SwrA as global modulator of the two-component system DegSU in Bacillus subtilis

The two-component system DegSU of Bacillus subtilis controls more than one hundred genes involved in several different cellular behaviours. Over the last four decades, the degU32^Hy allele, supposedly encoding a constitutively active mutant of the response regulator DegU, was exploited to define the impact of this system on cell physiology. Those studies concluded that phosphorylated DegU (DegU∼P) induced degradative enzyme expression while repressing flagellar motility and competence.Recent experiments, however, demonstrated that flagella expression is enhanced by DegU∼P if SwrA, a protein only encoded by wild strains, is present. Yet, to promote motility, SwrA must interact with DegU∼P produced by a wild-type degU allele, as it cannot correctly cooperate with the mutant DegU32^Hy protein.In this work, the impact of DegSU was reanalysed in the presence or absence of SwrA employing a DegS kinase mutant, degS200^Hy, to force the activation of the TCS. Our results demonstrate that the role of SwrA in B. subtilis physiology is wider than expected and affects several other DegSU targets. SwrA reduces subtilisin, cellulases and xylanases production while, besides motility, it also positively modulates competence for DNA uptake, remarkably relieving the inhibition caused by DegU∼P alone and restoring transformability in degS200^Hy strains.     

The phosphotransferase system gene ptsH plays an important role in MnSOD production,biofilm formation,swarming motility,and root colonization in Bacillus cereus 905

The rhizosphere bacterium Bacillus cereus 905 is capable of promoting plant growth through effective colonization on plant roots. The sodA2-encoding manganese-containing superoxide dismutase (MnSOD2) is important for survival of B. cereus 905 in the wheat rhizosphere. However, the genes involved in regulating sodA2 expression and the mechanisms of rhizosphere colonization of B. cereus 905 are not well elucidated. In this study, we found that the deletion of the ptsH gene, which encodes the histidine-phosphorylatable protein (HPr), a component of the phosphotransferase system (PTS), causes a decrease of about 60% in the MnSOD2 expression. Evidences indicate that the ptsH dramatically influences resistance to oxidative stress, glucose uptake, as well as biofilm formation and swarming motility of B. cereus 905. Root colonization assay demonstrated that ΔptsH is defective in colonizing wheat roots, while complementation of the sodA2 gene could partially restore the ability in utilization of arabinose, a non-PTS sugar, and root colonization caused by the loss of the ptsH gene. In toto, based on the current findings, we propose that PtsH contributes to root colonization of B. cereus 905 through multiple indistinct mechanisms, involving PTS and uptake of PTS-sugars, up-regulation of MnSOD2 production, and promotion of biofilm formation and swarming motility.     

Modulation of Pseudomonas aeruginosa surface-associated group behaviors by individual amino acids through c-di-GMP signaling

To colonize the cystic fibrosis lung, Pseudomonas aeruginosa establishes sessile communities referred to as biofilms. Although the signaling molecule c-di-GMP governs the transition from motile to sessile growth, the environmental signal(s) required to modulate biofilm formation remain unclear. Using relevant in vivo concentrations of the 19 amino acids previously identified in cystic fibrosis sputum, we demonstrated that arginine, ornithine, isoleucine, leucine, valine, phenylalanine and tyrosine robustly promoted biofilm formation in vitro. Among the seven biofilm-promoting amino acids, only arginine also completely repressed the ability of P. aeruginosa to swarm over semi-solid surfaces, suggesting that arginine may be an environmental cue favoring a sessile lifestyle. Mutating two documented diguanylate cyclases required for biofilm formation (SadC and RoeA) reduced biofilm formation and restored swarming motility on arginine-containing medium. Growth on arginine increased the intracellular levels of c-di-GMP, and this increase was dependent on the SadC and RoeA diguanylate cyclases. Strains mutated in sadC, roeA or both also showed a reduction in biofilm formation when grown with the other biofilm-promoting amino acids. Taken together, these results suggest that amino acids can modulate biofilm formation and swarming motility, at least in part, by controlling the intracellular levels of c-di-GMP.     

Two distinct pathways lead Bacillus thuringiensis to commit to sporulation in biofilm

The spore-forming bacterium Bacillus thuringiensis is an efficient biofilm producer, responsible for persistent contamination of industrial food processing systems. B. thuringiensis biofilms are highly heterogeneous bacterial structures in which three distinct cell types controlled by quorum sensing regulators were identified: PlcR-controlled virulent cells, NprR-dependent necrotrophic cells and cells committed to sporulation, a differentiation process controlled by Rap phosphatases and Spo0A-P. Interestingly, a cell lineage study revealed that, in LB medium or in insect larvae, only necrotrophic cells became spores. Here we analyzed cellular differentiation undertaken by cells growing in biofilm in a medium optimized for sporulation. No virulent cells were identified; surprisingly, two distinct routes could lead to differentiation as a spore in this growth condition: the NprR-dependent route, followed by the majority of cells, and the newly identified NprR-independent route, which is followed by 20% of sporulating cells.     

Burkholderia pseudomallei kynB plays a role in AQ production,biofilm formation,bacterial swarming and persistence

Kynurenine formamidase (KynB) forms part of the kynurenine pathway which metabolises tryptophan to anthranilate. This metabolite can be used for downstream production of 2-alkyl-4-quinolone (AQ) signalling molecules that control virulence in Pseudomonas aeruginosa. Here we investigate the role of kynB in the production of AQs and virulence-associated phenotypes of Burkholderia pseudomallei K96243, the causative agent of melioidosis. Deletion of kynB resulted in reduced AQ production, increased biofilm formation, decreased swarming and increased tolerance to ciprofloxacin. Addition of exogenous anthranilic acid restored the biofilm phenotype, but not the persister phenotype. This study suggests the kynurenine pathway is a critical source of anthranilate and signalling molecules that may regulate B. pseudomallei virulence.     

Characterization of sporulation histidine kinases of Paenibacillus polymyxa

Sporulation histidine kinases, which sense sporulation-specific signals and initiate phosphorelay reactions, are poorly conserved among Bacillus species. We found several putative genes for sporulation histidine kinases in the genome sequence of Paenibacillus polymyxa E681 and assayed the genes for complementation of sporulation mutants of Bacillus subtilis. One of these genes, Kin1377, significantly restored the sporulation deficiency of kinA kinB double mutant of B. subtilis, but not of B. subtilis spo0B mutant. These results indicated that Kin1377 requires B. subtilis Spo0B and possibly Spo0F to transfer phosphate to B. subtilis Spo0A. Another putative kinase, Kin1038, slightly restored the sporulation deficiencies of both kinA kinB double mutant and spo0B mutant of B. subtilis. However the sporulation deficiency of the B. subtilis spo0B mutant was significantly restored in the presence of both Kin1038 and P. polymyxa Spo0A. These results indicate that the overexpressed Kin1038 is able to interact directly with and activate P. polymyxa Spo0A, and that Spo0A can support spore formation in B. subtilis.     

RpoS differentially affects the general stress response and biofilm formation in the endophytic Serratia plymuthica G3

The σ^S subunit RpoS of RNA polymerase functions as a master regulator of the general stress response in Escherichia coli and related bacteria. RpoS has been reported to modulate biocontrol properties in the rhizobacterium Serratia plymuthica IC1270. However, the role of RpoS in the stress response and biofilm formation in S. plymuthica remains largely unknown. Here we studied the role of RpoS from an endophytic S. plymuthica G3 in regulating these phenotypes. Mutational analysis demonstrated that RpoS positively regulates the global stress response to acid or alkaline stresses, oxidative stress, hyperosmolarity, heat shock and carbon starvation, in addition to proteolytic and chitinolytic activities. Interestingly, rpoS mutations resulted in significantly enhanced swimming motility, biofilm formation and production of the plant auxin indole-3-acetic acid (IAA), which may contribute to competitive colonization and environmental fitness for survival. These findings provide further insight into the strain-specific role of RpoS in the endophytic strain G3 of S. plymuthica, where it confers resistance to general stresses encountered within the plant environment. The heterogeneous functionality of RpoS in rhizosphere and endophytic S. plymuthica populations may provide a selective advantage for better adaptation to various physiological and environmental stresses.     

Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Two-component systems play important roles in the physiology of many bacterial pathogens. Vibrio cholerae''s CarRS two-component regulatory system negatively regulates expression of vps (Vibrio polysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of the almEFG genes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of the almEFG operon. Similarly to a carR mutant, strains lacking almE, almF, and almG exhibited enhanced polymyxin B sensitivity. We also observed that strains lacking almE or the almEFG operon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance in V. cholerae.     

C-di-GMP turnover influences motility and biofilm formation in Bacillus amyloliquefaciens PG12

Bis-(3′→5′) cyclic dimeric guanosine monophosphate (c-di-GMP) is defined as a highly versatile secondary messenger in bacteria, coordinating diverse aspects of bacterial growth and behavior, including motility and biofilm formation. Bacillus amyloliquefaciens PG12 is an effective biocontrol agent against apple ring rot caused by Botryosphaeria dothidea. In this study, we characterized the core regulators of c-di-GMP turnover in B. amyloliquefaciens PG12. Using bioinformatic analysis, heterologous expression and biochemical characterization of knockout and overexpression derivatives, we identified and characterized two active diguanylate cyclases (which catalyze c-di-GMP biosynthesis), YhcK and YtrP and one active c-di-GMP phosphodiesterase (which degrades c-di-GMP), YuxH. Furthermore, we showed that elevating c-di-GMP levels up to a certain threshold inhibited the swimming motility of B. amyloliquefaciens PG12. Although yhcK, ytrP and yuxH knockout mutants did not display defects in biofilm formation, significant increases in c-di-GMP levels induced by YtrP or YuxH overexpression stimulated biofilm formation in B. amyloliquefaciens PG12. Our results indicate that B. amyloliquefaciens possesses a functional c-di-GMP signaling system that influences the bacterium's motility and ability to form biofilms. Since motility and biofilm formation influence the efficacy of biological control agent, our work provides a basis for engineering a more effective strain of B. amyloliquefaciens PG12.     

Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary.     

Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales

Some pseudomonads produce phenazines, a group of small, redox-active compounds with diverse physiological functions. In this study, we compared the phenotypes of Pseudomonas aeruginosa strain PA14 and a mutant unable to synthesize phenazines in flow cell and colony biofilms quantitatively. Although phenazine production does not impact the ability of PA14 to attach to surfaces, as has been shown for Pseudomonas chlororaphis Maddula et al., 2006, Maddula et al., 2008, it influences swarming motility and the surface-to-volume ratio of mature biofilms. These results indicate that phenazines affect biofilm development across a large range of scales, but in unique ways for different Pseudomonas species.