No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Surface-induced swarmer cell differentiation of Vibrio parahaemolyticus
Abstract
Vibrio parahaemolyticus distinguishes between life in a liquid environment and life on a surface. Growth on a surface induces differentiation from a swimmer cell to a swarmer cell type. Each cell type is adapted for locomotion under different circumstances. Swimmer cells synthesize a single polar flagellum (Fla) for movement in a liquid medium, and swarmer cells produce an additional distinct flagellar system, the lateral flagella (Laf), for movement across a solid substratum, called swarming. Recognition of surfaces is necessary for swarmer cell differentiation and involves detection of physical signals peculiar to that circumstance and subsequent transduction of information to affect expression of swarmer cell genes (laf). The polar flagellum functions as a tactile sensor controlling swarmer cell differentiation by sensing forces that restrict its movement. Surface recognition also involves a second signal, i.e. nutritional limitation for iron. Studying surface-induced differentiation could reveal a novel mechanism of gene control and lead to an understanding of the processes of surface colonization by pathogens and other bacteria.
... Hyperflagellation was supported by the transcriptional induction of flagellar gene expression. Transcriptional induction of flagellar gene expression in response to growth on hard agar media is also observed in other swarm bacteria including B. subtilis, Vibrio spp., Proteus mirabilis, and Serratia marcescens[36,[50][51][52][53]. An artificial increase in the number of flagella allows bacteria to swarm on harder agars or to swim through more viscous media in B. subtilis, Salmonera enterica, Proteus mirabilis, Pseudomonas aeruginosa[35][36][37][38]. ...
... Lateral flagella are driven by the H +dependent stator and provide the impetus for swarming motility[54]. The formation of lateral flagella and their stators is transcriptionally induced in response to growth on hard agar media[53]. However, unlike Vibrio spp., transcription of both motAB and motCD operons of Paenibacillus sp. was induced on hard agar. ...
Bacteria have developed various motility mechanisms to adapt to a variety of solid surfaces. A rhizosphere isolate, Paenibacillus sp. NAIST15-1, exhibited unusual motility behavior. When spotted onto 1.5% agar media, Paenibacillus sp. formed many colonies, each of which moved around actively at a speed of 3.6 μm/sec. As their density increased, each moving colony began to spiral, finally forming a static round colony. Despite its unusual motility behavior, draft genome sequencing revealed that both the composition and organization of flagellar genes in Paenibacillus sp. were very similar to those in Bacillus subtilis. Disruption of flagellar genes and flagellar stator operons resulted in loss of motility. Paenibacillus sp. showed increased transcription of flagellar genes and hyperflagellation on hard agar media. Thus, increased flagella and their rotation drive Paenibacillus sp. motility. We also identified a large extracellular protein, CmoA, which is conserved only in several Paenibacillus and related species. A cmoA mutant could neither form moving colonies nor move on hard agar media; however, motility was restored by exogenous CmoA. CmoA was located around cells and enveloped cell clusters. Comparison of cellular behavior between the wild type and cmoA mutant indicated that extracellular CmoA is involved in drawing water out of agar media and/or smoothing the cell surface interface. This function of CmoA probably enables Paenibacillus sp. to move on hard agar media.
... Both V. alginolyticus and its close relative V. parahaemolyticus use a single polar flagellum for swimming motility in liquid medium, but on solid surfaces they differentiate into elongated swarmer cells with multiple lateral flagella (11). Both species are among the most frequently encountered marine bacteria, are known to cause diseases in vertebrate and invertebrate marine animals, and are emerging human pathogens especially in food poisoning and wound infections (12,13). ...
... These strains rely on dual flagellar systems, whereby a single polar flagellum is the propulsion system for swimming activity, whereas lateral flagella are the driving force for swarming on solid media. The production of lateral flagella only is induced under high viscosity when the rotation of the polar flagellum is blocked (11,35). The best results for swarming activity were obtained with a commercial k-carrageenan mixture for gel preparations, which was used as standard method in this study, whereas i-carrageenan resulted in a similar but more diffuse swarming morphology. ...
Swarming represents a special case of bacterial behavior where motile bacteria migrate rapidly and collectively on surfaces. Swarming and swimming motility of bacteria has been studied well for rigid, self-propelled rods. In this study we report a strain of Vibrio alginolyticus, a species that exhibits similar collective motility but a fundamentally different cell morphology with highly flexible snake-like swarming cells. Investigating swarming dynamics requires high-resolution imaging of single cells with coverage over a large area: thousands of square microns. Researchers previously have employed various methods of motion analysis but largely for rod-like bacteria. We employ temporal variance analysis of a short time-lapse microscopic image series to capture the motion dynamics of swarming Vibrio alginolyticus at cellular resolution over hundreds of microns. Temporal variance is a simple and broadly applicable method for analyzing bacterial swarming behavior in two and three dimensions with both high-resolution and wide-spatial coverage. This study provides detailed insights into the swarming architecture and dynamics of Vibrio alginolyticus isolate B522 on carrageenan agar that may lay the foundation for swarming studies of snake-like, nonrod-shaped motile cell types.
... V. parahaemolyticus produces the polar flagellum either in liquid or on a surface, the lateral flagella are produced only under conditions in which V. parahaemolyticus is growing on the surface of solidified media [9,10]. Thus, the polar flagellar genes are believed to be expressed rather constitutively, however, the laf genes would only be activated under some specific circumstances, like touching the surface, or an increase in viscosity, or the addition of anti-polar flagellum antiserum to the culture medium [10,11]. ...
Vibrio parahaemolyticus, a significant food-borne pathogen that causes economic and public health problems worldwide, can produce two types of flagella, the single polar flagellum responsible for swimming in a liquid environment and the lateral flagella (laf) that enable the bacteria to swarm on the tops of solid surfaces. The polar flagellar genes are expressed either in liquid or on a surface, however, laf genes would only be activated by surface sensing. In this study, the molecular mechanism of surface sensing activating laf gene expression in V. parahaemolyticus was investigated. We found that the c-di-GMP concentration for liquid-grown cells was higher than the concentration for surface-grown cells and laf gene expression could be activated without touching surface if the intracellular level of c-di-GMP was decreased by overexpressing the scrABC operon in the wild-type strain. Surface sensing inhibits the transcription of those c-di-GMP-metabolizing enzymes which could negatively regulate swarming, and enhances those which could positively regulate swarming. Surface sensing also enhances the activity of quorum sensing (QS) of V. parahaemolyticus which plays an important role in regulating the transcription of components of the c-di-GMP network. Combined, the data indicate that V. parahaemolyticus enhances QS by surface sensing to down-regulate the intracellular level of c-di-GMP which results in producing the lateral flagella and swarming on a surface. What we found in this study suggests an important signal transduction pathway of regulating swarming motility by surface sensing in V. parahaemolyticus.
... V. parahaemolyticus genome contains approximately 50 genes for the polar flagellum and about 40 genes for the lateral flagella, located on its large and small chromosomes, respectively [3]. The expression of the polar flagellum is constitutive, whereas the production of lateral flagella depends on specific conditions, such as growth on surfaces and iron limitation [4]. Although there are numerous disparities in the genetic structure and expression patterns between the polar and lateral flagella of V. parahaemolyticus, it is fascinating to note that they can be regulated by the same set of regulators. ...
Vibrio parahaemolyticus is equipped with two distinct flagellar systems: a polar flagellum and numerous lateral flagella. The polar flagellum plays a role in propelling swimming in liquids, while the lateral flagella serve to enhance swarming on surfaces or in viscous environments. H-NS is a histone-like nucleoid structuring protein that plays a regulatory role in both the swimming and swarming motility of V. parahaemolyticus. However, the detailed mechanisms have not been fully understood. In this study, we have demonstrated that the deletion of hns hindered the growth rate of V. parahaemolyticus during the logarithmic growth phase and significantly decreased the swimming motility. H-NS directly activated the transcription of flgMN, flgAMN, flgBCDEFGHIJ, and flgKL-flaC located within the polar flagellar gene clusters. The expression of H-NS in Escherichia coli led to a marked elevation in the expression levels of flgM, flgA, flgB, and flgK, suggesting the positive effect of H-NS on the expression of polar flagellar genes in E. coli. This work demonstrates that the positive regulation of H-NS on the swimming motility in V. parahaemolyticus may be achieved through its regulation of polar flagellar gene expression and bacterial growth.
... While the molecular mechanisms of surface sensing are still unclear, it is well known that the flagella and type 4 pili (T4P) are required not only for detecting surface contact but also for signal transmission (2)(3)(4)(5)(6)(7). For example, pioneering work by McCarter and Silverman in 1988 (8,9), show that decreased rotation of the polar flagellum in Vibrio parahaemolyticus upon surface contact initiated a signal transduction pathway that triggered production of lateral flagella. Analogous work in Pseudomonas aeruginosa demonstrate that upon surface contact there is increased production of adherent pili (10). ...
To initiate biofilm formation, it is critical for bacteria to sense a surface and respond precisely to activate downstream components of the biofilm program. Type 4 pili (T4P) and increasing levels of c-di-GMP have been shown to be important for surface sensing and biofilm formation, respectively; however, mechanisms important in modulating the levels of this dinucleotide molecule to define a precise output response are unknown. Here, using macroscopic bulk assays and single-cell tracking analyses of Pseudomonas aeruginosa, we uncover a role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two-hybrid and bimolecular fluorescence complementation assays, combined with genetic studies, are consistent with a model whereby PilO interacts with SadC and that the PilO-SadC interaction inhibits SadC's activity, resulting in decreased biofilm formation and increased mo-tility. Using single-cell tracking, we monitor both the mean c-di-GMP and the variance of this dinucleotide in individual cells. Mutations that increase PilO-SadC interaction modestly, but significantly, decrease both the average and variance in c-di-GMP levels on a cell-by-cell basis, while mutants that disrupt PilO-SadC interaction increase the mean and variance of c-di-GMP levels. This work is consistent with a model wherein P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this dinucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement. bacterial biofilms | Pseudomonas aeruginosa | c-di-GMP | alignment complex | surface sensing
... While the molecular mechanisms of surface sensing are still unclear, it is well known that the flagella and type 4 pili (T4P) are required not only for detecting surface contact but also for signal transmission (2)(3)(4)(5)(6)(7). For example, pioneering work by McCarter and Silverman in 1988 (8,9), show that decreased rotation of the polar flagellum in Vibrio parahaemolyticus upon surface contact initiated a signal transduction pathway that triggered production of lateral flagella. Analogous work in Pseudomonas aeruginosa demonstrate that upon surface contact there is increased production of adherent pili (10). ...
Significance
Type 4 pili (T4P) of Pseudomonas aeruginosa are important for surface sensing and regulating intracellular c-di-GMP levels during biofilm formation. This work supports a role for the T4P alignment complex, previously known for supporting pili biogenesis, in surface-dependent signaling. Our findings indicate that P. aeruginosa uses a diguanylate cyclase, via a complex web of protein–protein interactions, to integrate signaling through the T4P and the flagellar motor to fine-tune c-di-GMP levels. A key implication of this work is that more than just regulating signal levels, cells must modulate the dynamic range of c-di-GMP to precisely control the transition to a biofilm lifestyle.
... It was suggested that this bacterial adaptation was due to loss of rotation of its polar flagellum probably due to attachment to a surface. This fact was supported by the experimental data in which flagellar rotation was blocked by treating with a drug phenamil, that disrupts ion transport in flagellar basal body thus inhibit flagellar rotation and appearance of lateral flagella without sensing a surface [16]. Likewise, similar phenomenon was well explained in Pseudomonas aeruginosa providing novel insights into biofilm development. ...
Biofilms are groups of microrganisms in which cells embedded in extracellular polysaccharide matrix (EPS) stick to each other on a surface. Bacterial polysaccharide is the major component of biofilm matrix and make it stable as well as resistant to disinfectants, biocides and antibiotics. Biofilms can be harmful or beneficial, however unfortunately negative effects outweighs the positive ones. Formation of biofilm may take place on both biotic and abiotic surfaces and may be monospecies or multispecies. Though multispecies biofilm are more prevalent in nature augmenting the need to communicate between inter and intra species. In fact, biofilm formation and quorum sensing (QS) are interconnected. Quorum sensing (QS) is a regulatory system that allows bacteria to produce and detect signal molecules and thereby communicate their behaviour in a cell-density dependent manner. Three main QS systems include: The acylhomoserine lactone (AHL) QS system in Gram-negative bacteria, the autoinducing peptide (AIP) QS system in Gram-positive bacteria and the autoinducer-2 (AI-2) QS system in both Gram-negative and-positive bacteria. Biofilm formation and associated problems are a serious emerging issue in household, industry and medical settings. Many antibiofilm strategies were suggested to cope with its formation rather than removal. Among these, four commonly include are: (1) use of broad spectrum antibiotics/biocides, (2) interference with bacterial EPS production, (3) quorum sensing (QS), and (4) flagellar assembly. A comprehensive knowledge of bacterial biofilm will help to develop effective control measures to prevent its formation at initial stages and to manage associated problems later, once developed and got mature completely.
... Also, the bacteria on the SS surface were stretched to some extent with an average cell length of 4.13 μm, while the morphology of the biofilm cells on the PS surface did not significantly change. Study showed that V. parahaemolyticus can produce a surface adaptation program when colonizes on the surface, which allows bacteria to transform from swimming cells (about 2 μm) to slender swarming cells (1-30 μm), thus facilitates effective cell colonization on the surface (Gode-Potratz et al., 2011;McCarter, 1999;McCarter and Silverman, 1990;Mizan et al., 2016). ...
Vibrio parahaemolyticus is a kind of gram-negative marine pathogen, which usually adheres to stainless steel (SS), glass (GS) and other abiotic surfaces in aquaculture and food processing in the form of biofilm and causes the spread of gastrointestinal illness. However, the deeply survival adaptation mechanism of V. parahaemolyticus biofilm cells on these contact surface remained unclear. Here, proteomics was used to investigated the physiological response of the V. parahaemolyticus biofilms cells to different abiotic surfaces (SS, GS and polystyrene (PS)). In addition, the effect of contact materials on the physical-chemical properties of biofilms are also characterized. Results showed that the expression of proteins of biofilm cells established on the SS surface were mainly related to the alleviation of metal ion stress and toxicity. The up-regulated proteins in the biofilm cells formed on the GS surface were mainly involved in the biological processes of sugar uptake, protein synthesis and bacterial chemotaxis. Meanwhile, the significantly expressed proteins in the biofilm cells formed on the PS surface were mainly involved in the cellular physiological activity of aromatic compound metabolism, osmotic stress and nutrient transport. All functional proteins mentioned above were closely related to the interaction characteristics of the contact surface and biofilm. This study provided an in-depth comparison of V. parahaemolyticus biofilm formation on these three abiotic surfaces, and presented a model in first time for the adaptation behavior of biofilm cells on different surfaces as affected by metal ion stress, nutrition, osmotic stress, and sugar utilization, which could facilitate an efficient control strategy for biofilm formation in industrial field.
... Swarmer cells are described as elongated and hyperflagellated cells, that are able to migrate towards the edge of a swarming plate or a nutrient-rich surface, away from the initial colony [35] [36] [37]. We have previously examined E. coli cells in a planktonic phase that lack or overexpress the cyclophilin PpiB and found that in both cases the present an impaired cell division [38]. ...
FklB is a member of the FK506-binding proteins (FKBPs), a family that consists of five genes in Escherichia coli. Little is known about the physiological and functional role of FklB in bacterial movement. In the present study, FklB knock-out mutant ΔfklB presented an increased swarming and swimming motility and biofilm formation phenotype, suggesting that FklB is a negative regulator of these cellular processes. Complementation with Peptidyl-prolyl isomerase (PPIase)-deficient fklB gene (Y181A) revealed that the defects in biofilm formation were not restored by Y181A, indicating that PPIase activity of FklB is modulating biofilm formation in E. coli. The mean cell length of ΔfklB swarming cells was significantly smaller as compared to the wild-type BW25113. Furthermore, the mean cell length of swarming and swimming wild-type and ΔfklB cells overexpressing fklB or Y181A was considerably larger, suggesting that PPIase activity of FklB plays a role in cell elongation and/or cell division. A multi-copy suppression assay demonstrated that defects in motility and biofilm phenotype were compensated by overexpressing sets of PPIase-encoding genes. Taken together, our data represent the first report demonstrating the involvement of FklB in cellular functions of E. coli.
... The V. parahaemolyticus contains two flagellar systems: polar flagellum and lateral flagellum. The polar flagellum is encoded by the polar flagellar genes responsible for the swimming motility, and the lateral flagellum is responsible for the swarming motility encoded by lateral flagellar genes [11]. A previous study also indicated that V. parahaemolyticus lacking flagellum could not adhere to either normal or degenerating tissue cells [12]. ...
Motility is important for virulence, biofilm formation, and the environmental adaptation of many bacteria. Vibrio parahaemolyticus (V. parahaemolyticus) contains two flagellar systems that are responsible for motility, and are tightly regulated by transcription regulators and sigma factors. In this study, we identified a novel transcription factor, VPA1701, which regulates the swarming motility of V. parahaemolyticus. The VPA1701 deletion mutant (ΔVPA1701) eliminated the swarming motility on the surface of BHI agar plates and reduced colonization in infant rabbits. RNA-seq assays, confirmed by qRT-PCR, indicated that VPA1701 regulated the expression of lateral flagellar cluster genes. Further analyses revealed that VPA1701 directly binds to the promoter region of the flgBCDEFGHIJKL cluster to regulate the expression of lateral flagellar genes. CalR was originally identified as a repressor for the swarming motility of V. parahaemolyticus, and it was inhibited by calcium. In this study, we found that VPA1701 could inhibit the expression of the calR gene to increase the swarming motility of V. parahaemolyticus. Calcium downregulated the expression of calR, indicating that calcium could increase swarming motility of ΔVPA1701 by inhibiting calR. Thus, this study illustrates how the transcription factor VPA1701 regulates the expression of lateral flagellar genes and calR to control the swarming motility of V. parahaemolyticus.
... Particularly, in liquid environments it exits as a short motile cell that is propelled by a single polar flagellum. However, when it attaches to solid surfaces, V. parahaemolyticus possesses the ability to induce a distinct differentiation program, which allows it to adapt to changes in its environment and colonize solid surfaces by means of swarm motility [1,[8][9][10][11][12]. One of the first steps in swarmer differentiation is inhibition of cell division, resulting in highly elongated rod-shaped filamentous swarmer cells (Fig. 1a). ...
Bacteria experience changes in their environment and have developed various strategies to respond accordingly. To accommodate environmental changes, certain bacteria differentiate between specialized cell types. Vibrio parahaemolyticus is a marine bacterium, a worldwide human pathogen and the leading agent of seafood-borne gastroenteritis. It exists as swimmer or swarmer cells, specialized for life in liquid and on solid environments, respectively. Swarmer cells are characteristically highly elongated—a morphology important for swarming behavior. When attached to surfaces it forms swarm colonies, however, it is not known how cells within swarming populations respond to changes in the external milieu and how its distinct life cycle influences its ecological dissemination. The worldwide distribution of V. parahaemolyticus accentuates the need for understanding the factors contributing to its dissemination. Here we determine the stage-wise development of swarm colonies and show how the swarm colony architecture fluctuates with changing environmental conditions. Swarm colonies act as a continuous source of cells that are released from the swarm colony into the environment. Surprisingly, the cell length distribution of released cells was very homogenous and almost no long cells were detected, indicating that swarmer cells are not released into the liquid environment but stay surface attached during flooding. Released cells comprise a distinct cell type that is morphologically optimized for swimming behavior and is capable of spreading in the liquid environment and attach to new surfaces. Release of this distinct cell type facilitates the dissemination of V. parahaemolyticus in the environment and likely influences the ecology of this bacterium.
... V. parahaemolyticus dsb mutant strains were still motile ( Figure 3B). We speculate that if the polar flagellum stops working, which might be the case in the dsb mutants, the lateral flagella will be activated and enable V. parahaemolyticus to remain motile (McCarter et al., 1988;McCarter and Silverman, 1989). ...
Bacterial pathogens maintain disulfide bonds for protein stability and functions that are required for pathogenesis. Vibrio parahaemolyticus is a Gram-negative pathogen that causes food-borne gastroenteritis and is also an important opportunistic pathogen of aquatic animals. Two genes encoding the disulfide bond formation protein A, DsbA, are predicted to be encoded in the V. parahaemolyticus genome. DsbA plays an important role in Vibrio cholerae virulence but its role in V. parahaemolyticus is largely unknown. In this study, the activities and functions of the two V. parahaemolyticus DsbA proteins were characterized. The DsbAs affected virulence factor expression at the post-translational level. The protein levels of adhesion factor VpadF (VP1767) and the thermostable direct hemolysin (TDH) were significantly reduced in the dsbA deletion mutants. V. parahaemolyticus lacking dsbA also showed reduced attachment to Caco-2 cells, decreased β-hemolytic activity, and less toxicity to both zebrafish and HeLa cells. Our findings demonstrate that DsbAs contribute to V. parahaemolyticus pathogenesis.
... Under suitable conditions, bacterial cells differentiate into hyperflagellated swarmers that are more motile on wet and viscous surfaces (Harshey, 2003). Biosurfactants produced by bacteria can change the viscosity of surfaces, thereby influencing cell differentiation and motility (McCarter and Silverman, 1990;Allison et al., 1993). Several studies showed that biosurfactants produced by pseudomonads and other bacteria can promote their surface motility. ...
Pseudomonas sp. 11K1, originally isolated from rhizosphere, possesses inhibitory activity against plant pathogenic fungi and bacteria. Herein, the genome of strain 11K1 was sequenced and subjected to in silico, mutational, and functional analyses. The 11K1 genome is 6,704,877 bp in length, and genome mining identified three potential cyclic lipopeptide (CLP) biosynthetic clusters, subsequently named brasmycin, braspeptin, and brasamide. Insertional and deletion mutants displayed impaired brasmycin and braspeptin production, and lost antifungal activity, but retained antibacterial activity against Xanthomonas oryzae. The structures of these two active CLPs were predicted based on adenylation (A) domains. Brasmycin is composed of nine amino acids and belongs to the syringomycin class, while braspeptin is a 22 amino acid cyclic peptide belonging to the tolaasin group. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry analysis revealed that brasmycin and braspeptin have different molecular weights compared with known syringomycin and tolaasin members, respectively. Mutation of brasmycin and braspeptin gene clusters affected both biofilm formation and colony morphology. Collectively, these results indicate that Pseudomonas sp. 11K1 produces two novel CLPs that may help bacteria compete for nutrients and niches in the environment.
... For bacteria such as V. parahaemolyticus, the increase in flagella number is differently regulated as it concerns the activation of lateral flagella system in addition to their polar flagellum (Partridge & Harshey, 2013). In V. parahaemolyticus bacteria, the transcription of the laf gene cluster is activated upon encountering solid environments, resulting in the formation of peritrichous lateral flagella and swarming motility (R. Belas, Simon, & Silverman, 1986;McCarter & Silverman, 1990;Shinoda & Okamoto, 1977). The transcription of the laf genes appears to be linked to the rotation rate of the polar flagellum. ...
Bacteria thrive in virtually all environments. Like all other living organisms, bacteria may encounter various types of stresses, to which cells need to adapt. In this chapter, we describe how cells cope with stressful conditions and how this may lead to dramatic morphological changes. These changes may not only allow harmless cells to withstand environmental insults but can also benefit pathogenic bacteria by enabling them to escape from the immune system and the activity of antibiotics. A better understanding of stress-induced morphogenesis will help us to develop new approaches to combat such harmful pathogens.
... It was suggested that this bacterial adaptation was due to loss of rotation of its polar flagellum probably due to attachment to a surface. This fact was supported by the experimental data in which flagellar rotation was blocked by treating with a drug phenamil, that disrupts ion transport in flagellar basal body thus inhibit flagellar rotation and appearance of lateral flagella without sensing a surface [16]. Likewise, similar phenomenon was well explained in Pseudomonas aeruginosa providing novel insights into biofilm development. ...
... Such a difference may be due to the concentration level of FtsZ in a cell and how it is regulated under various growth conditions. Both elongated V. parahaemolyticus swarmer cells and S. elongatus cells are induced by naturally occurring conditions such as surface attachment (McCarter and Silverman, 1990) and reduced light intensity, respectively. It has been shown that the FtsZ concentration in V. parahaemolyticus swarmer cells is maintained at a level sufficient to enable the formation of only one Z-ring per cell (Muraleedharan et al., 2018). ...
When resources are abundant, many rod-shaped bacteria reproduce through precise, symmetric divisions. However, realistic environments entail fluctuations between restrictive and permissive growth conditions. Here, we use time-lapse microscopy to study the division of the cyanobacterium Synechococcus elongatus as illumination intensity varies. We find that dim conditions produce elongated cells whose divisions follow a simple rule: cells shorter than ∼8 μm divide symmetrically, but above this length divisions become asymmetric, typically producing a short ∼3-μm daughter. We show that this division strategy is implemented by the Min system, which generates multi-node patterns and traveling waves in longer cells that favor the production of a short daughter. Mathematical modeling reveals that the feedback loops that create oscillatory Min patterns are needed to implement these generalized cell division rules. Thus, the Min system, which enforces symmetric divisions in short cells, acts to strongly suppress mid-cell divisions when S. elongatus cells are long.
... In free swimming, or planktonic culture, this rod-shaped bacterium has a single polar flagellum. However, upon surface attachment, V. parahaemolyticus sprouts several lateral flagella, which facilitates a type of surface motility called swarming (11,12). McCarter and Silverman demonstrated that the surface "signal" being perceived by the bacterium was impeded rotation of the polar flagellum (a consequence of being surface associated). ...
... Bacteria swim by rotating propellar-like organelles, called flagella, which extend from the cell surface (9). Pioneering work on the bacterium V. parahaemolyticus revealed that surface-sensing results in the differentiation from free-swimming bacteria that use the rotation of single polar flagellum to elongated bacteria that produce dozens of lateral flagella that facilitate surface colonization and spreading (10). Pili, also called fimbriae, are hair-like structures that extend from the cell surface and are usually associated with surface attachment. ...
Mechanosensing mechanisms for surface recognition by bacteria allow biofilm formation
... V. parahaemolyticus cells possess a single, polar flagellum that is constitutively produced and used for movement in a liquid. In addition, V. parahaemolyticus cells also produce peritrichous (lateral) flagella upon contact with a surface or under iron-limiting conditions [22][23][24][25]. These bacterial structures are known to be involved in surface adherence during biofilm formation and have been linked to virulence [26,27]. ...
The human pathogen Vibrio parahaemolyticus is a leading cause of seafood-borne illness in the USA, and infections with V. parahaemolyticus typically result from eating raw or undercooked oysters. V. parahaemolyticus has been shown to be highly resistant to oyster depuration, suggesting that the bacterium possesses specific mechanisms or factors for colonizing oysters and persisting during depuration. In this study, we characterized eight different V. parahaemolyticus strains for differences in resistance to oyster depuration, biofilm formation, and motility. While each strain exhibited distinct phenotypes in the various assays, we determined that biofilm formation on abiotic surfaces, such as glass or plastic, does not directly correlate with bacterial retention in oysters during depuration. However, we did observe that the motility phenotype of a strain appeared to be a better indicator for persistence in the oyster. Further studies examining the molecular mechanisms underlying the observed colonization differences by these and other V. parahaemolyticus strains may provide beneficial insights into what critical factors are required for proficient colonization of the Pacific oyster.
... donidellus) following grass carp reovirus (GCRV) infection [23]. The flagella of V. parahaemolyticus are of particular interest because this organism possesses two distinct types of flagella that propel the bacterium under different circumstances [27]. Flagellin is the main structural component of flagella in Gram positive and negative bacteria, where it appears as a filamentous appendage on the bacterial surface and it is involved in motility, attachment, and chemotaxis [28]. ...
Toll-like receptor 5 (TLR5) is a member of TLRs family responsible for the bacterial flagellin recognition in vertebrates. Herein, the TLR5M gene structure of Pacific red snapper (Lutjanus peru) was characterized. The full-length cDNA of LpTLR5M include an open reading frame (ORF) of 2715 bp encoding a polypeptide of 904 amino acids including 9 LRRs (residues 119–562) and one LRR-CT domain (residues 593–646) at the extracellular region, and a TIR domain (residues 710–904) in the cytoplasmic region. The amino acid sequence in L. peru TLR5 showed high identity (66–69%) with TLR5 from Paralichthys olivaceus and Scophthalmus maximus. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of LpTLR5M mRNA in all examined tissues with higher levels in intestine, liver and head-kidney. Furthermore, the expression of LpTLR5M and five cytokine genes was also investigated 24 h and one week post-stimulation in fish intraperitoneally injected with ToxA or Lysate antigens and live V. parahaemolyticus (Vp). TLR5M was significantly induced in fish infected with Vp. The pro-inflammatory cytokines IL-6, IL8 and IL-12 were significantly up-regulated in head-kidney in fish stimulated with Vp, while in intestine, these increases were observed after ToxA or Lysate injection. In contrast, IL-17 mRNA transcript was significantly up-regulated in fish infected with live Vp in intestine and sampled 24 h later. The results indicate that Lysate and Vp antigens can induce an immune response via TLR5M and that cytokines have an important role in the defense mechanisms against V. parahaemolyticus.
... The early reports of cellular differentiation in bacteria upon surface attachment (a switch from swimming mode to swarming mode) were related to the synthesis of extracellular structures like pili and multiple lateral flagella [32]. Besides pili and flagella, changes in cell shape and cell size have been monitored in the growing biofilms [33,34]. ...
Phenotypic changes or phase variation within biofilms is an important feature of bacterial dormant life. Enhanced resistance to antimicrobials is one of the distinct features displayed by a fraction of cells within biofilms. It is believed that persisters are mainly responsible for this phenotypic heterogeneity. However, there is still an unresolved debate on the formation of persisters. In this short review, we highlight all known genomic and proteomic changes encountered by bacterial cells within biofilms. We also describe all phenotypic changes displayed by bacterial cells within biofilms with particular emphasis on enhanced antimicrobial tolerance of biofilms with particular reference to persisters. In addition, all currently known models of persistence have been succinctly discussed.
... Cloning of the A. hydrophila AH-3 alternative sigma factor 54 (rpoN). In order to clone the A. hydrophila alternative sigma factor 54 (rpoN), the genomic library of A. hydrophila AH-3 (28) was transferred by mating into rifampin-resistant V. cholerae rpoN in-frame deletion mutant strain KKV56 (21). Transconjugants were selected for rifampin and tetracycline resistance and inoculated into LB agar (0.3% agar) plates supplemented with 2 mM glutamine and tetracycline. ...
... A distinct type of differentiation utilized by many bacteria, including species of Serratia (Alberti and Harshey, 1990), Aeromonas (Kirov et al., 2002), Salmonella (Harshey, 1994; Harshey and Matsuyama, 1994), Proteus (Rather, 2005), and Vibrio (McCarter, 2004), is the differentiation between a planktonic swimmer cell and a swarmer cell that is specialized for movement over solid surfaces or in viscous environments (McCarter, 2004). One organism that undergoes such differentiation between swimmer and swarmer cells is Vibrio parahaemolyticus, a worldwide human pathogen and major cause of seafood related gastroenteritis (McCarter and Silverman, 1990; McCarter, 1999 McCarter, , 2004 McCarter, , 2010 Makino et al., 2003; Stewart and McCarter, 2003;). In V. parahaemolyticus swimmer cells are short rod-shaped cells that – as the name suggests -are optimized for swimming in liquid environments. ...
When encountering new environments or changes to their external milieu, bacteria use elaborate mechanisms to respond accordingly. Here, we describe how Vibrio parahaemolyticus coordinates two such mechanisms – differentiation and chemotaxis. V. parahaemolyticus differentiates between two distinct cell types: short rod-shaped swimmer cells and highly elongated swarmer cells. We show that the intracellular organization of chemotactic signaling arrays changes according to the differentiation state. In swimmer cells chemotaxis arrays are strictly polarly localized, but in swarmer cells arrays form both at the cell poles and at irregular intervals along the entire cell length. Furthermore, the formation of lateral arrays increases with cell length of swarmer cells. Occurrence of lateral signaling arrays is not simply a consequence of the elongated state of swarmer cells, but is instead differentiation state-specific. Moreover, our data suggest that swarmer cells employ two distinct mechanisms for localization of polar and lateral signaling arrays, respectively. Furthermore, cells show a distinct differentiation and localization pattern of chemosensory arrays, depending on their location within swarm colonies, which likely allows for the organism to simultaneously swarm across surfaces while sustaining a pool of swimmers immediately capable of exploring new liquid surroundings.
... Moreover, 50% to 60% of mesophilic aeromonads are able to produce many unsheathed peritrichous lateral flagella when grown in viscous environments or over surfaces (58), which increase bacterial adherence and are required for swarming motility and biofilm formation (23). The expression of two distinct flagellar systems is relatively uncommon, although it has been observed with Vibrio parahaemolyticus (39), Azospirillum brasilense (45), Rhodospirillum centenum (32), Helicobacter mustelae (49), and Plesiomonas shigelloides (29). ...
Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.
... Salmonella enterica, the most frequent cause of food-borne disease outbreaks worldwide [12], is able to swarm on soft agar surfaces (0.5-0.8% agar) and is thus considered a temperate swarmer [13]. During swarming, the morphology of temperate swarmers does not significantly change, and processes such as elongation, the formation of multi-nucleoid cells, and hyperflagellation are not observed, unlike in robust swarmers such as Proteus and Vibrio species [14,15]. While swarming by Salmonella is clearly related to bacterial invasion and the that induction of the SOS response impairs swarming motility by reversibly bypassing chemoreptor polar array assembly, through a disturbance of the balance between RecA and CheW. ...
Swarming, a flagellar-driven multicellular form of motility, is associated with bacterial virulence and increased antibiotic resistance. In this work we demonstrate that activation of the SOS response reversibly inhibits swarming motility by preventing the assembly of chemoreceptor-signaling polar arrays. We also show that an increase in the concentration of the RecA protein, generated by SOS system activation, rather than another function of this genetic network impairs chemoreceptor polar cluster formation. Our data provide evidence that the molecular balance between RecA and CheW proteins is crucial to allow polar cluster formation in Salmonella enterica cells. Thus, activation of the SOS response by the presence of a DNA-injuring compound increases the RecA concentration, thereby disturbing the equilibrium between RecA and CheW and resulting in the cessation of swarming. Nevertheless, when the DNA-damage decreases and the SOS response is no longer activated, basal RecA levels and thus polar cluster assembly are reestablished. These results clearly show that bacterial populations moving over surfaces make use of specific mechanisms to avoid contact with DNA-damaging compounds.
... Iron-regulated genes, including those encoding the enterobactin and ferrichrome receptors, were also induced when the bacteria were grown on the surface of agar medium. This finding is consistent with data from previous studies showing that restricted diffusion of iron in agar media limits its availability to V. parahaemolyticus cells growing on surfaces, and iron is a key signal for swarmer cell differentiation (251). Differentiation in response to iron limitation requires Fur, but it is not known whether the effect of Fur is direct or indirect (L. ...
Iron is an essential element for
Vibrio
spp., but the acquisition of iron is complicated by its tendency to form insoluble ferric complexes in nature and its association with high-affinity iron-binding proteins in the host. Vibrios occupy a variety of different niches, and each of these niches presents particular challenges for acquiring sufficient iron.
Vibrio
species have evolved a wide array of iron transport systems that allow the bacteria to compete for this essential element in each of its habitats. These systems include the secretion and uptake of high-affinity iron-binding compounds (siderophores) as well as transport systems for iron bound to host complexes. Transporters for ferric and ferrous iron not complexed to siderophores are also common to
Vibrio
species. Some of the genes encoding these systems show evidence of horizontal transmission, and the ability to acquire and incorporate additional iron transport systems may have allowed
Vibrio
species to more rapidly adapt to new environmental niches. While too little iron prevents growth of the bacteria, too much can be lethal. The appropriate balance is maintained in vibrios through complex regulatory networks involving transcriptional repressors and activators and small RNAs (sRNAs) that act posttranscriptionally. Examination of the number and variety of iron transport systems found in
Vibrio
spp. offers insights into how this group of bacteria has adapted to such a wide range of habitats.
... V. parahaemolyticus possesses multiple cell types appropriate for life under different circumstances (Belas et al., 1986;McCarter, 2000McCarter, , 2001McCarter & Silverman, 1990;McCarter et al., 1988). The swimmer cell, with a single polar flagellum, is adapted to life in liquid environments. ...
The importance of combating infectious diseases has received international attention, p- viding the opportunity for a multidisciplinary approach that combines medicine with other scienti? candtechnologicalcapabilities,notablyinformationtechnology,nanotechnology,and biotechnology. In fact, it has been predicted that the future will bring a merging of these te- nologies with the cognitive and behavioral sciences-major forces that have the potential to balancetheworld'sinequities.Thescienti?ccommunityandworldleadersmustworktogether to use knowledge and its applications to improve the condition of the planet. The connection between infectious diseases and the oceans provides a paradigm for this perspective. A stark global context indisputably frames all human health issues in the twenty-?rst century: the world wide movement of people and goods. Throughout the past half century, international travel has skyrocketed; there are more than 500 million international arrivals per year. The greatest increase has taken place since the mid-1990s. The world has become integrated and global; consequently, the notion that it is possible to successfully eradicate a disease from the face of the planet has become simplistic. Infectious disease is a moving target and climate shifts will affect any disease that has an environmentally sensitive stage or vector. Recognizingsignalsfromclimatemodelsandincorporatingthemintohealthmeasurescanp videnewopportunitiesforproactive-ratherthanreactive-approachestopublichealth.Thus, careful attention to the role of the oceans in human health can offer new avenues of research that will provide new means of predicting and preventing those diseases that are rooted in the environment. In this volume, pathogens in the sea are reviewed by Colin Munn, who provides a broader perspective for the topic of pathogenic microorganisms associated with the world oceans. © 2005 Springer Science+Business Media, Inc. All rights reserved.
... H + -driven, in V. alginolyticus (35). However, the H + -dependent lateral flagella is induced in response to an increase in external viscosity (36). V. alginolyticus uses a distinct mechanism to maintain high protein export activity in different ionic environments by exchanging the SecDF paralogs (Fig. 7A). ...
Significance
Bacteria living in seawater must cope with low-sodium environments that they may encounter. Here we show an unexpected finding that remodeling of the Sec protein export machinery plays a pivotal role in this adaptation. Vibrio alginolyticus possesses alternative SecDF1 and SecDF2 homologs that use the transmembrane gradient of Na ⁺ and that of H ⁺ , respectively, to enhance protein export by cooperating with the SecYEG translocon. The synthesis of SecDF2 is induced in low-Na ⁺ environments, and this induction is essential for the bacterium to survive low salinity. Remarkably, the Vibrio species use a nascent polypeptide, VemP, to monitor the functional state of the Sec pathway and to up-regulate translation of SecDF2 when activity of the SecDF1-containing Sec machinery declines.
Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3′,5′-cyclic AMP (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the type IV pilus retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations in PilT, and in particular those impacting the ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its PilT retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current T4P-dependent surface sensing models for P. aeruginosa.
IMPORTANCE T4P are cellular appendages that allow P. aeruginosa to sense a surface, leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.
The importance of memory in bacterial decision-making is relatively unexplored. We show here that a prior experience of swarming is remembered when E. coli encounters a new surface, improving its future swarming efficiency. We conducted >10,000 single-cell swarm assays to discover that cells store memory in the form of cellular iron levels. This memory pre-exists in planktonic cells, but the act of swarming reinforces it. A cell with low iron initiates swarming early and is a better swarmer, while the opposite is true for a cell with high iron. The swarming potential of a mother cell, whether low or high, is passed down to its fourth-generation daughter cells. This memory is naturally lost by the seventh generation, but artificially manipulating iron levels allows it to persist much longer. A mathematical model with a time-delay component faithfully recreates the observed dynamic interconversions between different swarming potentials. We also demonstrate that iron memory can integrate multiple stimuli, impacting other bacterial behaviors such as biofilm formation and antibiotic tolerance.
Biofilm formation begins when bacteria contacting a surface induce cellular changes to become better adapted for surface growth. One of the first changes to occur for Pseudomonas aeruginosa after surface contact is an increase in the nucleotide second messenger 3’,5’-cyclic adenosine monophosphate (cAMP). It has been demonstrated that this increase in intracellular cAMP is dependent on functional Type IV pili (T4P) relaying a signal to the Pil-Chp system, but the mechanism by which this signal is transduced remains poorly understood. Here, we investigate the role of the Type IV pili retraction motor PilT in sensing a surface and relaying that signal to cAMP production. We show that mutations affecting the structure of PilT and in particular ATPase activity of this motor protein, reduce surface-dependent cAMP production. We identify a novel interaction between PilT and PilJ, a member of the Pil-Chp system, and propose a new model whereby P. aeruginosa uses its retraction motor to sense a surface and to relay that signal via PilJ to increased production of cAMP. We discuss these findings in light of current TFP-dependent surface sensing models for P. aeruginosa .
Importance
T4P are cellular appendages that allow P. aeruginosa to sense a surface leading to the production of cAMP. This second messenger not only activates virulence pathways but leads to further surface adaptation and irreversible attachment of cells. Here, we demonstrate the importance of the retraction motor PilT in surface sensing. We also present a new surface sensing model in P. aeruginosa whereby the T4P retraction motor PilT senses and transmits the surface signal, likely via its ATPase domain and interaction with PilJ, to mediate production of the second messenger cAMP.
We showed that Vibrio parahaemolyticus was able to naturally and reversibly switch between wrinkly and smooth phenotypes and disclosed the gene expression profiles related to wrinkly-smooth switching, showing that the significantly differentially expressed genes between the two colony morphology phenotypes were involved in various biological behaviors, including virulence factor production, biofilm formation, metabolism, adaptation, and colonization.
Vibrio parahaemolyticus can change their usual lifestyle of surviving in an aqueous environment attached to a host, wherein both swimming motility and swarming motility play important roles in lifestyle changes, respectively. VPA0041 is a novel transcription factor involved in regulating the swarming ability of V. parahaemolyticus. The deletion of the vpa0041 gene resulted in the loss of swarming motility in the brain heart infusion (BHI) agars, while the swimming motility was unaffected by VPA0041. Transmission electron microscope (TEM) assays showed that no flagellum was found around the bacterial cells. RNA-sequencing (RNA-Seq) analysis revealed that VPA0041 regulated 315 genes; 207 genes were up-regulated, and 108 genes were down-regulated. RNA-seq results indicated that the lateral flagellar genes were down-regulated by VPA0041, which was confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Electrophoretic mobility shift assays (EMSA) demonstrated that VPA0041 directly bound to the promoters of vpa0264, vpa1548, and vpa1550 to regulate the expression of the lateral flagellar genes. Our results demonstrated that the transcription factor VPA0041 could directly regulate the expression of lateral flagellar genes to mediate the swarming motility in V. parahaemolyticus.
Vibrio parahaemolyticus is a significant foodborne pathogen that causes economic and public health problems worldwide and has a high capacity to adapt to diverse environments and hosts. The second messenger cyclic diguanylate monophosphate (c-di-GMP) allows bacteria to shift from a planktonic form to a communal multicellular lifestyle and plays an important role in bacterial survival and transmission. Here, we characterized single-domain c-di-GMP synthetases in V. parahaemolyticus and identified a novel GGEEF domain-containing protein designated GefA that modulates bacterial swarming motility, biofilm formation, and virulence. GefA inhibits swarming motility by regulating the expression of lateral flagella, while it enhances biofilm formation by controlling exopolysaccharide biosynthesis. Under high-c-di- GMP conditions caused by scrABC knockout, we found that GefA is bifunctional, as it has no effect on swarming motility, but retains the ability to regulate biofilm formation. Subsequent studies suggested that GefA regulates the expression of type III secretion system 1 (T3SS1), which is an important virulence factor in V. parahaemolyticus. Here, we also revealed that the flagella participate in the infection of V. parahaemolyticus. We found that both the T3SS1 and flagella contribute to the GefAmediated virulence of V. parahaemolyticus in the zebrafish model. Our results expand the knowledge of the V. parahaemolyticus c-di-GMP synthetases and their roles in social behaviors and pathogenicity.
The survival and successful spread of many bacterial species hinges on their mode of motility. One of the most distinct of these is swarming, a collective form of motility where a dense consortium of bacteria employ flagella to propel themselves across a solid surface. Surface environments pose unique challenges, derived from higher surface friction/tension and insufficient hydration. Bacteria have adapted by deploying an array of mechanisms to overcome these challenges. Beyond allowing bacteria to colonize new terrain in the absence of bulk liquid, swarming also bestows faster speeds and enhanced antibiotic resistance to the collective. These crucial attributes contribute to the dissemination, and in some cases pathogenicity, of an array of bacteria.
This mini-review highlights; 1) aspects of swarming motility that differentiates it from other methods of bacterial locomotion. 2) Facilitatory mechanisms deployed by diverse bacteria to overcome different surface challenges. 3) The (often difficult) approaches required to cultivate genuine swarmers. 4) The methods available to observe and assess the various facets of this collective motion, as well as the features exhibited by the population as a whole.
Bacteria have developed a large array of motility mechanisms to exploit available resources and environments. These mechanisms can be broadly classified into swimming in aqueous media and movement over solid surfaces. Swimming motility involves either the rotation of rigid helical filaments through the external medium or gyration of the cell body in response to the rotation of internal filaments. On surfaces, bacteria swarm collectively in a thin layer of fluid powered by the rotation of rigid helical filaments, they twitch by assembling and disassembling type IV pili, they glide by driving adhesins along tracks fixed to the cell surface and, finally, non-motile cells slide over surfaces in response to outward forces due to colony growth. Recent technological advances, especially in cryo-electron microscopy, have greatly improved our knowledge of the molecular machinery that powers the various forms of bacterial motility. In this Review, we describe the current understanding of the physical and molecular mechanisms that allow bacteria to move around. In this Review, Wadhwa and Berg explore the most common bacterial motility mechanisms and summarize the current understanding of the molecular machines that enable bacteria to swim in aqueous media and move on solid surfaces.
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.
Spatial expansion of a population of cells can arise from growth of microorganisms, plant cells, and mammalian cells. It underlies normal or dysfunctional tissue development, and it can be exploited as the foundation for programming spatial patterns. This expansion is often driven by continuous growth and division of cells within a colony, which in turn pushes the peripheral cells outward. This process generates a repulsion velocity field at each location within the colony. Here we show that this process can be approximated as coarse-grained repulsive-expansion kinetics. This framework enables accurate and efficient simulation of growth and gene expression dynamics in radially symmetric colonies with homogenous z-directional distribution. It is robust even if cells are not spherical and vary in size. The simplicity of the resulting mathematical framework also greatly facilitates generation of mechanistic insights.
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.
To initiate biofilm formation it is critical for bacteria to sense a surface and respond precisely. Type 4 pili (T4P) have been shown to be important in surface sensing, however, mechanism(s) driving downstream changes important for the switch to biofilm growth have not been clearly defined. Here, using macroscopic bulk assays and single cell tracking analyses of Pseudomonas aeruginosa , we uncover a new role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two hybrid and bimolecular fluorescence complementation assays show that PilO physically interacts with SadC and that the PilO-SadC interaction inhibits SadC’s activity resulting in decreased biofilm formation and increased motility. We show that disrupting the PilO-SadC interaction contributes to greater variation of cyclic-di-GMP levels among cells, thereby increasing cell-to-cell heterogeneity in the levels of this signal. Thus, this work shows that P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this nucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement.
Significance Statement
T4P of P. aeruginosa are important for surface sensing and regulating intracellular cyclic-di-GMP levels. This work identifies a new role for the T4P alignment complex, previously known for its role in supporting pili biogenesis, in surface-dependent signaling. Furthermore, our findings indicate that P. aeruginosa uses a single DGC, via a complex web of protein-protein interactions, to integrate signaling through the T4P and the flagellar motor to fine-tune cyclic-di-GMP levels. A key implication of this work is that more than just regulating signal levels, cells must modulate the dynamic range of cyclic-di-GMP to precisely control the transition to a biofilm lifestyle.
H. pylori is one of the most common causes of chronic bacterial infection in man and its pathogenic role in the development of gastric and duodenal ulcers and gastric cancer is well documented. Chemotaxis is thought to be important in enabling H. pylori to reach the surface mucus layer in the stomach of infected subjects. Little is known about the mechanism of the chemotactic response or of its role played in the motility and virulence of H. pylori. Therefore the first step was to amplify and clone cheY and cheA homologues from H. pylori. Both CheY1 and CheAY had high identity to those found in other bacterial species. H. pylori N6 cheY1, cheAY, cheY, cheAY/Y1 and H. pylori SS1 cheY1 and cheAY isogenic mutants were constructed. The pattern of the chemotactic response was studied using swarm plates, capillary tube assays and computerised motility analysis. H. pylori CheAY seems likely to interact with the flagellar motor to bring about motor switching, whilst the CheY1 homologue acts to terminate the response. Mucin and urea were confirmed as chemoattractants and the results of the study were combined to produced a model of chemotaxis which differs from the E. coli paradigm in many respects. Finally the chemotaxis mutants were unable to colonise both the gnotobiotic piglet and mouse animal models confirming chemotaxis as a requirement for pathogenicity in H. pylori.
Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The “border-crossing” assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.
Bacteria constantly experience changes to their external milieu and need to adapt accordingly to ensure their survival. Certain bacteria adapt by means of cellular differentiation, resulting in the development of a specific cell type that is specialized for life in a distinct environment. Furthermore, to understand how bacteria adapt, it is essential to appreciate the significant changes that occur at the proteomic level. By analyzing the proteome of our model organism V. parahaemolyticus from distinct environmental conditions and cellular differential states, we demonstrate that the proteomic expression profile is highly flexible, which likely allows it to adapt to life in different environmental conditions and habitats. We show that, even within the same swarm colony there are specific zones of cells with distinct expression profiles. Furthermore, our data indicate that cell surface attachment and swarmer cell differentiation are distinct programs that require specific proteomic expression profiles. This likely allows V. parahaemolyticus to adapt to life in different environmental conditions and habitats. Finally, our analyses reveal that the expression profile of the essential protein pool is highly fluid, with significant fluctuations that dependent on the specific life‐style, environment and differentiation state of the bacterium. This article is protected by copyright. All rights reserved.
In order to adapt to different environments, Vibrio parahaemolyticus employed a complicated quorum sensing system to orchestrate gene expression and diverse colony morphology patterns. In this study, the function of the putative quorum sensing signal synthase gene cqsA (VPA0711 in V. Parahaemolyticus strain RIMD2210633 genome) was investigated. The cloning and expression of V. parahaemolyticus cqsA in Escherichia coli system induced the production of a new quorum sensing signal that was found in its culture supernatant. The signal was purified by high performance liquid chromatography methods and determined to be 3-hydroxyundecan- 4-one by indirect and direct mass spectra assays. The deletion of cqsA in RIMD2210633 changed V. parahaemolyticus colony morphology from the classical ‘fried-egg’ shape (thick and opaque in the center, while thin and translucent in the edge) of the wild-type colony to a ‘pancake’ shape (no significant difference between the centre and the edge) of the cqsA deleted colony. This morphological change could be restored by complementary experiment with cqsA gene or the signal extract. In addition, the expression of opaR, a well-known quorum sensing regulatory gene, could be up-regulated by cqsA deletion. Our results suggested that V. parahaemolyticus used cqsA to produce 3-hydroxyundecan-4-one signal and thereby regulated colony morphology and other quorum sensing-associated behaviors.
The bacterial flagellum is a helical filamentous organelle responsible for motility. In bacterial species possessing flagella at the cell exterior, the long helical flagellar filament acts as a molecular screw to generate thrust. Meanwhile, the flagella of spirochetes reside within the periplasmic space and not only act as a cytoskeleton to determine the helicity of the cell body, but also rotate or undulate the helical cell body for propulsion. Despite structural diversity of the flagella among bacterial species, flagellated bacteria share a common rotary nanomachine, namely the flagellar motor, which is located at the base of the filament. The flagellar motor is composed of a rotor ring complex and multiple transmembrane stator units and converts the ion flux through an ion channel of each stator unit into the mechanical work required for motor rotation. Intracellular chemotactic signaling pathways regulate the direction of flagella-driven motility in response to changes in the environments, allowing bacteria to migrate towards more desirable environments for their survival. Recent experimental and theoretical studies have been deepening our understanding of the molecular mechanisms of the flagellar motor. In this review article, we describe the current understanding of the structure and dynamics of the bacterial flagellum.
Atmospheric deposition represents a major vector of both macro-and micro-nutrients to the oligotrophic open oceans, potentially imposing a profound impact on the functioning of the microbial community. Whereas responses of the prokaryotes to atmospheric deposition are being studied at the community level, corresponding functional changes are essentially unknown. Here we used metatranscriptomic approaches to elucidate taxonomic and functional profiles of the prokaryotic community in response to Asian aerosols in the Western North Pacific Ocean. While Bacteria were downrepresented, Virus and Archaea were overrepresented in the aerosol treatment compared to the control. Within Bacteria, transcripts related to Cyanobacteria, including Prochlorococcus, Trichodesmium and Synechococcus, decreased dramatically, whereas transcripts related to Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes showed differential increases in the aerosol treatment. Nutrients and organic matters were enriched as evidenced by an overexpression of transporters for amino acids and utilization of various carbohydrates and a down-expression of transcripts related with phosphorus metabolism. Increased expression included transcripts involved in tricarboxylic acid cycle, pentose phosphate pathway, glycolysis and gluconeogenesis. Unexpectedly, the expression of transcripts associated with Fe metabolism suggested that Fe limitation was intensified. Transcripts associated with N fixation declined, corresponding to the decline of diazotroph-related transcripts. This result is against the paradigm of Fe fertilization from atmospheric deposition but may represent an extreme case that Fe was scavenged after aerosol addition. Negative effects included impaired sugar utilization and the stimulation of oxidative stress and heavy metal toxicity. All these changes lead the community to an energy-conserving lifestyle and promoted motility, chemotaxis and interspecies competition and interaction. The results provide new insights into the biogeochemical impacts of atmospheric deposition in the Western North Pacific Ocean. PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.3292v1 | CC BY 4.0 Open Access | rec
Vibrio parahaemolyticus exists as swimmer and swarmer cells, specialized for growth in liquid and on solid environments, respectively. Swarmer cells are characteristically highly elongated due to an inhibition of cell division, but still need to divide in order to proliferate and expand the colony. It is unknown how long swarmer cells divide without diminishing the population of long cells required for swarming behavior. Here we show that swarmer cells divide but the placement of the division site is cell length‐dependent; short swarmers divide at mid‐cell, while long swarmers switch to a specific non‐mid‐cell placement of the division site. Transition to non‐mid‐cell positioning of the Z‐ring is promoted by a cell length‐dependent switch in the localization‐dynamics of the division regulator MinD from a pole‐to‐pole oscillation in short swarmers to a multi‐node standing‐wave oscillation in long swarmers. Regulation of FtsZ levels restricts the number of divisions to one and SlmA ensures sufficient free FtsZ to sustain Z‐ring formation by preventing sequestration of FtsZ into division deficient clusters. By limiting the number of division‐events to one per cell at a specific non‐mid‐cell position, V. parahaemolyticus promotes the preservation of long swarmer cells and permits swarmer cell division without the need for dedifferentiation. This article is protected by copyright. All rights reserved. Vibrio parahaemolyticus swarmer cells are highly elongated – a morphology important for swarming behavior. A cell length‐dependent switch to non‐mid‐cell division is promoted by a transition in MinD localization dynamics to a multi‐node standing wave oscillation. By restricting division events to one per cell, this allows for swarmer cell‐division while preserving the presence of long cells, required for swarming behavior, within a proliferating population of swarmer cells.
Only a small fraction of bacteria can autonomously initiate growth on agar plates. Nongrowing bacteria typically enter a metabolically inactive dormant state and require specific chemical trigger factors or signals to exit this state and to resume growth. Micrococcus luteus has become a model organism for this important yet poorly understood phenomenon. Only a few resuscitation signals have been described to date, and all of them are produced endogenously by bacterial species. We report the discovery of a novel type of resuscitation signal that allows M. luteus to grow on agar but not agarose plates. Fractionation of the agar polysaccharide complex and sulfation of agarose allowed us to identify the signal as highly sulfated saccharides found in agar or carrageenans. Purification of hydrolyzed κ-carrageenan ultimately led to the identification of the signal as a small fragment of a large linear polysaccharide, i.e., an oligosaccharide of five or more sugars with a repeating disaccharide motif containing d-galactose-4-sulfate (G4S) 1,4-linked to 3,6-anhydro-α-d-galactose (DA), G4S-(DA-G4S)n≥2.
IMPORTANCE
Most environmental bacteria cannot initiate growth on agar plates, but they can flourish on the same plates once growth is initiated. While there are a number of names for and manifestations of this phenomenon, the underlying cause appears to be the requirement for a molecular signal indicating safe growing conditions. Micrococcus luteus has become a model organism for studying this growth initiation process, often called resuscitation, because of its apparent connection with the persistent or dormant form of Mycobacterium tuberculosis, an important human pathogen. In this report, we identify a highly sulfated saccharide from agar or carrageenans that robustly resuscitates dormant M. luteus on agarose plates. We identified and characterized the signal as a small repeating disaccharide motif. Our results indicate that signals inherent in or absent from the polysaccharide composition of solid growth media can have major effects on bacterial growth.
The degree to which surface motile bacteria explore their surroundings is influenced by aspects of their local environment. Accordingly, regulation of surface motility is controlled by numerous chemical, physical, and biological stimuli. Discernment of such regulation due to these multiple cues is a formidable challenge. Additionally inherent ambiguity and variability from the assays used to assess surface motility can be an obstacle to clear delineation of regulated surface motility behavior. Numerous studies have reported single environmental determinants of microbial motility and lifestyle behavior but the translation of these data to understand surface motility and bacterial colonization of human host or environmental surfaces is unclear. Here, we describe the current state of the field and our understanding of exogenous factors that influence bacterial surface motility.
Most marine bacteria secrete exopolysaccharide (EPS), which is important for bacterial survival in the marine environment. However, it is still unclear whether the self-secreted EPS is involved in marine bacterial motility. Here we studied the role of EPS in the lateral flagella-driven swarming motility of benthic bacterium Pseudoalteromonas sp. SM9913 (SM9913) by a comparison of wild SM9913 and ΔepsT, an EPS synthesis defective mutant. Reduction of EPS production in ΔepsT did not affect the growth rate or the swimming motility, but significantly decreased the swarming motility on a swarming plate, suggesting that the EPS may play a role in SM9913 swarming. However, the expression and assembly of lateral flagella in ΔepsT were not affected. Instead, ΔepsT had a different swarming behavior from wild SM9913. The swarming of ΔepsT did not have an obvious rapid swarming period, and its rate became much lower than that of wild SM9913 after 35 h incubation. An addition of surfactin or SM9913 EPS on the surface of the swarming plate could rescue the swarming level. These results indicate that the self-secreted EPS is required for the swarming of SM9913. This study widens our understanding of the function of the EPS of benthic bacteria.
Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.
A Salmonella typhimurium strain possessing a mutation in the fliF gene (coding for the component protein of the M ring of the flagellar basal body) swarmed poorly on a semisolid plate. However, cells grown in liquid medium swam normally and did not show any differences from wild-type cells in terms of swimming speed or tumbling frequency. When mutant cells were grown in a viscous medium, detached bundles of flagellar filaments as long as 100 microns were formed and the cells had impaired motility. Electron microscopy and immunoelectron microscopy revealed that the filaments released from the cells had the hook and a part of the rod of the flagellar basal body still attached. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis showed that the rod portion of the released structures consisted of the 30-kilodalton FlgG protein. Double mutants containing this fliF mutation and various che mutations were constructed, and their behavior in viscous media was analyzed. When the flagellar rotation of the mutants was strongly biased to either a counterclockwise or a clockwise direction, detached bundles were not formed. The formation of large bundles was most extreme in mutants weakly biased to clockwise rotation.
Vibrio parahaemolyticus has two distinct cell types, the swimmer cell and the swarmer cell, adapted for locomotion in different circumstances. The swimmer cell, produced when the bacterium is grown in liquid media, is a short rod with a single sheathed polar flagellum. The swarmer cell, produced when V. parahaemolyticus is grown on solidified media, is greatly elongated and synthesizes, in addition to the polar flagellum, numerous unsheathed lateral flagella which are responsible for translocation over surfaces. We are interested in understanding how this bacterium differentiates in response to contact with surfaces and have determined in earlier work that the polar flagellum acts as a tactile sensor which controls transcription of genes (laf) encoding the swarmer cell phenotype. Surface recognition involves sensing of forces that obstruct movement of the polar flagellum. In this report we show that a second signal, iron limitation, is also required for swarmer cell differentiation. Production of lateral flagella occurred only when polar flagellar function was perturbed and iron-limiting growth conditions were imposed. The same conditions were required to induce light production in strains of V. parahaemolyticus in which a laf gene was transcriptionally fused to the lux operon encoding the enzymes for bioluminescence. The lafA gene encoding the lateral flagellin subunit was cloned and used in Northern (RNA) blot measurements. Examination of mRNA levels revealed that transcription of lafA is dependent on growth in iron-depleted media. The control of differentiation by multiple environmental stimuli is discussed.
Two distinctly different organelles of locomotion are produced by Vibrio parahaemolyticus. The polar flagellum is responsible for motility in a liquid environment (swimming), and the lateral flagella enable the bacteria to move over surfaces (swarming). Synthesis of lateral flagella occurs when V. parahaemolyticus is grown on agar media but not when it is grown in liquid media. We used lux (luminescence gene) fusions to conveniently and sensitively analyze the factors which influence transcription of lateral flagella genes (laf). Transposon mini-Mu lux was used to mutagenize V. parahaemolyticus and to generate laf::lux transcriptional fusions. Mutants with insertions of mini-Mu lux in laf genes were defective in the swarming phenotype and produced light when the bacteria were propagated on agar media, but not when cells were grown in liquid media. Thus, surface-dependent expression of lateral flagella synthesis is controlled by regulation of transcription. Such fusion strains were also used to further define the environmental conditions which induce laf gene expression. Cultivation on media solidified by gelling agents other than agar also induced light production in fusion strains, as did growth on a variety of hydrophilic membrane filters suspended over liquid media. Growth at an air-surface interface was not necessary for expression since embedding the fusion strains in agar was also effective. Furthermore, induction of laf gene transcription could also be accomplished by increasing the viscosity of the liquid medium by the addition of a high-molecular-weight polymer such as polyvinylpyrrolidone. Increase in luminescence of the fusion strains was detected within 30 min of initiation of the inducing circumstance, and reversal of induction, e.g., by dilution of the viscous medium, resulted in a rapid decline in the rate of increase in luminescence. Conditions that induced luminescence in the fusion strains also induced the synthesis of lateral flagella in wild-type V. parahaemolyticus. The growth environment of the genes, and it appears that the signal that triggers laf expression is physical rather than chemical in nature. Possibilities for a sensing mechanism are discussed.
Sporulation of Bacillus subtilis is a primitive example of coupling between morphological changes and timing of gene expression during development. A major early control of transcriptional activity is dependent on a new sigma factor, sigma E, which is encoded by the sigE gene and synthesized as an inactive precursor, pro-sigma E. We show that mutations in the spoIIGA gene block the processing of pro-sigma E. Moreover, synthesis of both spoIIGA and sigE products in vegetative cells leads to expression of a sigma E-controlled promoter during growth, suggesting that SpoIIGA has pro-sigma E processing activity. The SpoIIGA polypeptide, which contains five potential transmembrane domains, is synthesized during sporulation 1 hr before processing activity can be detected. We propose that SpoIIGA processing activity is triggered by the presence of the sporulation septum, which is itself dependent on the spoIIAA and spoIIE products. These proteins are normally needed for pro-sigma E processing during sporulation but can be bypassed in vegetative cells. According to this model, a morphological structure would directly control the synthesis of a developmental sigma factor and would modify gene expression.
To evaluate the variation in the expression of cell wall antigens between Staphylococcus aureus grown in liquid medium and solid support, bacteria were harvested from liquid chemically defined medium and chemically defined medium in a 1% agar base. Cell wall proteins were then extracted by lysostaphin in a protoplast-stabilizing medium (30% raffinose). After separation of the cell wall antigens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blots, they were probed with chicken antiserum to an S. aureus strain grown on a solid support. For each of the 15 clinical strains analyzed, high-molecular-size bands (molecular size range, 120 to 220 kilodaltons) were either enhanced or distinctly present when compared with those from the cell wall extract of the same strain grown in liquid medium. Results of enzymatic treatment of whole staphylococci grown on solid medium suggested the proteinaceous nature and the surface location of these antigens. Limited passage studies demonstrated the ability of the staphylococci to alter these surface proteins when passaged alternately on liquid and solid media. These observations suggested the importance of the microenvironment to the expression of cell wall proteins in S. aureus. Correlations with observations in vivo may help identify the determinants of microbial pathogenicity in S. aureus.
Species of marine bacteria belonging to the genus Beneckea and strains of Photobacterium fischeri were negatively stained and examined by means of the electron microscope to determine the structure and arrangement of their flagella. All of the species of the genus Beneckea had single, polar, sheathed flagella when grown in liquid medium. When grown on solid medium, most strains of B. campbellii and B. neptuna and all strains of B. alginolytica and B. parahaemolytica had unsheathed, peritrichous flagella in addition to the single, sheathed, polar flagellum. The remaining species, B. nereida, B. pelagia, and B. natriegens, had a single, polar, sheathed flagellum when grown on solid medium. Strains of P. fischeri had sheathed flagella arranged in polar tufts. Only one group (B-2) of marine bacteria included in this study was found to have polar, unsheathed flagella.
A study of the ecology of Vibrio parahaemolyticus and related vibrios in the Rhode River area of Chesapeake Bay was carried out over the period December 1970 through August 1971. The incidence of V. parahaemolyticus and related vibrios was found to be correlated with water temperature. The vibrios could not be detected in the water column during the winter months, although they were present in sediment. From late spring to early summer, when water temperatures were 14 +/- 1 C, vibrios over-wintering in sediment were released from the bottom communities and attached to zooplankton, proliferating as the temperature rose. The number of vibrios in and on plankton was reflected in the water column bacterial population densities at water temperatures of ca. 19 C. Thus, temperature of the water column in the range of 14 to 19 C was found to be critical in the annual cycle of the vibrios. Interaction between sediment, water, and zooplankton was found to be essential in the natural estuarine ecosystem. Bacterial counts of zooplankton were found to be temperature dependent. The bacterial population associated with zooplankton was found to be predominantly on external surfaces and was specific, differing from that of the sediment. Vibrio spp. and related organisms comprised the total bacterial population associated with zooplankton in summer months. The ecological role of Vibrio spp., including V. parahaemolyticus, was found to be significant, with respect to their property of chitin digestion and in relation to the population dynamics of zooplankton in Chesapeake Bay.
The source of energy for bacterial motility is the intermediate in oxidative phosphorylation, not ATP directly. For chemotaxis, however, there is an additional requirement, presumably ATP. These conclusions are based on the following findings. (i) Unlike their parents, mutants of Escherichia coli and Salmonella typhimurium that are blocked in the conversion of ATP to the intermediate of oxidative phosphorylation failed to swim anaerobically, even when they produced ATP. When respiration was restored to the mutants, motility was simultaneously restored. (ii) Carbonylcyanide m-chlorophenylhydrazone, which uncouples oxidative phosphorylation, completely inhibited motility even though ATP remained present. (iii) Arsenate did not inhibit motility in the presence of an oxidizable substrate, though it did reduce ATP levels to less than 0.3% (iv) Arsenate completely inhibited chemotaxis under conditions where motility was normal.
Formation of the lateral flagella (L-flagella) of Vibrio parahaemolyticus was studied immunologically, using specific antiserum against L-flagella. On solid medium, L-flagella were formed at both high (37 degrees C) and low (25 degrees C) temperatures, although at high temperatures they became dissociated from the cells and decomposed in the medium. L-flagella were not formed in liquid or soft-agar medium. Formation of L-flagella was decreased by lowering the pH of the medium and repressed by transferring the cells from solid medium to liquid medium. Mutants possessing L-flagella but not a polar monotrichous flagellum (M-flagellum) swarmed on solid medium, whereas mutants were grown on solid medium and then transfered to liquid medium, the cells oscillated until they lost L-flagella. It is postulated that L-flagella are locomotive organelles on solid medium and in some cases also in liquid medium, whereas M-flagella are locomotive organelles only in liquid medium.
Two-component regulatory systems appear to be widespread in bacteria. Phosphorylation has been demonstrated in three of the known systems and correlated with in vivo function in two cases (Che and Ntr). Although phosphorylation of sensor and regulator proteins has so far been observed exclusively in vitro, transient protein phosphorylation could provide a basis for the mechanism of signal transduction in these bacterial systems. There is currently insufficient evidence, however, to establish the precise functional relationship(s) between the conserved sensor and regulator sequences, phosphorylation, and the detailed mechanism involved in signal transduction via the sensor and regulator proteins.
Bacterial flagellar motors are the reversible rotary engine which propels the cell by rotating a helical flagellar filament as a screw propeller. The motors are embedded in the cytoplasmic membrane, and the energy for rotation is supplied by the electrochemical potential of specific ions across the membrane. Thus, the analysis of motor rotation at the molecular level is linked to an understanding of how the living system converts chemical energy into mechanical work. Based on the coupling ions, the motors are divided into two types; one is the H+-driven type found in neutrophiles such as Bacillus subtilis and Escherichia coli and the other is the Na+-driven type found in alkalophilic Bacillus and marine Vibrio. In this review, we summarize the current status of research on the rotation mechanism of the Na+-driven flagellar motors, which introduces several new aspects in the analysis.
Light is produced by recombinant Escherichia coli that contain lux genes cloned from the marine bacterium Vibrio fischeri.
The bioluminescence phenotype requires genes for regulatory and biochemical functions, the latter encoded by five lux genes
contained in a single operon. These lux genes were disconnected from their native promoter and inserted into the transposon
mini-Mu. The resulting transposon, mini-Mulux, could induce mutations by insertional inactivation of a target gene, and the
lux DNA was oriented to align target gene transcription with that of the lux genes. Genes in Escherichia coli and Vibrio parahaemolyticus
were mutagenized, and mutants containing transposon-generated lux gene fusions produced light as a function of target gene
transcription. Light production offers a simple, sensitive, in vivo indicator of gene expression.
Swarmer cell genes, laf, are induced when V. parahaemolyticus is grown on the surface of solidified media, embedded in solidified media, suspended in viscous media, or agglutinated with antibody in liquid media. These conditions have in common the constraint of the movement of the polar flagellum. To test the hypothesis that the polar flagellum functions as a sensor necessary for control of swarmer cell formation, we have constructed a variety of mutations in genes encoding components of the polar flagellum, fla. The consequence of such mutations is the constitutive expression of laf genes. So, the performance of the polar flagellum is coupled to the transcription of laf genes such that when function is perturbed, either physically or genetically, swarmer cell genes are induced. Because the polar flagellum appears to be capable of sensing external forces influencing its motion, we suggest it is acting as a dynamometer.
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
The structure of sheathed flagella from Bdellovibrio bacteriovorus was investigated. The first three periods of these flagella were characterized by progressively smaller wavelengths and amplitudes in periods more distal to the cell. The damped appearance was due to a single nonrandom transition between two helical structures within each filament. The intersection of the two helices, one of which was a threefold-reduced miniature of the other, occurred at a fixed distance along the filament and resulted in a shift in the flagellar axis. Flagella increased in length as the cells aged and assumed a constant miniature waveform at their distal ends. The core filament was the principal determinant of flagellar morphology. It was composed of 28,000- and 29,500-dalton polypeptides. The 28,000-dalton subunits were located in the cell-proximal segment of the filament, and the 29,500-dalton subunits were located in the more distal region. The heteromorphous appearance of bdellovibrio flagella arose from the sequential assembly of these subunits. The basal complex associated with core filaments was examined because of its potential involvement in sheath formation. Bdellovibrio basal organelles were generally similar to those of other gram-negative species, but appeared to lack a disk analogous to the outer membrane-associated L ring which is a normal component of gram-negative basal complexes.
Polar monotrichous and peritrichous flagella of Vibrio parahaemolyticus were isolated and purified separately. On hydroxylapatite column chromatography, the flagellins of polar monotrichous flagella were eluted with a higher concentration of phosphate than those of peritrichous flagella. Gel diffusion tests showed an antigenic difference between the flagellins of polar monotrichous and peritrichous flagella. Electron microscope observations on cells stained with ferritin-conjugated antibodies demonstrated that polar monotrichous and peritrichous flagella reacted specifically with antimonotrichous flagellin and antiperitrichous flagellin antisera, respectively.
Vibrio parahaemolyticus, the flagellated nonswarming marine bacteria were induced to swarm on solid media under three different conditions: growth at 20–26°C on medium containing 1% NaCl, growth on a medium in a sealed Petridish and growth on H2O2-treated medium. The morphological transformations observed in cells during swarming of V. parahaemolyticus are similar to those found jor the naturally swarming Vibrio alginolyticus. The mechanism of swarming in both species involves massive formation of peritrichous flagella and a negative chemotactive response to metabolic byproducts.
BACTERIAL flagella are generally composed of three morphologically distinguishable regions: (a) the long flagellar filament which accounts for more than 95% of the flagellar protein; (b) the hook, which is generally 80-90 nm long and has a characteristic shape, and (c) the basal structure which is composed of an intricate set of disks and rods attaching the hook to the cell membrane and cell wall1-3.
The occurrence of bacterial flagella sheathed with an extension of the Gram-negative outer cell wall membrane poses problems for universal application of the rotary motor model for the bacterial flagellum mechanism. Two mechanisms are proposed which attempt to reconcile the existence of sheathed flagella with the rotary motor model, one of which involves rotation of both flagellar sheath and core, the other of which involves rotation of the core only, within a flexible wave-propagating sheath.