Substrate preference, RNA binding and active site versatility of Stenotrophomonas maltophilia nuclease SmNuc1, explained by a structural study.

Nucleases of the S1/P1 family have important applications in biotechnology and molecular biology. We have performed structural analyses of SmNuc1 nuclease from Stenotrophomonas maltophilia, including RNA cleavage product binding and mutagenesis in a newly discovered flexible Arg74-motif, involved in substrate binding and product release and likely contributing to the high catalytic rate. The Arg74Gln mutation shifts substrate preference towards RNA. Purine nucleotide binding differs compared to pyrimidines, confirming the plasticity of the active site. The enzyme-product interactions indicate a gradual, stepwise product release. The activity of SmNuc1 towards c-di-GMP in crystal resulted in a distinguished complex with the emerging product 5'-GMP. This enzyme from an opportunistic pathogen relies on specific architecture enabling high performance under broad conditions, attractive for biotechnologies.

Cyclic di-GMP as an antitoxin regulates bacterial genome stability and antibiotic persistence in biofilms.

Biofilms are complex bacterial communities characterized by a high persister prevalence, which contributes to chronic and relapsing infections. Historically, persister formation in biofilms has been linked to constraints imposed by their dense structures. However, we observed an elevated persister frequency accompanying the stage of cell adhesion, marking the onset of biofilm development. Subsequent mechanistic studies uncovered a comparable type of toxin-antitoxin (TA) module (TA-like system) triggered by cell adhesion, which is responsible for this elevation. In this module, the toxin HipH acts as a genotoxic deoxyribonuclease, inducing DNA double strand breaks and genome instability. While the second messenger c-di-GMP functions as the antitoxin, exerting control over HipH expression and activity. The dynamic interplay between c-di-GMP and HipH levels emerges as a crucial determinant governing genome stability and persister generation within biofilms. These findings unveil a unique TA system, where small molecules act as the antitoxin, outlining a biofilm-specific molecular mechanism influencing genome stability and antibiotic persistence, with potential implications for treating biofilm infections.

BpfD Is a c-di-GMP Effector Protein Playing a Key Role for Pellicle Biosynthesis in Shewanella oneidensis.

The aquatic γ-proteobacterium Shewanella oneidensis is able to form two types of biofilms: a floating biofilm at the air-liquid interface (pellicle) and a solid surface-associated biofilm (SSA-biofilm). S. oneidensis possesses the Bpf system, which is orthologous to the Lap system first described in Pseudomonas fluorescens. In the Lap systems, the retention of a large adhesin (LapA) at the cell surface is controlled by LapD, a c-di-GMP effector protein, and LapG, a periplasmic protease targeting LapA. Here, we showed that the Bpf system is mandatory for pellicle biogenesis, but not for SSA-biofilm formation, indicating that the role of Bpf is somewhat different from that of Lap. The BpfD protein was then proved to bind c-di-GMP via its degenerated EAL domain, thus acting as a c-di-GMP effector protein like its counterpart LapD. In accordance with its key role in pellicle formation, BpfD was found to interact with two diguanylate cyclases, PdgA and PdgB, previously identified as involved in pellicle formation. Finally, BpfD was shown to interact with CheY3, the response regulator controlling both chemotaxis and biofilm formation. Altogether, these results indicate that biofilm formation in S. oneidensis is under the control of a large c-di-GMP network.

A comparative genomic and phenotypic study of Vibrio cholerae model strains using hybrid sequencing.

Next-generation sequencing methods have become essential for studying bacterial biology and pathogenesis, often depending on high-quality, closed genomes. In this study, we utilized a hybrid sequencing approach to assemble the genome of C6706, a widely used Vibrio cholerae model strain. We present a manually curated annotation of the genome, enhancing user accessibility by linking each coding sequence to its counterpart in N16961, the first sequenced V. cholerae isolate and a commonly used reference genome. Comparative genomic analysis between V. cholerae C6706 and N16961 uncovered multiple genetic differences in genes associated with key biological functions. To determine whether these genetic variations result in phenotypic differences, we compared several phenotypes relevant to V. cholerae pathogenicity like genetic stability, acid sensitivity, biofilm formation and motility. Notably, V. cholerae N16961 exhibited greater motility and reduced biofilm formation compared to V. cholerae C6706. These phenotypic differences appear to be mediated by variations in quorum sensing and cyclic di-GMP signalling pathways between the strains. This study provides valuable insights into the regulation of biofilm formation and motility in V. cholerae.

The surface interface and swimming motility influence surface-sensing responses in Pseudomonas aeruginosa.

Bacterial biofilms have been implicated in several chronic infections. After initial attachment, a critical first step in biofilm formation is a cell inducing a surface-sensing response. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, two second messengers, cyclic diguanylate monophosphate (c-di-GMP) and cyclic adenosine monophosphate (cAMP), are produced by different surface-sensing mechanisms. However, given the disparate cellular behaviors regulated by these second messengers, how newly attached cells coordinate these pathways remains unclear. Some of the uncertainty relates to studies using different strains, experimental systems, and usually focusing on a single second messenger. In this study, we developed a tricolor reporter system to simultaneously gauge c-di-GMP and cAMP levels in single cells. Using PAO1, we show that c-di-GMP and cAMP are selectively activated in two commonly used experimental systems to study surface sensing. By further examining the conditions that differentiate a c-di-GMP or cAMP response, we demonstrate that an agarose-air interface activates cAMP signaling through type IV pili and the Pil-Chp system. However, a liquid-agarose interface favors the activation of c-di-GMP signaling. This response is dependent on flagellar motility and correlated with higher swimming speed. Collectively, this work indicates that c-di-GMP and cAMP signaling responses are dependent on the surface context.

GefB, a GGDEF domain-containing protein, affects motility and biofilm formation of Vibrio parahaemolyticus and is regulated by quorum sensing regulators.

Vibrio parahaemolyticus (V. parahaemolyticus) stands as the predominant etiological agent responsible for gastroenteritis associated with the consumption of seafood. Cyclic di-guanosine monophosphate (c-di-GMP), a secondary messenger in bacteria, controls multiple bacterial behaviors including pathogenesis, the development of biofilms, and motility. The protein GefB (VPA1478), characterized by the presence of a GGDEF domain, inhibits the swarming motility of V. parahaemolyticus. In this study, we showed that deletion of gefB remarkably reduced cellular c-di-GMP level and biofilm formation by V. parahaemolyticus, but significantly enhanced the swimming and swarming motility. In addition, GefB inhibited the polar and lateral flagellar genes but activated genes associated with exopolysaccharide production of V. parahaemolyticus. The data also demonstrated that vpa1477 and gefB are co-transcribed as a single transcriptional unit, designated as vpa1477-gefB. Transcription of vpa1477-gefB was under the collective regulation of the master quorum sensing (QS) regulators AphA and OpaR, which function at low (LCD) and high cell density (HCD), respectively. AphA positively regulated vpa1477-gefB transcription at LCD, whereas OpaR negatively regulated its transcription at HCD. The findings significantly enhance our comprehension of the metabolism and regulatory mechanisms of c-di-GMP in V. parahaemolyticus.

Small molecule communication of Legionella: the ins and outs of autoinducer and nitric oxide signaling.

SUMMARYLegionella pneumophila is a Gram-negative environmental bacterium, which survives in planktonic form, colonizes biofilms, and infects protozoa. Upon inhalation of Legionella-contaminated aerosols, the opportunistic pathogen replicates within and destroys alveolar macrophages, thereby causing a severe pneumonia termed Legionnaires' disease. Gram-negative bacteria employ low molecular weight organic compounds as well as the inorganic gas nitric oxide (NO) for cell-cell communication. L. pneumophila produces, secretes, and detects the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1, 3-hydroxypentadecane-4-one). LAI-1 is secreted by L. pneumophila in outer membrane vesicles and not only promotes communication among bacteria but also triggers responses from eukaryotic cells. L. pneumophila detects NO through three different receptors, and signaling through the volatile molecule translates into fluctuations of the intracellular second messenger cyclic-di-guanylate monophosphate. The LAI-1 and NO signaling pathways are linked via the pleiotropic transcription factor LvbR. In this review, we summarize current knowledge about inter-bacterial and inter-kingdom signaling through LAI-1 and NO by Legionella species.

VPA0198, a GGDEF domain-containing protein, affects the motility and biofilm formation of Vibrio parahaemolyticus and is regulated by quorum sensing associated regulators.

Cyclic di-GMP (c-di-GMP), a ubiquitous secondary messenger in bacteria, affects multiple bacterial behaviors including motility and biofilm formation. c-di-GMP is synthesized by diguanylate cyclase harboring a GGDEF domain and degraded by phosphodiesterase harboring an either EAL or HD-GYP domain. Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis, harbors more than 60 genes involved in c-di-GMP metabolism. However, roles of most of these genes including vpa0198, which encodes a GGDEF-domain containing protein, are still completely unknown. AphA and OpaR are the master quorum sensing (QS) regulators operating at low (LCD) and high cell density (HCD), respectively. QsvR integrates into QS to control gene expression via direct regulation of AphA and OpaR. In this study, we showed that deletion of vpa0198 remarkably reduced c-di-GMP production and biofilm formation, whereas promoted the swimming motility of V. parahaemolyticus. Overexpression of VPA0198 in the vpa0198 mutant strain significantly reduced the swimming and swarming motility and enhanced the biofilm formation ability of V. parahaemolyticus. In addition, transcription of vpa0198 was under the collective regulation of AphA, OpaR and QsvR. AphA activated the transcription of vpa0198 at LCD, whereas QsvR and OpaR coordinately and directly repressed vpa0198 transcription at HCD, thereby leading to a cell density-dependent expression of vpa0198. Therefore, this work expanded the knowledge of synthetic regulatory mechanism of c-di-GMP in V. parahaemolyticus.

BrfA functions as a bacterial enhancer-binding protein to regulate functional amyloid Fap-dependent biofilm formation in Pseudomonas fluorescens by sensing cyclic diguanosine monophosphate.

The functional amyloid of Pseudomonas (Fap) is essential for the formation of macrocolony biofilms, pellicles, and solid surface-associated (SSA) biofilms of Pseudomonas fluorescens PF07, an isolate from refrigerated marine fish. However, limited information on the expression regulation of fap genes is available. Herein, we found that a novel bacterial enhancer-binding protein (bEBP), BrfA, regulated Fap-dependent biofilm formation by directly sensing cyclic diguanosine monophosphate (c-di-GMP). Our in vivo data showed that the REC domain deletion of BrfA promoted fap gene expression and biofilm formation, and c-di-GMP positively regulated the transcription of fapA in a BrfA-dependent manner. In in vitro experiments, we found that the ATPase activity of BrfA was inhibited by the REC domain and was activated by c-di-GMP. BrfA and the sigma factor RpoN bound to the upstream region of fapA, and the binding ability of BrfA was not affected by either deletion of the REC domain or c-di-GMP. BrfA specifically bound to the three enhancer sites upstream of the fapA promoter, which contain the consensus sequence CA-(N4)-TGA(A/T)ACACC. In vivo experiments using a lacZ fusion reporter indicated that all three BrfA enhancer sites were essential for the activation of fapA transcription. Overall, these findings reveal that BrfA is a new type of c-di-GMP-responsive transcription factor that directly controls the transcription of Fap biosynthesis genes in P. fluorescens. Fap functional amyloids and BrfA-type transcription factors are widespread in Pseudomonas species. The novel insights into the c-di-GMP- and BrfA-dependent expression regulation of fap provided by this work will contribute to the development of antibiofilm strategies.

Comparative Genomic and Functional Analysis of c-di-GMP Metabolism and Regulatory Proteins in Bacillus velezensis LQ-3.

Bacillus velezensis is a promising candidate for biocontrol applications. A common second messenger molecule, bis-(3,5)-cyclic-dimeric-guanosine monophosphate (c-di-GMP), has the ability to regulate a range of physiological functions that impact the effectiveness of biocontrol. However, the status of the c-di-GMP signaling pathway in biocontrol strain LQ-3 remains unknown. Strain LQ-3, which was isolated from wheat rhizosphere soil, has shown effective control of wheat sharp eyespot and has been identified as B. velezensis through whole-genome sequencing analyses. In this study, we investigated the intracellular c-di-GMP signaling pathway of LQ-3 and further performed a comparative genomic analysis of LQ-3 and 29 other B. velezensis strains. The results revealed the presence of four proteins containing the GGDEF domain, which is the conserved domain for c-di-GMP synthesis enzymes. Additionally, two proteins were identified with the EAL domain, which represents the conserved domain for c-di-GMP degradation enzymes. Furthermore, one protein was found to possess a PilZ domain, indicative of the conserved domain for c-di-GMP receptors in LQ-3. These proteins are called DgcK, DgcP, YybT, YdaK, PdeH, YkuI, and DgrA, respectively; they are distributed in a similar manner across the strains and belong to the signal transduction family. We selected five genes from the aforementioned seven genes for further study, excluding YybT and DgrA. They all play a role in regulating the motility, biofilm formation, and colonization of LQ-3. This study reveals the c-di-GMP signaling pathway associated with biocontrol features in B. velezensis LQ-3, providing guidance for the prevention and control of wheat sharp eyespot by LQ-3.

c-di-GMP and AHL signals-triggered chemical communication under electrical signaling disruption restores Geobacter sulfurreducens biofilm formation.

Electrogenic biofilms, which have attracted considerable attention in simultaneous wastewater treatment and energy recovery in bioelectrochemical systems, are regulated by chemical communication and potassium channel-mediated electrical signaling. However, how these two communication pathways interact with each other has not been thoroughly investigated. This study first explored the roles of chemical communication, including intracellular bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) and extracellular N-acyl-homoserine lactone (AHL)-mediated quorum sensing, in electrogenic biofilm formation through an integrated analysis of transcriptomics and metabolomics. Electrical signaling disruption inhibited the formation and electroactivity of Geobacter sulfurreducens biofilm, which was mainly ascribed to the reduction in biofilm viability and extracellular protein/polysaccharide ratio. The upregulation of expression levels of genes encoding c-di-GMP and AHL synthesis by transcriptomic analysis, and the increased secretion of N-butanoyl-L-homoserine lactone by metabolomic analysis confirmed the enhancement of chemical communication under electrical signaling disruption, thus indicating a compensatory mechanism among different signaling pathways. Furthermore, protein-protein interaction network showed the convergence of different signaling pathways, with c-di-GMP-related genes acting as central bridges. This study highlights the interaction of different signaling pathways, especially the resilience of c-di-GMP signaling to adverse external stresses, thereby laying the foundation for facilitating electrogenic biofilm formation under adverse conditions in practical applications.

Divergent molecular strategies drive evolutionary adaptation to competitive fitness in biofilm formation.

Biofilm is a group of heterogeneously structured and densely packed bacteria with limited access to nutrients and oxygen. These intrinsic features can allow a mono-species biofilm to diversify into polymorphic subpopulations, determining the overall community's adaptive capability to changing ecological niches. However, the specific biological functions underlying biofilm diversification and fitness adaptation are poorly demonstrated. Here, we launched and monitored the experimental evolution of Pseudomonas aeruginosa biofilms, finding that two divergent molecular trajectories were adopted for adaptation to higher competitive fitness in biofilm formation: one involved hijacking bacteriophage superinfection to aggressively inhibit kin competitors, whereas the other induced a subtle change in cyclic dimeric guanosine monophosphate signaling to gain a positional advantage via enhanced early biofilm adhesion. Bioinformatics analyses implicated that similar evolutionary strategies were prevalent among clinical P. aeruginosa strains, indicative of parallelism between natural and experimental evolution. Divergence in the molecular bases illustrated the adaptive values of genomic plasticity for gaining competitive fitness in biofilm formation. Finally, we demonstrated that these fitness-adaptive mutations reduced bacterial virulence. Our findings revealed how the mutations intrinsically generated from the biofilm environment influence the evolution of P. aeruginosa.

The inhibition mechanism of co-cultured probiotics on biofilm formation of Klebsiella pneumoniae.

AIMS: Klebsiella pneumoniae, an important opportunistic pathogen of nosocomial inflection, is known for its ability to form biofilm. The purpose of the current study is to assess how co- or mono-cultured probiotics affect K. pneumoniae's ability to produce biofilms and investigate the potential mechanisms by using a polyester nonwoven chemostat and a Caco-2 cell line. METHODS AND RESULTS: Compared with pure cultures of Lactobacillus rhamnosus and Lactobacillus sake, the formation of K. pneumoniae biofilm was remarkably inhibited by the mixture of L. rhamnosus, L. sake, and Bacillus subtilis at a ratio of 5:5:1 by means of qPCR and FISH assays. In addition, Lactobacillus in combination with B. subtilis could considerably reduce the adherence of K. pneumoniae to Caco-2 cells by using inhibition, competition, and displacement assays. According to the RT-PCR assay, the adsorption of K. pneumoniae to Caco-2 cells was effectively inhibited by the co-cultured probiotics, leading to significant reduction in the expression of proinflammatory cytokines induced by K. pneumoniae. Furthermore, the HPLC and RT-PCR analyses showed that the co-cultured probiotics were able to successfully prevent the expression of the biofilm-related genes of K. pneumoniae by secreting plenty of organic acids as well as the second signal molecule (c-di-GMP), resulting in inhibition on biofilm formation. CONCLUSION: Co-culture of L. sake, L. rhamnosus, and B. subtilis at a ratio of 5:5:1 could exert an antagonistic effect on the colonization of pathogenic K. pneumoniae by down-regulating the expression of biofilm-related genes. At the same time, the co-cultured probiotics could effectively inhibit the adhesion of K. pneumoniae to Caco-2 cells and block the expression of proinflammatory cytokines induced by K. pneumoniae.

The global regulation of c-di-GMP and cAMP in bacteria.

Nucleotide second messengers are highly versatile signaling molecules that regulate a variety of key biological processes in bacteria. The best-studied examples are cyclic AMP (cAMP) and bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), which both act as global regulators. Global regulatory frameworks of c-di-GMP and cAMP in bacteria show several parallels but also significant variances. In this review, we illustrate the global regulatory models of the two nucleotide second messengers, compare the different regulatory frameworks between c-di-GMP and cAMP, and discuss the mechanisms and physiological significance of cross-regulation between c-di-GMP and cAMP. c-di-GMP responds to numerous signals dependent on a great number of metabolic enzymes, and it regulates various signal transduction pathways through its huge number of effectors with varying activities. In contrast, due to the limited quantity, the cAMP metabolic enzymes and its major effector are regulated at different levels by diverse signals. cAMP performs its global regulatory function primarily by controlling the transcription of a large number of genes via cAMP receptor protein (CRP) in most bacteria. This review can help us understand how bacteria use the two typical nucleotide second messengers to effectively coordinate and integrate various physiological processes, providing theoretical guidelines for future research.

Upscaling and Risk Evaluation of the Synthesis of the 3,5-Diamino-1H-Pyrazole, Disperazol.

This paper presents the work performed to transition a lab-scale synthesis (1 g) to a large-scale (400 g) synthesis of the 3-5-diamino-1H-Pyrazole Disperazol, a new pharmaceutical for treatment of antibiotic-resistant Pseudomonas aeruginosa biofilm infections. The potentially hazardous diazotisation step in the lab-scale synthesis was transformed to a safe and easy-to-handle flow chemistry step. Additionally, the paper presents an OSHA-recommended safety assessment of active compound E, as performed by Fauske and Associates, LLC, Burr Ridge, IL, USA.

Phylogenetic distribution and characterization of conserved C-di-GMP metabolizing proteins in filamentous cyanobacterium Arthrospira.

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger in bacteria that regulates multiple biological functions, including biofilm formation, virulence, and intercellular communication. However, c-di-GMP signaling is virtually unknown in economically important filamentous cyanobacteria, Arthrospira. In this study, we predicted 31 genes encoding GGDEF-domain proteins from A. platensis NIES39 as potential diguanylate cyclases (DGCs). Phylogenetic distribution analysis showed five genes (RS09460, RS04865, RS26155, M01840, and E02220) with highly conserved distribution across 25 Arthrospira strains. Adc1 encoded by RS09460 was further characterized as a typical DGC. By establishing the genetic transformation system of Arthrospira, we demonstrated that the overexpression of Adc1 promoted the production of extracellular polymeric substances (EPS), which in turn caused the aggregation of filaments. We also confirmed that RS04865 and RS26155 may encode active DGCs, while enzymatic activity assays showed that proteins encoded by M01840 and E02220 have phosphodiesterase (PDE) activity. Meta-analysis revealed that the expression profiles of RS09460 and RS04865 were unaffected under 31 conditions, suggesting that they may function as conserved genes in maintaining the basal level of c-di-GMP in Arthrospira. In summary, this report will provide the basis for further studies of c-di-GMP signal in Arthrospira.

NagZ modulates the virulence of E. cloacae by acting through the gene of unknown function, ECL_03795.

ß-N-acetylglucosaminidase (NagZ), a cytosolic glucosaminidase, plays a pivotal role in peptidoglycan recycling. Previous research demonstrated that NagZ knockout significantly eradicated AmpC-dependent ß-lactam resistance in Enterobacter cloacae. However, NagZ's role in the virulence of E. cloacae remains unclear. Our study, incorporating data on mouse and Galleria mellonella larval mortality rates, inflammation markers, and histopathological examinations, revealed a substantial reduction in the virulence of E. cloacae following NagZ knockout. Transcriptome sequencing uncovered differential gene expression between NagZ knockout and wild-type strains, particularly in nucleotide metabolism pathways. Further investigation demonstrated that NagZ deletion led to a significant increase in cyclic diguanosine monophosphate (c-di-GMP) levels. Additionally, transcriptome sequencing and RT-qPCR confirmed significant differences in the expression of ECL_03795, a gene with an unknown function but speculated to be involved in c-di-GMP metabolism due to its EAL domain known for phosphodiesterase activity. Interestingly, in ECL_03795 knockout strains, a notable reduction in the virulence was observed, and virulence was rescued upon complementation with ECL_03795. Consequently, our study suggests that NagZ's function on virulence is partially mediated through the ECL_03795→c-di-GMP pathway, providing insight into the development of novel therapies and strongly supporting the interest in creating highly efficient NagZ inhibitors.

IcmF2 of the type VI secretion system 2 plays a role in biofilm formation of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses two distinct type VI secretion systems (T6SS), namely T6SS1 and T6SS2. T6SS1 is predominantly responsible for adhesion to Caco-2 and HeLa cells and for the antibacterial activity of V. parahaemolyticus, while T6SS2 mainly contributes to HeLa cell adhesion. However, it remains unclear whether the T6SS systems have other physiological roles in V. parahaemolyticus. In this study, we demonstrated that the deletion of icmF2, a structural gene of T6SS2, reduced the biofilm formation capacity of V. parahaemolyticus under low salt conditions, which was also influenced by the incubation time. Nonetheless, the deletion of icmF2 did not affect the biofilm formation capacity in marine-like growth conditions, nor did it impact the flagella-driven swimming and swarming motility of V. parahaemolyticus. IcmF2 was found to promote the production of the main components of the biofilm matrix, including extracellular DNA (eDNA) and extracellular proteins, and cyclic di-GMP (c-di-GMP) in V. parahaemolyticus. Additionally, IcmF2 positively influenced the transcription of cpsA, mfpA, and several genes involved in c-di-GMP metabolism, including scrJ, scrL, vopY, tpdA, gefA, and scrG. Conversely, the transcription of scrA was negatively impacted by IcmF2. Therefore, IcmF2-dependent biofilm formation was mediated through its effects on the production of eDNA, extracellular proteins, and c-di-GMP, as well as its impact on the transcription of cpsA, mfpA, and genes associated with c-di-GMP metabolism. This study confirmed new physiological roles for IcmF2 in promoting biofilm formation and c-di-GMP production in V. parahaemolyticus.

Cyclic diguanylate differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in Clostridioides difficile.

Clostridioides difficile infection (CDI) caused by toxigenic C. difficile is the leading cause of antimicrobial and healthcare-associated diarrhea. The pathogenicity of C. difficile relies on the synergistic effect of multiple virulence factors, including spores, flagella, type IV pili (T4P), toxins, and biofilm. Spores enable survival and transmission of C. difficile, while adhesion factors such as flagella and T4P allow C. difficile to colonize and persist in the host intestine. Subsequently, C. difficile produces the toxins TcdA and TcdB, causing pseudomembranous colitis and other C. difficile-associated diseases; adhesion factors bind to the extracellular matrix to form biofilm, allowing C. difficile to evade drug and immune system attack and cause recurrent infection. Cyclic diguanylate (c-di-GMP) is a near-ubiquitous second messenger that extensively regulates morphology, the expression of virulence factors, and multiple physiological processes in C. difficile. In this review, we summarize current knowledge of how c-di-GMP differentially regulates the expression of virulence factors and pathogenesis-related phenotypes in C. difficile. We highlight that C. difficile spore formation and expression of toxin and flagella genes are inhibited at high intracellular levels of c-di-GMP, while T4P biosynthesis, cell aggregation, and biofilm formation are induced. Recent studies have enhanced our understanding of the c-di-GMP signaling networks in C. difficile and provided insights for the development of c-di-GMP-dependent strategies against CDI.

Auxin-mediated regulation of susceptibility to toxic metabolites, c-di-GMP levels, and phage infection in the rhizobacterium Serratia plymuthica.

The communication between plants and their microbiota is highly dynamic and involves a complex network of signal molecules. Among them, the auxin indole-3-acetic acid (IAA) is a critical phytohormone that not only regulates plant growth and development, but is emerging as an important inter- and intra-kingdom signal that modulates many bacterial processes that are important during interaction with their plant hosts. However, the corresponding signaling cascades remain largely unknown. Here, we advance our understanding of the largely unknown mechanisms by which IAA carries out its regulatory functions in plant-associated bacteria. We showed that IAA caused important changes in the global transcriptome of the rhizobacterium Serratia plymuthica and multidisciplinary approaches revealed that IAA sensing interferes with the signaling mediated by other pivotal plant-derived signals such as amino acids and 4-hydroxybenzoic acid. Exposure to IAA caused large alterations in the transcript levels of genes involved in amino acid metabolism, resulting in significant metabolic alterations. IAA treatment also increased resistance to toxic aromatic compounds through the induction of the AaeXAB pump, which also confers resistance to IAA. Furthermore, IAA promoted motility and severely inhibited biofilm formation; phenotypes that were associated with decreased c-di-GMP levels and capsule production. IAA increased capsule gene expression and enhanced bacterial sensitivity to a capsule-dependent phage. Additionally, IAA induced the expression of several genes involved in antibiotic resistance and led to changes in the susceptibility and responses to antibiotics with different mechanisms of action. Collectively, our study illustrates the complexity of IAA-mediated signaling in plant-associated bacteria. IMPORTANCE: Signal sensing plays an important role in bacterial adaptation to ecological niches and hosts. This communication appears to be particularly important in plant-associated bacteria since they possess a large number of signal transduction systems that respond to a wide diversity of chemical, physical, and biological stimuli. IAA is emerging as a key inter- and intra-kingdom signal molecule that regulates a variety of bacterial processes. However, despite the extensive knowledge of the IAA-mediated regulatory mechanisms in plants, IAA signaling in bacteria remains largely unknown. Here, we provide insight into the diversity of mechanisms by which IAA regulates primary and secondary metabolism, biofilm formation, motility, antibiotic susceptibility, and phage sensitivity in a biocontrol rhizobacterium. This work has important implications for our understanding of bacterial ecology in plant environments and for the biotechnological and clinical applications of IAA, as well as related molecules.