RESUMO
Vibrio (Aliivibrio) fischeri's initial rise to fame derived from its alluring production of blue-green light. Subsequent studies to probe the mechanisms underlying this bioluminescence helped the field discover the phenomenon now known as quorum sensing. Orthologs of quorum-sensing regulators (i.e., LuxR and LuxI) originally identified in V. fischeri were subsequently uncovered in a plethora of bacterial species, and analogous pathways were found in yet others. Over the past three decades, the study of this microbe has greatly expanded to probe the unique role of V. fischeri as the exclusive symbiont of the light organ of the Hawaiian bobtail squid, Euprymna scolopes. Buoyed by this optically amenable host and by persistent and insightful researchers who have applied novel and cross-disciplinary approaches, V. fischeri has developed into a robust model for microbe-host associations. It has contributed to our understanding of how bacteria experience and respond to specific, often fluxing environmental conditions and the mechanisms by which bacteria impact the development of their host. It has also deepened our understanding of numerous microbial processes such as motility and chemotaxis, biofilm formation and dispersal, and bacterial competition, and of the relevance of specific bacterial genes in the context of colonizing an animal host. Parallels in these processes between this symbiont and bacteria studied as pathogens are readily apparent, demonstrating functional conservation across diverse associations and permitting a reinterpretation of "pathogenesis." Collectively, these advances built a foundation for microbiome studies and have positioned V. fischeri to continue to expand the frontiers of our understanding of the microbial world inside animals.
Assuntos
Aliivibrio fischeri , Decapodiformes , Percepção de Quorum , Aliivibrio fischeri/genética , Aliivibrio fischeri/fisiologia , Animais , Decapodiformes/microbiologia , Simbiose , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Interações entre Hospedeiro e MicrorganismosRESUMO
Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.
Assuntos
Aliivibrio fischeri , Proteínas de Bactérias , Decapodiformes , Fosfatos , Simbiose , Aliivibrio fischeri/genética , Aliivibrio fischeri/fisiologia , Aliivibrio fischeri/enzimologia , Decapodiformes/microbiologia , Animais , Fosfatos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Desoxirribonucleases/metabolismo , Desoxirribonucleases/genéticaRESUMO
Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.
Assuntos
Aliivibrio fischeri , Biofilmes , Animais , Humanos , Aliivibrio fischeri/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Adesinas Bacterianas , Decapodiformes/microbiologia , Simbiose , PolissacarídeosRESUMO
The ninth American Society for Microbiology Conference on Biofilms was convened in-person on 13-17 November 2022 in Charlotte, NC. As the first of these conferences since prior to the start of the COVID-19 pandemic, the energy among the participants of the conference was clear, and the meeting was a tremendous success. The mixture of >330 oral and poster presentations resoundingly embodied the vitality of biofilm research across a wide range of topics and multiple scientific disciplines. Special activities, including a pre-conference symposium for early career researchers, further enhanced the attendee experience. As a general theme, the conference was deliberately structured to provide high levels of participation and engagement among early career scientists.
Assuntos
Pandemias , Sociedades Científicas , Humanos , Estados Unidos , BiofilmesRESUMO
The marine bacterium Vibrio fischeri initiates symbiotic colonization of its squid host, Euprymna scolopes, by forming and dispersing from a biofilm dependent on the symbiosis polysaccharide locus (syp). Historically, genetic manipulation of V. fischeri was needed to visualize syp-dependent biofilm formation in vitro, but recently, we discovered that the combination of two small molecules, para-aminobenzoic acid (pABA) and calcium, was sufficient to induce wild-type strain ES114 to form biofilms. Here, we determined that these syp-dependent biofilms were reliant on the positive syp regulator RscS, since the loss of this sensor kinase abrogated biofilm formation and syp transcription. These results were of particular note because loss of RscS, a key colonization factor, exerts little to no effect on biofilm formation under other genetic and medium conditions. The biofilm defect could be complemented by wild-type RscS and by an RscS chimera that contains the N-terminal domains of RscS fused to the C-terminal HPT domain of SypF, the downstream sensor kinase. It could not be complemented by derivatives that lacked the periplasmic sensory domain or contained a mutation in the conserved site of phosphorylation, H412, suggesting that these cues promote signaling through RscS. Lastly, pABA and/or calcium was able to induce biofilm formation when rscS was introduced into a heterologous system. Taken together, these data suggest that RscS is responsible for recognizing pABA and calcium, or downstream consequences of those cues, to induce biofilm formation. This study thus provides insight into signals and regulators that promote biofilm formation by V. fischeri. IMPORTANCE Bacterial biofilms are common in a variety of environments. Infectious biofilms formed in the human body are notoriously hard to treat due to a biofilm's intrinsic resistance to antibiotics. Bacteria must integrate signals from the environment to build and sustain a biofilm and often use sensor kinases that sense an external signal, which triggers a signaling cascade to elicit a response. However, identifying the signals that kinases sense remains a challenging area of investigation. Here, we determine that a hybrid sensor kinase, RscS, is crucial for Vibrio fischeri to recognize para-aminobenzoic acid and calcium as cues to induce biofilm formation. This study thus advances our understanding of the signal transduction pathways leading to biofilm formation.
Assuntos
Ácido 4-Aminobenzoico , Cálcio , Humanos , Ácido 4-Aminobenzoico/metabolismo , Cálcio/metabolismo , Aliivibrio fischeri/genética , Proteínas de Bactérias/genética , Biofilmes , Fosfotransferases/metabolismoRESUMO
Anti-sigma factor antagonists SpoIIAA and RsbV from Bacillus subtilis are the archetypes for single-domain STAS proteins in bacteria. The structures and mechanisms of these proteins along with their cognate anti-sigma factors have been well studied. SpoIIAA and RsbV utilize a partner-switching mechanism to regulate gene expression through protein-protein interactions to control the activity of their downstream anti-sigma factor partners. The Vibrio fischeri STAS domain protein SypA is also proposed to employ a partner-switching mechanism with its partner SypE, a serine kinase/phosphatase that controls SypA's phosphorylation state. However, this regulation appears opposite to the canonical pathway, with SypA being the more downstream component rather than SypE. Here we explore the commonalities and differences between SypA and the canonical single-domain STAS proteins SpoIIAA and RsbV. We use a combination of AlphaFold 2 structure predictions and computational modeling to investigate the SypA-SypE binding interface. We then test a subset of our predictions in V.fischeri by generating and expressing SypA variants. Our findings suggest that, while SypA shares many sequence and structural traits with anti-sigma factor antagonist STAS domain proteins, there are significant differences that may account for SypA's distinct regulatory output.
RESUMO
Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates after 72 h. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research. IMPORTANCE From soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between V. fischeri and its squid host, Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.
Assuntos
Aliivibrio fischeri , Decapodiformes , Animais , Aliivibrio fischeri/genética , Decapodiformes/microbiologia , Simbiose , Fosfatos , BiofilmesRESUMO
During colonization of the Hawaiian bobtail squid (Euprymna scolopes), Vibrio fischeri bacteria undergo a lifestyle transition from a planktonic motile state in the environment to a biofilm state in host mucus. Cyclic diguanylate (c-di-GMP) is a cytoplasmic signaling molecule that is important for regulating motility-biofilm transitions in many bacterial species. V. fischeri encodes 50 proteins predicted to synthesize and/or degrade c-di-GMP, but a role for c-di-GMP regulation during host colonization has not been investigated. We examined strains exhibiting either low or high levels of c-di-GMP during squid colonization and found that while a low-c-di-GMP strain had no colonization defect, a high c-di-GMP strain was severely impaired. Expression of a heterologous c-di-GMP phosphodiesterase restored colonization, demonstrating that the effect is due to high c-di-GMP levels. In the constitutive high-c-di-GMP state, colonizing V. fischeri exhibited reduced motility, altered biofilm aggregate morphology, and a regulatory interaction where transcription of one polysaccharide locus is inhibited by the presence of the other polysaccharide. Our results highlight the importance of proper c-di-GMP regulation during beneficial animal colonization, illustrate multiple pathways regulated by c-di-GMP in the host, and uncover an interplay of multiple exopolysaccharide systems in host-associated aggregates. IMPORTANCE There is substantial interest in studying cyclic diguanylate (c-di-GMP) in pathogenic and environmental bacteria, which has led to an accepted paradigm in which high c-di-GMP levels promote biofilm formation and reduce motility. However, considerably less focus has been placed on understanding how this compound contributes to beneficial colonization. Using the Vibrio fischeri-Hawaiian bobtail squid study system, we took advantage of recent genetic advances in the bacterium to modulate c-di-GMP levels and measure colonization and track c-di-GMP phenotypes in a symbiotic interaction. Studies in the animal host revealed a c-di-GMP-dependent genetic interaction between two distinct biofilm polysaccharides, Syp and cellulose, that was not evident in culture-based studies: elevated c-di-GMP altered the composition and abundance of the in vivo biofilm by decreasing syp transcription due to increased cellulose synthesis. This study reveals important parallels between pathogenic and beneficial colonization and additionally identifies c-di-GMP-dependent regulation that occurs specifically in the squid host.
Assuntos
Aliivibrio fischeri , GMP Cíclico , Aliivibrio fischeri/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Celulose/metabolismo , GMP Cíclico/metabolismo , Decapodiformes/microbiologia , Regulação Bacteriana da Expressão Gênica , SimbioseRESUMO
The symbiont Vibrio fischeri uses motility to colonize its host. In numerous bacterial species, motility is negatively controlled by cyclic-di-GMP (c-di-GMP), which is produced by diguanylate cyclases (DGCs) with GGDEF domains and degraded by phosphodiesterases with either EAL or HD-GYP domains. To begin to decode the functions of the 50 Vibrio fischeri genes with GGDEF, EAL, and/or HD-GYP domains, we deleted each gene and assessed each mutant's migration through tryptone broth salt (TBS) soft agar medium containing or lacking magnesium (Mg) and calcium (Ca), which are known to influence V. fischeri motility. We identified 6, 13, and 16 mutants with altered migration in TBS-Mg, TBS, and TBS-Ca soft agar, respectively, a result that underscores the importance of medium conditions in assessing gene function. A biosensor-based assay revealed that Mg and Ca affected c-di-GMP levels negatively and positively, respectively; the severe decrease in c-di-GMP caused by Mg addition correlates with its strong positive impact on bacterial migration. A mutant defective for VF_0494, a homolog of V. cholerae rocS, exhibited a severe defect in migration across all conditions. Motility of a VF_1603 VF_2480 double mutant was also severely defective and could be restored by expression of "c-di-GMP-blind" alleles of master flagellar regulator flrA. Together, this work sheds light on the genes and conditions that influence c-di-GMP-mediated control over motility in V. fischeri and provides a foundation for (i) assessing roles of putative c-di-GMP-binding proteins, (ii) evaluating other c-di-GMP-dependent phenotypes in V. fischeri, (iii) uncovering potential redundancy, and (iv) deciphering signal transduction mechanisms. IMPORTANCE Critical bacterial processes, including motility, are influenced by c-di-GMP, which is controlled by environment-responsive synthetic and degradative enzymes. Because bacteria such as Vibrio fischeri use motility to colonize their hosts, understanding the roles of c-di-GMP-modulating enzymes in controlling motility has the potential to inform on microbe-host interactions. We leveraged recent advances in genetic manipulation to generate 50 mutants defective for putative c-di-GMP synthetic and degradative enzymes. We then assessed the consequences on motility, manipulating levels of magnesium and calcium, which inversely influenced motility and levels of c-di-GMP. Distinct subsets of the 50 genes were required under the different conditions. Our data thus provide needed insight into the functions of these enzymes and environmental factors that influence them.
Assuntos
Proteínas de Escherichia coli , Vibrio cholerae , Ágar , Aliivibrio fischeri/genética , Aliivibrio fischeri/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , Cálcio/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Magnésio/metabolismo , Vibrio cholerae/metabolismoRESUMO
Colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation, motility, and bioluminescence. Previous work has demonstrated an inhibitory role for the small RNA (sRNA) Qrr1 in quorum-induced bioluminescence of V. fischeri, but the contribution of the corresponding sRNA chaperone, Hfq, was not examined. We thus hypothesized that V. fischeri Hfq similarly functions to inhibit bacterial bioluminescence as well as regulate other key steps of symbiosis, including bacterial biofilm formation and motility. Surprisingly, deletion of hfq increased luminescence of V. fischeri beyond what was observed for the loss of qrr1 sRNA. Epistasis experiments revealed that, while Hfq contributes to the Qrr1-dependent regulation of light production, it also functions independently of Qrr1 and its downstream target, LitR. This Hfq-dependent, Qrr1-independent regulation of bioluminescence is also independent of the major repressor of light production in V. fischeri, ArcA. We further determined that Hfq is required for full motility of V. fischeri in a mechanism that partially depends on the Qrr1/LitR regulators. Finally, Hfq also appears to function in the control of biofilm formation: loss of Hfq delayed the timing and diminished the extent of wrinkled colony development, but did not eliminate the production of SYP-polysaccharide-dependent cohesive colonies. Furthermore, loss of Hfq enhanced production of cellulose and resulted in increased Congo red binding. Together, these findings point to Hfq as an important regulator of multiple phenotypes relevant to symbiosis between V. fischeri and its squid host.
Assuntos
Aliivibrio fischeri/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Interferente Pequeno/metabolismo , Aliivibrio fischeri/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Celulose/metabolismo , Regulação Bacteriana da Expressão Gênica , Luminescência , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , FenótipoRESUMO
The marine bacterium Vibrio fischeri colonizes its host, the Hawaiian bobtail squid, in a manner requiring both bacterial biofilm formation and motility. The decision to switch between sessile and motile states is often triggered by environmental signals and regulated by the widespread signaling molecule c-di-GMP. Calcium is an environmental signal previously shown to affect both biofilm formation and motility by V. fischeri. In this study, we investigated the link between calcium and c-di-GMP, determining that calcium increases intracellular c-di-GMP dependent on a specific diguanylate cyclase, calcium-sensing protein A (CasA). CasA is activated by calcium, dependent on residues in an N-terminal sensory domain, and synthesizes c-di-GMP through an enzymatic C-terminal domain. CasA is responsible for calcium-dependent inhibition of motility and activation of cellulose-dependent biofilm formation. Calcium regulates cellulose biofilms at the level of transcription, which also requires the transcription factor VpsR. Finally, the Vibrio cholerae CasA homolog, CdgK, is unable to complement CasA and may be inhibited by calcium. Collectively, these results identify CasA as a calcium-responsive regulator, linking an external signal to internal decisions governing behavior, and shed light on divergence between Vibrio spp. IMPORTANCE Biofilm formation and motility are often critical behaviors for bacteria to colonize a host organism. Vibrio fischeri is the exclusive colonizer of its host's symbiotic organ and requires both biofilm formation and motility to initiate successful colonization, providing a relatively simple model to explore complex behaviors. In this study, we determined how the environmental signal calcium alters bacterial behavior through production of the signaling molecule c-di-GMP. Calcium activates the diguanylate cyclase CasA to synthesize c-di-GMP, resulting in inhibition of motility and activation of cellulose production. These activities depend on residues in CasA's N-terminal sensory domain and C-terminal enzymatic domain. These findings thus identify calcium as a signal recognized by a specific diguanylate cyclase to control key bacterial phenotypes. Of note, CasA activity is seemingly inverse to that of the homologous V. cholerae protein, CdgK, providing insight into evolutionary divergence between closely related species.
Assuntos
Aliivibrio fischeri/metabolismo , Biofilmes , Cálcio/metabolismo , Celulose/metabolismo , Fósforo-Oxigênio Liases/metabolismo , Aliivibrio fischeri/enzimologia , Proteínas de Bactérias/metabolismo , Sinalização do Cálcio , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Havaí , Fatores de Transcrição/metabolismo , Vibrio cholerae/genéticaRESUMO
The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP). In vitro, however, wild-type strain ES114 fails to form SYP-dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study this phenomenon. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and yeast extract; supplementation with calcium is required to induce biofilm formation by a binK mutant. Here, through our discovery that yeast extract inhibits biofilm formation, we uncover signals and underlying mechanisms that control V. fischeri biofilm formation. In contrast to its inability to form a biofilm on unsupplemented LBS, a binK mutant formed cohesive, SYP-dependent colony biofilms on tTBS, modified LBS that lacks yeast extract. Moreover, wild-type strain ES114 became proficient to form cohesive, SYP-dependent biofilms when grown in tTBS supplemented with both calcium and the vitamin para-aminobenzoic acid (pABA); neither molecule alone was sufficient, indicating that this phenotype relies on coordinating two cues. pABA/calcium supplementation also inhibited bacterial motility. Consistent with these phenotypes, cells grown in tTBS with pABA/calcium were enriched in transcripts for biofilm-related genes and predicted diguanylate cyclases, which produce the second messenger cyclic-di-GMP (c-di-GMP). They also exhibited elevated levels of c-di-GMP, which was required for the observed phenotypes, as phosphodiesterase overproduction abrogated biofilm formation and partially rescued motility. This work thus provides insight into conditions, signals, and processes that promote biofilm formation by V. fischeri. IMPORTANCE Bacteria integrate environmental signals to regulate gene expression and protein production to adapt to their surroundings. One such behavioral adaptation is the formation of a biofilm, which can promote adherence and colonization and provide protection against antimicrobials. Identifying signals that trigger biofilm formation and the underlying mechanism(s) of action remain important and challenging areas of investigation. Here, we determined that yeast extract, commonly used for growth of bacteria in laboratory culture, inhibits biofilm formation by Vibrio fischeri, a model bacterium used for investigating host-relevant biofilm formation. Omitting yeast extract from the growth medium led to the identification of an unusual signal, the vitamin para-aminobenzoic acid (pABA), that when added together with calcium could induce biofilm formation. pABA increased the concentrations of the second messenger, c-di-GMP, which was necessary but not sufficient to induce biofilm formation. This work thus advances our understanding of signals and signal integration controlling bacterial biofilm formation.
Assuntos
Ácido 4-Aminobenzoico/metabolismo , Aliivibrio fischeri/metabolismo , Biofilmes , Cálcio/metabolismo , GMP Cíclico/análogos & derivados , Polissacarídeos Bacterianos/metabolismo , Aliivibrio fischeri/genética , Aliivibrio fischeri/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Decapodiformes/microbiologia , Decapodiformes/fisiologia , Regulação Bacteriana da Expressão Gênica , SimbioseRESUMO
Bacterial motility is critical for symbiotic colonization by Vibrio fischeri of its host, the squid Euprymna scolopes, facilitating movement from surface biofilms to spaces deep inside the symbiotic organ. While colonization has been studied traditionally using strain ES114, others, including KB2B1, can outcompete ES114 for colonization for a variety of reasons, including superior biofilm formation. We report here that KB2B1 also exhibits an unusual pattern of migration through a soft agar medium: whereas ES114 migrates rapidly and steadily, KB2B1 migrates slowly and then ceases migration. To better understand this phenomenon, we isolated and sequenced five motile KB2B1 suppressor mutants. One harbored a mutation in the gene for the cAMP receptor protein (crp); because this strain also exhibited a growth defect, it was not characterized further. Two other suppressors contained mutations in the quorum sensing pathway that controls bacterial bioluminescence in response to cell density, and two had mutations in the diguanylate cyclase (DGC) gene VF_1200. Subsequent analysis indicated that (1) the quorum sensing mutations shifted KB2B1 to a perceived low cell density state and (2) the high cell density state inhibited migration via the downstream regulator LitR. Similar to the initial point mutations, deletion of the VF_1200 DGC gene increased migration. Consistent with the possibility that production of the second messenger c-di-GMP inhibited the motility of KB2B1, reporter-based measurements of c-di-GMP revealed that KB2B1 produced higher levels of c-di-GMP than ES114, and overproduction of a c-di-GMP phosphodiesterase promoted migration of KB2B1. Finally, we assessed the role of viscosity in controlling the quorum sensing pathway using polyvinylpyrrolidone and found that viscosity increased light production of KB2B1 but not ES114. Together, our data indicate that while the two strains share regulators in common, they differ in the specifics of the regulatory control over downstream phenotypes such as motility.
RESUMO
As our understanding of the human microbiome progresses, so does the need for natural experimental animal models that promote a mechanistic understanding of beneficial microorganism-host interactions. Years of research into the exclusive symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri have permitted a detailed understanding of those bacterial genes underlying signal exchange and rhythmic activities that result in a persistent, beneficial association, as well as glimpses into the evolution of symbiotic competence. Migrating from the ambient seawater to regions deep inside the light-emitting organ of the squid, V. fischeri experiences, recognizes and adjusts to the changing environmental conditions. Here, we review key advances over the past 15 years that are deepening our understanding of these events.
Assuntos
Aliivibrio fischeri/fisiologia , Decapodiformes/microbiologia , Interações entre Hospedeiro e Microrganismos , Simbiose , Animais , Decapodiformes/anatomia & histologia , Evolução Molecular , Havaí , Água do Mar/microbiologiaRESUMO
Vibrio species, including the squid symbiont Vibrio fischeri, become competent to take up DNA under specific conditions. For example, V. fischeri becomes competent when grown in the presence of chitin oligosaccharides or upon overproduction of the competence regulatory factor TfoX. While little is known about the regulatory pathway(s) that controls V. fischeri competence, this microbe encodes homologs of factors that control competence in the well-studied V. cholerae To further develop V. fischeri as a genetically tractable organism, we evaluated the roles of some of these competence homologs. Using TfoX-overproducing cells, we found that competence depends upon LitR, the homolog of V. cholerae master quorum-sensing and competence regulator HapR, and upon homologs of putative pilus genes that in V. cholerae facilitate DNA uptake. Disruption of genes for negative regulators upstream of LitR, namely, the LuxO protein and the small RNA (sRNA) Qrr1, resulted in increased transformation frequencies. Unlike LitR-controlled light production, however, competence did not vary with cell density under tfoX overexpression conditions. Analogous to the case with V. cholerae, the requirement for LitR could be suppressed by loss of the Dns nuclease. We also found a role for the putative competence regulator CytR. Finally, we determined that transformation frequencies varied depending on the TfoX-encoding plasmid, and we developed a new dual tfoX and litR overexpression construct that substantially increased the transformation frequency of a less genetically tractable strain. By advancing the ease of genetic manipulation of V. fischeri, these findings will facilitate the rapid discovery of genes involved in physiologically relevant processes, such as biofilm formation and host colonization.IMPORTANCE The ability of bacteria to take up DNA (competence) and incorporate foreign DNA into their genomes (transformation) permits them to rapidly evolve and gain new traits and/or acquire antibiotic resistances. It also facilitates laboratory-based investigations into mechanisms of specific phenotypes, such as those involved in host colonization. Vibrio fischeri has long been a model for symbiotic bacterium-host interactions as well as for other aspects of its physiology, such as bioluminescence and biofilm formation. Competence of V. fischeri can be readily induced upon overexpression of the competence factor TfoX. Relatively little is known about the V. fischeri competence pathway, although homologs of factors known to be important in V. cholerae competence exist. By probing the importance of putative competence factors that control transformation of V. fischeri, this work deepens our understanding of the competence process and advances our ability to genetically manipulate this important model organism.
Assuntos
Aliivibrio fischeri/genética , Aliivibrio fischeri/metabolismo , Proteínas de Bactérias/genética , DNA/metabolismo , Transativadores/genética , Transformação BacterianaRESUMO
N-Acetylmuramoyl-l-alanine amidases are periplasmic hydrolases that cleave the amide bond between N-acetylmuramic acid and alanine in peptidoglycan (PG). Unlike many Gram-negative bacteria that encode redundant periplasmic amidases, Vibrio fischeri appears to encode a single protein that is homologous to AmiB of Vibrio cholerae We screened a V. fischeri transposon mutant library for strains altered in biofilm production and discovered a biofilm-overproducing strain with an insertion in amiB (VF_2326). Further characterization of biofilm enhancement suggested that this phenotype was due to the overproduction of cellulose, and it was dependent on the bcsA cellulose synthase. Additionally, the amiB mutant was nonmotile, perhaps due to defects in its ability to septate during division. The amidase mutant was unable to compete with the wild type for the colonization of V. fischeri's symbiotic host, the squid Euprymna scolopes In single-strain inoculations, host squid inoculated with the mutant eventually became colonized but with a much lower efficiency than in squid inoculated with the wild type. This observation was consistent with the pleiotropic effects of the amiB mutation and led us to speculate that motile suppressors of the amiB mutant were responsible for the partially restored colonization. In culture, motile suppressor mutants carried point mutations in a single gene (VF_1477), resulting in a partial restoration of wild-type motility. In addition, these point mutations reversed the effect of the amiB mutation on cellulosic biofilm production. These data are consistent with V. fischeri AmiB possessing amidase activity; they also suggest that AmiB suppresses cellulosic biofilm formation but promotes successful host colonization.IMPORTANCE Peptidoglycan (PG) is a critical microbe-associated molecular pattern (MAMP) that is sloughed by cells of V. fischeri during symbiotic colonization of squid. Specifically, this process induces significant remodeling of a specialized symbiotic light organ within the squid mantle cavity. This phenomenon is reminiscent of the loss of ciliated epithelium in patients with whooping cough due to the production of PG monomers by Bordetella pertussis Furthermore, PG processing machinery can influence susceptibility to antimicrobials. In this study, we report roles for the V. fischeri PG amidase AmiB, including the beneficial colonization of squid, underscoring the urgency to more deeply understand PG processing machinery and the downstream consequences of their activities.
Assuntos
Aliivibrio fischeri/enzimologia , Amidoidrolases/fisiologia , Proteínas de Bactérias/fisiologia , Aliivibrio fischeri/citologia , Aliivibrio fischeri/genética , Aliivibrio fischeri/fisiologia , Amidoidrolases/genética , Proteínas de Bactérias/genética , Biofilmes , Divisão Celular , Mutação , SimbioseRESUMO
Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species. The bacterium has been used as a model organism to study symbiosis in the context of its association with its host, the Hawaiian bobtail squid Euprymna scolopes. The genetic tractability of this bacterium has facilitated the mapping of pathways that mediate interactions between these organisms. The protocols included here describe methods for genetic manipulation of V. fischeri. Following these protocols, the researcher will be able to introduce linear DNA via transformation to make chromosomal mutations, to introduce plasmid DNA via conjugation and subsequently eliminate unstable plasmids, to eliminate antibiotic resistance cassettes from the chromosome, and to randomly or specifically mutagenize V. fischeri with transposons. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Transformation of V. fischeri with linear DNA Basic Protocol 2: Plasmid transfer into V. fischeri via conjugation Support Protocol 1: Removing FRT-flanked antibiotic resistance cassettes from the V. fischeri genome Support Protocol 2: Eliminating unstable plasmids from V. fischeri Alternate Protocol 1: Introduction of exogenous DNA using a suicide plasmid Alternate Protocol 2: Site-specific transposon insertion using a suicide plasmid Alternate Protocol 3: Random transposon mutagenesis using a suicide plasmid.
Assuntos
Aliivibrio fischeri/genética , Técnicas Genéticas , Animais , Conjugação Genética , DNA Fúngico , Decapodiformes/microbiologia , Plasmídeos , Simbiose , Transformação GenéticaRESUMO
Efficient symbiotic colonization of the squid Euprymna scolopes by the bacterium Vibrio fischeri depends on bacterial biofilm formation on the surface of the squid's light organ. Subsequently, the bacteria disperse from the biofilm via an unknown mechanism and enter through pores to reach the interior colonization sites. Here, we identify a homolog of Pseudomonas fluorescens LapG as a dispersal factor that promotes cleavage of a biofilm-promoting adhesin, LapV. Overproduction of LapG inhibited biofilm formation and, unlike the wild-type parent, a ΔlapG mutant formed biofilms in vitro. Although V. fischeri encodes two putative large adhesins, LapI (near lapG on chromosome II) and LapV (on chromosome I), only the latter contributed to biofilm formation. Consistent with the Pseudomonas Lap system model, our data support a role for the predicted c-di-GMP-binding protein LapD in inhibiting LapG-dependent dispersal. Furthermore, we identified a phosphodiesterase, PdeV, whose loss promotes biofilm formation similar to that of the ΔlapG mutant and dependent on both LapD and LapV. Finally, we found a minor defect for a ΔlapD mutant in initiating squid colonization, indicating a role for the Lap system in a relevant environmental niche. Together, these data reveal new factors and provide important insights into biofilm dispersal by V. fischeri.
Assuntos
Adesinas Bacterianas/metabolismo , Aliivibrio fischeri/metabolismo , Biofilmes/crescimento & desenvolvimento , Aliivibrio fischeri/genética , Animais , Proteínas de Bactérias/metabolismo , Decapodiformes/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Transdução de Sinais , SimbioseRESUMO
Vibrio fischeri is a nonpathogenic organism related to pathogenic Vibrio species that can be readily grown and stored with common laboratory equipment. In this article, protocols for routine growth, storage, and phenotypic assessment of V. fischeri, as well as recipes for useful media, are included. Specifically, this article describes procedures and considerations for growth of this microbe in complex and minimal media. It also describes assays for biofilm formation, motility, and bioluminescence, three commonly assessed phenotypes of V. fischeri. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth of V. fischeri from frozen stocks Basic Protocol 2: Growth of V. fischeri in rich, undefined liquid medium Alternate Protocol 1: Growth of V. fischeri in minimal medium Basic Protocol 3: Storage of V. fischeri in frozen stocks Basic Protocol 4: Biofilm assay on solid agar Alternate Protocol 2: Biofilm assay in shaking liquid culture Alternate Protocol 3: Biofilm assay in static liquid culture Basic Protocol 5: Motility assay Basic Protocol 6: Luminescence assay.