Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3’,5’-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.


Introduction
analyzed by WFL and the Pel-specific antibody (Fig. 6C). This revealed that deletion of cdgE led 3 4 7 to increased binding of both WFL and the α-Pel antibody versus the wild-type strain, with 3 4 8 material appearing in the secreted fraction of the Δ cdgE mutant that was not present in the wild-3 4 9 type (Fig. 6C). In contrast, no signal was detected from the Δ cdgF mutant using WFL or the α- Pel antibody, similar to the Δ pelF mutant (Fig. 6C). This suggests that CgdE and CdgF play a Identification of pel operons and the production of the Pel polysaccharide have not been  (Table S1; [25]). Characterization of the pelDEA DA FG operon 3 6 1 from B. cereus ATCC 10987 has validated these findings and shown that this strain uses the Pel 3 6 2 polysaccharide for biofilm formation and that polymer production is regulated post- translationally by the binding of c-di-GMP to PelD. Furthermore, we show that Pel biosynthesis 3 6 4 is reciprocally regulated by the DGC CdgF, and the PDE CdgE. BCE_5586, and BCE_5587, which we have termed pelA DA , pelF, and pelG herein, led to an 3 6 9 impairment in biofilm formation and based on these results, Okshevsky et al. hypothesized that 3 7 0 the BCE_5583 -BCE_5587 operon (herein pelDEA DA FG) may be involved in the production of 3 7 1 an extracellular polysaccharide [42]. Two distinct transposon insertions were also observed in 3 7 2 cdgF, which was the only DGC identified in the screen, as well as an insertion in BCE_5588 that al. confirms that this gene is required for biofilm formation in ATCC 10987. As this screen 3 7 8 focused on mutants with impaired biofilm formation, genes whose deletion leads to hyper- biofilm formation, such as BCE_5582 (pelA H ) and cdgE, were not identified. [52-54]. As is the case amongst a diverse array of biofilm forming bacteria, eDNA has been 3 8 7 shown to be crucial for biofilm formation in ATCC 14579 [52], the environmental B. cereus 3 8 8 isolate AR156 [55], and ATCC 10987 [42]. First characterized in Bacillus subtilis [56], the 3 8 9 exopolysaccharide producing epsA-O operon is also present in B. cereus, although its 3 9 0 contribution to biofilm formation appears to be strain dependent. In the plant associated B. in P. aeruginosa, where Pel and/or the Psl polysaccharide are variably required [21]. Finally, B. ATCC 10987 utilizes TasA and/or CalY for biofilm formation is currently unknown. Our bioinformatics analysis and validation of Pel production in ATCC 10987 described and Streptococcus salivarius through a transposon mutagenesis screen [59]. Interestingly, the 4 0 4 authors noted that 75% of S. thermophilus genomes (9 of 12) available at the time of their 4 0 5 analysis did not contain the locus which we have attributed to Pel production, and suggested that 4 0 6 this operon may be a remnant of commensal life that has been lost due to the adaptation of S. thermophilus to domestication and its use as a fermentation starter in the dairy industry [59]. Similarly, we note that this locus is only present in ~26% (9 of 34) fully-sequenced S. with fully-sequenced genomes contained a pel operon. However, given the scarcity of 4 1 9 experimental data regarding biofilm formation by Bifidobacterium breve generally, we are 4 2 0 unable to speculate as to why the pel locus is so well conserved in this species. The motile to sessile transition in bacteria is regulated largely by c-di-GMP. To date, the 4 2 2 mechanisms of signal regulation, signal transduction, and the phenotypic consequences of c-di- Gram-positive PelD orthologs, which suggests a spectrum of activities associated with these 4 4 4 proteins that is not limited to c-di-GMP recognition (Fig. S2). In fact, the GGDEF domain is 4 4 5 completely absent in Streptococcal PelD orthologs, implying that if Streptococci produce Pel, 4 4 6 they must utilize a different mechanism for the post-translational regulation of Pel biosynthesis 4 4 7 than that described here for B. cereus. Alternatively, it is possible that Pel biosynthesis is not 4 4 8 regulated post-translationally in Streptococci. Interestingly, we noted that PelD from S. thermophilus JIM8232 contained only the transmembrane region and GAF domain, likely due to 4 5 0 interruption of this gene by a transposon (Fig. S2, S6). However, despite this truncated pelD 4 5 1 gene, JIM8232 produces significant amounts of biofilm biomass that is dependent on the pel 4 5 2 locus [59]. In contrast, S. salivarius JIM8777, which also utilizes the pel locus [59], has an intact 4 5 3 epimerase region at the amino-terminus of its PelD protein (Fig. S2). This suggests either that the  Amongst the Streptococcal, Clostridial, and Actinobacterial species identified in this presence of a well-conserved set of accessory genes located immediately downstream of and 4 6 0 2 2 contiguous with the pelDEA DA FG operon (Fig. S4, S5). The protein products of these accessory 4 6 1 genes are predicted to encode, among other things, an ortholog of the spore coat protein CotH.

6 2
While CotH functions as a protein kinase within the spore coat of endospore forming bacteria 4 6 3 [35], its identification here amongst non-spore forming Streptococci suggests that, within the 4 6 4 context of the putative pelDEA DA FG locus and the adjoining accessory genes, the cotH-like gene 4 6 5 may be performing a different function. One possibility is that it may play a role in the regulation Streptococci as they would not be able to regulate Pel biosynthesis via a canonical c-di-GMP-  Taken together, the data and analyses we have presented here suggests that the Pel  that there is still a great deal to be uncovered regarding the mechanism of Pel biosynthesis and its  Table S2. Generation of plasmids was performed using E. coli DH5α as a host for replication. growth of all strains. LB contained, per litre of ultrapure water, 10 g tryptone, 5 g NaCl, and 10 g 4 9 3 yeast extract. To prepare solid media, 1.5% (w/v) agar were added to LB. Where appropriate, 4 9 4 antibiotics were added to growth media. For E. coli, 100 μ g/mL carbenicillin, 50 μ g/mL 4 9 5 kanamycin, or 100 μ g/mL spectinomycin was used, and for B. cereus, 3 μ g/mL erythromycin or 4 9 6 10 μ g/mL chloramphenicol were used, as necessary. All basic microbiological and molecular biological techniques were performed using 5 0 0 standard protocols. Genomic DNA was isolated using BioRad InstaGene Matrix. Plasmid BioBasic. Restriction enzymes, DNA ligase, alkaline phosphatase, and DNA polymerase were  (Table S3). Sanger sequencing to confirm the sequence of plasmids and chromosomal mutations 5 0 5 (described below) was performed at The Center for Applied Genomics, Toronto.  To generate electrocompetent B. cereus, 2 mL of an overnight LB culture grown to 5 2 1 saturation was used to inoculate 500 mL of LB, which was then grown at 37 °C to an OD 600nm of 5 2 2 0.2 -0.4. Cells were collected by centrifugation at 4000 × g for 10 min at 4 °C and washed in 10 5 2 3 mL of cold electroporation buffer (1 mM Tris pH 8.0, 10% (w/v) sucrose, 15% (v/v) glycerol).

4 8
Plasmids were then isolated from individual colonies and verified by Sanger sequencing using pMAD-SEQ-F and pMAD-SEQ-R primers (Table S3).

0
To generate deletion mutants (Table S2), galactopyranoside (X-Gal) and grown at 30 °C for up to five days, or until blue colonies began to 5 5 5 appear corresponding to single crossover mutants [61]. Blue colonies were then pooled and used 5 5 6 to inoculate a 5 mL LB culture without antibiotic, which was grown at 37 °C overnight to 5 5 7 saturation. Three more cycles of growth were subsequently performed by subculturing the of the final saturated culture were then plated onto LB agar to obtain single colonies, which were white on X-Gal agar and were erythromycin sensitive, colony PCR was performed using primers 5 6 3 that targeted the outside, flanking regions of the gene of interest (Table S3) to identify unmarked 5 6 4 gene deletions. These PCR products were then Sanger sequenced using the same primers to 5 6 5 confirm the correct deletion.

6 6
To generate R363A, D366A, and R395A chromosomal point variants (Table S2) (Thermo Scientific). The upstream reverse and downstream forward primers (Table S3) (Table S3) were tailed with restriction enzyme cleavage sites to 5 7 3 enable ligation-dependent cloning of the spliced PCR products as described above for gene 5 7 4 deletion alleles. Allelic exchange was subsequently performed as described above, and 5 7 5 2 7 chromosomal point variants were confirmed by Sanger sequencing of PCR products generated 5 7 6 by colony PCR using primers flanking the region of pelD that was mutated (Table S3). To express genes in B. cereus for complementation of chromosomal deletions, the vector was subsequently constructed immediately downstream of the xylAR promoter using primers 5 8 4 tailed with restriction enzyme cleavage sites (Table S3) to generate pAD123-P xyl .

8 5
To generate complementation vectors, the gene of interest was amplified from B. cereus  (Table S3). The forward primer in each case was tailed with the synthetic ribosome binding site 5 8 9 5'-TAAGGAGGAAGCAGGT-3'. The gene of interest was then ligated into pAD123-P xyl using 5 9 0 T4 DNA ligase (Thermo Scientific). The resulting complementation vectors (Table S2) transformed into E. coli DH5α and selected on LB agar containing 100 μ g/mL carbenicillin.

9 4
To generate complemented B. cereus strains (Table S2), expression vectors were 5 9 5 transformed into ATCC 10987 mutants using the protocol outline above, and transformants were 5 9 6 selected on LB agar containing 10 μ g/mL chloramphenicol. Expression of genes from pAD123-5 9 7 P xyl was induced using 0.001 -1 % (w/v) xylose, as indicated. Crystal violet microtiter plate assay 6 0 0 As an indirect measure of biofilm formation, the crystal violet assay was performed to 6 0 1 assess the ability of B. cereus mutant strains to adhere to the wells of a plastic 96-well microtiter clean, standard 96-well microtiter plate (Grenier) and absorbance was measured at 550 nm. To assess the ability of B. cereus strains to form an air-liquid interface biofilm, or shaking. To make direct comparisons with complemented strains, empty pAD123-P xyl was 6 2 2 transformed into wild-type ATCC 10987 and deletion mutants as a vector control, as appropriate, 6 2 3 and 10 μ g/mL chloramphenicol was added to all cultures. The next day, the density of the culture 6 2 4 was adjusted to OD 600nm = 1.0 and 30 μ L of the resulting culture was used to inoculate 3 mL of  To visualize biomass adherent to the walls of borosilicate glass tubes, pellicles were 6 3 0 generated as described above and media was gently removed by pipetting. The glass tube was 6 3 1 washed twice with 4 mL of water, followed by staining for 10 min with 4 mL of 0.1% (w/v) 6 3 2 crystal violet at room temperature. The crystal violet was then gently removed, followed by three 6 3 3 washes with 4 mL water. The adherent biomass stained by crystal violet was then photographed. the air-liquid interface. The preparation for SEM was done as reported previously and is briefly Denton DCP-1 and sputter coated in gold using the Denton Desk V TSC. Samples then imaged 6 4 8 using the Quanta FEG 250 SEM, operated at 10 kV under high vacuum. For fluorescence microscopy, cultures were diluted from overnight cultures to 10 5 cfu/mL 6 5 2 and inoculated into 96-well plates in the same manner as for SEM biofilm growth. Glass an ORCA-R 2 C10600 digital camera (Hamamatsu). Pel was obtained as previously described in Colvin et al [31]. Cells were harvested by 6 6 4 centrifugation (16,000 × g for 2 min) from 1 mL aliquots of B. cereus ATCC 10987 grown occasional vortexing, and centrifuged (16,000 × g for 10 min) to harvest the supernatant 6 6 8 containing cell associated Pel. Cell-associated and secreted Pel were treated with proteinase K 6 6 9 (final concentration, 0.5 mg/mL) for 60 min at 60 °C, followed by 30 min at 80 °C to inactivate 6 7 0 proteinase K. [41]. 5 μL of cell associated and secreted Pel, prepared as described above, were pipetted onto a 6 7 3 nitrocellulose membrane and left to air dry for 10 min. The membrane was blocked with 5% at room temperature with shaking, and washed again. All immunoblots were developed using 6 7 9 SuperSignal West Pico (Thermo Scientific) following the manufacturer's recommendations. For WFL-HRP immunoblots, 5 μL of cell associated and secreted Pel, prepared as 6 8 1 described above, were pipetted onto a nitrocellulose membrane and left to air dry for 10 min. The washed twice for 5 min and once for 10 min with TBS-T, then developed as described above. ORF, excluding the predicted signal sequence (residues 1-21), was amplified from B. cereus tailed with restriction enzyme cleavage sites for ligation-dependent cloning (Table S3). The gene 6 9 2 of interest was then ligated into pET24a (Novagen), which encodes a C-terminal hexahistidine-6 9 3 tag, using T4 DNA ligase (Thermo Scientific). The resulting PelA H Bc expression vector ( Table   6 9 4 S2), was transformed into E. coli DH5α and selected on LB agar containing 50 μ g/mL 6 9 5 kanamycin. Plasmids were then isolated from individual colonies and verified by Sanger sequencing using T7 and T7ter primers (Table S3). Phusion DNA polymerase (Thermo Scientific). The upstream reverse and downstream forward 7 0 0 primers (Table S3) were tailed with an ~15-20 bp segment centered on E213 that contained the  (Table S3) were tailed with restriction enzyme cleavage sites to 7 0 3 enable ligation-dependent cloning of the spliced PCR product as described above for wild-type were grown overnight at 18 °C and harvested the next day via centrifugation at 5,000 × g for 20 7 1 0 min at 4 °C. Cell pellets were used immediately for purification. Preparation of c-di-GMP production-troublemakers revealed. Microbiologyopen. 2017;6: e00487. role of cepacian exopolysaccharide in resistance to stress conditions. Appl Environ Antibody to a conserved antigenic target is protective against diverse prokaryotic and     The equivalent B. cereus residues to Arg367, Asp370, and Arg402 of P. aeruginosa were 1 0 6 1 identified as Arg363, Asp366, and Arg395, and are represented as green sticks for clarity. The   phosphodiesterase that converts c-di-GMP to 5'-pGpG and thus negatively regulates Pel 1 0 9 2 biosynthesis. GalE epimerizes UDP-GlcNAc to UDP-GalNAc, which is presumably utilized by drawn to scale. The length of each protein is indicated to the right of the respective diagram.            Figure S1: Model of P. aeruginosa Pel biosynthesis. PelF is a glycosyltransferase that polymerizes UDP-GalNAc and UDP-GlcNAc to generate the Pel polysaccharide (light pink triangles are acetyl groups, red hexagons are GalN/GlcN monosaccharide units, dark red teardrops are UDP). Once synthesized, Pel is transported across the inner membrane via PelD, PelE, and/or PelG. In the periplasm, PelA interacts with the soluble tetratricopeptide repeat (TPR) domain of PelB to partially deacetylate the polymer and generate mature Pel, which is subsequently exported across the outer membrane through the β-barrel porin domain of PelB. PelC forms a dodecameric funnel that helps to channel Pel towards the PelB pore. Pel biosynthesis requires binding of the nucleotide second messenger c-di-GMP to the inhibitory site of the cytoplasmic GGDEF domain of PelD. C, cytoplasm; IM, inner membrane; P, periplasmic space; PG, peptidoglycan; OM, outer membrane; c-di-GMP, cyclic-3',5'-dimeric guanosine monophosphate.