Cyclic Dimeric GMP Research Articles

Tyrosol blocks E. coli anaerobic biofilm formation via YbfA and FNR to increase antibiotic susceptibility

Bacteria within mature biofilms are highly resistant to antibiotics than planktonic cells. Oxygen limitation contributes to antibiotic resistance in mature biofilms. Nitric oxide (NO) induces biofilm dispersal; however, low NO levels stimulate biofilm formation, an underexplored process. Here, we introduce a mechanism of anaerobic biofilm formation by investigating the antibiofilm activity of tyrosol, a component in wine. Tyrosol inhibits E. coli and Pseudomonas aeruginosa biofilm formation by enhancing NO production. YbfA is identified as a target of tyrosol and its downstream targets are sequentially determined. YbfA activates YfeR, which then suppresses the anaerobic regulator FNR. This suppression leads to decreased NO production, elevated bis-(3’−5’)-cyclic dimeric GMP levels, and finally stimulates anaerobic biofilm formation in the mature stage. Blocking YbfA with tyrosol treatment renders biofilm cells as susceptible to antibiotics as planktonic cells. Thus, this study presents YbfA as a promising antibiofilm target to address antibiotic resistance posed by biofilm-forming bacteria, with tyrosol acting as an inhibitor.

OpaR Exerts a Dynamic Control over c-di-GMP Homeostasis and cpsA Expression in Vibrio parahaemolyticus through Its Regulation of ScrC and the Trigger Phosphodiesterase TpdA.

The second messenger cyclic dimeric GMP (c-di-GMP) plays a central role in controlling decision-making processes that are vitally important for the environmental survival of the human pathogen Vibrio parahaemolyticus. The mechanisms by which c-di-GMP levels and biofilm formation are dynamically controlled in V. parahaemolyticus are poorly understood. Here, we report the involvement of OpaR in controlling c-di-GMP metabolism and its effects on the expression of the trigger phosphodiesterase (PDE) TpdA and the biofilm-matrix related gene cpsA. Our results revealed that OpaR negatively modulates the expression of tpdA by maintaining a baseline level of c-di-GMP. The OpaR-regulated PDEs ScrC, ScrG, and VP0117 enable the upregulation of tpdA, to different degrees, in the absence of OpaR. We also found that TpdA plays the dominant role in c-di-GMP degradation under planktonic conditions compared to the other OpaR-regulated PDEs. In cells growing on solid medium, we observed that the role of the dominant c-di-GMP degrader alternates between ScrC and TpdA. We also report contrasting effects of the absence of OpaR on cpsA expression in cells growing on solid media compared to cells forming biofilms over glass. These results suggest that OpaR can act as a double-edged sword to control cpsA expression and perhaps biofilm development in response to poorly understood environmental factors. Finally, using an in-silico analysis, we indicate outlets of the OpaR regulatory module that can impact decision making during the motile-to-sessile transition in V. parahaemolyticus. IMPORTANCE The second messenger c-di-GMP is extensively used by bacterial cells to control crucial social adaptations such as biofilm formation. Here, we explore the role of the quorum-sensing regulator OpaR, from the human pathogen V. parahaemolyticus, on the dynamic control of c-di-GMP signaling and biofilm-matrix production. We found that OpaR is crucial to c-di-GMP homeostasis in cells growing on Lysogeny Broth agar and that the OpaR-regulated PDEs TpdA and ScrC alternate in the dominant role over time. Furthermore, OpaR plays contrasting roles in controlling the expression of the biofilm-related gene cpsA on different surfaces and growth conditions. This dual role has not been reported for orthologues of OpaR, such as HapR from Vibrio cholerae. It is important to investigate the origins and consequences of the differences in c-di-GMP signaling between closely and distantly related pathogens to better understand pathogenic bacterial behavior and its evolution.

A Library of Promoter-gfp Fusion Reporters for Studying Systematic Expression Pattern of Cyclic-di-GMP Metabolism-Related Genes in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa is an environmental microorganism and is a model organism for biofilm research. Cyclic dimeric GMP (c-di-GMP) is a bacterial second messenger that plays critical roles in biofilm formation. P. aeruginosa contains approximately 40 genes that encode enzymes that participate in the metabolism of c-di-GMP (biosynthesis or degradation), yet it lacks tools that aid investigation of the systematic expression pattern of those genes. In this study, we constructed a promoter-gfp fusion reporter library that consists of 41 reporter plasmids. Each plasmid contains a promoter of corresponding c-di-GMP metabolism-related (CMR) genes from P. aeruginosa reference strain PAO1; thus, each promoter-gfp fusion reporter can be used to detect the promoter activity as well as the transcription of corresponding gene. The promoter activity was tested in P. aeruginosa and Escherichia coli. Among the 41 genes, the promoters of 26 genes showed activity in both P. aeruginosa and E. coli. The library was applied to determine the influence of different temperatures, growth media, and subinhibitory concentrations of antibiotics on the transcriptional profile of the 41 CMR genes in P. aeruginosa. The results showed that different growth conditions did affect the transcription of different genes, while the promoter activity of a few genes was kept at the same level under several different growth conditions. In summary, we provide a promoter-gfp fusion reporter library for systematic monitoring or study of the regulation of CMR genes in P. aeruginosa. In addition, the functional promoters can also be used as a biobrick for synthetic biology studies. IMPORTANCE The opportunistic pathogen P. aeruginosa can cause acute and chronic infections in humans, and it is one of the main pathogens in nosocomial infections. Biofilm formation is one of the most important causes for P. aeruginosa persistence in hosts and evasion of immune and antibiotic attacks. c-di-GMP is a critical second messenger to control biofilm formation. In P. aeruginosa reference strain PAO1, 41 genes are predicted to participate in the making and breaking of this dinucleotide. A major missing piece of information in this field is the systematic expression profile of those genes in response to changing environment. Toward this goal, we constructed a promoter-gfp transcriptional fusion reporter library that consists of 41 reporter plasmids, each of which contains a promoter of corresponding c-di-GMP metabolism-related genes in P. aeruginosa. This library provides a helpful tool to understand the complex regulation network related to c-di-GMP and to discover potential therapeutic targets.

Dual Regulatory Role Exerted by Cyclic Dimeric GMP To Control FsnR-Mediated Bacterial Swimming.

ABSTRACTBacterial motility has great medical and ecological significance because of its essential role in bacterial survival and pathogenesis. Cyclic dimeric GMP (c-di-GMP), a second messenger in bacteria, is the predominant regulator of flagellar synthesis and motility and possesses turnover mechanisms that have been thoroughly investigated. Therefore, much attention has been focused on identifying the upstream stimulatory signals and downstream modules that respond to altered c-di-GMP levels. Here, we systematically analyzed c-di-GMP cyclases and phosphodiesterases in Stenotrophomonas maltophilia to screen for motility regulators. Of these enzymes, we identified and characterized a new phosphodiesterase named SisP, which was found to facilitate bacterial swimming upon stimulation with ferrous iron. SisP-mediated degradation of c-di-GMP leads to FsnR-dependent transcription of flagellar genes. Remarkably, c-di-GMP controls FsnR via two independent mechanisms: by direct binding and indirectly by modulating its phosphorylation state. In this study, we deciphered a novel “one stone, two birds” regulatory strategy of c-di-GMP and uncovered the signal that stimulates c-di-GMP hydrolysis. Facilitation of bacterial swimming motility by ferrous iron might contribute to the higher risk of bacterial infection in acutely ill patients.

Signal Transduction Network Principles Underlying Bacterial Collective Behaviors.

Bacteria orchestrate collective behaviors and accomplish feats that would be unsuccessful if carried out by a lone bacterium. Processes undertaken by groups of bacteria include bioluminescence, biofilm formation, virulence factor production, and release of public goods that are shared by the community. Collective behaviors are controlled by signal transduction networks that integrate sensory information and transduce the information internally. Here, we discuss network features and mechanisms that, even in the face of dramatically changing environments, drive precise execution of bacterial group behaviors. We focus on representative quorum-sensing and second-messenger cyclic dimeric GMP (c-di-GMP) signal relays. We highlight ligand specificity versus sensitivity, how small-molecule ligands drive discrimination of kin versus nonkin, signal integration mechanisms, single-input sensory systems versus coincidence detectors, and tuning of input-output dynamics via feedback regulation. We summarize how different features of signal transduction systems allow groups of bacteria to successfully interpret and collectively react to dynamically changing environments.

Thermosynechococcus switches the direction of phototaxis by a c-di-GMP-dependent process with high spatial resolution.

Many cyanobacteria, which use light as an energy source via photosynthesis, show directional movement towards or away from a light source. However, the molecular and cell biological mechanisms for switching the direction of movement remain unclear. Here, we visualized type IV pilus-dependent cell movement in the rod-shaped thermophilic cyanobacterium Thermosynechococcus vulcanus using optical microscopy at physiological temperature and light conditions. Positive and negative phototaxis were controlled on a short time scale of 1 min. The cells smoothly moved over solid surfaces towards green light, but the direction was switched to backward movement when we applied additional blue light illumination. The switching was mediated by three photoreceptors, SesA, SesB, and SesC, which have cyanobacteriochrome photosensory domains and synthesis/degradation activity of the bacterial second messenger cyclic dimeric GMP (c-di-GMP). Our results suggest that the decision-making process for directional switching in phototaxis involves light-dependent changes in the cellular concentration of c-di-GMP. Direct visualization of type IV pilus filaments revealed that rod-shaped cells can move perpendicular to the light vector, indicating that the polarity can be controlled not only by pole-to-pole regulation but also within-a-pole regulation. This study provides insights into previously undescribed rapid bacterial polarity regulation via second messenger signalling with high spatial resolution.

Persulfide-Responsive Transcription Factor SqrR Regulates Gene Transfer and Biofilm Formation via the Metabolic Modulation of Cyclic di-GMP in Rhodobacter capsulatus.

Bacterial phage-like particles (gene transfer agents—GTAs) are widely employed as a crucial genetic vector in horizontal gene transfer. GTA-mediated gene transfer is induced in response to various stresses; however, regulatory mechanisms are poorly understood. We found that the persulfide-responsive transcription factor SqrR may repress the expression of several GTA-related genes in the photosynthetic bacterium Rhodobacter capsulatus. Here, we show that the sqrR deletion mutant (ΔsqrR) produces higher amounts of intra- and extracellular GTA and gene transfer activity than the wild type (WT). The transcript levels of GTA-related genes are also increased in ΔsqrR. In spite of the presumption that GTA-related genes are regulated in response to sulfide by SqrR, treatment with sulfide did not alter the transcript levels of these genes in the WT strain. Surprisingly, hydrogen peroxide increased the transcript levels of GTA-related genes in the WT, and this alteration was abolished in the ΔsqrR strain. Moreover, the absence of SqrR changed the intracellular cyclic dimeric GMP (c-di-GMP) levels, and the amount of c-di-GMP was correlated with GTA activity and biofilm formation. These results suggest that SqrR is related to the repression of GTA production and the activation of biofilm formation via control of the intracellular c-di-GMP levels.

Dysregulation of Cytosolic c-di-GMP in Edwardsiella piscicida Promotes Cellular Non-Canonical Ferroptosis.

Programmed cell death plays an important role in modulating host immune defense and pathogen infection. Ferroptosis is a type of inflammatory cell death induced by intracellular iron-dependent accumulation of toxic lipid peroxides. Although ferroptosis has been associated with cancer and other sterile diseases, very little is known about the role of ferroptosis in modulating host-pathogen interactions. We show that accumulation of the secondary messenger bis-(3′,5′)-cyclic dimeric GMP (c-di-GMP) in the pathogenic bacterium Edwardsiella piscicida (E. piscicida) triggers a non-canonical ferroptosis pathway in infected HeLa cells. Moreover, we observed that the dysregulation of c-di-GMP in E. piscicida promotes iron accumulation, mitochondrial dysfunction, and production of reactive oxygen species, all of which that can be blocked by iron chelator. Importantly, unlike classical ferroptosis that is executed via excess lipid peroxidation, no lipid peroxidation was detected in the infected cells. Furthermore, lipoxygenases inhibitors and lipophilic antioxidants are not able to suppress morphological changes and cell death induced by E. piscicida mutant producing excess c-di-GMP, and this c-di-GMP dysregulation attenuates bacterial virulence in vivo. Collectively, our results reveal a novel non-canonical ferroptosis pathway mediated by bacterial c-di-GMP and provide evidence for a role of ferroptosis in the regulation of pathogen infection.

Pseudomonas aeruginosa Oligoribonuclease Controls Tolerance to Polymyxin B by Regulating Pel Exopolysaccharide Production.

Polymyxins are considered as the last resort antibiotics to treat infections caused by multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is an opportunistic pathogen that causes various infections in humans. Proteins involved in lipopolysaccharide modification and maintaining inner and outer membrane integrities have been found to contribute to the bacterial resistance to polymyxins. Oligoribonuclease (Orn) is an exonuclease that regulates the homeostasis of intracellular (3'-5')-cyclic dimeric GMP (c-di-GMP), thereby regulating the production of extracellular polysaccharide in P. aeruginosa. Previously, we demonstrated that Orn affects the bacterial resistance to fluoroquinolone, β-lactam and aminoglycoside antibiotics. In this study, we found that mutation of orn increased the bacterial survival following polymyxin B treatment in a wild-type P. aeruginosa strain PA14. Overexpression of c-di-GMP degradation enzymes in the orn mutant reduced the bacterial survival. By using a fluorescence labeled polymyxin B, we found that mutation of orn increased the bacterial surface bound polymyxin B. Deletion of the Pel synthesis genes or treatment with a Pel hydrolase reduced the surface bound polymyxin B and bacterial survival. We further demonstrated that Pel binds to extracellular DNA (eDNA), which traps polymyxin B and thus protects the bacterial cells. Collectively, our results revealed a novel defense mechanism against polymyxin in P. aeruginosa.

Diguanylate Cyclase and Phosphodiesterase Interact To Maintain the Specificity of Cyclic di-GMP Signaling in the Regulation of Antibiotic Synthesis in Lysobacter enzymogenes.

Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. A large number of c-di-GMP-related diguanylate cyclases (DGCs), phosphodiesterases (PDEs), and effectors are responsible for the complexity and dynamics of c-di-GMP signaling. Some of these components employ various methods to avoid undesired cross talk to maintain signaling specificity. The synthesis of the antibiotic HSAF (heat-stable antifungal factor) in Lysobacter enzymogenes is regulated by a specific c-di-GMP signaling pathway that includes a PDE, LchP, and a c-di-GMP effector, Clp (also a transcriptional regulator). In the present study, from among 19 DGCs, we identified a diguanylate cyclase, LchD, that participates in this pathway. Subsequent investigation indicates that LchD and LchP physically interact and that the catalytic center of LchD is required for both the formation of the LchD-LchP complex and HSAF production. All the detected phenotypes support that LchD and LchP display local c-di-GMP signaling to regulate HSAF biosynthesis. Although direct evidence is lacking, our investigation, which shows that the interaction between a DGC and a PDE maintains the specificity of c-di-GMP signaling, suggests the possibility of the existence of local c-di-GMP pools in bacteria. IMPORTANCE Cyclic dimeric GMP (c-di-GMP) is a universal second messenger in bacteria. The signaling of c-di-GMP is complex and dynamic, and it is mediated by a large number of components, including c-di-GMP synthases (diguanylate cyclases [DGCs]), c-di-GMP-degrading enzymes (phosphodiesterases [PDEs]), and c-di-GMP effectors. These components deploy various methods to avoid undesired cross talk to maintain signaling specificity. In the present study, we identified a DGC that interacted with a PDE to specifically regulate antibiotic biosynthesis in L. enzymogenes. We provide direct evidence to show that the DGC and PDE form a complex and also indirect evidence to argue that they may balance a local c-di-GMP pool to control antibiotic production. These results represent an important finding regarding the mechanism of a DGC and PDE pair to control the expression of specific c-di-GMP signaling pathways.

An Intracellular Sensing and Signal Transduction System That Regulates the Metabolism of Polycyclic Aromatic Hydrocarbons in Bacteria.

ABSTRACTMany bacteria utilize polycyclic aromatic hydrocarbon (PAH) as carbon and energy sources for growth. These bacteria play an important role in the amelioration of PAH pollution in various environments. However, it is unclear how bacteria sense PAHs and how PAH degradation pathways are regulated via signal transduction. Here, we investigated these mechanisms in Cycloclasticus, a ubiquitous PAH-degrading bacterium in marine environments. We identified the key genes involved in intracellular PAH sensing, signal transduction, and the differential regulation of degradation pathways for each PAH examined. Our results showed that PAHs bind specifically to a diguanylate cyclase PdgC, leading to the generation of cyclic dimeric GMP (c-di-GMP), which subsequently binds to two CRP/FNR family regulators, DPR-1 and DPR-2. c-di-GMP activates the transcription of DPR-1 and DPR-2 to positively regulate degradation pathways specific to pyrene and phenanthrene/naphthalene, respectively. This is the first report of an intracellular signal transduction pathway associated with PAH degradation in bacteria. Our results improve our understanding of the intracellular responses to PAHs. The existence of the identified genes in other bacteria indicates that the strategy described here is widely used by other PAH-degrading bacteria.IMPORTANCE Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and have been found indoors, in the atmosphere, in terrestrial soils, in marine waters and sediments, and even in outer space. Bacteria degrade PAHs via degradation pathways. PAH signal sensing and transduction, as well as the regulation of PAH degradation pathways, are crucial for bacterial PAH biodegradation. However, prior to this study, these processes were poorly known. This study employed multiple molecular approaches to better understand the regulatory networks controlling PAH metabolism in bacteria. This report illustrates, for the first time, PAH-specific intracellular sensing, signal transduction, and metabolic regulatory pathways. Our results will help to increase our understanding of the hydrocarbon-metabolism regulatory network as well as the regulatory intricacies that control microbial biodegradation of organic matter. These key data should be considered to improve the rational design and efficiency of recombinant biodegradable, bacterial biosensors, and biocatalysts in modern green chemistry.

The predatory soil bacterium Lysobacter reprograms quorum sensing system to regulate antifungal antibiotic production in a cyclic-di-GMP-independent manner

Soil bacteria often harbour various toxins to against eukaryotic or prokaryotic. Diffusible signal factors (DSFs) represent a unique group of quorum sensing (QS) chemicals that modulate interspecies competition in bacteria that do not produce antibiotic-like molecules. However, the molecular mechanism by which DSF-mediated QS systems regulate antibiotic production for interspecies competition remains largely unknown in soil biocontrol bacteria. In this study, we find that the necessary QS system component protein RpfG from Lysobacter, in addition to being a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE), regulates the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF), which does not appear to depend on the enzymatic activity. Interestingly, we show that RpfG interacts with three hybrid two-component system (HyTCS) proteins, HtsH1, HtsH2, and HtsH3, to regulate HSAF production in Lysobacter. In vitro studies show that each of these proteins interacted with RpfG, which reduced the PDE activity of RpfG. Finally, we show that the cytoplasmic proportions of these proteins depended on their phosphorylation activity and binding to the promoter controlling the genes implicated in HSAF synthesis. These findings reveal a previously uncharacterized mechanism of DSF signalling in antibiotic production in soil bacteria.

Identification of LuxR Family Regulators That Integrate Into Quorum Sensing Circuit in Vibrio parahaemolyticus.

Vibrio parahaemolyticus is one of the most important food-borne pathogens that cause economic and public health problems worldwide. Quorum sensing (QS) is a way for the cell-cell communication between bacteria that controls a wide spectrum of processes and phenotypic behaviors. In this study, we performed a systematic research of LuxR family regulators in V. parahaemolyticus and found that they influence the bacterial growth and biofilm formation. We then established a QS reporter plasmid based on bioluminescence luxCDABE operon of Vibrio harveyi and demonstrated that several LuxR family regulators integrated into QS circuit in V. parahaemolyticus. Thereinto, a novel LuxR family regulator, named RobA, was identified as a global regulator by RNA-sequencing analyses, which affected the transcription of 515 genes in V. parahaemolyticus. Subsequent studies confirmed that RobA regulated the expression of the exopolysaccharides (EPS) synthesis cluster and thus controlled the biofilm formation. In addition, bioluminescence reporter assays showed that RobA plays a key role in the QS circuit by regulating the expression of opaR, aphA, cpsQ-mfpABC, cpsS, and scrO. We further demonstrated that the regulation of RobA to EPS and MfpABC depended on OpaR and CpsQ, which combined the QS signal with bis-(3′-5′)-cyclic dimeric GMP to construct a complex regulatory network of biofilm formation. Our data provided new insights into the bacterial QS mechanisms and biofilm formation in V. parahaemolyticus.

Three PilZ Domain Proteins, PlpA, PixA, and PixB, Have Distinct Functions in Regulation of Motility and Development in Myxococcus xanthus.

ABSTRACTIn bacteria, the nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pilus-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth, while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility.IMPORTANCE c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain, while the intact protein and c-di-GMP binding are essential for PixB to support development. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

A SNP in the Cache 1 Signaling Domain of Diguanylate Cyclase STM1987 Leads to Increased In Vivo Fitness of Invasive Salmonella Strains.

Nontyphoidal Salmonella (NTS) strains are associated with gastroenteritis worldwide but are also the leading cause of bacterial bloodstream infections in sub-Saharan Africa. The invasive NTS (iNTS) strains that cause bloodstream infections differ from standard gastroenteritis-causing strains by >700 single-nucleotide polymorphisms (SNPs). These SNPs are known to alter metabolic pathways and biofilm formation and to contribute to serum resistance and are thought to signify iNTS strains becoming human adapted, similar to typhoid fever-causing Salmonella strains. Identifying SNPs that contribute to invasion or increased virulence has been more elusive. In this study, we identified a SNP in the cache 1 signaling domain of diguanylate cyclase STM1987 in the invasive Salmonella enterica serovar Typhimurium type strain D23580. This SNP was conserved in 118 other iNTS strains analyzed and was comparatively absent in global S Typhimurium isolates associated with gastroenteritis. STM1987 catalyzes the formation of bis-(3',5')-cyclic dimeric GMP (c-di-GMP) and is proposed to stimulate production of cellulose independent of the master biofilm regulator CsgD. We show that the amino acid change in STM1987 leads to a 10-fold drop in cellulose production and increased fitness in a mouse model of acute infection. Reduced cellulose production due to the SNP led to enhanced survival in both murine and human macrophage cell lines. In contrast, loss of CsgD-dependent cellulose production did not lead to any measurable change in in vivo fitness. We hypothesize that the SNP in stm1987 represents a pathoadaptive mutation for iNTS strains.

Alkaline pH Increases Swimming Speed and Facilitates Mucus Penetration for Vibrio cholerae.

Intestinal mucus is the first line of defense against intestinal pathogens. It acts as a physical barrier between epithelial tissues and the lumen that enteropathogens must overcome to establish a successful infection. We investigated the motile behavior of two Vibrio cholerae strains (El Tor C6706 and Classical O395) in mucus using single-cell tracking in unprocessed porcine intestinal mucus. We determined that V. cholerae can penetrate mucus using flagellar motility and that alkaline pH increases swimming speed and, consequently, improves mucus penetration. Microrheological measurements indicate that changes in pH between 6 and 8 (the physiological range for the human small intestine) had little effect on the viscoelastic properties of mucus. Finally, we determined that acidic pH promotes surface attachment by activating the mannose-sensitive hemagglutinin (MshA) pilus in V. cholerae El Tor C6706 without a measurable change in the total cellular concentration of the secondary messenger cyclic dimeric GMP (c-di-GMP). Overall, our results support the hypothesis that pH is an important factor affecting the motile behavior of V. cholerae and its ability to penetrate mucus. Therefore, changes in pH along the human small intestine may play a role in determining the preferred site for V. cholerae during infection.IMPORTANCE The diarrheal disease cholera is still a burden for populations in developing countries with poor sanitation. To develop effective vaccines and prevention strategies against Vibrio cholerae, we must understand the initial steps of infection leading to the colonization of the small intestine. To infect the host and deliver the cholera toxin, V. cholerae has to penetrate the mucus layer protecting the intestinal tissues. However, the interaction of V. cholerae with intestinal mucus has not been extensively investigated. In this report, we demonstrated using single-cell tracking that V. cholerae can penetrate intestinal mucus using flagellar motility. In addition, we observed that alkaline pH improves the ability of V. cholerae to penetrate mucus. This finding has important implications for understanding the dynamics of infection, because pH varies significantly along the small intestine, between individuals, and between species. Blocking mucus penetration by interfering with flagellar motility in V. cholerae, reinforcing the mucosa, controlling intestinal pH, or manipulating the intestinal microbiome will offer new strategies to fight cholera.

Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from Streptomyces ghanaensis ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis.

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3′→5′) GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis.

A Two-Component System Acquired by Horizontal Gene Transfer Modulates Gene Transfer and Motility via Cyclic Dimeric GMP

Bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important intracellular signaling molecule that affects diverse physiological processes in bacteria. The intracellular levels of c-di-GMP are controlled by proteins acting as diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that synthesize and degrade c-di-GMP, respectively. In the alphaproteobacterium Rhodobacter capsulatus, flagellar motility and gene exchange via production of the gene transfer agent RcGTA are regulated by c-di-GMP. One of the R. capsulatus proteins involved in this regulation is Rcc00620, which contains an N-terminal two-component system response regulator receiver (REC) domain and C-terminal DGC and PDE domains. We demonstrate that the enzymatic activity of Rcc00620 is regulated through the phosphorylation status of its REC domain, which is controlled by a cognate histidine kinase protein, Rcc00621. In this system, the phosphorylated form of Rcc00620 is active as a PDE enzyme and stimulates gene transfer and motility. In addition, we discovered that the rcc00620 and rcc00621 genes are present in only one lineage within the genus Rhodobacter and were acquired via horizontal gene transfer from a distantly related alphaproteobacterium in the order Sphingomonadales. Therefore, a horizontally acquired regulatory system regulates gene transfer in the recipient organism.

A novel c-di-GMP signal system regulates biofilm formation in Pseudomonas aeruginosa.

The bacterial second messenger cyclic-di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. The human pathogen Pseudomonas aeruginosa encodes 17 diguanylate cyclase (DGCs) proteins which are required for c-di-GMP synthesis. Therefore, the c-di-GMP regulatory system in P. aeruginosa is highly sophisticated. SiaD, one of the DGC enzymes, is co-transcribed with SiaA/B/C and has been shown to be essential for bacterial aggregate formation in response to environmental stress. However, the detailed function of this operon remains unknown. In our recent paper (Chen et al., doi: 10.15252/embj.2019103412), we have demonstrated that the siaABCD operon encodes a signaling network that regulates biofilm and aggregate formation by modulating the enzymatic activity of SiaD. Among this signaling system, SiaC interaction with SiaD promotes the diguanylate cyclase activity of SiaD and subsequently facilities the intracellular c-di-GMP synthesis; SiaB is a unique protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD. This report unveils a novel signaling system that controls biofilm formation, which may provide a potential target for developing antimicrobial drugs.

Cyclic di-GMP-Mediated Regulation of Gene Transfer and Motility in Rhodobacter capsulatus.

Gene transfer agents (GTAs) are bacteriophage-like particles produced by several bacterial and archaeal lineages that contain small pieces of the producing cells' genomes that can be transferred to other cells in a process similar to transduction. One well-studied GTA is RcGTA, produced by the alphaproteobacterium Rhodobacter capsulatus RcGTA gene expression is regulated by several cellular regulatory systems, including the CckA-ChpT-CtrA phosphorelay. The transcription of multiple other regulator-encoding genes is affected by the response regulator CtrA, including genes encoding putative enzymes involved in the synthesis and hydrolysis of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). To investigate whether c-di-GMP signaling plays a role in RcGTA production, we disrupted the CtrA-affected genes potentially involved in this process. We found that disruption of four of these genes affected RcGTA gene expression and production. We performed site-directed mutagenesis of key catalytic residues in the GGDEF and EAL domains responsible for diguanylate cyclase (DGC) and c-di-GMP phosphodiesterase (PDE) activities and analyzed the functions of the wild-type and mutant proteins. We also measured RcGTA production in R. capsulatus strains where intracellular levels of c-di-GMP were altered by the expression of either a heterologous DGC or a heterologous PDE. This adds c-di-GMP signaling to the collection of cellular regulatory systems controlling gene transfer in this bacterium. Furthermore, the heterologous gene expression and the four gene disruptions had similar effects on R. capsulatus flagellar motility as found for gene transfer, and we conclude that c-di-GMP inhibits both RcGTA production and flagellar motility in R. capsulatusIMPORTANCE Gene transfer agents (GTAs) are virus-like particles that move cellular DNA between cells. In the alphaproteobacterium Rhodobacter capsulatus, GTA production is affected by the activities of multiple cellular regulatory systems, to which we have now added signaling via the second messenger dinucleotide molecule bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). Similar to the CtrA phosphorelay, c-di-GMP also affects R. capsulatus flagellar motility in addition to GTA production, with lower levels of intracellular c-di-GMP favoring increased flagellar motility and gene transfer. These findings further illustrate the interconnection of GTA production with global systems of regulation in R. capsulatus, providing additional support for the notion that the production of GTAs has been maintained in this and related bacteria because it provides a benefit to the producing organisms.