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Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases.

ABSTRACT: Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of Bacillus subtilis strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in Clostridium difficile using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative C. difficile 630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in Bacillus subtilis.

SUBMITTER: Gao X

PROVIDER: S-EPMC4178680 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases.

Gao Xiaohui X Dong Xiao X Subramanian Sundharraman S Matthews Paige M PM Cooper Caleb A CA Kearns Daniel B DB Dann Charles E CE

Applied and environmental microbiology 20140801 19

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often enco ...[more]

PMID: 25085482

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Project description:Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro, which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro, showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient.IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis Thus, local-pool signal transduction operates extremely efficiently and specifically.

Project description:Intracellular concentration of cyclic diguanylate monophosphate (c-di-GMP), a second messenger molecule, is regulated in bacteria by diguanylate cyclases (DGCs) (synthesizing c-di-GMP) and phosphodiesterases (PDEs) (degrading c-di-GMP). c-di-GMP concentration ([c-di-GMP]) affects motility and sessility in a reciprocal fashion; high [c-di-GMP] typically inhibits motility and promotes sessility. A c-di-GMP sensor domain, PilZ, also regulates motility and sessility. Uropathogenic Escherichia coli regulates these processes during infection; motility is necessary for ascending the urinary tract, while sessility is essential for colonization of anatomical sites. Here, we constructed and screened 32 mutants containing deletions of genes encoding each PDE (n = 11), DGC (n = 13), PilZ (n = 2), and both PDE and DGC (n = 6) domains for defects in motility, biofilm formation, and adherence for the prototypical pyelonephritis isolate E. coli CFT073. Three of 32 mutations affected motility, all of which were in genes encoding enzymatically inactive PDEs. Four PDEs, eight DGCs, four PDE/DGCs, and one PilZ regulated biofilm formation in a medium-specific manner. Adherence to bladder epithelial cells was regulated by [c-di-GMP]. Four PDEs, one DGC, and three PDE/DGCs repress adherence and four DGCs and one PDE/DGC stimulate adherence. Thus, specific effectors of [c-di-GMP] and catalytically inactive DGCs and PDEs regulate adherence and motility in uropathogenic E. coli. IMPORTANCE Uropathogenic Escherichia coli (UPEC) contains several genes annotated as encoding enzymes that increase or decrease the abundance of the second messenger molecule, c-di-GMP. While this class of enzymes has been studied in an E. coli K-12 lab strain, these proteins have not been comprehensively examined in UPEC. UPEC utilizes both swimming motility and adherence to colonize and ascend the urinary tract; both of these processes are hypothesized to be regulated by the concentration of c-di-GMP. Here, for the first time, in a uropathogenic strain, E. coli CFT073, we have characterized mutants lacking each protein and demonstrated that the uropathogen has diverged from E. coli K-12 to utilize these enzymes to regulate adherence and motility by distinct mechanisms.

Project description:Cyclic diguanosine monophosphate (c-di-GMP) is an important second messenger involved in bacterial switching from motile to sessile lifestyles. In the opportunistic pathogen Pseudomonas aeruginosa, at least 40 genes are predicted to encode proteins for the making and breaking of this signal molecule. However, there is still paucity of information concerning the systemic expression pattern of these genes and the functions of uncharacterized genes. In this study, we analyzed the phylogenetic distribution of genes from P. aeruginosa that were predicted to have a GGDEF domain and found five genes (PA5487, PA0285, PA0290, PA4367, and PA5017) with highly conserved distribution across 52 public complete pseudomonad genomes. PA5487 was further characterized as a typical diguanylate cyclase (DGC) and was named dgcH A systemic analysis of the gene expression data revealed that the expression of dgcH is highly invariable and that dgcH probably functions as a conserved gene to maintain the basal level of c-di-GMP, as reinforced by gene expression analyses. The other four conserved genes also had an expression pattern similar to that of dgcH The functional analysis suggested that PA0290 encoded a DGC, while the others functioned as phosphodiesterases (PDEs). Our data revealed that there are five DGC and PDE genes that maintain the basal level of c-di-GMP in P. aeruginosa IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that can cause infections in animals, humans, and plants. The formation of biofilms by P. aeruginosa is the central mode of action to persist in hosts and evade immune and antibiotic attacks. Cyclic-di-GMP (c-di-GMP) is an important second messenger involved in the regulation of biofilm formation. In P. aeruginosa PAO1 strain, there are around 40 genes that encode enzymes for making and breaking this dinucleotide. A major missing piece of information in this field is the phylogeny and expression profile of those genes. Here, we took a systemic approach to investigate this mystery. We found that among 40 c-di-GMP metabolizing genes, 5 have well-conserved phylogenetic distribution and invariable expression profiles, suggesting that there are enzymes required for the basal level of c-di-GMP in P. aeruginosa This study thus provides putative therapeutic targets against P. aeruginosa infections.

Project description:BackgroundMost of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily.ResultsWe found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively.ConclusionsIn this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

Dataset Information

Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases.

Publications

Engineering of Bacillus subtilis strains to allow rapid characterization of heterologous diguanylate cyclases and phosphodiesterases.

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