><td>unknown</td><td>94</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154212.1</td><td>ribosome recycling factor</td><td>frr</td><td>ribosomal large subunit binding</td><td>translation</td><td>cytoplasm</td><td>94</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154214.1</td><td>translation elongation factor Ts</td><td>tsf</td><td>translation elongation factor activity</td><td>translational elongation</td><td>cytoplasm</td><td>94</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388789.1</td><td>methionine-tRNA ligase</td><td>metG</td><td>methionine-tRNA ligase activity</td><td>methionyl-tRNA aminoacylation</td><td>cytoplasm</td><td>94</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_071543361.1</td><td>translation initiation factor IF-3</td><td>infC</td><td>ribosome binding</td><td>protein biosynthesis</td><td>cytoplasm</td><td>94</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153515.1</td><td>asparagine-tRNA ligase</td><td>asnS</td><td>asparagine-tRNA ligase activity</td><td>protein biosynthesis</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388783.1</td><td>glutamate-tRNA ligase</td><td>gltX</td><td>aminoacyl-tRNA synthetase</td><td>protein biosynthesis</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156478.1</td><td>50S ribosomal protein L16</td><td>rplP</td><td>structural constituent of ribosome</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>96</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_004392983.1</td><td>30S ribosomal protein S18</td><td>rpsR</td><td>structural constituent of ribosome</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>96</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_007609878.1</td><td>Asp-tRNA(Asn)/Glu-tRNA(Gln) amidotransferase subunit GatA</td><td>gatA</td><td>glutaminyl-tRNA synthase (glutamine-hydrolyzing) activity</td><td>translation</td><td>cytoplasm</td><td>96</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388311.1</td><td>preQ(1) synthase</td><td>queF</td><td>preQ1 synthase activity</td><td>queuosine biosynthesis</td><td>cytoplasm</td><td>96</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153123.1</td><td>elongation factor P</td><td>efp</td><td>translation elongation factor activity</td><td>protein biosynthesis</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156499.1</td><td>50S ribosomal protein L15</td><td>rplO</td><td>structural constituent of ribosome</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003154182.1</td><td>30S ribosomal protein S15</td><td>rpsO</td><td>rRNA binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156556.1</td><td>50S ribosomal protein L13</td><td>rplM</td><td>structural constituent of ribosome</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156430.1</td><td>50S ribosomal protein L11</td><td>rplK</td><td>large ribosomal subunit rRNA binding</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156467.1</td><td>50S ribosomal protein L23</td><td></td><td>structural constituent of ribosome</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156472.1</td><td>30S ribosomal protein S19</td><td>rpsS</td><td>structural constituent of ribosome</td><td>translation/response to antibiotic</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154544.1</td><td>peptide deformylase</td><td>defB</td><td>peptide deformylase activity</td><td>co-translational protein modification</td><td>unknown</td><td>91</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156489.1</td><td>30S ribosomal protein S8</td><td>rpsH</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>92</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154163.1</td><td>ribonuclease J2</td><td>rnjB</td><td>5'-3' RNA exonuclease activity/RNA endonuclease activity</td><td>mRNA processing</td><td>cytoplasm</td><td>93</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152406.1</td><td>30S ribosomal protein S4</td><td>rpsD</td><td>structural constituent of ribosome</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152493.1</td><td>50S ribosomal protein L35</td><td>n bL35</td><td>structural constituent of ribosome</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153426.1</td><td>30S ribosomal protein S1</td><td>ypfD</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156492.1</td><td>50S ribosomal protein L18</td><td>rplR</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_004392977.1</td><td>30S ribosomal protein S6</td><td>rpsF</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388227.1</td><td>polyribonucleotide nucleotidyltransferase</td><td>npn</td><td>3'-5'-RNA exonuclease activity</td><td>RNA catabolic process</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388229.1</td><td>translation initiation factor IF-2</td><td>infB</td><td>translation initiation factor activity</td><td>translational initiation</td><td>cytoplasm</td><td>95</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156484.1</td><td>30S ribosomal protein S17</td><td>rpsQ</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>96</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156490.1</td><td>50S ribosomal protein L6</td><td>rplF</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>96</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152660.1</td><td>50S ribosomal protein L21</td><td>rplU</td><td>structural constituent of ribosome</td><td>translation</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154550.1</td><td>ribonuclease J1</td><td>rnjA</td><td>5'-3' RNA exonuclease activity/RNA endonuclease activity</td><td>mRNA processing</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156465.1</td><td>50S ribosomal protein L3</td><td>rplC</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156466.1</td><td>50S ribosomal protein L4</td><td>rplD</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>97</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156476.1</td><td>30S ribosomal protein S3</td><td>rpsC</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156487.1</td><td>50S ribosomal protein L5</td><td>rplE</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156545.1</td><td>30S ribosomal protein S13</td><td>rpsM</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003225846.1</td><td>30S ribosomal protein S9</td><td>rpsI</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>98</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154215.1</td><td>30S ribosomal protein S2</td><td>rpsB</td><td>structural constituent of ribosome</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156445.1</td><td>30S ribosomal protein S12</td><td>rpsL</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156447.1</td><td>30S ribosomal protein S7</td><td>rpsG</td><td>structural constituent of ribosome/ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003156470.1</td><td>50S ribosomal protein L2</td><td>rplB</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156485.1</td><td>50S ribosomal protein L14</td><td>rplN</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156493.1</td><td>30S ribosomal protein S5</td><td>rpsE</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156547.1</td><td>30S ribosomal protein S11</td><td>rpsK</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156550.1</td><td>50S ribosomal protein L17</td><td>rplQ</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>99</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156475.1</td><td>50S ribosomal protein L22</td><td>rplV</td><td>structural constituent of ribosome/ ARN binding</td><td>translation</td><td>cytoplasm</td><td>100</td><td>J</td><td>x</td><td></td><td></td></tr><tr><td colspan="11"><strong>Unknown</strong></td></tr><tr><td>WP_003152178.1</td><td>DUF1016 domain-containing protein/biofilm surface layer hydrophobin BslA</td><td>bslA/ yuaB</td><td>biofilm formation</td><td>unknown/biofilm formation</td><td>extracellular region</td><td>74</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015387422.1</td><td>DUF1259 domain-containing protein</td><td>ycxD</td><td>unknown/transaminase activity</td><td>unknown/alpha-amino acid metabolic process</td><td>unknown</td><td>27</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003151908.1</td><td>hypothetical protein</td><td>mpr</td><td>unknown/serine-type endopeptidase activity</td><td>unknown/proteolysis</td><td>unknown/ extracellular region</td><td>31</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388185.1</td><td>hypothetical protein</td><td>cotC</td><td>unknown</td><td>unknown/sporulation</td><td>unknown</td><td>45</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_014306056.1</td><td>S8 family serine peptidase</td><td>pnbA</td><td>unknown/cholinesterase activity</td><td>unknown</td><td>unknown</td><td>63</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388008.1</td><td>SipW-dependent-type signal peptide-containing protein/biofilm matrix protein TasA</td><td>tasA</td><td>major protein component of the biofilm extracellular matrix/amyloid fibers that bind cells together in the biofilm/antibacterial/spore coat assembly</td><td>sporulation</td><td>extracellular region/ Forespore intermembrane space</td><td>84</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003154111.1</td><td>outer spore coat protein CotE</td><td>cotE</td><td>identical protein binding</td><td>sporulation</td><td>spore coat</td><td>96</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_014304250.1</td><td>DUF4879 domain-containing protein</td><td>yolA</td><td>unknown</td><td>unknown</td><td>extracellular</td><td>27</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388007.1</td><td>amyloid fiber anchoring/assembly protein TapA</td><td>tapA</td><td>unknown/required for the proper anchoring and polymerization of TasA amyloid fibers at the cell surface</td><td>unknown</td><td>region/ bacterial biofilm matrix</td><td>47</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388281.1</td><td>hypothetical protein</td><td>ylaE</td><td>unknown</td><td>unknown</td><td>unknown</td><td>52</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003155052.1</td><td>peptide-binding protein</td><td>appA</td><td>peptide transmembrane transporter activity</td><td>peptide transport/ sporulation</td><td>periplasmic space</td><td>73</td><td>unknown</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_014305020.1</td><td>insulinase family protein</td><td>ymfH</td><td>metallopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>86</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388142.1</td><td>IseA DL-endopeptidase inhibitor family protein</td><td>yoeB</td><td>unknown</td><td>unknown</td><td>unknown</td><td>77</td><td>unknown</td><td>x</td><td></td><td>x</td></tr><tr><td>WP_187,650,984.1</td><td>hypothetical protein</td><td>ywmD</td><td>unknown</td><td>unknown</td><td>unknown</td><td>20</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_014305702.1</td><td>hydrolase</td><td>yddQ</td><td>unknown/hydrolase activity</td><td>unknown</td><td>unknown</td><td>31</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387848.1</td><td>SGNH/GDSL hydrolase family protein</td><td>yddQ</td><td>unknown/hydrolase activity</td><td>unknown</td><td>unknown</td><td>31</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387689.1</td><td>M28 family peptidase</td><td>ywaD</td><td>unknown/peptidase activity</td><td>unknown/proteolysis</td><td>unknown/ extracellular region</td><td>48</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_024084955.1</td><td>YdhK family protein</td><td>ydhK</td><td>unknown</td><td>unknown</td><td>unknown</td><td>61</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387670.1</td><td>carboxylesterase/lipase family protein</td><td>pnbA</td><td>unknown/cholinesterase activity</td><td>unknown</td><td>unknown</td><td>63</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151188.1</td><td>VWA domain-containing protein</td><td>ywmC</td><td>unknown</td><td>unknown</td><td>unknown</td><td>66</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003152178.1</td><td>DUF1016 domain-containing protein</td><td>bslA/ yuaB</td><td>biofilm formation</td><td>unknown/Biofilm formation</td><td>extracellular region</td><td>74</td><td>unknown</td><td>x</td><td>x</td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_015387776.1</td><td>YukJ family protein</td><td>yukJ</td><td>unknown</td><td>unknown</td><td>unknown</td><td>81</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388356.1</td><td>phage tail sheath family protein</td><td>xkdK</td><td>unknown</td><td>unknown</td><td>unknown</td><td>86</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003152143.1</td><td>type 1 glutamine amidotransferase</td><td>yraA</td><td>unknown/peptidase activity</td><td>unknown/proteolysis</td><td>unknown</td><td>38</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388651.1</td><td>hypothetical protein</td><td>floT</td><td>unknown</td><td>unknown/regulation of cell shape</td><td>unknown</td><td>28</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154036.1</td><td>hypothetical protein</td><td>ssuD</td><td>unknown/alkanesulfonate monooxygenase activity</td><td>unknown/alkanesulfonate catabolic process</td><td>unknown</td><td>30</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387655.1</td><td>ribonuclease</td><td>yvaE</td><td>unknown/amino-acid betaine transmembrane transporter activity</td><td>unknown/transport</td><td>unknown/plasma membrane</td><td>43</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003155828.1</td><td>DUF1906 domain-containing protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>49</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003154023.1</td><td>lytic polysaccharide monooxygenase</td><td>ctc</td><td>unknown/Ribonucleoprotein</td><td>unknown/translation</td><td>unknown/cytoplasm</td><td>50</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003153543.1</td><td>hypothetical protein</td><td>yqgA</td><td>unknown</td><td>unknown</td><td>unknown/extracellular region</td><td>52</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003156431.1</td><td>50S ribosomal protein L1</td><td>yqgA</td><td>unknown</td><td>unknown</td><td>unknown/extracellular region</td><td>52</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015387631.1</td><td>flagellin</td><td>hag</td><td>structural molecule activity</td><td>bacterial-type flagellum</td><td>unknown/extracellular region</td><td>56</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_004393267.1</td><td>hypothetical protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>66</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003151740.1</td><td>hypothetical protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>81</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_003154135.1</td><td>stage V sporulation protein SpoVS</td><td>spoVS</td><td>nucleic acid binding</td><td>sporulation //Cell division</td><td>unknown</td><td>100</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_021734090.1</td><td>hypothetical protein</td><td>cotG</td><td>unknown</td><td>sporulation</td><td>unknown</td><td>100</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388720.1</td><td>GTP-binding protein</td><td>yciC</td><td>GTP binding/hydrolase activity</td><td>unknown</td><td>unknown</td><td>84</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388552.1</td><td>LPXTG cell wall anchor domain-containing protein</td><td>apr</td><td>serine-type endopeptidase activity</td><td>protrolysis</td><td>unknown/extracellular region</td><td>22</td><td>unknown</td><td>x</td><td>x</td><td>x</td></tr><tr><td>WP_015388714.1</td><td>aminotransferase class I/II-fold pyridoxal phosphate-dependent enzyme</td><td>aspB</td><td>unknown/L-aspartate:2-oxoglutarate aminotransferase activity</td><td>unknown/biosynthetic process</td><td>unknown/cytoplasm</td><td>25</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388335.1</td><td>glycosyltransferase</td><td>sunS</td><td>unknown</td><td>bacteriocin</td><td>unknown</td><td>28</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015388287.1</td><td>serine hydrolase</td><td>pbpE</td><td>hydrolase</td><td>biosynthetic process</td><td>unknown</td><td>29</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003152402.1</td><td>hypothetical protein</td><td>era</td><td>unknown/ribosomal small subunit binding</td><td>Unknown/positive regulation of cell division/ribosomal small subunit assembly</td><td>unknown/Cytoplasm/Cell membrane</td><td>30</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_014304383.1</td><td>hypothetical protein</td><td>yral</td><td>unknown</td><td>unknown</td><td>unknown/spore coat</td><td>30</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387690.1</td><td>NAD(P)-dependent oxidoreductase</td><td>ytcB</td><td>dTDP-glucose 4,6-dehydratase activity</td><td>UDP-rhamnose biosynthetic process</td><td>unknown</td><td>31</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_015387616.1</td><td>glycosyltransferase family 2 protein</td><td>ggaA</td><td>glycosyltransferase</td><td>cell wall organization</td><td>unknown</td><td>33</td><td>unknown</td><td>x</td><td>x</td><td></td></tr><tr><td>WP_003153276.1</td><td>cytochrome P450</td><td>yjiB</td><td>unknown/cholest-4-en-3-one 26-monooxygenase activity</td><td>unknown/steroid hydroxylase activity</td><td>unknown</td><td>38</td><td>unknown</td><td>x</td><td>x</td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003155515.1</td><td>barstar family protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown/cytoplasm</td><td>49</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388034.1</td><td>ACP S-malonyltransferase</td><td>pksE</td><td>[acyl-carrier-protein] S-malonyltransferase activity</td><td>Unknown/antibiotic biosynthetic process/fatty acid biosynthetic process</td><td>unknown/cytoplasm</td><td>59</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152644.1</td><td>SPOR domain-containing protein</td><td>spoIIB</td><td>peptidoglycan binding</td><td>sporulation</td><td>plasma membrane</td><td>60</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388149.1</td><td>non-ribosomal peptide synthetase</td><td>ppsD</td><td>phosphopantetheine binding</td><td>antibiotic biosynthetic process/amino acid activation for nonribosomal peptide biosynthetic process</td><td>unknown</td><td>63</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388212.1</td><td>non-ribosomal peptide synthetase</td><td></td><td>Unknown/fatty acid synthase activity</td><td>unknown/fatty acid biosynthetic process</td><td>unknown</td><td>63</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388186.1</td><td>hypothetical protein</td><td>yvgO</td><td></td><td>unknown</td><td>unknown</td><td>63</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155509.1</td><td>nitroreductase</td><td>yfhC</td><td>Unknown/oxidoreductase activity</td><td>unknown</td><td>unknown</td><td>67</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155277.1</td><td>YheC/YheD family protein</td><td>yheD</td><td>Unknown</td><td>unknown/sporulation</td><td>Unknown spore coat</td><td>67</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156592.1</td><td>RDD family protein</td><td>yckC</td><td>unknown</td><td>unknown</td><td>unknown/plasma membrane</td><td>68</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_007409232.1</td><td>enoyl-CoA hydratase</td><td>yhaR</td><td>unknown</td><td>unknown</td><td>unknown/plasma membrane</td><td>69</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387781.1</td><td>(S)-benzoin forming benzil reductase</td><td>yueD</td><td>sepiapterin reductase activity</td><td>tetrahydrobiopterin biosynthetic process</td><td>unknown/cytoplasm</td><td>69</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154085.1</td><td>sporulation protein</td><td>nucB</td><td>nuclease activity</td><td>sporulation</td><td>extracellular region</td><td>70</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_046341317.1</td><td>hypothetical protein</td><td>cgeC</td><td>unknown</td><td>unknown</td><td>unknown</td><td>71</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388097.1</td><td>hypothetical protein</td><td>yodI</td><td>unknown</td><td>unknown</td><td>cell membrane</td><td>71</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152478.1</td><td>putative sporulation protein YtxC</td><td>ytxC</td><td>unknown</td><td>unknown</td><td>unknown</td><td>75</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154479.1</td><td>hypothetical protein</td><td>ylbA</td><td>unknown</td><td>unknown</td><td>unknown</td><td>75</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154632.1</td><td>hypothetical protein</td><td>ykwD</td><td>unknown</td><td>unknown</td><td>unknown</td><td>76</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155695.1</td><td>VOC family protein</td><td>yetH</td><td>unknown</td><td>unknown</td><td>unknown</td><td>76</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156589.1</td><td>competence protein ComJ</td><td>nin</td><td>unknown</td><td>establishment of competence for transformation</td><td>plasma membrane</td><td>78</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387536.1</td><td>spore coat protein GerQ</td><td>gerQ</td><td>isopeptide cross-linking via N6-(L-isoglutamyl)-L-lysine</td><td>sporulation</td><td>spore coat</td><td>79</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153148.1</td><td>stage III sporulation protein AG</td><td>spoIIIAG</td><td>unknown</td><td>sporulation</td><td>plasma membrane</td><td>80</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003151446.1</td><td>DUF4097 domain-containing protein</td><td>yvlB</td><td>unknown</td><td>unknown</td><td>plasma membrane</td><td>81</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388178.1</td><td>CoA-binding protein</td><td>yneT</td><td>unknown</td><td>unknown</td><td>unknown</td><td>85</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154178.1</td><td>insulinase family protein</td><td>ymxG</td><td>metalloendopeptidase activity</td><td>proteolysis</td><td>unknown</td><td>90</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152380.1</td><td>YtxH domain-containing protein</td><td>ytxH</td><td>unknown</td><td>unknown</td><td>unknown</td><td>92</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_004264624.1</td><td>VWA domain-containing protein</td><td>yabS</td><td>unknown</td><td>unknown</td><td>unknown</td><td>93</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152958.1</td><td>GatB/YqeY domain-containing protein</td><td>yqeY</td><td>carbon-nitrogen ligase activity, with glutamine as amido-N-donor</td><td>unknown</td><td>unknown</td><td>93</td><td>unknown</td><td>x</td><td></td><td></td></tr></tbody></table>

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<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003153984.1</td><td>small acid-soluble spore protein P</td><td>sspP</td><td>asexual sporulation</td><td>sporulation</td><td>spore coat</td><td>97</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003218330.1</td><td>protein Veg</td><td>veg</td><td>regulation of DNA-templated transcription</td><td>sporulation</td><td>cytoplasm/ spore core</td><td>100</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154969.1</td><td>YjcG family protein</td><td>yjcG</td><td>hydrolase activity, acting on ester bonds</td><td>unknown</td><td>unknown</td><td>80</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387693.1</td><td>PIG-L family deacetylase</td><td>bshB1</td><td>unknown/hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds, in linear amides</td><td>unknown/bacillithiol biosynthetic process</td><td>unknown</td><td>26</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153268.1</td><td>SDR family oxidoreductase</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>37</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003155166.1</td><td>YbjQ family protein</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>37</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_014304855.1</td><td>pyocin knob domain-containing protein</td><td>xkdV</td><td>unknown</td><td>unknown</td><td>unknown</td><td>37</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388166.1</td><td>non-ribosomal peptide synthetase</td><td>ppsE</td><td>unknown</td><td>unknown</td><td>unknown</td><td>37</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003156618.1</td><td>antimicrobial peptide LCI</td><td>yoqM</td><td>unknown</td><td>unknown</td><td>unknown</td><td>38</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388164.1</td><td>non-ribosomal peptide synthetase</td><td>ppsD</td><td>unknown/phosphopantetheine binding</td><td>unknown/Antibiotic biosynthesis</td><td>unknown/ Cytoplasm</td><td>40</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388165.1</td><td>non-ribosomal peptide synthetase</td><td>srfAB</td><td>unknown/ligase activity</td><td>unknown/Antibiotic biosynthesis</td><td>unknown/ Cytoplasm</td><td>41</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388199.1</td><td>SMP-30/gluconolactonase/LRE family protein</td><td>pksN</td><td>unknown/fatty acid synthase activity</td><td>Unknown/fatty acid biosynthetic process</td><td>unknown/ Cytoplasm</td><td>41</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_021734242.1</td><td>aldo/keto reductase</td><td>yvgN</td><td>unknown/aldo-keto reductase (NADP) activity</td><td>unknown/small molecule metabolic process</td><td>unknown</td><td>43</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388035.1</td><td>SDR family NAD(P)-dependent oxidoreductase</td><td></td><td>unknown</td><td>unknown</td><td>unknown</td><td>45</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_175,415,675.1</td><td>spore coat protein</td><td>cotO</td><td>unknown</td><td>unknown/sporulation</td><td>unknown</td><td>50</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_014306116.1</td><td>pectate lyase</td><td>pelB</td><td>unknown/pectin lyase activity</td><td>unknown</td><td>unknown/ extracellular region</td><td>55</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388499.1</td><td>5'-nucleotidase C-terminal domain-containing protein</td><td>yhcR</td><td>unknown/5'-nucleotidase activity</td><td>unknown/nucleotide catabolic process</td><td>unknown/ extracellular region</td><td>64</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387766.1</td><td>S9 family peptidase</td><td>yuxL</td><td>unknown/serine-type endopeptidase activity</td><td>unknown/proteolysis</td><td>unknown</td><td>68</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387578.1</td><td>VWA domain-containing protein</td><td>ywmD</td><td>unknown</td><td>unknown</td><td>unknown</td><td>72</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388152.1</td><td>family 10 glycosylhydrolase</td><td>yngK</td><td>hydrolase activity, acting on glycosyl bonds</td><td>metabolic process</td><td>unknown</td><td>73</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154450.1</td><td>sporulation-specific transcriptional regulator GerR</td><td>ylbO</td><td>DNA-binding</td><td>unknown</td><td>unknown</td><td>79</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_014304356.1</td><td>aldo/keto reductase family oxidoreductase</td><td>ycsN</td><td>oxidoreductase activity</td><td>unknown</td><td>cytoplasm</td><td>81</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153362.1</td><td>DUF1002 domain-containing protein</td><td>ypuA</td><td>unknown</td><td>unknown</td><td>plasma membrane</td><td>84</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015387548.1</td><td>cupin domain-containing protein</td><td>bacB</td><td>intramolecular oxidoreductase activity, transposing C = C bond/ cobalt ion binding</td><td>antibiotic biosynthetic process</td><td>cytoplasm</td><td>84</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_015388696.1</td><td>pyridoxamine 5'-phosphate oxidase family protein</td><td>ydaG</td><td>unknown</td><td>stress response</td><td>unknown</td><td>86</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153156.1</td><td>Asp23/Gls24 family envelope stress response protein</td><td>yqhY</td><td>unknown</td><td>unknown</td><td>unknown</td><td>91</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154415.1</td><td>YlmC/YmxH family sporulation protein</td><td>ylmC</td><td>unknown</td><td>sporulation</td><td>unknown</td><td>92</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154717.1</td><td>alpha/beta-type small acid-soluble spore protein</td><td>sspD</td><td>double-stranded DNA binding</td><td>sporulation</td><td>unknown</td><td>92</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_007408043.1</td><td>alpha/beta-type small acid-soluble spore protein</td><td>sspA</td><td>double-stranded DNA binding</td><td>DNA topological change/Sporulation</td><td>unknown</td><td>92</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003152961.1</td><td>flotillin-like protein FloA</td><td>floA</td><td>unknown</td><td>unknown</td><td>plasma membrane</td><td>93</td><td>unknown</td><td>x</td><td></td><td></td></tr></tbody></table>

(continued on next page)
<table><caption>Table 2 (continued)</caption><thead><tr><th>Accession N<sup>a</sup></th><th>Protein Name<sup>b</sup></th><th>Gene<sup>c</sup></th><th>Molecular function<sup>d</sup></th><th>Biological process<sup>e</sup></th><th>Subcellular localization<sup>f</sup></th><th>% Identity<sup>g</sup> with B. subtilis 168 proteins</th><th>COG symbol<sup>h</sup></th><th>CFLP<sup>i</sup></th><th>LB-fraction<sup>j</sup></th><th>TB-fraction<sup>k</sup></th></tr></thead><tbody><tr><td>WP_003154908.1</td><td>DUF1641 domain-containing protein</td><td>yjlC</td><td>unknown</td><td>unknown</td><td>unknown</td><td>95</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003153449.1</td><td>trp RNA-binding attenuation protein MtrB</td><td>mtrB</td><td>DNA-templated transcription termination/RNA binding</td><td>negative regulation of translational initiation</td><td>unknown</td><td>96</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_003154837.1</td><td>phage tail tube protein</td><td>xkdM</td><td>unknown</td><td>unknown</td><td>unknown</td><td>97</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_004264638.1</td><td>sporulation protein YabP</td><td>yabP</td><td>unknown</td><td>sporulation</td><td>membrane/endospore-forming forespore</td><td>98</td><td>unknown</td><td>x</td><td></td><td></td></tr><tr><td>WP_004264630.1</td><td>RNA-binding protein S1</td><td>yabR</td><td>unknown</td><td>unknown</td><td>unknown</td><td>99</td><td>unknown</td><td>x</td><td></td><td></td></tr></tbody></table>

a Accession number.

b Protein name in the NCBI database for *B. amyloliquefaciens* L-17.

c Gene designation.

d Subcellular localization.

e molecular function and f biological process data obtained from the UniProtKB database after BLAST sequence similarity searches.

g Identity (%) in UniProt Protein BLAST: percentage of identical amino acids between the *B. amyloliquefaciens* L-17 proteins sequence and that of *B. subtilis* 168.

h COG category symbol: functional categories according Clusters of Orthologous Genes (COG) database.

i CFLP: proteins extracted on the liquid phase underlying the pellicle.

j LB-fraction: proteins loosely bound to the matrix that were detached by mechanical disruption of the pellicle.

k TB-fraction: proteins tightly bound to the matrix that were chemically extracted of the pellicle.

In green, the identified proteins in L-17 strain having low % identity (<70 %) with that of *B. subtilis* 168.
fibers cannot be formed and the level of TasA protein is reduced (Romero et al., 2011). The interaction network showed that TapA would interact with BsIA protein (WP_003152178.1), one of the major proteins of the L-17 strain pellicle (Section 3.1). This last was described in *Bacillus* species as a major contributor to surface cohesiveness and water repellency for growing biofilms exposed to air (Kalamara et al., 2021; Morris et al., 2017). It also acts synergistically with TasA fibers and exopoly-saccharide to generate structural complexity in the biofilm (Ostrowski et al., 2011). In addition, the interaction network emphasis the interaction between BsIA and the extracellular triacylglycerol lipase (EstA, WP_003156672.1). However, the implication of this lipase in the biofilm formation is not described in the literature.

### 3.2.4. Extracellular enzymes

Several extracellular enzymes were identified in this study (Table 1). Some exhibit interactions according to the prediction of STRING database (Fig. 3c). For example, the *aprE* gene product, S8 family serine peptidase (WP_003155195), was identified among the major proteins of LB-fraction (Fig. 2a). This protein exhibits 84 % sequence identity with its homologous subtilisin BPN' in *B. subtilis* 168. This serine protease is widely used in a variety of various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications (Azrin et al., 2022). We also identified two other extracellular S8 family serine peptidase WP_015388265.1 and WP_014306056.1, homologous to Bpr and Vpr proteases of *B. subtilis* 168, respectively (Table 1 shows the % of sequence identity). Vpr protease is one of the most abundant proteins in the TB-fraction, while Bpr protease was among the most prevalent proteins in the LB and TB fractions.

The abundance of proteases in the matrix and the predicted interaction network highlights the importance of these enzymes in the pellicle of *B. amyloliquefaciens* L-17. It's known that the extracellular proteases support growth of bacteria and play a crucial role in bacterial feeding: the degradation of cellular and environmental proteins as a source of amino acids and peptides (Harwood and Kikuchi, 2022; Rosazza et al., 2024). In *B. subtilis*, Rosazza et al. (2024) highlighted that the extracellular proteases promote colony biofilm formation and modulate its architecture, even when glutamic acid provides a freely available nitrogen source.

Other extracellular enzymes, such as chitosanase (WP_015387749.1), csn gene product which appears to be abundant in LB-fraction may be involved in the defense of the biofilm bacterial community against exogenous micro-organisms. In fact, the chitosanase isolated from *B. licheniformis* (Muslim et al., 2016) and *B. cereus* (Gao et al., 2008) exhibits an antibiofilm and antifungal activity, respectively.

The identification of the glycoside hydrolase family 68 protein (WP_014306184.1) homologous of the levansucrase encoded by *sacB* in *B. subtilis* 168 (Table 1) may suggest the probable presence of levan (homopolymer of fructose) in the EPSs polysaccharidic fraction in the pellicle matrix of *B. amyloliquefaciens* L-17. This enzyme is implicated in the levan biosynthesis (Benigar et al., 2014). Dogsa et al. (2013) showed that Levan, although not essential for biofilm formation, can be a structural and possibly stabilizing component of *B. subtilis* floating biofilms and may also serve as a nutritional reserve. The same hypothesis may be put forward in the pellicle of L-17 strain, but additional analyses are necessary to clarify the presence and the role of the Levan in *B. amyloliquefaciens* L-17 pellicle matrix.

### 3.2.5. Spore related proteins

We identified about 10 proteins related to sporulation (Table 1) showing a predicted interaction on STRING map (Fig. 3c). Examples include CotE, CotR, CotF, CotJC, SpoVS, and SpoIVA. We have emphasized that the CotE and CotR proteins are among the 10 most abundant proteins in the TB-fraction (Fig. 2b) as of SopIVA protein in the LB-fraction (Fig. 2a). In addition, the following proteins: hypothetical protein (WP_021734090.1), FAD-binding oxidoreductase

(WP_003156110.1), gamma-type small acid-soluble spore protein (WP_003155477.1), multicopper oxidase family protein (WP_003155789.1) and glycoside hydrolase family 18 protein (WP_032865795.1) showed strong homology with CotG, YvdP, SspE, CotA, and SleL of *B. subtilis* 168 (Table 1). The role of all these proteins in sporulation is well discussed in the literature (Hilbert and Piggot, 2004; McBride and Haldenwang, 2004; Mckenney et al., 2013; Riley et al., 2020). This suggests the presence of spores in the *B. amyloliquefaciens* L-17 pellicle. Overall, our results suggest cellular specialization and division of labor within the *B. amyloliquefaciens* L-17 pellicle, as reported in *B. subtilis* biofilm. Indeed, in the *B. subtilis* biofilm communities, different groups of cells fulfill distinct functions. Some bacteria produce EPSs while other members of a biofilm community can regain motility to explore the environment for new sources of nutrients. Furthermore, cells within the biofilm can differentiate into spores when the community gets older and nutrients are limiting (Gerwig and Stülke, 2014; Vlamakis et al., 2008). Little knowledge is available regarding the regulatory mechanisms that govern this sophisticated organization within the community.

## 3.3. Analysis of CFLP associated proteins

### 3.3.1. Identification of CFLP associated proteins

During biofilm growth, the coexistence of planktonic and sessile cells can lead to dynamic exchanges between the two populations (El-Khoury et al., 2021). However, the interchanges between bacteria in these two states have been little explored. A molecular characterization of the liquid phase underlying the pellicle appears to be essential to decipher this coordination. In this study, we carried out for the first time a large-scale proteomic analysis of the CFLP of *B. amyloliquefaciens* L-17 (Fig. 1b). We identified 429 CFLP associated proteins (Table 2). Flagellin is the most abundant protein in CFLP (Fig. 4a). It is normal for planktonic *B. amyloliquefaciens* L-17 to produce this protein to build their flagella and ensure locomotion. Nevertheless, the over-presence of this protein in CFLP could suggest that a subpopulation of planktonic cells is propelled by flagella to tunnel deep within biofilm structure generating the biofilm irrigation and increasing nutrient flow in the matrix as it was demonstrated in *B. thuringiensis* 407 (Houry et al., 2012). Once again, we believe that confocal laser microscopy at regular intervals will be necessary to verify and explore this phenomenon in *B. amyloliquefaciens* strain L-17.

### 3.3.2. Comparison of pellicle proteins and CFLP associated proteins identified in *B. amyloliquefaciens* L-17

A total of 118 proteins were concomitantly identified in the pellicle and CFLP (Pell-CFLP proteins). Most of them (53 %) are from the LB-fraction. This is not surprising since the proteins of this fraction are weakly linked to the pellicle matrix, facilitating their transition to CFLP. However, 31 % of the Pell-CFLP proteins were found in the TB-fraction and 16 % are found both in the LB, TB and in CFLP samples (Table 2). These results support the possible exchange relationships that could exist between planktonic *B. amyloliquefaciens* L-17, and their counterparts engaged in pellicle lifestyle. Indeed, this dynamic relationship could involve the globality of matrix proteins and would not be limit to those of the EPSs weakly bound layer.

We focused then on Pell-CFLP proteins that are most abundant in the pellicle (TB and/or LB fractions) and are also prevalent in CFLP (i.e. Tas A and BsIA proteins, flagellin or Bpr serine peptidase) (Fig. 4). Fig. 4b shows a comparison of the abundance of these proteins in LB, TB and CFLP samples. Tas A is over presented in the pellicle and seems to be tightly bound to the matrix. Its identification in the CFLP is unsurprising. The role of TasA in the structure of the *B. subtilis* biofilm is well documented, but it is also produced at the beginning of spore formation, when the cells enter stationary phase, and is secreted into the medium as well as deposited in the spore (Stöver and Driks, 1999). BsIA appears to follow the same scenario. This secreted protein forms surface
<figure><img src="image_6.png" /><figcaption>Fig. 4. Label-free quantification based on spectral counting showing a) the abundance of the identified proteins in the cell free liquid phase (CFLP) of *B. amyloliquefaciens* L-17 and b) the differential abundance of TasA protein, BsIA hydrophobin, flagellin and Bpr serine peptidase in the loosely bound (LB), tightly bound (TB) and CFLP fractions. Statistical significance for NS: no significant, \*p < 0.05, \*\*p < 0.01, and \*\*\*p < 0.001. LB fraction contains proteins detached by mechanical disruption of the pellicle. TB fraction includes proteins chemically extracted from the pellicle.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>b)</figcaption></figure>

hydrophobic layers around the biofilm of *Bacillus* species and renders it water-repellent (Morris et al., 2017). The Bpr extracellular protease is also more abundant in the pellicle than the CFLP reflecting a particular importance of Bpr in the pellicle matrix. Flagellin is over presented both in the LB-fraction and CFLP. This supports the hypothesis of the flagellin importance in the pellicle either to provide mobility to cells that leaves biofilm or to specialize the subpopulation of stealth swimmers giving the pellicle elasticity (Section 3.1).

### 3.3.3. Subcellular localization and biological function of CFLP associated proteins

The subcellular localization and functional categories of each protein are reported in Table 2 and Fig. 4a shows the distributions of CFLP proteins based on these data.

The investigation of cellular location showed that 8 % of CFLP proteins were predicted as strictly extracellular or extracellular with another possible location (Fig. 5a). Of the 10 most CFLP abundant proteins (Fig. 4a), 8 fit into this scenario (Table 2). We noted that this category includes 10 proteins (27 %) identified only in CFLP and were not found in pellicle (Table 2). These are mainly enzymes, which would

<figure><img src="image_8.png" /><figcaption>Fig. 5. (a) Subcellular localization and (b) functional categories of the cell free liquid phase associated proteins identified in *B. amyloliquefaciens* L-17. The proteins with dual localization can be associated to membrane/cytoplasm, cell membrane/spore or cytoplasm/spore. The Cell membrane localization groups together plasma membrane, periplasmic space and bacterial flagellum. The category "Others" groups together functional categories with <3 proteins (i.e. mobilome: prophages, transposons; Intracellular trafficking, secretion, and vesicular transport).</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>b)</figcaption></figure>
be produced and released into the extracellular environment by plank-
tonic bacteria. Half of the proteins are originally from the cytoplasm, the
periplasmic space or the plasma membrane. This indicates significant
cell lysis given that the culture is 72 h old, and the environment is no
longer very favorable to bacterial growth. The residence of predicted
cytoplasmic and cell membrane proteins in the extracellular proteome
has also been reported and discussed in B. subtilis by Tjalsma et al.
(2004). In addition to the obvious hypothesis of cell lysis, these authors
support that these proteins would leave the cellular compartments via
flagellar export machinery, the holin systems, or other unidentified
export systems. We also found a few proteins (2 %) related to the spore
in CFLP but most of them were also identified in the pellicle. Finely, the
subcellular localization of 164 proteins (38 %) remains unknown.
Among these proteins, 105 (24 % of CFLP identified proteins) have not
described localization in the databases and 59 proteins (14 % of CFLP
identified proteins) present <70 % sequence identity with their ho-
mologous in B. subtilis 168 (Table 2). Because of this low homology, we
cannot transpose the information available on the proteins of B. subtilis
to that of B. amyloliquefaciens. We have therefore included these proteins
in the unknown category.

All the identified 429 CFLP proteins were classified in clusters of
orthologous genes (COGs) functional categories (Fig. 5b). CFLP identi-
fied proteins are involved in various physiological processes. We then
focused on the 310 proteins only identified in the CFLP (Table 2),
admitting that the remainder of proteins that simultaneously found in
CFLP and pellicle have already been discussed in the previous para-
graphs. We noted that the molecular function and biological process of a
large proportion (23 %) of proteins are not known. We identified 5 non-
ribosomal peptide synthases (WP_015388164.1, WP_015388149.1,
WP_015388166.1, WP_015388212.1, WP_015388165.1) which reflect
the presence of non-ribosomally synthesized antimicrobial peptides in
CFLP (Table 2). In addition, we identified the amino acid adenylation
domain-containing protein (WP_197,923,070.1) homologous of surfac-
tin synthase A of B. subtilis 168 (72 % sequence identity). This suggests
the presence of surfactin, a well-studied biosurfactant with antifungal
properties, produced by various Bacillus species such as
B. amyloliquefaciens, B. subtilis, and B. pumilus (Jacques, 2011; Ranjan
et al., 2023). This may also be supported by the presence of the peptide
ABC transporter substrate-binding protein (WP_015388449.1), the oppA
gent product, among the most abundant CFLP identified proteins
(Fig. 4a). Indeed, OppA plays a key role in surfactin synthesis regulation
in B. subtilis (Wang et al., 2019). A targeted mass spectrometry analysis,
aimed at identifying antimicrobial peptides in CFLP, would be necessary
to characterize these bioactive molecules. Moreover, we identified a
panel of enzymes in the CFLP in which the trifunctional nucleotide
phosphoesterase YfkN, glucuronoxylanase xynC and the catalase KatA
were over presented (Fig. 4a). These enzymes were also characterized in
the cell free supernatant of the B. subtilis planktonic (Chambert et al.,
2003; Naclerio et al., 1995; St. John et al., 2006). Other enzymes
including proteases, lipases, aminotransferases, peroxidases and phytase
were also characterized in CFLP (Table 2). This result is not surprising
since B. amyloliquefaciens and other Bacillus species have long been
recognized to produce commercial enzymes having biotechnological
applications including textile applications, feed industry, food industry,
and organic synthesis (Farhat-Khemakhem et al., 2018; Ngalimat et al.,
2021; Rizwanuddin et al., 2023).

Based on our findings, we can conclude that the liquid phase un-
derlying the pellicle of L-17 strain should not be overlooked. It requires
particular interest since (i) it seems to be the site of collaboration and
regulation between the mobile and sessile bacterial subpopulations and
(ii) it could be a source of commercial enzymes and natural antimicro-
bial agents applicable in various fields.

4. Conclusion

The present study provides the first global picture of proteins both in

the pellicle matrix and in the liquid phase of *B. amyloliquefaciens* L-17
occurred by high-throughput proteomic approach. For the pellicle ma-
matrix proteins characterization, the combination of physical (ULTRA-
TURRAX grinding) and chemical (NaOH treatment) technique es of EPSs
extraction followed by the MS analysis allowed to establish a list of 131
pellicle associated proteins which can loosely or tightly bound to the
matrix. The identified proteins are involved in various physiological
processes. The main identified proteins in the pellicle of L-17 strain are
TasA, TapA, and BsIA whose roles in the biofilm architecture and pro-
prieties are well described in *B. subtilis*. The literature available data and
the interaction network highlighted that these proteins deserve to be
genetically and biochemically explored to provide more explanations
and information on their role in the physiology of the air-liquid interface
biofilm in L-17 strain.

The identification of the flagellin and the related-sporulation pro-
teins suggests a cellular specialization and a repartition of labor within
the pellicle. Further microscopy studies are necessary to explore this
hypothesis. Since the protein composition of the matrix is continually
evolving, it would be interesting to perform proteomic analysis at
different stages of pellicle life. This should be completed by the prote-
omic characterization of cellular proteins extracted from bacteria asso-
ciated pellicle. Together, these investigations would allow us to
highlight important molecular players in the formation, maintenance
and regulation of the pellicle.

Proteomic characterization of the liquid phase underlying the pellicle showed that this fraction is rich in proteins since 423 proteins were identified. Among them, some have also been identified in the matrix of the pellicle. This strengthens the idea of dynamic exchanges between the sessile and planktonic populations. Liquid phase specific proteins include enzymes involved in the biosynthesis process of non-ribosomal peptides. Studies, such as targeted MS, seem to be necessary to characterize these bioactive agents. A variety of commercial enzymes are also characterized. This finding can promote the application of liquid phase in biocontrol, and in different industrial and agricultural fields.

Ultimately, this study constitutes the foundation for further biolog-
ical studies to (i) draw conclusions regarding the pellicle formation and
regulation (ii) understand its spatial and the architectural organization
(iii) elucidate social interactions and the division of labor in the mi-
crobial community (iv) decipher the molecular interaction network
between the sessile and planktonic population (v) highlight the appli-
cation of the cell-free liquid phase in industry and agriculture.

CRediT authorship contribution statement

**Tassadit Ouidir:** Writing – original draft, Methodology, Data cura-
tion, Conceptualization. **Julie Hardouin:** Investigation, Data curation,
Formal analysis. **Claire-Emmanuelle Marcato-Romain:** Writing – re-
view & editing. **Elisabeth Girbal-Neuhauser:** Visualization, Writing –
review & editing, Investigation. **Yassine Nait Chabane:** Conceptuali-
zation, Methodology, Writing – review & editing, Supervision, Project
administration.

Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgement

This research is supported by a grant from the Occitanie Regional
Council and funding from Toulouse University (Grant n 000268/
2017001895). The authors are grateful to Meriem ZAIDI-AIT SALEM for
carrying out the bacterial culture and proteins extraction and to the staff
of the PISSARO (Normandy University, Rouen, France) for proteomic
analysis.