# Genetic Clearness Novel Strategy of Group IBacillus Species Isolated from Fermented Food and Beverages by Using Fibrinolytic Enzyme Gene Encoding a Serine-Like Enzyme

Authors: Moïse Doria Kaya-Ongoto; Christian Aimé Kayath; Etienne Nguimbi; Aimé Augustin Lebonguy; Stech Anomène Eckzechel Nzaou; Paola Sandra Elenga Wilson; Gabriel Ahombo
Journal: Journal of Nucleic Acids
 (2019)

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## Abstract

Fibrinolytic enzyme gene (fibE) is widely conserved amongBacillus spp. belonging to group I species. This is encoding a serine-like enzyme (FibE) secreted in extracellular medium. This present work aims to assess the molecular usefulness of this novel conserved housekeeping gene among group IBacillus spp. to identify and discriminate some related strains in traditional fermented food and beverages in Republic of Congo. First of all 155 isolates have been screened for enzymatic activities using caseinolytic assays. PCR techniques and nested PCR method using specific primers and correlated with 16S RNA sequencing were used. Blotting techniques have been performed for deep comparison with molecular methods. As a resultB. amyloliquefaciens (1),B. licheniformis (1),B. subtilis (1),B. pumilus (3),B. altitudinis (2),B. atrophaeus (1), andB. safensis (3)have been specifically identified among 155 isolates found in fermented food and beverages. Genetic analysis and overexpression of glutathione S-transferases (GSTs) fused to mature protein of FibE inEscherichia coli BL21 and TOP10 showed 2-fold higher enzymatic activities by comparison with FibE wild type one. Immunodetection should be associated but this does not clearly discriminateBacillus belonging to group I.


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## Body

## 1. Introduction

From north to south of Republic of Congo fermented foods and beverages diversify and do contain a very impressive microorganism biodiversity. The most common microorganisms are the genus ofBacillus, lactic acid bacteria, non-lactic acid bacteria, molds, and yeast that play very important role in local fermented foods and beverages [1–4].Bacillus strains are ubiquitous bacteria that exploit about twenty wide varieties of Congo Brazzaville fermented food and beverages including “Toba mbody” which is a traditional food obtained by alkaline fermentation of cassava leaves. Not only leaves but also cassava tubers could be fermented after three to four days by producing a local product much consumed in the south of the Republic of Congo called “Bikedi”. Ripe cassava tubers can be mixed with peanut paste allowing obtaining “Mbala-mpinda”. In addition more popular beverage is palm wine which is an alcoholic beverage created from the sap of palm tree called “Samba”. Another beverage found is “Loungouila”,a sugar cane wine, and “Mbavu”,a local banana wine. A very recent study is focusing on “Pandé” which is a local fermented food ofRaphia taedigera (Mart.) Mart. (1838) [3–5]. Members of the genusBacillus are rod-shaped spore-forming bacteria belonging to the firmicutes with DNA base composition around 50 to 54% G+C. Besides, bacilli are more stable during processing and storage of fermented food and beverages [6] and pharmaceutical preparations. These have been shown suitable candidates for health promoting formulations [7]. An analysis of twenty completeBacillus genomes has been demonstrated and drafted.B. subtilis, B. pumilus, B. licheniformis,andB. amyloliquefaciensare more genome related based on core genome, Genomic Similarity Score (GSS) [8], and Cluster of Orthologous Groups (COGs) [9]. Conserved signature indels (CSIs) which are inserts or deletions within conserved regions of homologous proteins [10] have been used to discriminate in silicoBacillus spp. Several ways have been described to split thisBacillusgroup according to biochemistry, lifestyles, and/or growth on different substrates [9].Bacillus spp. may be divided into nine groups (groups I–IX), based on their high level phylogeny or phenotypic features linked to 16S rRNA gene sequence similarity.B. subtilis, B. pumilus, B. licheniformis,B. amyloliquefaciens, B. altitudinis, B. mojavensis,B. safensis, B. circulans,andB. atrophaeusbelong to group I [10, 11]. Interestingly, most of the aforementioned bacteria are related species and share a remarkably high level of 16S rRNA gene sequence similarity toB. subtilis which is around 99% or greater even though DNA–DNA hybridization values with the latter fall below 70 % [12]. Moreover very few molecular discrimination, phenotypic, or biochemical characteristics that differentiate these species from each other can be found.Sometimes the genusBacillus is phylogenetically incoherent taxon with members of the group lacking a common evolutionary matter [10]. In this way genetic discrimination must be used. Some prokaryotic housekeeping genes playing various vital key roles for cell survival have been used to discriminate bacteria including 16S rRNA gene. Housekeeping genes are molecularly important to assess a rapid and reliable result in terms of identification of closely relatedBacillus strains. Among housekeeping genesrpoB,gyrB,nifD,recA, andatpD are the most cited [13, 14]. These are considered like the bright and first choices in terms of multilocus sequence typing [13]. 16S rRNA gene is highly conserved and is an essential standard for bacteria taxonomy [15]. Partial sequencing of the 16S rRNA gene has been also used for the rapid identification ofBacillus bacteria [16]. However, this criterion seems to be insufficient for some species [17]. Indeed, the classification of closely related bacterial species seems to be sometimes difficult [18]. It has been shown that the 16S rRNA gene can undergo occasional transfer or lateral recombination [19]. The other drawback related to the use of the 16S rRNA gene is that this gene often has a variable number of copies. Some species may have one to fifteen ribosomal operons in their genome [20], which does not allow a quantitative study of the species.Bacteria belonging to theBacillusgroup I [11] includingB. subtilis, B. pumilus, B. licheniformis,B. amyloliquefaciens, B. mojavensis, B. safensis, B. altitudinis, andB. atrophaeus harbor, a wildly conserved gene encoding the “so-called” fibrinolytic enzyme (fibE), a serine-like enzyme. This enzyme should play a prime role in the treatment of cardiovascular disease. Homologues of this gene have been isolated.B. subtilis natto producing nattokinase (NK) has been isolated from a Japanese fermented food made from soybeans [21].B. amyloliquefaciens CH5,B. amyloliquefaciens DC-4, andBacillus sp. CK were isolated from fermented soybean [22–25].B. amyloliquefaciens NM76, producing an enzyme with high fibrinolytic activity, was isolated from Ntoba Mbodi, a fermented food from Republic of Congo [26]. In NCBI databases, thefibE gene has been named according to theBacillus species. This has been designated as peptidase S8 inB. pumilus 3-19 [27] andB. safensis JPL_MERTA8-2 [28], while inB. mojavensis, A21, the product of this gene, was nominated as subtilisin BM1 and this gene was calledaprE inB. licheniformis F5. In this work, this gene has been nominatedfibE(fibrinolytic enzyme gene). The product of this gene is encoded as pre-pro-protein (28kDa). The presequence corresponds to a signal sequence of SEC pathways, with a characteristic motif AXA cleavable by the signal peptidase [29]. The prosequence is a sequence that is cleaved for the protein maturation processing [30]. The size of the gene corresponding from the cleavable motif is about 850 bp depending on the species. This gene is highly conserved through group IBacillus and this has a sequence homology close to 90%. As part of this work, we targeted this gene for its nucleotide variability at the start point of mature protein incliding AQSV amino acides. We are focusing on some important troubleshooting to identify species within the complexity ofBacillus spp. belonging to group I.


## 2. Material and Methods

### 2.1. Isolation and Characterization of Strains

10g of each collected sample from fermented foods, beverages, soil, and intestine of Guppy fish was aseptically sampled into a sterile falcon tube. Using sterile water the sample was homogenized and distributed in ten flacon sterile tubes. Dilutions were done and bacterial suspension was streaked on Mossel agar medium supplemented with 4.2 mL of polymyxin B. Enumeration of colonies was done in triplicate on the same medium. The plates were incubated at 37°C for 24 h to 48°C. Microscope has been used for morphological characterization and the Gram status confirmation was determined using 3% KOH. Sporulation, hydrogen peroxide (H2O2), and oxidases tests were used for biochemical characterization.


### 2.2. Enzymatic Activities

In order to discriminate at the genera level ofBacillus strains, enzymatic activities were performed by using casein as a substrate. Briefly 1 g of agarose was weighed and mixed with 100 mL of PBS. The mixture was heated in a microwave for 3 min until agarose was completely dissolved and then cooled in a water bath at 40°C. Then 10 mL of skim milk was added to the mixture. After homogenization, the mixture was poured into the Petri dishes. Once solidified, wells were carefully and aseptically generated into the gels. An overnight culture was first initiated at 37°C with stirring. The culture was then centrifuged at 6000 rpm for 10 min. A volume of 50 μL of the supernatant is deposited in the wells made on the agar medium composed of 1% agarose gel, 0.01 M PBS, pH 7.4, and skimmed milk. The Petri dishes are incubated at 37°C for 24 hours. The presence of caseinolytic activity is detected by a clear halo around colonies indicating hydrolysis of casein. The halo diameter was measured.


### 2.3. Genomic DNA Extraction, Sequencing, and DNA Technology Identification

In order to directly identify the isolates by using DNA technology, thefibE gene encoding for the fibrinolytic enzyme has been found in the NCBI (National Center for Biotechnology Information, http://www.ncbi.gov/Blast.cgi) genomic database of targeted strains likeB. amyloliquefaciens,B. subtilis, B. pumilus, B. licheniformis, B. altitudinis, B. mojavensis, B. safensis,andB. atrophaeus. The pDRAW32 software has been used for bioinformatic analysis. The primers were rigorously selected from the mature protein and internal primers for nested PCR method were used to generate 461 pb amplicon (Table 1). Extraction and purification of isolate genomic DNA were performed according to the NucleoSpin Microbial DNA (Macherey-NAGEL) kit. Briefly, isolates were grown in 5 mL LB broth for 24 h at 37°C with stirring. The DNA purity was assessed by electrophoresis on agarose gel and by the ratio of optical densities 260/280 nm. PCR products have been sequenced. By using universal primers of16S rRNA fD1 (5′- AGAGTTTGATCCTGGCTCAG -3′) and rP2 (5′- ACGGCTACCTTGTTACGACTT -3′), confirmation of isolates has been done. 5 μL of each amplification product was mixed with 2 μL of loading buffer (BIOKE). Mixtures were subjected to electrophoresis on 1% agarose gel (w/v). The 10 kb 2-Log (BIOKE) was used as a molecular weight marker. The PCR products were purified using the solution for Gel Extraction kit (Omega Bio-tek), the purified products were subjected to sequencing by the Sanger technique (3130xl Genetic Analyzer, Applied Biosystems). The sequences obtained were aligned with the software Bio Numerics 7.5 (Applied Maths, Belgium) and corrected manually to resolve discrepancies between the sense and antisense strands. Sequences were compared with homologous sequences contained in the sequence databanks through the portal NCBI using the BLASTn program based on the identification criterion published by Drancourt [31].Table 1
Primers used in this study in terms of identification of strains.
Oligos names 5′- - - -3′ Size Targeted species For Group 1 Bacillus Identification Ba.IdMa-F GCGCAGTCCGTGCCTTACGGCGT 828 pb B. amyloliquefaciens Ba.IdMa-R TTACTGAGCTGCCGCCTGTACG Ba.SHMa-F TACAACTCTCACGGAACTCACGTTGCC 461 pb Ba.SHMa-R TATTTGTTTCCAGGAAGCGTG  Bl.Id.Ma-F GCGCAAACCGTTCCTTACGGCAT 825pb B. licheniformis Bl.Id.Ma-R TTATTGAGCGGCAGCTTCGAC Bl.SHMa-F GGCAACGGACACGGCACACATGTTGC 461 pb Bl.SHMa-R TAAGTGTTCGTTGGGTAAGTG  Bs.Id.Ma-F GCGCAATCTGTTCCTTATGGCAT 835 pb B. subtilis Bs.Id.Ma-R TTATTGTGCAGCTGCTTGTACGTTGA Bs.SHMa-F GGCAGTTCTCACGGTACGCATGTAGCC 461 pb Bs.SHMa-R TAAGTGCCTCCAGGAAGTGTG  Bp.Id.Ma-F GCACAAACCGTCCCTTATGGAAT 828 pb B. pumilus Bp.Id.Ma-R TTAGTTAGAAGCCGCTTGAGCG Bp.SHMa-F TTTCAATCACATGGAACTCACGTAGCC 461 pb Bp.SHMa-R TATCCACTGCTTGGTACTGTA  Bm.Id.Ma-F GCGCAATCTGTTCCTTACGGCAT 837 pb B. mojavensis Bm.Id.Ma-R TTATTGTGCAGCTGCCTGCAC Bm.SHMa-F GGCAGTTCTCACGGCACGCATGTAGCC 461 pb Bm.SHMa-R TATGTGCCGCCAGGAAGTGTG  Bsa.Id.Ma-F GCACAAACCGTCCCTTATGGAAT 828 pb B. safensis Bsa.Id.Ma-R TTAGTTAGAAGCCGCTTGAACGTTG Bsa.SHMa-F TTTCAATCACATGGAACTCACGTAGCA 461 pb Bsa.SHMa-R TATCCACTGCTTGGCACTGTA  Bat.Id.Ma-F GCTCAGTCA GTACCTTATG GCAT 828 pb B. atrophaeus Bat.Id.Ma-R TTATTGCGCTGCTGCCTGAACG Bat.SHMa-F GGAAATTCACACGGAACTCACG 461 pb Bat.SHMa-R TAGCTGCTGCCCGGAAGTGTG  Bal.Id.Ma-F GGTCAAAGCGTCCCTTATGGTA 828 pb B. altitudinis Bal.Id.Ma-R TTATCGTGCAGCTTTTTGTAC Bal.SHMa-F CCGCATGAACACGGAACCCACG 461 pb Bal.SHMa-R TATTCGTTATGAGGAATGGTA


### 2.4. Plasmids Construction and Expression of Recombinant Proteins

Primers used for constructions are listed in Table2 and plasmids in Table 3. After identifying strains using DNA technology at the species level, all thefibE fragments were amplified from chromosome ofBacillusspp. genomic DNA. PCR fragments were amplified by using OneTaq DNA polymerase (Bioke) and inserted in frame withgst in pGEX-4T-1 digested byBamHI andEcoRI of gene amplified fromB. amyloliquefaciens,B. pumilus,B. mojavensis,B. subtilis,B. atrophaeus, andB. altitudinis, BamHI andXhoI forB. licheniformis, andEcoR1 andXho1 forB. safensis(see Table 3). The resulting plasmids were named pSNZ1, pDOK2, pDOK3, pDOK5, pDOK6, pDOK7, pDOK8, and pDOK9 (Table 3). All constructions were randomly checked by DNA sequencing.E. coli strains BL21 and TOP10, harboring pGEX-4T-1 or its derivatives (see Table 3) expressing GST-FibE from different eight strains of group IBacillus, were cultured in LB broth with 100 μg/mL ampicillin for 2 h at 37°C, and then IPTG was added to a final concentration of 0.1 mM. After incubation for 3 h at 30°C, bacteria were harvested and GST fusion proteins were purified as described by the manufacturer of Glutathione Sepharose 4B (Amersham Pharmacia Biotech). Proteins production and purification were monitored by SDS-PAGE and Coomassie blue. Purified proteins were also analyzed by immunoblotting using anti-GST antibodies (GE Healthcare). Enzymatic activities were then assessed by using a volume of 50 μL of the supernatant coming fromE. coli TOP10 and BL21 with pGEX-4T-1 or its derivatives. The diameter of clear zone of enzymatic activities was then measured.Table 2
Primers used in this study for plasmid constructions.
bamACK1F TGGATCCAGAGGCAAAAAGGTATGGATCAG B. amyloliquefaciens ACK1 TGGATCCGCGCAGTCCGTGCCTTACGGCGT ecoACK2R AGAATTCTTACTGAGCTGCCGCCTGTACG  bamACK3F TGGATCCATGAGGAAAAAGAGTTTTTGGCTTGGG B. licheniformis ACK2 TGGATCC__GCGCAAACCGTTCCTTACGGCAT xhoACK4R ACTCGAGTTATTGAGCGGCAGCTTCGAC  bamACK5F TGGATCCAGAAGCAAAAAATTGTGGATCAGC B. subtilis ACK3 TGGATCCGCGCAATCTGTTCCTTATGGCAT ecoACK6R AGAATTCTTATTGTGCAGCTGCTTGTACGTTGA  bamACK7F AGGATCCAAAA AGAAAAATGT GATGACAAGT B. pumilus ACK4 A G G A T C C _ _GCACAAACCGTCCCTTATGGAAT ecoACK8R AGAATTCTTAGTTAGAAGCCGCTTGAGCG  bamACK9F AGGATCCAGAAGCAAAAAATTGTGGATCAGC B. mojavensis ACK5 AGGATCC__GCGCAATCTGTTCCTTACGGCAT ecoACK10R AGAATTCTTATTGTGCAGCTGCCTGCAC  ecoACK11F T G A A T T C _ _TGCGTGAAAAAGAAAAATGTGATGACA B. safensis ACK6 T G A A T T C _ _GCACAAACCGTCCCTTATGGAAT xhoACK12R ACTCGAGTTAGTTAGAAGCCGCTTGAACGTTG  bamACK13F TGGATCCAGAAGCA GGAAATTATG GATCGG B. atrophaeus ACK7 T GGATCCGCTCAGTCA GTACCTTATG GCAT ecoACK14R AGAATTCTTATTGCGCTGCTGCCTGAACG  bamACK15F TGGATCCAAGGTCA AATCATTTGG AGCAGGG B. altitudinis ACK8 T G G A T C C _ _GGTCAAAGCGTCCCTTATGGTA ecoACK16R AGAATTCTTATCGTGCAGCTTTTTGTACTable 3
List of constructions used and nominated strains as well.
Plasmids Constructions Strains used to transform constructions Given names expressing pGEX-4T-1 and its derivatives References pGEX-4T-1  E. coli BL21 APK1 This study E. coli TOP10 APK2 This study  pSNZ1 pGEX-4T-1:fibEma-Ba E. coli BL21 QEK1 This study E coli TOP10 KE1 This study  pDOK2 pGEX-4T-1:fibEma-Bs E. coli BL21 QEK2 This study E. coli TOP10 KE2 This study  pDOK3 pGEX-4T-1:fibEma-Bl E. coli BL21 QEK3 This study E. coli TOP10 KE3 This study  pDOK5 pGEX-4T-1:fibEma-Bp E. coli BL21 QEK4 This study E. coli TOP10 KE4 This study  pDOK6 pGEX-4T-1:fibE-Bm E. coli BL21 QEK5 This study E. coli TOP10 KE5 This study  pDOK7 pGEX-4T-1:fibEma-Bsa E. coli BL21 QEK6 This Study E. coli TOP10 KE6 This study  pDOK8 pGEX-4T-1:fibEma-Bal E. coli BL21 QEK7 This study E. coli TOP10 KE7 This study  pDOK9 pGEX-4T-1:fibEma-Bat E. coli BL21 QEK8 This Study E. coli TOP10 KE8 This study


### 2.5. Generating Polyclonal Antibodies and Bacillus Immunodetection

Polyclonal antibodies were generated by using five-week-old female specific-pathogen-free BALB/c mice. 10μg of GST-FibE derivatives has been injected into naïve BALB/c mice in the first week. A reminder on intraperitoneal injection was performed two weeks later to elicit the production of antibodies. All purified variants of fusion proteins including GST-FibE-Bs, GST-FibE-Ba, GST-FibE-Bl, GST-FibE-Bp, GST-FibE-Bsa, GST-FibE-Bat, and GST-FibE-Bal were exhausted against the background of Crude extracts and culture supernatants ofE. coli TOP10 producing GST alone. Overnight pure cultures using LB medium ofB. amyloliquefaciens, B. licheniformis, B. subtilis, B. pumilus, B. mojavensis, B. safensis, B. atrophaeus,andB. altitudinishave been performed. Supernatants were mixed with 80% NH4SO4 at +4°C. Cultures were centrifuged at 6000 rpm, during 1 hour. Pellets were collected with 500 μL of PBS. SDS-PAGE stained with Coomassie blue and immunodetection were carried out using polyclonal antibodies anti-FibE variants.


## 2.1. Isolation and Characterization of Strains

10g of each collected sample from fermented foods, beverages, soil, and intestine of Guppy fish was aseptically sampled into a sterile falcon tube. Using sterile water the sample was homogenized and distributed in ten flacon sterile tubes. Dilutions were done and bacterial suspension was streaked on Mossel agar medium supplemented with 4.2 mL of polymyxin B. Enumeration of colonies was done in triplicate on the same medium. The plates were incubated at 37°C for 24 h to 48°C. Microscope has been used for morphological characterization and the Gram status confirmation was determined using 3% KOH. Sporulation, hydrogen peroxide (H2O2), and oxidases tests were used for biochemical characterization.


## 2.2. Enzymatic Activities

In order to discriminate at the genera level ofBacillus strains, enzymatic activities were performed by using casein as a substrate. Briefly 1 g of agarose was weighed and mixed with 100 mL of PBS. The mixture was heated in a microwave for 3 min until agarose was completely dissolved and then cooled in a water bath at 40°C. Then 10 mL of skim milk was added to the mixture. After homogenization, the mixture was poured into the Petri dishes. Once solidified, wells were carefully and aseptically generated into the gels. An overnight culture was first initiated at 37°C with stirring. The culture was then centrifuged at 6000 rpm for 10 min. A volume of 50 μL of the supernatant is deposited in the wells made on the agar medium composed of 1% agarose gel, 0.01 M PBS, pH 7.4, and skimmed milk. The Petri dishes are incubated at 37°C for 24 hours. The presence of caseinolytic activity is detected by a clear halo around colonies indicating hydrolysis of casein. The halo diameter was measured.


## 2.3. Genomic DNA Extraction, Sequencing, and DNA Technology Identification

In order to directly identify the isolates by using DNA technology, thefibE gene encoding for the fibrinolytic enzyme has been found in the NCBI (National Center for Biotechnology Information, http://www.ncbi.gov/Blast.cgi) genomic database of targeted strains likeB. amyloliquefaciens,B. subtilis, B. pumilus, B. licheniformis, B. altitudinis, B. mojavensis, B. safensis,andB. atrophaeus. The pDRAW32 software has been used for bioinformatic analysis. The primers were rigorously selected from the mature protein and internal primers for nested PCR method were used to generate 461 pb amplicon (Table 1). Extraction and purification of isolate genomic DNA were performed according to the NucleoSpin Microbial DNA (Macherey-NAGEL) kit. Briefly, isolates were grown in 5 mL LB broth for 24 h at 37°C with stirring. The DNA purity was assessed by electrophoresis on agarose gel and by the ratio of optical densities 260/280 nm. PCR products have been sequenced. By using universal primers of16S rRNA fD1 (5′- AGAGTTTGATCCTGGCTCAG -3′) and rP2 (5′- ACGGCTACCTTGTTACGACTT -3′), confirmation of isolates has been done. 5 μL of each amplification product was mixed with 2 μL of loading buffer (BIOKE). Mixtures were subjected to electrophoresis on 1% agarose gel (w/v). The 10 kb 2-Log (BIOKE) was used as a molecular weight marker. The PCR products were purified using the solution for Gel Extraction kit (Omega Bio-tek), the purified products were subjected to sequencing by the Sanger technique (3130xl Genetic Analyzer, Applied Biosystems). The sequences obtained were aligned with the software Bio Numerics 7.5 (Applied Maths, Belgium) and corrected manually to resolve discrepancies between the sense and antisense strands. Sequences were compared with homologous sequences contained in the sequence databanks through the portal NCBI using the BLASTn program based on the identification criterion published by Drancourt [31].Table 1
Primers used in this study in terms of identification of strains.
Oligos names 5′- - - -3′ Size Targeted species For Group 1 Bacillus Identification Ba.IdMa-F GCGCAGTCCGTGCCTTACGGCGT 828 pb B. amyloliquefaciens Ba.IdMa-R TTACTGAGCTGCCGCCTGTACG Ba.SHMa-F TACAACTCTCACGGAACTCACGTTGCC 461 pb Ba.SHMa-R TATTTGTTTCCAGGAAGCGTG  Bl.Id.Ma-F GCGCAAACCGTTCCTTACGGCAT 825pb B. licheniformis Bl.Id.Ma-R TTATTGAGCGGCAGCTTCGAC Bl.SHMa-F GGCAACGGACACGGCACACATGTTGC 461 pb Bl.SHMa-R TAAGTGTTCGTTGGGTAAGTG  Bs.Id.Ma-F GCGCAATCTGTTCCTTATGGCAT 835 pb B. subtilis Bs.Id.Ma-R TTATTGTGCAGCTGCTTGTACGTTGA Bs.SHMa-F GGCAGTTCTCACGGTACGCATGTAGCC 461 pb Bs.SHMa-R TAAGTGCCTCCAGGAAGTGTG  Bp.Id.Ma-F GCACAAACCGTCCCTTATGGAAT 828 pb B. pumilus Bp.Id.Ma-R TTAGTTAGAAGCCGCTTGAGCG Bp.SHMa-F TTTCAATCACATGGAACTCACGTAGCC 461 pb Bp.SHMa-R TATCCACTGCTTGGTACTGTA  Bm.Id.Ma-F GCGCAATCTGTTCCTTACGGCAT 837 pb B. mojavensis Bm.Id.Ma-R TTATTGTGCAGCTGCCTGCAC Bm.SHMa-F GGCAGTTCTCACGGCACGCATGTAGCC 461 pb Bm.SHMa-R TATGTGCCGCCAGGAAGTGTG  Bsa.Id.Ma-F GCACAAACCGTCCCTTATGGAAT 828 pb B. safensis Bsa.Id.Ma-R TTAGTTAGAAGCCGCTTGAACGTTG Bsa.SHMa-F TTTCAATCACATGGAACTCACGTAGCA 461 pb Bsa.SHMa-R TATCCACTGCTTGGCACTGTA  Bat.Id.Ma-F GCTCAGTCA GTACCTTATG GCAT 828 pb B. atrophaeus Bat.Id.Ma-R TTATTGCGCTGCTGCCTGAACG Bat.SHMa-F GGAAATTCACACGGAACTCACG 461 pb Bat.SHMa-R TAGCTGCTGCCCGGAAGTGTG  Bal.Id.Ma-F GGTCAAAGCGTCCCTTATGGTA 828 pb B. altitudinis Bal.Id.Ma-R TTATCGTGCAGCTTTTTGTAC Bal.SHMa-F CCGCATGAACACGGAACCCACG 461 pb Bal.SHMa-R TATTCGTTATGAGGAATGGTA


## 2.4. Plasmids Construction and Expression of Recombinant Proteins

Primers used for constructions are listed in Table2 and plasmids in Table 3. After identifying strains using DNA technology at the species level, all thefibE fragments were amplified from chromosome ofBacillusspp. genomic DNA. PCR fragments were amplified by using OneTaq DNA polymerase (Bioke) and inserted in frame withgst in pGEX-4T-1 digested byBamHI andEcoRI of gene amplified fromB. amyloliquefaciens,B. pumilus,B. mojavensis,B. subtilis,B. atrophaeus, andB. altitudinis, BamHI andXhoI forB. licheniformis, andEcoR1 andXho1 forB. safensis(see Table 3). The resulting plasmids were named pSNZ1, pDOK2, pDOK3, pDOK5, pDOK6, pDOK7, pDOK8, and pDOK9 (Table 3). All constructions were randomly checked by DNA sequencing.E. coli strains BL21 and TOP10, harboring pGEX-4T-1 or its derivatives (see Table 3) expressing GST-FibE from different eight strains of group IBacillus, were cultured in LB broth with 100 μg/mL ampicillin for 2 h at 37°C, and then IPTG was added to a final concentration of 0.1 mM. After incubation for 3 h at 30°C, bacteria were harvested and GST fusion proteins were purified as described by the manufacturer of Glutathione Sepharose 4B (Amersham Pharmacia Biotech). Proteins production and purification were monitored by SDS-PAGE and Coomassie blue. Purified proteins were also analyzed by immunoblotting using anti-GST antibodies (GE Healthcare). Enzymatic activities were then assessed by using a volume of 50 μL of the supernatant coming fromE. coli TOP10 and BL21 with pGEX-4T-1 or its derivatives. The diameter of clear zone of enzymatic activities was then measured.Table 2
Primers used in this study for plasmid constructions.
bamACK1F TGGATCCAGAGGCAAAAAGGTATGGATCAG B. amyloliquefaciens ACK1 TGGATCCGCGCAGTCCGTGCCTTACGGCGT ecoACK2R AGAATTCTTACTGAGCTGCCGCCTGTACG  bamACK3F TGGATCCATGAGGAAAAAGAGTTTTTGGCTTGGG B. licheniformis ACK2 TGGATCC__GCGCAAACCGTTCCTTACGGCAT xhoACK4R ACTCGAGTTATTGAGCGGCAGCTTCGAC  bamACK5F TGGATCCAGAAGCAAAAAATTGTGGATCAGC B. subtilis ACK3 TGGATCCGCGCAATCTGTTCCTTATGGCAT ecoACK6R AGAATTCTTATTGTGCAGCTGCTTGTACGTTGA  bamACK7F AGGATCCAAAA AGAAAAATGT GATGACAAGT B. pumilus ACK4 A G G A T C C _ _GCACAAACCGTCCCTTATGGAAT ecoACK8R AGAATTCTTAGTTAGAAGCCGCTTGAGCG  bamACK9F AGGATCCAGAAGCAAAAAATTGTGGATCAGC B. mojavensis ACK5 AGGATCC__GCGCAATCTGTTCCTTACGGCAT ecoACK10R AGAATTCTTATTGTGCAGCTGCCTGCAC  ecoACK11F T G A A T T C _ _TGCGTGAAAAAGAAAAATGTGATGACA B. safensis ACK6 T G A A T T C _ _GCACAAACCGTCCCTTATGGAAT xhoACK12R ACTCGAGTTAGTTAGAAGCCGCTTGAACGTTG  bamACK13F TGGATCCAGAAGCA GGAAATTATG GATCGG B. atrophaeus ACK7 T GGATCCGCTCAGTCA GTACCTTATG GCAT ecoACK14R AGAATTCTTATTGCGCTGCTGCCTGAACG  bamACK15F TGGATCCAAGGTCA AATCATTTGG AGCAGGG B. altitudinis ACK8 T G G A T C C _ _GGTCAAAGCGTCCCTTATGGTA ecoACK16R AGAATTCTTATCGTGCAGCTTTTTGTACTable 3
List of constructions used and nominated strains as well.
Plasmids Constructions Strains used to transform constructions Given names expressing pGEX-4T-1 and its derivatives References pGEX-4T-1  E. coli BL21 APK1 This study E. coli TOP10 APK2 This study  pSNZ1 pGEX-4T-1:fibEma-Ba E. coli BL21 QEK1 This study E coli TOP10 KE1 This study  pDOK2 pGEX-4T-1:fibEma-Bs E. coli BL21 QEK2 This study E. coli TOP10 KE2 This study  pDOK3 pGEX-4T-1:fibEma-Bl E. coli BL21 QEK3 This study E. coli TOP10 KE3 This study  pDOK5 pGEX-4T-1:fibEma-Bp E. coli BL21 QEK4 This study E. coli TOP10 KE4 This study  pDOK6 pGEX-4T-1:fibE-Bm E. coli BL21 QEK5 This study E. coli TOP10 KE5 This study  pDOK7 pGEX-4T-1:fibEma-Bsa E. coli BL21 QEK6 This Study E. coli TOP10 KE6 This study  pDOK8 pGEX-4T-1:fibEma-Bal E. coli BL21 QEK7 This study E. coli TOP10 KE7 This study  pDOK9 pGEX-4T-1:fibEma-Bat E. coli BL21 QEK8 This Study E. coli TOP10 KE8 This study


## 2.5. Generating Polyclonal Antibodies and Bacillus Immunodetection

Polyclonal antibodies were generated by using five-week-old female specific-pathogen-free BALB/c mice. 10μg of GST-FibE derivatives has been injected into naïve BALB/c mice in the first week. A reminder on intraperitoneal injection was performed two weeks later to elicit the production of antibodies. All purified variants of fusion proteins including GST-FibE-Bs, GST-FibE-Ba, GST-FibE-Bl, GST-FibE-Bp, GST-FibE-Bsa, GST-FibE-Bat, and GST-FibE-Bal were exhausted against the background of Crude extracts and culture supernatants ofE. coli TOP10 producing GST alone. Overnight pure cultures using LB medium ofB. amyloliquefaciens, B. licheniformis, B. subtilis, B. pumilus, B. mojavensis, B. safensis, B. atrophaeus,andB. altitudinishave been performed. Supernatants were mixed with 80% NH4SO4 at +4°C. Cultures were centrifuged at 6000 rpm, during 1 hour. Pellets were collected with 500 μL of PBS. SDS-PAGE stained with Coomassie blue and immunodetection were carried out using polyclonal antibodies anti-FibE variants.


## 3. Results

### 3.1. Bacteria Isolation

A qualifying assessment included Mossel medium culture, macroscopic analysis, microscopic analysis (Gram-positive bacilli), Gram-positive bacteria with 3% KOH, spore-forming bacteria test, and the positive catalase reaction had allowed the classification of the working bacterium as theBacillus genus. A total of 155 pure cultured isolates were obtained from different raw material palm wine (30), Ntoba Mbodi (21), Mbala-mpinda (19), Pandé (12), banana (35), sugar cane wine (13), soil (15), and intestine of Guppy fish (10). All bacteria were spore-forming colonies, catalase positive and Gram-positive staining. To easily discriminateBacillus spp. at the genera level, pure cultured isolates were assessed for their ability to produce caseinolytic proteases. The clear halo shows activity and the absence of the clear halo means the absence of caseinolytic activity. The rate of isolates with enzymatic activities as follows: palm wine (56%), Ntoba Mbodi (47%), Mbala-mpinda (63%), Pandé (33%), banana wine (54%), sugar cane wine (69%), soil (73%), and intestine of Guppy fish (80%).


### 3.2. Direct Amplification offibE Gene

Identification up to the species level was carried out by directly targetingfibE gene amplification ofBacillus spp. belonging to group I. Among 155 isolates, 73% of bacteria that were able to degrade casein have been randomly selected. A total of 720 PCR reactions have been done by using primers targeting the sequence encoding the mature protein (Figure 1(a)) including Ba.IdMa-F/Ba.IdMa-R (B. amyloliquefaciens), Bl.Id.Ma-F/Bl.Id.Ma-R (B. licheniformis), Bs.Id.Ma-F/Bs.Id.Ma-R (B. subtilis), Bp.Id.Ma-F/Bp.Id.Ma-R (B. pumilus), Bm.Id.Ma-F/Bm.Id.Ma-R (B. mojavensis), and Bsa.Id.Ma-F/Bsa.Id.Ma-R (B. safensis). Among 155 pure cultured isolates, 12 positive PCR fragments have been amplified. As a result, one strain ofB. amyloliquefaciens, one ofB. licheniformis, one ofB. subtilis, three ofB. pumilus, three ofB. safensis,one ofB. altitudinis,one ofB. atrophaeus,and three ofB. pumilus have been specifically obtained (Figure 1(b)). No fragment has been amplified in terms ofB. mojavensis.Figure 1
(a) Schematic representation offibE gene and its corresponding regions, presequence, prosequence, and AQSV motifs by following the mature sequence and internal sequence. (b) Direct identification of the mature sequence fibEma ofBacillus belonging to group 1. MW: molecular weight. (c) Amplification of internal sequence of fibE (fibEss). Bs:B. subtilis. Ba:B. amyloliquefaciens, Bl:B. licheniformis, Bp:Bacillus pumilus, Bal:B. altitudinis, Bsa:Bacillus safensis, Bat:Bacillus atrophaeus, andBm: B. mojavensis.
(a) (b) (c)In addition, by using Ba.SHMa-F/Ba.SHMa-R, Bl.SHMa-F/Bl.SHMa-R, Bs.SHMa-F/Bs.SHMa-R, Bp.SHMa-F/Bp.SHMa-R, Bm.SHMa-F/Bm.SHMa-R, and Bsa.SHMa-F/Bsa.SHMa-R primers, the internal fragments corresponding to 461 pb have been specifically amplified includingB. mojavensis (Figure 1(c)). B. mojavensis has been amplified by directly using genomic DNA (Figure 1(c)). The sequencing of 11 PCR products of maturefibE fragments and analysis on NCBI showed 100% of identities corresponding to the targeted strains. To ensure those strains were the good ones, 16S ribosomal RNA genes were used to more confirm in parallel. 16S ribosomal RNA sequence has been submitted in GenBank. Rates of identities have been reported (Table 4).Table 4
Comparison between 16S rRNA analysis and specific gene analysis.
Strains % of Identities GenBank numbers fibE encoding mature protein fibEss 16SRNA B. subtilis strain GL48 100% 100% 99% MK099888.1  B. licheniformis 100% 100% nd nd  B. mojavensis. nd 100% nd nd  B. atrophaeus 100% 100% nd nd  B. amyloliquefaciens LULU2 100% 100% 99% MK156314.1  B. altitudinisstrain YUYU1 100% 100% 99% MK156313.1  Bacillus altitudinisstrain GL42 100% 100% 99% MK099889.1  B. pumilus strain DOK3 100% 100% 99% MK156166.1  B. pumilus strain DOK2 100% 100% 99% MK156165.1  B. pumilus strain DOK1 100% 100% 99% MK156164.1  Bacillus safensisstrain DOK9 100% 100% 99% MK184540.1  B. safensis strain DOK8 100% 100% 99% MK184539.1  B. safensis strain DOK7 100% 100% 99% MK184538.1


### 3.3. Expression in E. coli BL21 and TOP10

The wholefibE gene encompassing pre- and prosequence products from seven strains ofBacillus belonging to group I species was cloned in frame with the vector pGEX-4T-1 and expressed inE. coli strains BL21 and TOP10. The purification of GST fused to recombinant serine-like enzyme had shown that the fusion has been cleaved (data not shown). Then mature sequence from AQSVPY peptide has been fused with GST. The same molecular weight, 52kDa, for fused proteins has been seen for identifiedBacillus spp. includingB. amyloliquefaciens, B. licheniformis, B. subtilis, B. pumilus, B. safensis, B. atrophaeus,andB. altitudinis (Figure 2: top). Using monoclonal anti-GST, the fused proteins have been identified (Figure 2: bottom).Figure 2
Coomassie blue staining of GST fused to derivatives (TOP) and anti-GST immunodetection.To easily characterizefibE gene and to assess whether the expression of fused recombinant FibE protein has the same characteristics as wild type one, construction of pGEX-4T-1 and its derivatives has been done. TheE. coli transformants including TOP10 group (KE1, KE2, KE3, KE4, KE6, KE7, and KE8; see Table 3) and BL21 group (QEK1, QEK2, QEK3, QEK4, QEK6, QEK7, and QEK8; see Table 3) showed high enzymatic activities of identifiedBacillus spp. (Figure 3). It was done from the soluble lysate of induced cells harboring pGEX-4T-1 and derivatives. The activity was 2-fold higher compared with the wild type (Figure 4).Figure 3
Enzymatic activities of identified strains belonging to group I ofBacillus spp. BS:B. subtilis, Ba:B. amyloliquefaciens, Bl:B. licheniformis, Bp:B. pumilus, Bal:B. altitudinis, Bsa:B. safensis, Bat:B. atrophaeus. KE1 (pGEX-4T-1: fibEma-Ba /TOP10), KE2 (pGEX-4T-1: fibEma-Bs/TOP10), KE3 (pGEX-4T-1: fibEma-Bl/TOP10), KE4 (pGEX-4T-1: fibEma-Bp), KE6 (pGEX-4T-1: fibEma-Bsa/TOP10), KE7 (pGEX-4T-1: fibEma-Bal/TOP10), and KE8 (pGEX-4T-1: fibEma-Bat/TOP10). QEK1 (pGEX-4T-1: fibEma-Ba /BL21), QEK2 (pGEX-4T-1: fibEma-Bs/BL21), QEK3 (pGEX-4T-1: fibEma-Bl/BL21):, QEK4 (pGEX-4T-1: fibEma-Bp/BL21), QEK6 (pGEX-4T-1: fibEma-Bsa/BL21), QEK7 (pGEX-4T-1: fibEma-Bal/BL21), and QEK8 (pGEX-4T-1: fibEma-Bat/BL21).Figure 4
Western blotting using antibody antifibrinolytic enzymes ofBacillusbelonging to group I. Bs:B. subtilis, Ba:B. amyloliquefaciens, Bl:B. licheniformis,Bsa: B. safensis, Bal:B. altitudinis, Bp: B. pumilus, Bat:B. atrophaeus.


### 3.4. Immunodetection of FibE

In order to compare polyclonal antibody and genetic amplification, we generated antibodies by using BALB/c mice for GST-FibE derivatives from eight strains includingB. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. altitudinis, B. safensis, andB. atrophaeus. Immunoblotting of α-FibE-Bs, α-FibE-Ba, α-FibE-Bl, α-FibE-Bsa, and α-FibE-Bat showed that each antibody can easily recognize secreted proteins of randomly chosenBacillus wild type belonging to group I. All polyclonal antibodies can detect wild type secreted proteins fromB. mojavensis except for α-FibE-Bsa. α-FibE-Bp cannot recognize secreted protein fromB. licheniformis. α-FibE-Bsa cannot detectB. atrophaeus,B. safensis, andB. pumilus (Figure 4).


## 3.1. Bacteria Isolation

A qualifying assessment included Mossel medium culture, macroscopic analysis, microscopic analysis (Gram-positive bacilli), Gram-positive bacteria with 3% KOH, spore-forming bacteria test, and the positive catalase reaction had allowed the classification of the working bacterium as theBacillus genus. A total of 155 pure cultured isolates were obtained from different raw material palm wine (30), Ntoba Mbodi (21), Mbala-mpinda (19), Pandé (12), banana (35), sugar cane wine (13), soil (15), and intestine of Guppy fish (10). All bacteria were spore-forming colonies, catalase positive and Gram-positive staining. To easily discriminateBacillus spp. at the genera level, pure cultured isolates were assessed for their ability to produce caseinolytic proteases. The clear halo shows activity and the absence of the clear halo means the absence of caseinolytic activity. The rate of isolates with enzymatic activities as follows: palm wine (56%), Ntoba Mbodi (47%), Mbala-mpinda (63%), Pandé (33%), banana wine (54%), sugar cane wine (69%), soil (73%), and intestine of Guppy fish (80%).


## 3.2. Direct Amplification offibE Gene

Identification up to the species level was carried out by directly targetingfibE gene amplification ofBacillus spp. belonging to group I. Among 155 isolates, 73% of bacteria that were able to degrade casein have been randomly selected. A total of 720 PCR reactions have been done by using primers targeting the sequence encoding the mature protein (Figure 1(a)) including Ba.IdMa-F/Ba.IdMa-R (B. amyloliquefaciens), Bl.Id.Ma-F/Bl.Id.Ma-R (B. licheniformis), Bs.Id.Ma-F/Bs.Id.Ma-R (B. subtilis), Bp.Id.Ma-F/Bp.Id.Ma-R (B. pumilus), Bm.Id.Ma-F/Bm.Id.Ma-R (B. mojavensis), and Bsa.Id.Ma-F/Bsa.Id.Ma-R (B. safensis). Among 155 pure cultured isolates, 12 positive PCR fragments have been amplified. As a result, one strain ofB. amyloliquefaciens, one ofB. licheniformis, one ofB. subtilis, three ofB. pumilus, three ofB. safensis,one ofB. altitudinis,one ofB. atrophaeus,and three ofB. pumilus have been specifically obtained (Figure 1(b)). No fragment has been amplified in terms ofB. mojavensis.Figure 1
(a) Schematic representation offibE gene and its corresponding regions, presequence, prosequence, and AQSV motifs by following the mature sequence and internal sequence. (b) Direct identification of the mature sequence fibEma ofBacillus belonging to group 1. MW: molecular weight. (c) Amplification of internal sequence of fibE (fibEss). Bs:B. subtilis. Ba:B. amyloliquefaciens, Bl:B. licheniformis, Bp:Bacillus pumilus, Bal:B. altitudinis, Bsa:Bacillus safensis, Bat:Bacillus atrophaeus, andBm: B. mojavensis.
(a) (b) (c)In addition, by using Ba.SHMa-F/Ba.SHMa-R, Bl.SHMa-F/Bl.SHMa-R, Bs.SHMa-F/Bs.SHMa-R, Bp.SHMa-F/Bp.SHMa-R, Bm.SHMa-F/Bm.SHMa-R, and Bsa.SHMa-F/Bsa.SHMa-R primers, the internal fragments corresponding to 461 pb have been specifically amplified includingB. mojavensis (Figure 1(c)). B. mojavensis has been amplified by directly using genomic DNA (Figure 1(c)). The sequencing of 11 PCR products of maturefibE fragments and analysis on NCBI showed 100% of identities corresponding to the targeted strains. To ensure those strains were the good ones, 16S ribosomal RNA genes were used to more confirm in parallel. 16S ribosomal RNA sequence has been submitted in GenBank. Rates of identities have been reported (Table 4).Table 4
Comparison between 16S rRNA analysis and specific gene analysis.
Strains % of Identities GenBank numbers fibE encoding mature protein fibEss 16SRNA B. subtilis strain GL48 100% 100% 99% MK099888.1  B. licheniformis 100% 100% nd nd  B. mojavensis. nd 100% nd nd  B. atrophaeus 100% 100% nd nd  B. amyloliquefaciens LULU2 100% 100% 99% MK156314.1  B. altitudinisstrain YUYU1 100% 100% 99% MK156313.1  Bacillus altitudinisstrain GL42 100% 100% 99% MK099889.1  B. pumilus strain DOK3 100% 100% 99% MK156166.1  B. pumilus strain DOK2 100% 100% 99% MK156165.1  B. pumilus strain DOK1 100% 100% 99% MK156164.1  Bacillus safensisstrain DOK9 100% 100% 99% MK184540.1  B. safensis strain DOK8 100% 100% 99% MK184539.1  B. safensis strain DOK7 100% 100% 99% MK184538.1


## 3.3. Expression in E. coli BL21 and TOP10

The wholefibE gene encompassing pre- and prosequence products from seven strains ofBacillus belonging to group I species was cloned in frame with the vector pGEX-4T-1 and expressed inE. coli strains BL21 and TOP10. The purification of GST fused to recombinant serine-like enzyme had shown that the fusion has been cleaved (data not shown). Then mature sequence from AQSVPY peptide has been fused with GST. The same molecular weight, 52kDa, for fused proteins has been seen for identifiedBacillus spp. includingB. amyloliquefaciens, B. licheniformis, B. subtilis, B. pumilus, B. safensis, B. atrophaeus,andB. altitudinis (Figure 2: top). Using monoclonal anti-GST, the fused proteins have been identified (Figure 2: bottom).Figure 2
Coomassie blue staining of GST fused to derivatives (TOP) and anti-GST immunodetection.To easily characterizefibE gene and to assess whether the expression of fused recombinant FibE protein has the same characteristics as wild type one, construction of pGEX-4T-1 and its derivatives has been done. TheE. coli transformants including TOP10 group (KE1, KE2, KE3, KE4, KE6, KE7, and KE8; see Table 3) and BL21 group (QEK1, QEK2, QEK3, QEK4, QEK6, QEK7, and QEK8; see Table 3) showed high enzymatic activities of identifiedBacillus spp. (Figure 3). It was done from the soluble lysate of induced cells harboring pGEX-4T-1 and derivatives. The activity was 2-fold higher compared with the wild type (Figure 4).Figure 3
Enzymatic activities of identified strains belonging to group I ofBacillus spp. BS:B. subtilis, Ba:B. amyloliquefaciens, Bl:B. licheniformis, Bp:B. pumilus, Bal:B. altitudinis, Bsa:B. safensis, Bat:B. atrophaeus. KE1 (pGEX-4T-1: fibEma-Ba /TOP10), KE2 (pGEX-4T-1: fibEma-Bs/TOP10), KE3 (pGEX-4T-1: fibEma-Bl/TOP10), KE4 (pGEX-4T-1: fibEma-Bp), KE6 (pGEX-4T-1: fibEma-Bsa/TOP10), KE7 (pGEX-4T-1: fibEma-Bal/TOP10), and KE8 (pGEX-4T-1: fibEma-Bat/TOP10). QEK1 (pGEX-4T-1: fibEma-Ba /BL21), QEK2 (pGEX-4T-1: fibEma-Bs/BL21), QEK3 (pGEX-4T-1: fibEma-Bl/BL21):, QEK4 (pGEX-4T-1: fibEma-Bp/BL21), QEK6 (pGEX-4T-1: fibEma-Bsa/BL21), QEK7 (pGEX-4T-1: fibEma-Bal/BL21), and QEK8 (pGEX-4T-1: fibEma-Bat/BL21).Figure 4
Western blotting using antibody antifibrinolytic enzymes ofBacillusbelonging to group I. Bs:B. subtilis, Ba:B. amyloliquefaciens, Bl:B. licheniformis,Bsa: B. safensis, Bal:B. altitudinis, Bp: B. pumilus, Bat:B. atrophaeus.


## 3.4. Immunodetection of FibE

In order to compare polyclonal antibody and genetic amplification, we generated antibodies by using BALB/c mice for GST-FibE derivatives from eight strains includingB. subtilis, B. amyloliquefaciens, B. licheniformis, B. pumilus, B. altitudinis, B. safensis, andB. atrophaeus. Immunoblotting of α-FibE-Bs, α-FibE-Ba, α-FibE-Bl, α-FibE-Bsa, and α-FibE-Bat showed that each antibody can easily recognize secreted proteins of randomly chosenBacillus wild type belonging to group I. All polyclonal antibodies can detect wild type secreted proteins fromB. mojavensis except for α-FibE-Bsa. α-FibE-Bp cannot recognize secreted protein fromB. licheniformis. α-FibE-Bsa cannot detectB. atrophaeus,B. safensis, andB. pumilus (Figure 4).


## 4. Discussion

This work aims to contribute to the identification with cheaper equipment and rapid method of Bacillus spp. belonging to the phylogenetic group I [11] along withB. amyloliquefaciens, B. licheniformis, B. subtilis, B. pumilus, B. mojavensis, B. safensis, B. atrophaeus, andB. altitudinis.The phenotypic and biochemical characteristics of the 155 isolates were related and oriented towardsBacillus genera. Republic of Congo harbors several fermented foods and beverages containing unidentified microorganisms. Seven samples including local fermented foods (Samba, Ntoba Mbodi, Pandé, Loungouila, and Mbala-mpinda), soil, and intestine of Guppy fish [32] were used for the isolation of these bacteria. The genusBacillus can be isolated from different fermented foods together with Ntoba Mbodi [5], Mbala-mpinda, Pandé, banana wine (mbavu), and sugar cane wine (Loungouila) but also from other diverse environments such as rocks, soil, the aquatic environments, and gut of various insects and animals [33]. The genusBacillus is also known for its ability to produce extracellular proteases. In this work, we have shown that, among 155 isolates, 73% of the characterized bacteria have proteolytic activities. The diameter of the light halo amounting to casein degradation varied between 1.5 ± 0.05 cm and 2.7 ± 0.03 cm. If isolates do not degrade casein, this does not necessarily mean that they do not have secreted protease; each species has an optimal temperature at which it degrades a given type of macromolecule [26]. Phenotypic tests only focused on the genera level. No species has been identified on the basis of the microbiology classical tests in this work. These methods have limitations in terms of microorganism’s identification. Close species such asB. pumilus andB. safensis are biochemically difficult to discriminate. These bacteria share a strong homology close to 99 to 100% [34]. It seems obvious that the use of molecular identification methods has more advantages. These methods are more reliable [35] compared to conventional one. Here, we exploited the polymorphism of thefibE gene. By targeting this new generation of highly conserved gene, we showed thatB. pumilus (3),B. amyloliquefaciens (1),B. licheniformis (1),B. subtilis (1),B. safensis(3),B. altitudinis (2), andB. atrophaeus have been amplified. The agarose gel electrophoresis of this amplification clearly showed bands about 850 bp and 450 bp as internal sequence, consistent with the size of the amplicons obtained in in silico studies. The amplification of thefibE-Bm gene usingB. mojavensis-specific oligos was negative with 850 bp but positive with 450 bp when targetingB. mojavensis genomic implication.B. mojavensis has been largely isolated through several environments such as marine water [36], but also from soil and plants [37]. Previously it was shown thatBacillus species belonging to group I may have several copies of the genes encoding fibrinolytic enzymes. One pair of primers can amplify fibrinolytic genes of different sizes in different species ofBacillus, but these strains must have highly variable regions for amplification of these primers [26]. In this work we were able to only amplify one band. This could testify the reliability of the method. The strategy here allows us to identify the species in a very reliable and specific way, because, unlike the housekeeping genes generally used in the identification of these species, the sequences used in this study were targeted in a region containing very diversifying, degenerate codons. This increases the specificity of the hybridization of the primers used [38].It is worth remembering that polymorphisms result from three types of DNA sequence variation [39]. In the “forward” and “reverse” oligonucleotide sequences that we had aligned, “single nucleotide polymorphisms (SNPs)” correspond to nucleotide substitutions. The in silico analysis of the genotyping of the forward sequences made it possible to highlight the presence of the nucleotide substitutions at the level of the primers (1 to 28 base pairs) (Figure 5). By comparing targeted sequences of eight strains in which primers have been chosen, we found nucleotide variabilities from 1 to 23 at positions 2, 3, 4, 5, 6, 8, 7, 11, 17, 20, and 21. In Bl, Bs, Bp, Ba, Bat, Bs, and Bm, the guanine nucleotide was, respectively, replaced by cytosine (2G<C) in Bal. More change can be detected at position 3, (Bal, Bat) 3T<A (Bsa, Bp) <G (Bl, Ba, Bs). In position 6 adenine was replaced by guanine (Bl, Bs, Bp, Bat, Bm) 6A<G (Bat, Ba), (Bal, Bl, Bp, Bsa) 7A<T (Ba, Bat, Bs, Bm). In position 8 guanine is replaced by a cytosine (Bal) 8G<C (Bl, Bsa, Bp, Ba, Bat, Bm, Bs), and in position 21 (Bal) 21T<C (Bl, Bs, Bp, Bat, Bm) <A (Bsa, Ba). Based on this analysis, modifications could be viewed from 180 to 202, from 521 to 541, and from 808 to 828. These SNPs are largely sufficient to specifically amplify thefibE gene (Figure 5).Figure 5
Sequence alignment for randomly chosen primers.Construction in frame with the whole sequence showed that the fusion protein can be easily expressed inE. coli TOP10 and BL21. Previous studies have demonstrated that fibrinolytic enzyme can be expressed inE. coli [40–43]. In this work we showed GST fused to the whole protein sequence of FibE can be easily cleaved in the presequence domain encompassing the signal peptides (data not shown). We show that the protein overexpression of the mature sequence fused to the GST increased the fibrinolytic activity.Polyclonal antibodies of FibE proteins variants are able to detect each FibE variant belonging to theBacillus Group I. The predicted peptides encompassing six amino acid motifs (AQSVPY) are of wild type, conserved amongBacillus spp. belonging to group I. This also includes the epitope S87. These epitopes were confirmed by using Antibody Epitope Prediction (http://tools.immuneepitope.org/bcell/) using BepiPred Linear Epitope Prediction. It was particularly interesting to understand that identification using PCR amplification and sequencing seems to be the best lesson learnt in terms ofBacillus group I identification. The purpose of this part of the study described here was to assess a genetic discrimination and specific immunoidentification assay for detection ofBacillus belonging to group I in order to develop techniques that would avoid preenrichment and costly equipment so that it can be used for microorganisms identification isolated from local fermented food and beverages. Although it has been previously demonstrated that polyclonal antibodies of outer membrane proteins (OMP) fromV. cholerae O1 can be easily detected [44], we do consider that identification at the level of the gene should be a better approach because the homology percentage of the FibE protein is very high (70%) and this would generate more difficulties for an immunological approach.To our knowledge there is no other study related to the identification ofBacillus belonging to group I targeting the highly conserved gene encoding fibrinolytic enzymes. Since 1987 nobody had postulated this gene could be used to discriminateBacillus. This strategy is the first one in this regard since the discovery of the fibrinolytic enzymes. However, several studies by using a specific gene of interest for the direct identification of strains have been done it as well. Bacteria such asB. thuringiensis can be detected by the sequences of thecry gene primers without sequencing. These primers hybridize specifically inB. thuringiensis [45]. Using a direct PCR approach, a yeast study identified species ofSaccharomyces arboricola,S. bayanus, S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and S. pastorianus by targeting specific genes to identify these species [38]. In additionShigella flexneri andSalmonella typhimurium can be easily identified by, respectively, targetingicsB and invG genes [32]. These methods have a major and direct advantage for the identification of these strains.


## 5. Conclusion

Overall, this study is part of the development of new approach to easily, quickly, and reliably identifyBacillus bacteria belonging to group I species. We have isolated and characterizedBacillus species from different samples by using classical microbiology techniques and molecular biology one. Our new strategy, using a highly conserved gene encoding a fibrinolytic enzyme, allows us to identify isolates includingB. amyloliquefaciens, B. licheniformis, B. subtilis, B. pumilus, B. mojavensis, B. atrophaeus, B. altitudinis, andB. safensis.This identification was confirmed in parallel by using the amplification and sequencing of the 16S rRNA gene, which is recognized as the standard for the identification of bacteria. Our identification strategy is direct and less expensive and has promising future. This could open windows and doors for various perspectives such as assessment of cell-cell interactions by understanding quorum sensing, quorum quenching, and biofilms during fermentation process.



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*Source: 5484896-2019-05-20.xml*