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Genetic analysis in Vibrio
Abstract
Bacteria of the genus Vibrio are remarkably diverse, and until recently the methodology for genetic analysis consisted of a patchwork of different approaches, many of which were narrowly applicable to a single species. The invention of the recombinant DNA technology and the subsequent innovations in transposon mutagenesis and in transductive and conjugative gene transfer techniques have led to the development of very powerful and general strategies for genetic analysis of species of Vibrio. The striking synergy of combining recombinant DNA, transposon, and gene transfer methods is particularly evident in the construction of transposons which generate gene fusions and of broad host range plasmids which deliver transposons and mutated genes and which mobilize chromosomes. With such tools it should be possible to perform advanced genetic analysis on the many undomesticated species of Vibrio still to be explored.
... Phosphodiesterase activity has been demonstrated to be associated with the EAL domain (6,13,29,60,65). Purified composite proteins with both domains-as the six enzymes from G. xylinus-generally display either diguanylate cyclase or phosphodiesterase activity (61). This has resulted in the hypothesis that only one domain is catalytically active, whereas the second domain may be nonfunctional or regulatory (12,56). ...
... Chromosomal DNA was prepared according to the protocol of Woo et al. (71). Conjugation and allelic replacement methods for V. parahaemolyticus have been described (61). Allelic replacements were confirmed by Southern blot analysis. ...
In this work, we describe a new gene controlling lateral flagellar gene expression. The gene encodes ScrG, a protein containing
GGDEF and EAL domains. This is the second GGDEF-EAL-encoding locus determined to be involved in the regulation of swarming:
the first was previously characterized and named scrABC (for “swarming and capsular polysaccharide regulation”). GGDEF and EAL domain-containing proteins participate in the synthesis
and degradation of the nucleotide signal cyclic di-GMP (c-di-GMP) in many bacteria. Overexpression of scrG was sufficient to induce lateral flagellar gene expression in liquid, decrease biofilm formation, decrease cps gene expression, and suppress the ΔscrABC phenotype. Removal of its EAL domain reversed ScrG activity, converting ScrG to an inhibitor of swarming and activator of
cps expression. Overexpression of scrG decreased the intensity of a 32P-labeled nucleotide spot comigrating with c-di-GMP standard, whereas overexpression of scrGΔEAL enhanced the intensity of the spot. Mutants with defects in scrG showed altered swarming and lateral flagellin production and colony morphology (but not swimming motility); furthermore,
mutation of two GGDEF-EAL-encoding loci (scrG and scrABC) produced cumulative effects on swarming, lateral flagellar gene expression, lateral flagellin production and colony morphology.
Mutant analysis supports the assignment of the primary in vivo activity of ScrG to acting as a phosphodiesterase. The data
are consistent with a model in which multiple GGDEF-EAL proteins can influence the cellular nucleotide pool: a low concentration
of c-di-GMP favors surface mobility, whereas high levels of this nucleotide promote a more adhesive Vibrio parahaemolyticus cell type.
... The precise point of insertion was defined by DNA sequencing. The procedures for conjugation and gene replacement in V. parahaemolyticus have been described elsewhere (52). General DNA manipulations were adapted from the methods of Sambrook et al. (47). ...
... Retrieval of clones and plasmid construction. The fliF locus was identified by cloning a tetracycline-resistant transposon from the mini-Mu-induced Fla Ϫ mutant strain ML199 according to procedures described previously (52). The segment of chromosomal DNA contiguous with the transposon was sequenced and then used as a probe to retrieve cosmid pLM2047 from a V. parahaemolyticus library constructed using DNA prepared from strain BB22. ...
Vibrio parahaemolyticus possesses two types of flagella, polar and lateral, powered by distinct energy sources, which are derived from the sodium and proton motive forces, respectively. Although proton-powered flagella in Escherichia coli and Salmonella enterica serovar Typhimurium have been extensively studied, the mechanism of torque generation is still not understood. Molecular knowledge of the structure of the sodium-driven motor is only now being developed. In this work, we identify the switch components, FliG, FliM, and FliN, of the sodium-type motor. This brings the total number of genes identified as pertinent to polar motor function to seven. Both FliM and FliN possess charged domains not found in proton-type homologs; however, they can interact with the proton-type motor of E. coli to a limited extent. Residues known to be critical for torque generation in the proton-type motor are conserved in the sodium-type motor, suggesting a common mechanism for energy transfer at the rotor-stator interface regardless of the driving force powering rotation. Mutants representing a complete panel of insertionally inactivated switch and motor genes were constructed. All of these mutants were defective in sodium-driven swimming motility. Alkaline phosphatase could be fused to the C termini of MotB and MotY without abolishing motility, whereas deletion of the unusual, highly charged C-terminal domain of FliM disrupted motor function. All of the mutants retained proton-driven, lateral motility over surfaces. Thus, although central chemotaxis genes are shared by the polar and lateral systems, genes encoding the switch components, as well as the motor genes, are distinct for each motility system.
... Recombinants were retransformed into DH5␣, screened for appropriate drug markers, and analyzed by restriction digest. The insertion-deletion constructs were then conjugated into V. parahaemolyticus, and the standard allelic replacement procedure followed (58). All allelic replacements and transposon mutants were confirmed with Southern blot analysis by using Hybond-NX membranes (Amersham) and [ 32 P]dCTP-labeled DNA probes (Pharmacia). ...
... To recreate transposon mutants, the transposon-junction plasmid was linearized by digestion with the appropriate restriction enzyme, ligated into the compatibly digested gene replacement vector, pLAFRII, and transformed into DH5␣ with selection for kanamycin and tetracycline. The transposon-junction-pLAFRII clone could then be used in standard gene replacement procedure (58). ...
Movement on surfaces, or swarming motility, is effectively mediated by the lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf is induced by conditions inhibiting rotation of the polar flagellum, which is used for swimming in liquid. However, not all
V. parahaemolyticus isolates swarm proficiently. The organism undergoes phase variation between opaque (OP) and translucent (TR) cell types.
The OP cell produces copious capsular polysaccharide and swarms poorly, whereas the TR type produces minimal capsule and swarms
readily. OP↔TR switching is often the result of genetic alterations in the opaR locus. Previously, OpaR, a Vibrio harveyi LuxR homolog, was shown to activate expression of the cpsA locus, encoding capsular polysaccharide biosynthetic genes. Here, we show that OpaR also regulates swarming by repressing
laf gene expression. However, in the absence of OpaR, the swarming phenotype remains tightly surface regulated. To further investigate
the genetic controls governing swarming, transposon mutagenesis of a TR (ΔopaR1) strain was performed, and SwrT, a TetR-type regulator, was identified. Loss of swrT, a homolog of V. harveyi luxT, created a profound defect in swarming. This defect could be rescued upon isolation of suppressor mutations that restored
swarming. One class of suppressors mapped in swrZ, encoding a GntR-type transcriptional regulator. Overexpression of swrZ repressed laf expression. Using reporter fusions and quantitative reverse transcription-PCR, SwrT was demonstrated to repress swrZ transcription. Thus, we have identified the regulatory link that inhibits swarming of OP strains and have begun to elucidate
a regulatory circuit that modulates swarming in TR strains.
... Molecular genetic techniques. Plasmids and oligonucleotides are listed in Table S1, and the former were constructed using standard techniques and materials as described previously (45). Deletion/insertion mutations were made with a l Red recombinase system in E. coli (46) to introduce mutations into the cosmid containing lafV to create pLM3700 using the antibiotic resistance cassettes found in plasmid pKD3. ...
Vibrio parahaemolyticus rapidly colonizes surfaces using swarming motility. Surface contact induces the surface-sensing regulon, including lateral flagellar genes, spurring dramatic shifts in physiology and behavior. The bacterium can also adopt a sessile, surface-associated lifestyle and form robust biofilms. These alternate colonization strategies are influenced reciprocally by the second messenger c-di-GMP. Although V. parahaemolyticus possesses 43 predicted proteins with the c-di-GMP-forming GGDEF domain, none have been previously been identified as contributors to surface colonization. We sought to explore this knowledge gap by using a suppressor transposon screen to restore the swarming motility of a nonswarming, high-c-di-GMP strain. Two diguanylate cyclases, ScrJ and ScrL, each containing tetratricopeptide repeat-coupled GGDEF domains, were demonstrated to contribute additively to swarming gene repression. Both proteins required an intact catalytic motif to regulate. Another suppressor mapped in lafV, the last gene in a lateral flagellar operon. Containing a degenerate phosphodiesterase (EAL) domain, LafV repressed transcription of multiple genes in the surface sensing regulon; its repressive activity required LafK, the primary swarming regulator. Mutation of the signature EAL motif had little effect on LafV’s repressive activity, suggesting that LafV belongs to the subclass of EAL-type proteins that are regulatory but not enzymatic. Consistent with these activities and their predicted effects on c-di-GMP, scrJ and scrL but not lafV, mutants affected the transcription of the c-di-GMP-responsive biofilm reporter cpsA::lacZ. Our results expand the knowledge of the V. parahaemolyticus GGDEF/EAL repertoire and its roles in this surface colonization regulatory network.
IMPORTANCE
A key survival decision, in the environment or the host, is whether to emigrate or aggregate. In bacteria, c-di-GMP signaling almost universally influences solutions to this dilemma. In V. parahaemolyticus, c-di-GMP reciprocally regulates swarming and sticking (i.e., biofilm formation) programs of surface colonization. Key c-di-GMP-degrading phosphodiesterases responsive to quorum and nutritional signals have been previously identified. c-di-GMP binding transcription factors programming biofilm development have been studied. Here, we further develop the blueprint of the c-di-GMP network by identifying new participants involved in dictating the complex decision of whether to swarm or stay. These include diguanylate cyclases with tetratricopeptide domains and a degenerate EAL protein that, analogously to the negative flagellar regulator RflP/YdiV of enteric bacteria, serves to regulate swarming.
... Molecular genetic techniques. Plasmids and oligonucleotides are listed in Table S1 and the former were constructed using standard techniques and materials as described previously (30). Deletion/ insertion mutations were made with a Red recombinase system in E. coli (31) to introduce mutations into cosmids containing cpsS, cpsSQ, scrMNO, and scrO to create pLM3769, pLM3765, pLM4194, and pLM4185, respectively, using the antibiotic resistance cassettes found in plasmids pKD4 (Kan; pLM3765 and pLM4194) and pKD3 (Cam; pLM3769 and pLM4185). ...
Vibrio parahaemolyticus can inhabit open ocean, chitinous shells, and the human gut. Such varied habitats and the transitions between them require adaptable regulatory networks controlling energetically expensive behaviors, including swarming motility and biofilm formation, which are promoted by low and high concentrations of the signaling molecule c-di-GMP, respectively. Here, we describe four homologous c-di-GMP-binding Scr transcription factors in V. parahaemolyticus . Members of this family of regulators are present in many vibrios, yet their numbers and the natures of their activities differ across species. Our work highlights the distinctive roles that these transcription factors play in dynamically controlling biofilm formation and architecture in V. parahaemolyticus and serves as a powerful example of regulatory network evolution and diversification.
... Mutant construction was verified by PCR. Conjugation and allelic replacement methods for V. parahaemolyticus have been previously described (Silverman et al., 1991). ...
Motile bacteria are proficient at finding optimal environments for colonization. Often, they use chemotaxis to sense nutrient availability and dangerous concentrations of toxic chemicals. For many bacteria, the repertoire of chemoreceptors is large, suggesting they possess a broad palate with respect to sensing. However, knowledge of the molecules detected by chemotaxis signal transduction systems is limited. Some bacteria, like Vibrio parahaemolyticus, are social and swarm in groups on surfaces. This marine bacterium and human pathogen secretes the S signal autoinducer, which cues degradation of intracellular c‐di‐GMP leading to transcription of the swarming program. Here, we report that the S signal also directs motility at a behavioral level by serving as a chemoattractant. The data demonstrate that V. parahaemolyticus senses the S signal using SscL and SscS, homologous methyl‐accepting chemotaxis proteins. SscL is required by planktonic bacteria for S signal chemotaxis. SscS plays a role during swarming, and mutants lacking this chemoreceptor swarm faster and produce colonies with more deeply branched swarming fronts than the wild type or the sscL mutant. Other Vibrio species can swim towards the S signal, suggesting a recruitment role for this cell‐cell communication molecule in the context of polymicrobial marine communities.
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... Kanamycin-resistant pDSW361 and derivatives were transferred to V. parahaemolyticus by electroporation; cosmids were transferred by conjugation. Conjugation and allelic replacement methods for V. parahaemolyticus have been previously described (34). Allelic replacements were confirmed by PCR. ...
The Vibrio parahaemolyticus Scr system modulates decisions pertinent to surface colonization by affecting the cellular level of cyclic dimeric GMP (c-di-GMP).
In this work, we explore the scope and mechanism of this regulation. Transcriptome comparison of ΔscrABC and wild-type strains revealed expression differences with respect to ∼100 genes. Elevated c-di-GMP repressed genes in the
surface-sensing regulon, including those encoding the lateral flagellar and type III secretion systems and N-acetylglucosamine-binding protein GpbA while inducing genes encoding other cell surface molecules and capsular polysaccharide.
The transcription of a few regulatory genes was also affected, and the role of one was characterized. Mutations in cpsQ suppressed the sticky phenotype of scr mutants. cpsQ encodes one of four V. parahaemolyticus homologs in the CsgD/VpsT family, members of which have been implicated in c-di-GMP signaling. Here, we demonstrate that
CpsQ is a c-di-GMP-binding protein. By using a combination of mutant and reporter analyses, CpsQ was found to be the direct,
positive regulator of cpsA transcription. This c-di-GMP-responsive regulatory circuit could be reconstituted in Escherichia coli, where a low level of this nucleotide diminished the stability of CpsQ. The molecular interplay of additional known cps regulators was defined by establishing that CpsS, another CsgD family member, repressed cpsR, and the transcription factor CpsR activated cpsQ. Thus, we are developing a connectivity map of the Scr decision-making network with respect to its wiring and output strategies
for colonizing surfaces and interaction with hosts; in doing so, we have isolated and reproduced a c-di-GMP-sensitive regulatory
module in the circuit.
... (10). This procedure as well as the procedures for conjugation and gene replacement in V. parahaemolyticus has been described elsewhere (50). Mutations were also constructed in vitro. ...
Vibrio parahaemolyticus possesses two alternate flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (swimming), while multiple lateral flagella move the bacterium over surfaces (swarming). Energy to rotate the polar flagellum is derived from the sodium membrane potential, whereas lateral flagella are powered by the proton motive force. Lateral flagella are arranged peritrichously, and the unsheathed filaments are polymerized from a single flagellin. The polar flagellum is synthesized constitutively, but lateral flagella are produced only under conditions in which the polar flagellum is not functional, e.g., on surfaces. This work initiates characterization of the sheathed, polar flagellum. Four genes encoding flagellins were cloned and found to map in two loci. These genes, as well as three genes encoding proteins resembling HAPs (hook-associated proteins), were sequenced. A potential consensus polar flagellar promoter was identified by using upstream sequences from seven polar genes. It resembled the enterobacterial sigma 28 consensus promoter. Three of the four flagellin genes were expressed in Escherichia coli, and expression was dependent on the product of the fliA gene encoding sigma 28. The fourth flagellin gene may be different regulated. It was not expressed in E. coli, and inspection of upstream sequence revealed a potential sigma 54 consensus promoter. Mutants with single and multiple defects in flagellin genes were constructed in order to determine assembly rules for filament polymerization. HAP mutants displayed new phenotypes, which were different from those of Salmonella typhimurium and most probably were the result of the filament being sheathed.
... harveyi (3,4,21,38). We thus chose the Mu derivative Mu dl 1681 (7) to mutagenize a symbiosis-competent strain of V fischeri, isolate and identify several classes of motility mutants, and test their ability to colonize juvenile squids. ...
Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.
... Mutagenesis with ATnphoA (from C. Manoil [28]) was performed by infecting a culture of E. coli LLM1717. This procedure and the procedures for conjugation and gene replacement in V parahaemolyticus have been described elsewhere (39). All strain constructions were confirmed by Southern blot analysis of restricted genomic DNA (29, 32) on 0.45-,um Magna Charge nylon membranes (Micron Separations Inc., Westborough, Mass.). ...
Energy to power the rotation of bacterial flagella can be derived from the proton or sodium transmembrane potential. Until now, genes encoding a bacterial sodium-type flagellar motor have not been defined. A gene, motY, encoding one component of the sodium-type flagellar motor of Vibrio parahaemolyticus was cloned by complementation of a Mot- mutant strain. Sequencing revealed an open reading frame of 879 nucleotides in which a transposon conferring a motility defect mapped. Overexpression of motY in Escherichia coli allowed identification of a product 33 kDa in apparent size on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This size correlated well with the predicted molecular mass of 33,385 Da. Unlike mot genes identified in other bacteria, localized transposon mutagenesis suggested that the locus was not an extended region containing multiple genes required for swimming motility. Sequencing upstream and downstream of motY confirmed that the gene maps alone and placed it within a locus homologous to the E. coli rnt locus. Although data bank searches failed to reveal significant similarity to known motility components, the carboxyl terminus of MotY showed extensive homology to a number of outer membrane proteins known to interact with peptidoglycan, including OmpA and peptidoglycan-associated lipoproteins. To a limited extent, this domain could also be identified in the Bacillus subtilis MotB protein. This finding suggests that MotY plays the role of a stator in the sodium flagellar motor, stabilizing the force-generating unit through direct interaction with the cell wall.
... Cosmid DNA was prepared and used to transform strain CC118, selecting for tetracycline and kanamycin resistance. The procedure for gene replacement in V. parahaemolyticus has been outlined elsewhere (49). For mobilization of the cosmids carrying the transposon mutations into V. parahaemolyticus, the tra donor was pRK2013 (from Gary Ditta [15]); for selecting gene replacement, the "kick-out" plasmid was pPHIJI (from Ron Taylor [6]). ...
Vibrio parahaemolyticus possesses two distinct motility systems, the polar system used for swimming in liquid environments and the lateral system used for swarming over surfaces. Growth on surfaces induces swarmer cell differentiation and expression of the lateral motility system. Mutants, created by transposon mutagenesis of a clone expressing lateral flagellin and gene disruption in V. parahaemolyticus, were unable to swarm and failed to make lateral flagellin; therefore, unlike the case for the polar system, there is one gene (lafA) encoding lateral flagellin. In addition to lafA, other genes required for swarming but not for swimming were identified by gene replacement mutagenesis. The nucleotide sequence of the clone determined open reading frames (ORFs) and deduced amino acid sequences showed similarities to flagellar components of other bacteria: flagellin, hook-associated protein (HAP2), motor components, and flagellar sigma factor (sigma 28). Many sigma 28 factors have been shown to recognize cognate promoters; however, expression of lafA in Escherichia coli required LafS, and E. coli sigma 28 did not substitute. Also, there were no sequences preceding genes encoding flagellin or HAP2 resembling the sigma 28 consensus promoter. The product of the sigma-like gene seems to be a unique member of the sigma 28 cluster. It appears the result of requiring expression for immunodetection of flagellin clones was that the sigma locus was fortuitously cloned, since the sigma and lafA loci were not contiguous in the chromosome. This work initiates identification and placement of genes in a scheme of control for swarmer cell differentiation; three levels have been identified in the transcriptional hierarchy.
... Blots were washed in 6ϫ SSC three times for 15 min each time at room temperature. Conjugative transfer of plasmids to vibrios has been described previously (43). ...
Vibrio parahaemolyticus differentiates from a polarly flagellated, short, rod-shaped cell known as the swimmer to the elongated, hyperflagellated, and multinucleated swarmer cell type when it is grown on a surface. The swarmer is adapted to movement over and colonization of surfaces. To understand the signal transduction mechanism by which the bacterium recognizes surfaces and reprograms gene expression, we isolated a new class of mutants defective in surface sensing. These mutants were constitutive for swarmer cell gene expression, inappropriately expressing high levels of a swarmer cell gene fusion product when grown in liquid. They showed no defect in the swimming motility system, unlike all previously isolated constitutive mutants which have defects in the alternate, polar motility system. The lesions in the majority of the newly isolated mutants were found to be in a gene, lonS, which encodes a polypeptide exhibiting 81% sequence identity to the Escherichia coli Lon protein, an ATP-dependent protease. Upstream sequences preceding the lonS coding region resemble a heat shock promoter, and the homology extends to sequences flanking lonS. The gene order appears to be clpX lonS hupB, like the organization of the E. coli locus. V. parahaemolyticus lonS complemented E. coli lon mutants to restore UV resistance and capsular polysaccharide regulation to that of the wild type. Vibrio lonS mutants were UV sensitive. In addition, when grown in liquid and examined in a light microscope, lonS mutant cells were extremely long and thus resembled swarmer cells harvested from a surface.
... Transposon mutagenesis with Tn5 has been described elsewhere (29). Procedures for the transfer of clones from Escherichia coli to V. parahaemolyticus via conjugation and gene replacement techniques have also been described elsewhere (31). General DNA manipulations were adapted from the methods of Sambrook et al. (27). ...
Vibrio parahaemolyticus is an organism well adapted to communal life on surfaces. When grown on a surface or in a viscous layer, the bacterium induces a large gene system and differentiates to swarmer cells capable of movement over and colonization of surfaces. V. parahaemolyticus displays additional phenotypic versatility manifested as variable colony morphology, switching between translucent and opaque colony types. Although not itself luminescent, V. parahaemolyticus produces autoinducer molecules capable of inducing luminescence in Vibrio harveyi. To examine the role of quorum signaling in the lifestyles of V. parahaemolyticus, the functional homolog of the gene encoding the V. harveyi autoinducer-controlled transcriptional regulatory protein LuxR was cloned. Sequence analysis of the clone predicted an open reading frame with a deduced product 96% identical to LuxR. Introduction of the clone carrying the luxR-like locus into V. parahaemolyticus dramatically affected colony morphology, converting a translucent strain to an opaque one. When the coding sequence for the luxR homolog was placed under the control of the Ptac promoter, conversion to the opaque phenotype became inducible by isopropyl-beta-D-thiogalactopyranoside. Allelic disruption of the luxR-like gene on the chromosome of an opaque strain produced a translucent strain proficient in swarming ability. Primer extension mapping demonstrated opaR transcription in opaque but not translucent cell types. It is postulated that this gene, which has been named opaR, encodes a transcription factor controlling cell type. The underlying genetic basis for opaque-translucent variation may be the consequence of a genomic alteration detected in the opaR locus of opaque and translucent strains.
... General DNA manipulations were adapted from the methods of Sambrook et al. (52). Transposon mutagenesis with mini-Mu lac (Tet r ) and the strategy for cloning the targeted gene have been described previously (58). The V. parahaemolyticus cosmid library was prepared by using the pLAFRII vector (40). ...
Vibrio parahaemolyticus has dual flagellar systems adapted for locomotion under different circumstances. A single, sheathed polar flagellum propels
the swimmer cell in liquid environments. Numerous unsheathed lateral flagella move the swarmer cell over surfaces. The polar
flagellum is produced continuously, whereas the synthesis of lateral flagella is induced under conditions that impede the
function of the polar flagellum, e.g., in viscous environments or on surfaces. Thus, the organism possesses two large gene
networks that orchestrate polar and lateral flagellar gene expression and assembly. In addition, the polar flagellum functions
as a mechanosensor controlling lateral gene expression. In order to gain insight into the genetic circuitry controlling motility
and surface sensing, we have sought to define the polar flagellar gene system. The hierarchy of regulation appears to be different
from the polar system of Caulobacter crescentus or the peritrichous system of enteric bacteria but is pertinent to many Vibrio andPseudomonas species. The gene identity and organization of 60 potential flagellar and chemotaxis genes are described. Conserved sequences
are defined for two classes of polar flagellar promoters. Phenotypic and genotypic analysis of mutant strains with defects
in swimming motility coupled with primer extension analysis of flagellar and chemotaxis transcription provides insight into
the polar flagellar organelle, its assembly, and regulation of gene expression.
... To test the idea that the lateral flagellar regulator was responsible for polar flagellar gene expression, a mutant with defects in both flaK and lafK was constructed. The lafK1::Gen r allele and methods for introduction onto the chromosome via allelic exchange have been described (19,20). Introduction of lafK1::Gen r into a swarming-competent strain abolishes swarming motility (20) and production of lateral flagella (Fig. 3A, compare the mutant in lane 2 with the parent in lane 1). ...
Gene organization and hierarchical regulation of the polar flagellar genes of Vibrio parahaemolyticus, Vibrio cholerae, and Pseudomonas aeruginosa appear highly similar, with one puzzling difference. Two σ54-dependent regulators are required to direct different classes of intermediate flagellar gene expression in V. cholerae and P. aeruginosa, whereas the V. parahaemolyticus homolog of one of these regulators, FlaK, appears dispensable. Here we demonstrate that there is compensatory activation
of polar flagellar genes by the lateral flagellar regulator LafK.
... cosmids carrying 40-kb insertions of wild-type V. parahaemolyticus DNA (53) in E. coli. Conjugation and gene replacement methods for V. parahaemolyticus have been described elsewhere (77). All gene replacements were confirmed by Southern blot analysis performed using Hybond-NX membranes (Amersham) and Primer extension experiments were conducted as previously described (34) using RNA (ϳ15 g/reaction) prepared from plate-and liquid-grown LM5674. ...
Vibrio parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the
bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral
flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production
of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions
in the structural and regulatory laf genes. A Tn5-based lux transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the
laf regulon. Twenty-nine independent insertions were distributed within 16 laf genes. DNA sequence analysis identified 38 laf genes in two loci. Among the mutants isolated, 11 contained surface-induced lux fusions. A hierarchy of laf gene expression was established following characterization of the laf::lux transcriptional fusion strains and by mutational and primer extension analyses of the laf regulon. The laf system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however, laf regulation was different from the Salmonella-Escherichia coli paradigm. There is no apparent flhDC counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria.
A potential σ54-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific σ28 factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like
product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.
... General molecular biology methods were adapted from Sambrook et al. (43). Conjugation and allelic replacement methods for V. parahaemolyticus have been previously described (45). All allelic replacements were confirmed by PCR. ...
In Vibrio parahaemolyticus, scrC participates in controlling the decision to be a highly mobile swarmer cell or a more adhesive, biofilm-proficient cell type.
scrC mutants display decreased swarming motility over surfaces and enhanced capsular polysaccharide production. ScrC is a cytoplasmic
membrane protein that contains both GGDEF and EAL conserved protein domains. These domains have been shown in many organisms
to respectively control the formation and degradation of the small signaling nucleotide cyclic dimeric GMP (c-di-GMP). The
scrC gene is part of the three-gene scrABC operon. Here we report that this operon influences the cellular nucleotide pool and that c-di-GMP levels inversely modulate
lateral flagellar and capsular polysaccharide gene expression. High concentrations of this nucleotide prevent swarming and
promote adhesiveness. Further, we demonstrate that ScrC has intrinsic diguanylate cyclase and phosphodiesterase activities,
and these activities are controlled by ScrAB. Specifically, ScrC acts to form c-di-GMP in the absence of ScrA and ScrB; whereas
ScrC acts to degrade c-di-GMP in the presence of ScrA and ScrB. The scrABC operon is specifically induced by growth on a surface, and the analysis of mutant phenotypes supports a model in which the
phosphodiesterase activity of ScrC plays a dominant role during surface translocation and in biofilms.
... The red recombinase technique was adapted from Datsenko and Wanner (12) to introduce mutations onto V. parahaemolyticus cosmid DNA in E. coli. The recombinant cosmids bearing the gene disruptions were then used for allelic replacement in V. parahaemolyticus as previously described (42). ...
Vibrio parahaemolyticus, a biofouling marine bacterium and human pathogen, undergoes phase variation displaying translucent (TR) and opaque (OP)
colony morphologies. Prior studies demonstrated that OP colonies produce more capsular polysaccharide (CPS) than TR colonies
and that opacity is controlled by the Vibrio harveyi LuxR-type transcriptional activator OpaR. CPS has also been shown to be regulated by the scrABC signaling pathway, which involves a GGDEF-EAL motif-containing sensory protein. The present study identifies cps genes and examines their regulation. Transposon insertions in the cps locus, which contains 11 genes, abolished opacity. Such mutants failed to produce CPS and were defective in pellicle formation
in microtiter wells and in a biofilm attachment assay. Reporter fusions to cpsA, the first gene in the locus, showed ∼10-fold-enhanced transcription in the OP (opaR+) strain compared to a TR (ΔopaR) strain. Two additional transcriptional regulators were discovered. One potential activator, CpsR, participates in the scrABC GGDEF-EAL-signaling pathway; CpsR was required for the increased cps expression observed in scrA ΔopaR strains. CpsR, which contains a conserved module found in members of the AAA+ superfamily of ATP-interacting proteins, is
homologous to Vibrio cholerae VpsR; however, unlike VpsR, CpsR was not essential for cps expression. CpsS, the second newly identified regulator, contains a CsgD-type DNA-binding domain and appears to act as a
repressor. Mutants with cpsS defects have greatly elevated cps transcription; their high level of cpsA expression was CpsR dependent in ΤR strains and primarily OpaR dependent in OP strains. Thus, a network of positive and negative
regulators modulates CPS production in V. parahaemolyticus.
IntroductionIntroduction of Genetic Material into Gram Negative Bacteria: The Binary Broad Host Range Conjugation SystemMethods for Genomic Integration of Cloned DNAIn vivo Manipulation of Gram Negative Bacteria Using TransposonsSpecific Example: Identification and Characterization of LPS Biosynthesis Genes in Rhizobium leguminosarum bv. viciaeConcluding Remarks
This chapter discusses the exploitation of bacteriophages for genetic analysis and in vitro recombinant DNA techniques in diverse bacterial species. Bacteriophages are obligate intracellular parasites and are uniquely adapted to growth in their hosts. They will inevitably, therefore, encode genetic elements that are easily accessible as tools for the genetic analysis of their hosts. The numbers of papers relating to phage research would indicate that the interest in phage has not peaked. Indeed the numbers of papers reporting new integrases are increasing at an apparently exponential rate. As microbiology moves into detailed analysis of diverse species, examining their evolution, ecology and physiology, phages are both integral to these processes (e.g. the movement of pathogenicity islands in the evolution of harmful bacteria) and easily exploitable. Phages are the most numerous and most diverged entities on this planet. This must be a good place to look for new genetic tools.
IntroductionIntroduction of Genetic Material into Gram Negative Bacteria: The Binary Broad Host Range Conjugation SystemMethods for Genomic Integration of Cloned DNAIn vivo Manipulation of Gram Negative Bacteria Using TransposonsSpecific Example: Identification and Characterization of LPS Biosynthesis Genes in Rhizobium leguminosarum bv. viciaeConcluding Remarks
Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum. The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions. Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum. A gene, motX, encoding one component of the torque generator has been cloned and sequenced. The deduced protein sequence is 212 amino acids in length. MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor. Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli. The rate of lysis caused by induction of motX was proportional to the sodium ion concentration. Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis. Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride. The data suggest that MotX forms a sodium channel. The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains.
Density-dependent expression of luminescence in Vibrio harveyi is regulated by the concentration of an extracellular signal molecule (autoinducer) in the culture medium. A recombinant clone that restored function to one class of spontaneous dim mutants was found to encode functions necessary for the synthesis of, and response to, a signal molecule. Sequence analysis of the region encoding these functions revealed three open reading frames, two (luxL and luxM) that are required for production of an autoinducer substance and a third (luxN) that is required for response to this signal substance. The LuxL and LuxM proteins are not similar in amino acid sequence to other proteins in the database, but the LuxN protein contains regions of sequence resembling both the histidine protein kinase and the response regulator domains of the family of two-component, signal transduction proteins. The phenotypes of mutants with luxL, luxM and luxN defects indicated that an additional signal-response system controlling density-dependent expression of luminescence remains to be identified.
The flaA locus of Vibrio parahaemolyticus encodes one of the four polar flagellin genes, the flagellum-capping protein HAP2, and three additional flagellar genes. Sequence analysis downstream of the gene encoding HAP2 revealed the region to be similar to the fliD (HAP2) locus of Pseudomonas aeruginosa. The deduced protein sequences for the newly identified genes suggest that one protein belongs to the family of transcriptional regulatory proteins known to interact with sigma (54), one may be a rod component of the flagellum, and one resembles the FliS protein. fliS is an essential flagellar gene in many bacteria; however its function is not clear. The V. parahaemolyticus polar flaC flagellin gene was poorly expressed in Escherichia coli Production of FlaC was stimulated by provision of the flaA locus in trans. Dissection of this locus revealed that the fliS-like gene, flaJ, was required for increased expression of flaC. Stimulation by FlaJ occurred in E.coli mutants defective in either the master flagellar-controlling operon or the gene encoding the flagellar sigma (28). Therefore the effect of FlaJ was not mediated through flagellar proteins. Nor was it mediated through sigma (54) for enhanced FlaC production was observed in mutants with defects in the gene encoding sigma (54).
Although the study of microbe-host interactions has been traditionally dominated by an interest in pathogenic associations, there is an increasing awareness of the importance of cooperative symbiotic interactions in the biology of many bacteria and their animal and plant hosts. This review examines a model system for the study of such symbioses, the light organ association between the bobtail squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri. Specifically, the initiation, establishment, and persistence of the benign bacterial infection of the juvenile host light organ are described, as are efforts to understand the mechanisms underlying this specific colonization program. Using molecular genetic techniques, mutant strains of V. fischeri have been constructed that are defective at specific stages of the development of the association. Some of the lessons that these mutants have begun to teach us about the complex and long-term nature of this cooperative venture are summarized.
This chapter describes bicistronic mammalian expression vector, pHaMDR-DHFR that is used to study the effect of mutations introduced into the human multidrug resistant (MDR)1 gene. Mutagenesis is one of the most common approaches employed in the structure–function study of proteins both in somatic cells 1,2 and in germ cells. To analyze the function of the mutagenized gene, it is generally subcloned into an expression vector that usually has two different promoters for the gene of interest and the selective marker. Studies of the internal ribosomal entry site (IRES) have made it possible to develop a bicistronic or polycistronic vector in which a fusion transcript can be obtained from two or more genes linked together by one or more IRES sequences. The second gene downstream from the IRES guarantees the expression of the first gene.
Swarming is an adaptation of many bacteria to growth on surfaces. A search for genes controlling swarmer cell differentiation
of Vibrio parahaemolyticus identified a novel three-gene operon that potentially encodes a pyridoxal-phosphate-dependent enzyme, an extracellular solute-binding
protein, and a membrane-bound GGDEF- and EAL-motif sensory protein. The functions of these motifs, which are named after conserved
amino acid sequences, are unknown, although the domains are found singly and in combination in a variety of bacterial signaling
proteins. Studies with translational fusions supported the predicted localization of the gene products. When the operon was
overexpressed, swarmer cell gene transcription was induced in liquid culture. Mutants with defects in any of the three genes
exhibited decreased swarming and lateral flagellar (laf) gene expression. Complementation studies confirmed an operon organization and suggested that all three genes participated
in laf regulation. The lesions that decreased swarming increased capsular polysaccharide (CPS) production, and overexpression of
the operon inhibited transcription of the CPS gene cpsA. Thus, the scrABC locus appears to inversely regulate two gene systems that are pertinent to colonization of surface swarming and CPS.
Three pleiotropic, quorum sensing-defective Vibrio harveyi mutants were observed to precipitate soluble Pb2+ as an insoluble compound. The compound was purified and subjected to X-ray diffraction and elemental analyses. These assays
identified the precipitated compound as Pb9(PO4)6, an unusual and complex lead phosphate salt that is produced synthetically at temperatures of ca. 200°C. Regulation of the
precipitation phenotype was also examined. Introduction of a luxO::kan allele into one of the mutants abolished lead precipitation, indicating that the well-characterized autoinducer 1 (AI1)-AI2
quorum-sensing system can block lead precipitation in dense cell populations. Interestingly, the V. harveyi D1 mutant, a strain defective for secretion of both AI1 and AI2, was shown to be an effective trans inhibitor of lead precipitation. This suggests that a previously undescribed V. harveyi autoinducer, referred to as AI3, can also negatively regulate lead precipitation. Experiments with heterologous bacterial
populations demonstrated that many different species are capable of trans regulating the V. harveyi lead precipitation phenotype. Moreover, one of the V. harveyi mutants in this study exhibited little or no response to intercellular signals from other V. harveyi inocula but was quite responsive to some of the heterologous bacteria. Based on these observations, we propose that V. harveyi carries at least one quorum sensor that is specifically dedicated to receiving cross-species communication.
EI Tor strains of Vibrio cholerae are capable of producing a haemolysin which they actively secrete into the growth medium. This requires translation to produce the protein at the surface of the cytoplasmic membrane and translocation across this membrane, the periplasmic space and the outer membrane. The mechanism by which this occurs is poorly understood.
In addition to the structural gene for the haemolysin ( hlyA ), we have cloned a second adjacent gene, hlyB. By site‐directed mutagenesis, specific hlyB mutants have been constructed. These mutants are defective in the secretion of HlyA in the early to mid‐exponential phase of growth and the haemolysin becomes trapped within the cell and is only released in stationary phase. Nucleotide sequence analysis and cell fractionations reveal HlyB to be a 60.3 kD putative outer membrane‐associated protein.
Fourteen mutants have been identified in which the frequency of spontaneous mutations in mitochondrial DNA is increased. As well as increasing the frequency of mutations to resistance to erythromycin, oligomycin and spiramycin, all the mutants also show changes in the frequency of spontaneous petite induction. None of the mutants has any effect on the frequency of spontaneous nuclear mutations. Nine of the mutants are in one complementation group and five are in another. The phenotype of both groups is caused by a single nuclear mutation.
The nucleotide sequence of the trp operon of the marine enteric bacterium Vibrio parahemolyticus is presented. The gene order E, G, D, C(F), B, A is identical to that of other enterics. The structural genes of the operon are preceded by a long leader region encoding a 41-residue peptide containing five tryptophan residues. The organization of the leader region suggests that transcription of the operon is subject to attenuation control. The promoter-operator region of the V. parahemolyticus trp operon is almost identical to the corresponding promoter-operator of E. coli. The similarities suggest that promoter strength and operator function are identical in the two species, and that transcription initiation is regulated by repression. The operon appears to lack the internal promoter within trpD that is common in terrestrial enteric species.
The relationship between phenotypic variation and nucleotide sequence variation of the gene encoding Vibrio parahaemolyticus thermostable direct haemolysin (tdh gene) was examined. Strains showing a typical haemolysin-positive phenotype carried two chromosomal gene copies (designated tdh1 and tdh2) while tdh-gene-positive strains showing a weakly positive or negative haemolysin phenotype possessed only a single chromosomal gene copy. Both gene copies from a typical haemolysin-positive strain were cloned and sequenced and possessed 97.2% homology. Comparison of the amino acid sequence predicted from the nucleotide sequence with the protein sequence determined by Edman degradation as well as construction of a tdh1-deficient yet haemolytic strain of V. parahaemolyticus suggest that the tdh2 locus is primarily responsible for the haemolytic phenotype. Two other tdh gene copies were cloned from a phenotypically negative strain which was unusual in that it contained one gene copy on a plasmid (designated tdh4) in addition to a single copy on the chromosome (tdh3). Both tdh3 and tdh4 were expressed in Escherichia coli and TDHs with haemolytic activity were produced. These gene copies were sequenced and shared 96.7% homology with the tdh1 gene. The V. parahaemolyticus strain carrying tdh3 and tdh4 gene copies did not produce detectable amount of tdh-specific RNA transcript. It seems, therefore, that differences in the transcriptional control are primarily responsible for the differences seen in haemolytic phenotype.
We have previously identified a unique site, pac, from which packaging of precursor concatameric viral DNA into proheads starts during the maturation process of bacteriophage CP-T1. The direction of this packaging was determined from restriction enzyme cleavage patterns of CP-T1 DNA. A restriction enzyme generated fragment containing pac was cloned and the surrounding DNA region sequenced. Analysis of the nucleotide sequence revealed numerous repeat regions related to the consensus sequence PuagttGAT.AAT.aa.t. Within the sequenced region an open reading frame encoding a 12260 Mr protein was also identified. This protein appears to share homology with the binding domains of known DNA binding proteins and may represent a putative Pac terminase possessing the specific endonuclease activity required for cleavage at the pac site. Minicell analysis of deletion derivatives of the pac-containing clone revealed a protein of approximately 12,900 Mr encoded within this same region, confirming that this Pac protein is phage encoded.
The cellular location of the haemolysin of Vibrio cholerae El Tor strain 017 has been analyzed. This protein is found both in the periplasmic space and the extracellular medium in Vibrio cholerae. However, when the cloned gene, present on plasmid pPM431, is introduced into E. coli K-12 this protein remains localized predominantly in the periplasmic space with no activity detected in the extracellular medium. Mutants of E. coli K-12 (tolA and tolB) which leak periplasmic proteins mimic excretion and release the haemolysin into the growth medium. Secretion of haemolysin into the periplasm is independent of perA (envZ) and in fact, mutants in perA (envZ) harbouring pPM431 show hyperproduction of periplasmic haemolysin. These results in conjunction with those for other V. cholerae extracellular proteins suggest that although E. coli K-12 can secrete these proteins into the periplasm, it lacks a specific excretion mechanism, present in V. cholerae, for the release of soluble proteins into the growth medium.
The 25-kDa peptidoglycan-associated outer-membrane protein and most likely porin of Vibrio cholerae is a major immunogenic species. It has been purified by ion-exchange elution on hydroxyapatite followed by gel filtration on Bio-Gel P150 both in the presence of sodium dodecyl sulfate. This protein, of greater than 90% purity as judged by Western blotting, has been used to raise antibodies in rabbits. The antisera were then used to screen V. cholerae gene banks, constructed in Escherichia coli K12, and this has enabled us to isolate several colonies harbouring the cloned gene. The plasmids in these colonies have been designated pPM451, pPM455 and pPM472. These plasmids have a 5.3 X 10(3)-base BamHI fragment of V. cholerae DNA in common. Restriction endonuclease mapping of these plasmids has been performed and the protein identified both by Western blot analysis and in E. coli K12 minicells. The protein is not efficiently expressed in E. coli K12. It is proposed to use the name ompV to describe the structural gene, present in the cloned DNA, for this V. cholerae outer membrane protein.