# Short genome report of cellulose-producing commensal Escherichia coli 1094

Joaquin Bernal-Bayard, Laura Gomez-Valero, Aimee Wessel, Varun Khanna,

Christiane Bouchier, Jean-Marc Ghigo

► To cite this version:

Joaquin Bernal-Bayard, Laura Gomez-Valero, Aimee Wessel, Varun Khanna, Christiane Bouchier, et al.. Short genome report of cellulose-producing commensal Escherichia coli 1094. Standards in Genomic Sciences, 2018, 13 (1), pp.13. 10.1186/s40793-018-0316-0. pasteur-02015655

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SHORT GENOME REPORT

Open Access

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CrossMark

Short genome report of cellulose-
producing commensal *Escherichia coli* 1094

Joaquin Bernal-Bayard<fnref n="1" />*, Laura Gomez-Valero²,³, Aimee Wessel<fnref n="1" />, Varun Khanna⁴, Christiane Bouchier⁵
and Jean-Marc Ghigo<fnref n="1" />* ID

**Abstract**

Bacterial surface colonization and biofilm formation often rely on the production of an extracellular polymeric matrix that mediates cell-cell and cell-surface contacts. In *Escherichia coli* and many *Betaproteobacteria* and *Gammaproteobacteria* cellulose is often the main component of the extracellular matrix. Here we report the complete genome sequence of the cellulose producing strain *E. coli* 1094 and compare it with five other closely related genomes within *E. coli* phylogenetic group A. We present a comparative analysis of the regions encoding genes responsible for cellulose biosynthesis and discuss the changes that could have led to the loss of this important adaptive advantage in several *E. coli* strains.
Data deposition: The annotated genome sequence has been deposited at the European Nucleotide Archive under the accession number PRJEB21000.

**Keywords:** *E. coli*, Commensal, Biofilm, Cellulose, Extracellular matrix, Bcs operon

**Introduction**

Biofilms are ubiquitous microbial communities growing in close association with surfaces present in natural and anthropic environments [1]. Biofilm bacteria often self-assemble by producing a cohesive extracellular matrix that protects these multicellular aggregates against environmental changes and maintains the integrity of the biofilm structure [2]. Cellulose is a homo-polysaccharide composed of glucose units linked by β-1 → 4 glycosidic bonds and is the most common organic compound on Earth [3]. Initially studied in *Gluconacetobacter xylinus*, cellulose production is a widespread phenomenon shared by many commensal and pathogenic *Betaproteobacteria* and *Gammaproteobacteria*, including *Salmonella enterica serovar Typhimurium*, *Klebsiella pneumoniae*, *Burkholderia mallei*, *Shigella boydii*, *Yersinia enterocolitica*, *Vibrio fischeri*, *Pseudomonas putida* and many *Escherichia coli* strains [4, 5]. Here we report the complete genome sequence of *E. coli* 1094, a biofilm forming and cellulose-producing strain isolated from the feces of a healthy human male. *E. coli* 1094 lacks virulence factors commonly associated with pathogenic *E. coli* and is a member of *E. coli* phylogenetic group A [6, 7].

**Organism information**

**Classification and features**

*Escherichia coli* is a Gram-negative, rod-shaped, non-spore forming and facultative anaerobic species belonging to the *Enterobacteriaceae* family. They are commonly found in the intestines of endotherms and are taxonomically placed within the *Gammaproteobacteria* of the *Proteobacteria* phylum (Table 1).

*E. coli* 1094 is a commensal strain isolated from feces of a healthy human male. Like many other natural *E. coli* isolates, 1094 produces cellulose as the main component of its biofilm extracellular polymeric matrix (Fig. 1) [8], and has been used as a model for studying both transcriptional regulation and function of cellulose biosynthesis genes (bcs genes) [8, 9], as well as to analyze the structure of the cellulose secretion machinery [10]. Here, we investigate global genomic differences between representative members of *E. coli* phylogenetic group A and discuss their functional consequences on cellulose production.

*E. coli* 1094 was previously classified as a member of the phylogenetic group A based on triplex PCR with a combination of primers amplifying two genes (*chuA* and *yjaA*), and an anonymous DNA fragment designated TSPE4.C2 [7]. Using all available complete genomes of phylogenetic group A, we performed a core genome

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<footnote>* Correspondence: joaquin.bernal@pasteur.fr; jean-marc.ghigo@pasteur.fr</footnote>

<footnote n="1">¹Département de Microbiologie, Unité de Génétique des Biofilms, Institut Pasteur, 25-28 rue du Dr. Roux, F-75015 Paris, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
<table><caption>**Table 1** Classification and general features of *Escherichia coli* strain 1094</caption><thead><tr><th>MIGS ID</th><th>Property</th><th>Term</th><th>Evidence code<sup>a</sup></th></tr></thead><tbody><tr><td></td><td>Classification</td><td>Domain Bacteria</td><td>TAS [33]</td></tr><tr><td></td><td></td><td>Phylum Proteobacteria</td><td>TAS [34]</td></tr><tr><td></td><td></td><td>Class Gammaproteobacteria</td><td>TAS [34]</td></tr><tr><td></td><td></td><td>Order "Enterobacteriales"</td><td>TAS [34, 35]</td></tr><tr><td></td><td></td><td>Family Enterobacteraceae</td><td>TAS [36]</td></tr><tr><td></td><td></td><td>Genus Escherichia</td><td>TAS [37, 38]</td></tr><tr><td></td><td></td><td>Species Escherichia coli</td><td>TAS [37, 38]</td></tr><tr><td></td><td></td><td>Type strain: 1094</td><td>TAS</td></tr><tr><td></td><td>Gram stain</td><td>Negative</td><td>IDA, TAS [39]</td></tr><tr><td></td><td>Cell shape</td><td>Rod</td><td>IDA, TAS [39]</td></tr><tr><td></td><td>Motility</td><td>Motile</td><td>IDA, TAS [39]</td></tr><tr><td></td><td>Sporulation</td><td>Non</td><td>TAS [39]</td></tr><tr><td></td><td>Temperature range</td><td>10 °C–45 °C</td><td>NAS</td></tr><tr><td></td><td>Optimum Temperature</td><td>37 °C</td><td>IDA</td></tr><tr><td></td><td>pH range; optimum</td><td>5.5–8.0; 7</td><td></td></tr><tr><td></td><td>Carbon source</td><td>Glucose</td><td>IDA</td></tr><tr><td>MIGS-6</td><td>Habitat</td><td>Human gut</td><td></td></tr><tr><td>MIGS-6,3</td><td>Salinity</td><td>Not reported</td><td></td></tr><tr><td>MIGS-22</td><td>Oxygen requirement</td><td>Facultative anaerobe</td><td>IDA, TAS [39, 40]</td></tr><tr><td>MIGS-15</td><td>Biotic relationship</td><td>Human specimen</td><td>NAS</td></tr><tr><td>MIGS-14</td><td>Pathogenicity</td><td>Nonpathogenic</td><td>NAS</td></tr><tr><td>MIGS-4</td><td>Geographic location</td><td>France</td><td></td></tr><tr><td>MIGS-5</td><td>Sample collection</td><td>1999</td><td></td></tr><tr><td>MIGS-4,1</td><td>Latitude</td><td>Not reported</td><td></td></tr><tr><td>MIGS-4,2</td><td>Longitude</td><td>Not reported</td><td></td></tr><tr><td>MIGS-4,4</td><td>Altitude</td><td>Not reported</td><td></td></tr></tbody></table>

alignment to produce a phylogenetic tree, and found it to be in agreement with previous phylogenetic classification [11], showing that *E. coli* strain 1094 is closely related to strains ATCC 8739 and HS (Fig. 2).

## Genome sequencing information

### Genome project history

*E. coli* 1094 has been used as a model to study different aspects of cellulose biosynthesis and biofilm formation [8, 9]. Cellulose production requires the expression of bacterial cellulose synthesis genes clustered in two divergent operons, as well as genes involved in general glucose metabolism [8], In order to further elucidate the genetic bases of cellulose synthesis, we chose to sequence *E. coli* 1094 using two approaches: Illumina and PacBio sequencing. While Illumina sequencing and subsequent downstream analysis generated 204 contigs, PacBio sequencing and assembly produced 4 contigs. A summary of the project information and its association

with "Minimum Information about a Genome Sequence" [12] are provided in Table 2.

### Growth conditions and genomic DNA preparation

*E. coli* 1094 was cultivated in LB medium overnight at 37 °C. High quality genomic DNA for sequencing was extracted using the Wizard Genomic DNA Kit (Promega) (for Illumina approach), or the QiaAMP DNA Mini Kit (QIAGEN) (for PacBio approach).

### Genome sequencing and assembly

#### Illumina sequencing

Whole genome library preparation (NEXTflex PCR-Free DNA-Seq kit, Bioo Scientific) and sequencing following standard protocols developed by the supplier were performed at the Genomics platform at the Pasteur Institute. Single reads averaging 110 base pairs were collected on a HiSeq2000 (Illumina, San Diego, CA). Approximately 8,285,636 reads were assembled using CLC Bio (version 4.

<footnote><sup>a</sup>Evidence codes - TAS Traceable Author Statement, NAS Non-traceable Author Statement, IDA Inferred from Direct Assay. These evidence codes are from the Gene Ontology project [12]</footnote>
<figure><img src="image_4.png" /><figcaption>**Fig. 1** Scanning electron microscopy of cellulose producing strain *E. coli* 1094. *E. coli* 1094, Bacteria with cellulose labeled by CBM29-1-2 and immunogold antibodies. Scanning electron microscopy and immunogold labelling were performed at LBCME facility, Faculte de Medecine de Tours, Tours, France)</figcaption></figure>

20) giving a total of 204 contigs. The final Illumina-based sequence includes 4,982,209 bases with a G + C content of 50.81%.

## PacBio sequencing

Library preparation, sequencing, and assembly were performed by the Earlham Institute. PacBio sequencing libraries were prepared from 10 µg DNA using standard Pacific Biosciences protocols (Pacific Biosciences, Menlo Park, CA). Following construction, libraries were size selected, from 7 to 50 kb, using the Sage Science BluePippin™ system with a 0.75% gel cassette. Libraries were run on the Pacific Biosciences RSII sequencer at 350pM using P6/C4 chemistry. A Single Molecule Real Time (SMRT) cell was used, yielding 150,292 reads, and 1213 megabases of sequence data. Reads were assembled using PacBio's hierarchical genome-assembly process (HGAP.3), with filtering and adaptor trimming performed as previously described [13]. The minimum seed read length used was 6 kb, with a quality score cutoff of 0.8. The Celera Assembler was used to produce 4 large contigs, using pre-assembled, error-corrected reads. The maximum contig length produced was 4,903,991 bases.

## Illumina & PacBio comparison

Illumina single-end reads were mapped against the four large contigs generated by PacBio reads using the single-end mode of Bwa mem v0.7.4 [14] with default parameters (Fig. 3). Output SAM files were converted to BAM

<figure><img src="image_5.png" /><figcaption>**Fig. 2** Phylogenomic comparison of *E. coli* strains from phylogenetic group A. The tree was inferred using Parsnp, a fast core-genome multi-aligner. Color-coded stars represent relevant genetic events related with the loss of cellulose production: orange, premature stop codon in *bcsQ*; purple, deletion of a large *bcs* region; green, premature stop codon in *bcsE*</figcaption></figure>

files using SAMtools v0.1.19 [15]. Sequencing coverage was computed using BEDtools v2.17.0 0 [16] and values were normalized to 1 (genomes are haploids), from the median coverage over the large contigs (354×). The mapping coverage along the four PacBio contigs validated the sequence assemblies. Some peaks of high coverage are observed in unitig_0, unitig_1 and unitig_2 (Fig. 3a), which suggests that multiple copies of these regions exist. By contrast, the coverage of the complete unitig_3 indicates that there is likely more than one copy per chromosome within each cell.

SPAdes v 3.9.1 [17, 18] was used for assembling Illumina reads, for detecting putative plasmids sequence with the options '-plasmid', '-k 21,51,71', 'cut-off auto' and '-careful'. Illumina reads were re-mapped onto the contigs using the single-end mode of Bwa mem v0.7.4 with default parameters. After converting output SAM files to BAM files by SAMtools, putative plasmid reads were extracted using
<table><caption>**Table 2** Project information</caption><thead><tr><td>MIGS ID</td><td>Property</td><td>Term</td></tr></thead><tbody><tr><td>MIGS 31</td><td>Finishing quality</td><td>High quality drafts</td></tr><tr><td>MIGS-28</td><td>Libraries used</td><td>Two genomic libraries; one Illumina library, one PacBio standard library</td></tr><tr><td>MIGS 29</td><td>Sequencing platforms</td><td>Illumina Miseq2000, Pacific Biosciences RSII</td></tr><tr><td>MIGS 31.2</td><td>Fold coverage</td><td>354x Illumina, 114.3x PacBio</td></tr><tr><td>MIGS 30</td><td>Assemblers</td><td>CLC Bio (version 4.20), HGAP.3</td></tr><tr><td>MIGS 32</td><td>Gene calling method</td><td>GLIMMER2</td></tr><tr><td></td><td>Locus Tag</td><td>EC1094V2</td></tr><tr><td></td><td>Genbank ID</td><td>LT883139-LT883142</td></tr><tr><td></td><td>GenBank Date of Release</td><td>05-DEC-2017</td></tr><tr><td></td><td>GOLD ID</td><td>Gp0267270</td></tr><tr><td></td><td>BIOPROJECT</td><td>PRJEB21000</td></tr><tr><td>MIGS 13</td><td>Source Material Identifier</td><td>CRBIP19.182</td></tr><tr><td></td><td>Project relevance</td><td>Human commensal</td></tr></tbody></table>

SAMtools (option 'view -F 4') and recorded in a Fastq file by picardtools 'SamToFastq' [19]. Putative plasmid reads were mapped against the four large PacBio contigs using the single-end mode of Bwa mem v0.7.4 with default parameters. The coverage computed by BEDtools (Fig. 3b) indicates that the complete unitig_3 is classified as a putative plasmid sequence and also appears to exist in high copy number. For unitig_0, unitig_1 and unitig_2, the mapping coverage shows some portions of large contigs classified as putative plasmids. This may correspond to plasmids with similar coverage to the chromosome, due to low copy number, or to misclassification by plasmidSPAdes.

## Sequence circularization

PacBio scaffold sequences were compared against themselves with the bl2seq BLASTN algorithm [20], and ACT [21] was used for synteny visualization. The resulting overlapping sequences were easily identified between the beginning and the end of each large contigs, suggesting that all four PacBio large contigs are circular. To determine the size of the chromosome and each plasmid, the size of the overlapping region (Unitig_0: 15,155; Unitig_1: 8,919; Unitig_2: 13,971; Unitig_3: 9,380) was subtracted from the length of each contig (Unitig_0: 4,903,991; Unitig_1: 123,705; Unitig_2: 118,720; Unitig_3: 30,364); the final sizes are reported in Table 3.

## Genome annotation

The complete genome sequence was annotated with the RAST server [22] which predicted 5028 coding sequences and 110 RNAs.

## Genome properties

A summary of the genome of *E. coli* 1094 is included in Table 3. The genome statistics are provided in Table 4.

Three putative plasmids were identified, and found to be circular. The genome of strain 1094 has a total length of 5,176,780 base pairs and a G+C content of 50.9%. The majority of the protein-coding genes were assigned a putative function (78.8%) while the remaining ones were annotated as hypothetical proteins. Genes in internal clusters were detected using BLASTclust with thresholds of 70% covered length and 30% sequence identity [23]. CRISPR, transmembrane helice, signalP and Pfam protein families predictions were done using CRISPRFinder [24], TMHMM Server v2.0 [25], SignalP 4.0 [26] and Pfam 29.0 [27], respectively. The distribution of genes into COGs functional categories is presented in Table 5.

## Insights from the genome sequence

*E. coli* 1094 sequence was aligned against selected genomes belonging to *E. coli* phylogenetic group A. Alignment and posterior NJ (neighbor joining) phylogenetic reconstruction was carried out with Parsnp, a fast core-genome multi-aligner, using default parameters [28]. Alignment and tree visualization was done with Gingrich, a dynamic visual platform (Fig. 2) [28].

We selected five *E. coli* genomes that are representative of the multiple clades within phylogenetic group A, and performed orthologous clustering to examine the genomic differences of *E. coli* 1094. [*E. coli* K12 W3110 (accession n°: AP009048), *E. coli* BL21-Gold(DE3)pLysS AG' (accession CP001665), *E. coli* HS (accession n°: CP000802), *E. coli* ATCC 8739 (accession n°: CP000946) and the prototypical enterotoxigenic strain of *E. coli* ETEC H10407 (accession FN649414)] (Fig. 4). Our analysis reveals a core genome of 3409 genes shared among all strains (Fig. 4). *E. coli* ETEC H10407 possesses the highest number of specific genes (834), followed by *E. coli* 1094 (499), *E. coli*
<figure><img src="image_6.png" /><figcaption>**Table 3** Summary of 1094 genome: one chromosome and 3 putative plasmids</figcaption></figure>

<table><thead><tr><td>Label</td><td>Size (Mb)</td><td>Topology</td><td>INSDC identifier</td><td>RefSeq ID</td></tr></thead><tbody><tr><td>Chromosome</td><td>4.888</td><td>Circular</td><td>LT883139</td><td></td></tr><tr><td>Plasmid 1</td><td>0.115</td><td>Circular</td><td>LT883140</td><td></td></tr><tr><td>Plasmid 2</td><td>0.105</td><td>Circular</td><td>LT883141</td><td></td></tr><tr><td>Plasmid 3</td><td>0.021</td><td>Circular</td><td>LT883142</td><td></td></tr></tbody></table>

HS (440), *E. coli* BL21-Gold(DE3)pLysS AG' (292), *E. coli* K12 W3110 (241) and *E. coli* ATCC8739 (216).

The analysis of the location of genes only present in *E. coli* 1094 identified eight 1094-specific regions, most of which are prophage or phage-associated proteins; two regions represent a putative complete prophage, containing genes encoding the phage capsid tail and replication proteins (Fig. 4). Other 1094-specific regions of interest contain CRISPR associated proteins. We also identified clusters only present in some, but not all *E. coli* strains
<table><caption>**Table 4** Genome statistics</caption><thead><tr><th>Attribute</th><th>Value</th><th>% of Total</th></tr></thead><tbody><tr><td>Genome size (bp)</td><td>5,176,780</td><td>100.0</td></tr><tr><td>DNA coding (bp)</td><td>4,503,495</td><td>86.9</td></tr><tr><td>DNA G + C (bp)</td><td>2,340,599</td><td>50.9</td></tr><tr><td>DNA scaffolds</td><td>4</td><td>100.0</td></tr><tr><td>Total genes</td><td>5138</td><td>100.0</td></tr><tr><td>Protein coding genes</td><td>5028</td><td>97.8</td></tr><tr><td>RNA genes</td><td>110</td><td>2.1</td></tr><tr><td>Pseudo genes</td><td>0</td><td>0</td></tr><tr><td>Genes in internal clusters</td><td>232</td><td>4.5</td></tr><tr><td>Genes with function prediction</td><td>4390</td><td>85.4</td></tr><tr><td>Genes assigned to COGs</td><td>4827</td><td>93.9</td></tr><tr><td>Genes with Pfam domains</td><td>4377</td><td>85.2</td></tr><tr><td>Genes with signal peptides</td><td>398</td><td>7.7</td></tr><tr><td>Genes with transmembrane helices</td><td>1099</td><td>21.4</td></tr><tr><td>CRISPR repeats</td><td>11</td><td>0.2</td></tr></tbody></table>

analyzed, including (i) a putative complete type VI secretion system (present in 1094 and HS), (ii) a type III secretion system cluster (absent in BL21-Gold(DE3) and K12 W3110 strains of *E. coli*) (iii) a cluster of genes encoding invasins and an iron acquisition system, (present in 1094 and H10407), and (iv) several cellulose biosynthesis genes present in all strains, except for *E. coli* HS.

We performed a basic BLAST (BLASTN 2.6.1+, [29]) of each smaller contig identified, and report the following results. Unitig_1 shows sequence homology to *Salmonella enterica subsp. enterica serovar Senftenberg* strain 775 W plasmid *pSSE-ATCC-43845* (Accession: CP016838.1), with 33% of the contig showing 99% identity. Unitig_2 displays sequence homology to *Klebsiella pneumoniae subsp. pneumoniae* strain 234-12 plasmid *pKpn23412-362*, with 39% of the contig showing 99% identity (Accession: CP011314.1); other regions of the contig show homology to multiple *E. coli* strains and plasmids. Unitig_3 displays sequence homology to *Citrobacter freundii* strain B38 plasmid pOZ182; 68% of the contig shows 96% identity (Accession: CP016765.1); in addition, two separate regions of 7.5 and 2.1 kb are highly homologous to *E. coli* plasmid pV004-a DNA and pV001-a DNA (Accession: LC056140.1 and

<table><caption>**Table 5** Number of genes associated with general COG functional categories</caption><thead><tr><th>Code</th><th>Value</th><th>%age</th><th>Description</th></tr></thead><tbody><tr><td>J</td><td>179</td><td>3.5</td><td>Translation, ribosomal structure and biogenesis</td></tr><tr><td>A</td><td>3</td><td>0.05</td><td>RNA processing and modification</td></tr><tr><td>K</td><td>315</td><td>6.3</td><td>Transcription</td></tr><tr><td>L</td><td>254</td><td>5.05</td><td>Replication, recombination and repair</td></tr><tr><td>B</td><td>0</td><td>0</td><td>Chromatin structure and dynamics</td></tr><tr><td>D</td><td>45</td><td>0.9</td><td>Cell cycle control, Cell division, chromosome partitioning</td></tr><tr><td>V</td><td>58</td><td>1.15</td><td>Defense mechanisms</td></tr><tr><td>T</td><td>159</td><td>3.16</td><td>Signal transduction mechanisms</td></tr><tr><td>M</td><td>285</td><td>5.66</td><td>Cell wall/membrane biogenesis</td></tr><tr><td>N</td><td>62</td><td>1.23</td><td>Cell motility</td></tr><tr><td>U</td><td>116</td><td>2.3</td><td>Intracellular trafficking and secretion</td></tr><tr><td>O</td><td>172</td><td>3.42</td><td>Posttranslational modification, protein turnover, chaperones</td></tr><tr><td>C</td><td>308</td><td>6.12</td><td>Energy production and conversion</td></tr><tr><td>G</td><td>771</td><td>15.3</td><td>Carbohydrate transport and metabolism</td></tr><tr><td>E</td><td>318</td><td>6.32</td><td>Amino acid transport and metabolism</td></tr><tr><td>F</td><td>109</td><td>2.16</td><td>Nucleotide transport and metabolism</td></tr><tr><td>H</td><td>136</td><td>2.7</td><td>Coenzyme transport and metabolism</td></tr><tr><td>I</td><td>139</td><td>2.76</td><td>Lipid transport and metabolism</td></tr><tr><td>P</td><td>280</td><td>5.56</td><td>Inorganic ion transport and metabolism</td></tr><tr><td>Q</td><td>67</td><td>1.33</td><td>Secondary metabolites biosynthesis, transport and catabolism</td></tr><tr><td>R</td><td>-</td><td>-</td><td>General function prediction only</td></tr><tr><td>S</td><td>1051</td><td>20.9</td><td>Function unknown</td></tr><tr><td>-</td><td>-</td><td>-</td><td>Not in COGs</td></tr></tbody></table>

The total is based on the total number of protein coding genes in the genome
<figure><img src="image_7.png" /></figure>

LC056078.1). Taken together, this suggests that 1094 expresses 3 distinct circular plasmids.

## Comparative analysis of the bcs (bacterial cellulose synthesis) region

We compared the region corresponding to the *E. coli* 1094 *bcs* operon with corresponding regions in the strains *E. coli* W3110, HS, BL21-Gold(DE3)pLysS AG', ATCC 8739, and H10407, which are representative of phylogenetic group A. This analysis shows that whereas only partial fragments of the external genes of the *bcs* operon (*bcsC* and *bcsG*) exist in *E. coli* HS, the five other strains analyzed contain all genes within the *bcs* operon (Fig. 5). K12 derivative strains do not produce cellulose, as they contain a premature stop codon in the gene *bcsQ*, due to a single nucleotide polymorphism (SNP) in the region TTG/TAG (17 T > A) (Fig. 5) [30]. Serra et al., repaired this SNP, which resulted in cellulose production in *E. coli* K12 W3110, suggesting that the premature stop codon in *bcsQ* could either affect the function of BcsQ, or has a polar effect on neighboring genes [30]. The other SNPs (relative to *E. coli* 1094)

observed in the *bcs* operons of *E. coli* K-12 strains result either in synonymous codons or in conservative amino acid exchanges. By contrast, comparison of the *bcs* operon sequence between *E. coli* 1094 and *E. coli* ATCC8739 revealed 100% sequence identity between *bcsQ*, *bcsR*, *bcsA* and *bcsB* genes. Finally, the sequence of the *bcs* operon in *E. coli* ATCC8739 contains one SNP in *bcsE* that leads to a premature stop codon, and may also have a polar effect on *bcsF* and *bcsG*, which are essential for cellulose production [8] (Fig. 5). *E. coli* strains BL21-Gold(DE3)pLysS AG' and H10407 contain multiple SNPs in *bcs* genes that are essential for cellulose biosynthesis. Some of these SNPs lead to amino acid changes that could negatively affect the function of these proteins. *bcsA* is particularly interesting, as a pairwise genetic comparison in all six *E. coli* strains analyzed revealed the existence of multiple *bcsA* SNPs (between 21 and 29, depending on the strain pair compared). None of the SNPs resulted in amino acid exchange or a premature stop codon, suggesting a strong selection pressure for this specific amino acid composition. Our phylogenetic analysis of the
<figure><img src="image_8.png" /><figcaption>**Fig. 5** Genomic organization and Blastx comparison of the bacterial cellulose synthesis (*bcs*) region in six *E. coli* strains. The name of the corresponding genes are indicated. The gray color code represents the pairwise Blast matches between one strain, and the strain directly below it; darkness corresponds to higher similarity between the two strains, and strains were ordered based on phylogenetic relatedness. The star in red represents a SNP leading to a premature stop codon. The figure was generated using the Easyfig software [41]</figcaption></figure>

phylogenetic group A allowed us to localize these evolutionary events, by following a parsimony criterion, which allowed us to infer that mutations leading to premature stop codons took place in two separate evolutionary events: the branch of the strain ATCC8739 and in the K12 common ancestor (Fig. 2). These mutations resulted in sequence interruption of *bcsE* and *bcsQ* respectively.

### Cellulose production of *E. coli* strains

Cellulose production was tested in the six *E. coli* strains analyzed in this study. We monitored colony fluorescence on LB-calcofluor plates, which is a common assay used to detect cellulose biosynthesis [31] (Fig. 6). This analysis revealed that only *E. coli* 1094 strain produced detectable levels of cellulose. As was previously shown, laboratory *E. coli* K12 strains do not produce cellulose

<figure><img src="image_9.png" /><figcaption>**Fig. 6** Cellulose production in six *E. coli* strains. Cellulose secretion was evaluated by the binding of calcofluor (CF), as visualized by colony fluorescence under ultraviolet light (fluorescence appears white in image). Five microliters of an overnight culture was spotted onto LB agar plates, containing 0.02% of CF and incubated for 24 h at 30 °C. Images were acquired using a G:BOX imaging system (Syngene, Frederick, MD)</figcaption></figure>
[30]. Here we show that, as expected, *E. coli* HS is not able to produce this polymer, as it was known to lack all *bcs* genes. Interestingly, cellulose production was not detected in *E. coli* ATCC 8739, suggesting that the SNP in the region TGT/TGA (303 T > A) of *bcsE* described above could have a polar effect on the downstream genes. While the *bcsEFG* operon is essential for cellulose production [8], *bcsE* is only necessary for maximal cellulose production [32], suggesting that the inability to synthetize detectable levels of cellulose may be due to a polar effect of the *bcsEFG* operon. Finally, cellulose production was not detected in strains BL21-Gold(DE3) pLysS AG' and H10407, which could be explained by the presence of multiple amino acid changes that potentially affect the biological activity of BcsB, BcsC and BcsQ proteins, which are essential for cellulose production.

## Conclusions

The human commensal intestinal isolate *Escherichia coli* strain 1094 naturally produces cellulose, a polysaccharide known to contribute to adhesion and biofilm development. We compared genomic content and cellulose production of closely related *E. coli* strains in phylogenetic group A, and found 1094 to be the sole strain to produce measureable levels of cellulose, and conclude that these strains lack the capacity to produce cellulose due to one or several SNPs in cellulose biosynthesis genes: non-synonymous SNPs in *bcsB*, *bcsC*, *bcsQ*, and a nonsense mutation in *bcsE*. The genome sequencing and annotation here provides valuable information for future study of the *bcs* genes and cellulose production.

## Abbreviations

Bcs: Bacterial cellulose synthesis; CF: Calcofluor; RAST: Rapid Annotation using Subsystem Technology; SNPs: Single nucleotide polymorphism

## Acknowledgements

*E. coli* strain 1094 was a generous gift from Chantal Le Bouguennec. We would like to thank Magali Tichit for his technical help and Evelyne Begaud and the CRBIP (Centre de Ressources Biologiques de l'Institut Pasteur) for providing the *E. coli* ATCC8739 strain. High-throughput sequencing was performed by the Genomics Platform of the Institut Pasteur, a member of the France Génomique consortium [ANR10-INBS-09-08].

## Funding

This work was supported by the Institut Pasteur and grants from the French Government's Investissement d'Avenir program, Laboratoire d'Excellence: Integrative Biology of Emerging Infectious Diseases [grant n°ANR-10-LABX-62-IBEID], and from the Fondation pour la Recherche Médicale grant, [Equipe FRM DEQ20140329508].

## Authors' contributions

JB and JMG conceived the project. JB and LGV conducted phylogenomic studies. JB, LGV, AKW and JMG wrote the manuscript. JB and AKW performed the laboratory experiments. CB performed Illumina library preparations, and Illumina sequencing runs. JB and LGV assembled and annotated the genome. VK performed comparative analysis of Illumina and PacBio sequencing, and identified plasmids. All authors read and approved the final version of the manuscript.

## Competing interests

The authors declare that they have no competing interests.

## Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

## Author details

¹Département de Microbiologie, Unité de Génétique des Biofilms, Institut Pasteur, 25-28 rue du Dr. Roux, F-75015 Paris, France. ²Département de Génomes et Génétique, Unité de Biologie des Bactéries Intracellularies, Institut Pasteur, 25-28 rue du Dr. Roux, F-75015 Paris, France. ³Centre National de la Recherche Scientifique (CNRS). UMR 3525, 75724 Paris, France. ⁴Institut Pasteur – Hub Bioinformatique et Biostatistique – C3BI, USR 3756 IP CNRS, Paris, France. ⁵Institut Pasteur, Plate-forme Génomique, Pôle Biomics, CITECH 25-28 rue du Dr. Roux, F-75015 Paris, France.

Received: 21 August 2017 Accepted: 13 April 2018

Published online: 09 May 2018

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