Chemotaxis of Silicibacter sp. strain TM1040 toward dinoflagellate products

The alpha-proteobacteria phylogenetically related to the Roseobacter clade are predominantly responsible for the degradation of organosulfur compounds, including the algal osmolyte dimethylsulfoniopropionate (DMSP). Silicibacter sp. strain TM1040, isolated from a DMSP-producing Pfiesteria piscicida dinoflagellate culture, degrades DMSP, producing 3-methylmercaptopropionate. TM1040 possesses three lophotrichous flagella and is highly motile, leading to a hypothesis that TM1040 interacts with P. piscicida through a chemotactic response to compounds produced by its dinoflagellate host. A combination of a rapid chemotaxis screening assay and a quantitative capillary assay were used to measure chemotaxis of TM1040. These bacteria are highly attracted to dinoflagellate homogenates; however, the response decreases when homogenates are preheated to 80 degrees C. To help identify the essential attractant molecules within the homogenates, a series of pure compounds were tested for their ability to serve as attractants. The results show that TM1040 is strongly attracted to amino acids and DMSP metabolites, while being only mildly responsive to sugars and the tricarboxylic acid cycle intermediates. Adding pure DMSP, methionine, or valine to the chemotaxis buffer resulted in a decreased response to the homogenates, indicating that exogenous addition of these chemicals blocks chemotaxis and suggesting that DMSP and amino acids are essential attractant molecules in the dinoflagellate homogenates. The implication of Silicibacter sp. strain TM1040 chemotaxis in establishing and maintaining its interaction with P. piscicida is discussed.     

Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function.

Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.     

Production of antibacterial compounds and biofilm formation by Roseobacter species are influenced by culture conditions

Bacterial communities associated with marine algae are often dominated by members of the Roseobacter clade, and in the present study, we describe Roseobacter phenotypes that may provide this group of bacteria with selective advantages when colonizing this niche. Nine of 14 members of the Roseobacter clade, of which half were isolated from cultures of the dinoflagellate Pfiesteria piscicida, produced antibacterial compounds. Many non-Roseobacter marine bacteria were inhibited by sterile filtered supernatants of Silicibacter sp. TM1040 and Phaeobacter (formerly Roseobacter) strain 27-4, which had the highest production of antibacterial compound. In contrast, Roseobacter strains were susceptible only when exposed to concentrated compound. The production of antibacterial compound was influenced by the growth conditions, as production was most pronounced when bacteria were grown in liquid medium under static conditions. Under these conditions, Silicibacter sp. TM1040 cells attached to one another, forming rosettes, as has previously been reported for Phaeobacter 27-4. A spontaneous Phaeobacter 27-4 mutant unable to form rosettes was also defective in biofilm formation and the production of antibacterial compound, indicating a possible link between these phenotypes. Rosette formation was observed in 8 of 14 Roseobacter clade strains examined and was very pronounced under static growth in 5 of these strains. Attachment to surfaces and biofilm formation at the air-liquid interface by these five strains was greatly facilitated by growth conditions that favored rosette formation, and rosette-forming strains were 13 to 30 times more efficient in attaching to glass compared to strains under conditions where rosette formation was not pronounced. We hypothesize that the ability to produce antibacterial compounds that principally inhibit non-Roseobacter species, combined with an enhancement in biofilm formation, may give members of the Roseobacter clade a selective advantage and help to explain the dominance of members of this clade in association with marine algal microbiota.     

Flagella, motility and invasive virulence of Pseudomonas aeruginosa

The role of motility as a virulence factor in Pseudomonas aeruginosa burn wound sepsis was examined using mutants deficient in the Fla or Mot phenotype. Physiological profiles of parental strains and Fla- and Mot- mutants were similar with respect to antibiograms, O antigen types, growth rates, and proteolytic, exotoxin A and phospholipase activities, providing evidence for isogenicity. Lethality studies using a subcutaneous mouse burn model showed that three Fla- mutants and one Mot- mutant were much less virulent (10(2) to 10(5) times) than the parent wild-type. Topical challenges in the flame burn model showed that a Fla- mutant of strain M-2 was approximately tenfold less virulent. A reduction in virulence, although somewhat less than tenfold, was also observed in the scald burn model for M-2 Fla-, and Mot- strains. Tissue colonization experiments revealed a characteristic, rapidly systemic infection in burned mice challenged with wild-type organisms. Nonmotile mutants similarly proliferated in the burn wound, but the characteristic bacteraemia and systemic invasion were markedly absent. The infection remained localized in the skin wound and the mice survived. The pattern of infection by nonmotile mutants in the colonization studies was very similar to that obtained with Fla+ cells in burned animals passively treated with antiflagellar antibody. These results add substantial support to the concept of motility as a P. aeruginosa virulence factor in invasive infections.     

Inactivation of Campylobacter jejuni flagellin genes by homologous recombination demonstrates that flaA but not flaB is required for invasion.

The role of the Campylobacter jejuni flagella in adhesion to, and penetration into, eukaryotic cells was investigated. We used homologous recombination to inactivate the two flagellin genes flaA and flaB of C. jejuni, respectively. Mutants in which flaB but not flaA is inactivated remain motile. In contrast a defective flaA gene leads to immotile bacteria. Invasion studies showed that mutants without motile flagella have lost their potential to adhere to, and penetrate into, human intestinal cells in vitro. Invasive properties could be partially restored by centrifugation of the mutants onto the tissue culture cells, indicating that motility is a major, but not the only, factor involved in invasion.     

Chemotaxis of Silicibacter sp. Strain TM1040 toward Dinoflagellate Products

The α-proteobacteria phylogenetically related to the Roseobacter clade are predominantly responsible for the degradation of organosulfur compounds, including the algal osmolyte dimethylsulfoniopropionate (DMSP). Silicibacter sp. strain TM1040, isolated from a DMSP-producing Pfiesteria piscicida dinoflagellate culture, degrades DMSP, producing 3-methylmercaptopropionate. TM1040 possesses three lophotrichous flagella and is highly motile, leading to a hypothesis that TM1040 interacts with P. piscicida through a chemotactic response to compounds produced by its dinoflagellate host. A combination of a rapid chemotaxis screening assay and a quantitative capillary assay were used to measure chemotaxis of TM1040. These bacteria are highly attracted to dinoflagellate homogenates; however, the response decreases when homogenates are preheated to 80°C. To help identify the essential attractant molecules within the homogenates, a series of pure compounds were tested for their ability to serve as attractants. The results show that TM1040 is strongly attracted to amino acids and DMSP metabolites, while being only mildly responsive to sugars and the tricarboxylic acid cycle intermediates. Adding pure DMSP, methionine, or valine to the chemotaxis buffer resulted in a decreased response to the homogenates, indicating that exogenous addition of these chemicals blocks chemotaxis and suggesting that DMSP and amino acids are essential attractant molecules in the dinoflagellate homogenates. The implication of Silicibacter sp. strain TM1040 chemotaxis in establishing and maintaining its interaction with P. piscicida is discussed.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

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.     

Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.     

铜绿假单胞菌泳动能力相关新基因的筛选及鉴定

从Mu转座突变子文库中经过表型筛选,得到12株泳动(Swimming motility)能力缺陷的突变子,经Mu转座子插入位点的确认、基因克隆及测序分析发现其中10个突变子中Mu转座子分别插入到10个不同的与鞭毛运动和功能相关的基因中,2个突变子中Mu转座子插入到功能未知的新基因(PA2950和PA5022)中,电镜观察结果表明这2个突变株均具有完整的鞭毛,初步推测这2个基因可能是参与鞭毛泳动的能量代谢、趋化作用或信息传递的新基因。     

Comparative ultrastructural and functional studies of Helicobacter pylori and Helicobacter mustelae flagellin mutants: both flagellin subunits, FlaA and FlaB, are necessary for full motility in Helicobacter species.

Helicobacter mustelae causes chronic gastritis and ulcer disease in ferrets. It is therefore considered an important animal model of human Helicobacter pylori infection. High motility even in a viscous environment is one of the common virulence determinants of Helicobacter species. Their sheathed flagella contain a complex filament that is composed of two distinctly different flagellin subunits, FlaA and FlaB, that are coexpressed in different amounts. Here, we report the cloning and sequence determination of the flaA gene of H. mustelae NCTC12032 from a PCR amplification product. The FlaA protein has a calculated molecular mass of 53 kDa and is 73% homologous to the H. pylori FlaA subunit. Isogenic flaA and flaB mutants of H. mustelae F1 were constructed by means of reverse genetics. A method was established to generate double mutants (flaA flaB) of H. mustelae F1 as well as H. pylori N6. Genotypes, motility properties, and morphologies of the H. mustelae flagellin mutants were determined and compared with those of the H. pylori flaA and flaB mutants described previously. The flagellar organizations of the two Helicobacter species proved to be highly similar. When the flaB genes were disrupted, motility decreased by 30 to 40%. flaA mutants retained weak motility by comparison with strains that were devoid of both flagellin subunits. Weakly positive motility tests of the flaA mutants correlated with the existence of short truncated flagella. In H. mustelae, lateral as well as polar flagella were present in the truncated form. flaA flaB double mutants were completely nonmotile and lacked any form of flagella. These results show that the presence of both flagellin subunits is necessary for complete motility of Helicobacter species. The importance of this flagellar organization for the ability of the bacteria to colonize the gastric mucosa and to persist in the gastric mucus remains to be proven.     

Mutational analysis of flagellum-independent surface spreading of Serratia marcescens 274 on a low-agar medium.

In a previous study (J. O'Rear, L. Alberti, and R. M. Harshey, J. Bacteriol. 174:6125-6137, 1992) we reported the isolation of several transposon mutants of Serratia marcescens 274 that were defective either in swarming alone or in both swimming and swarming motility. All the nonflagellate (Fla-) mutants, while defective in both types of motility, were able to spread rapidly on the surface of low-agar (0.35%) media. We show here that some of the swarming-defective mutants are defective in the production of serrawettin W1, an extracellular cyclic lipopeptide produced by S. marcescens 274. When combined with a Fla defect, the serrawettin (Swt) mutants are deficient in spreading on low-agar media. The spreading deficiency can be overcome by serrawettin supplied extracellularly. Introduction of Fla defects into chemotaxis mutants does not affect this mode of surface translocation. These results suggest that spreading may be a passive form of translocation. We also report that swarming defects in all mutants showing a Dps phenotype (able to swarm within the inoculated area but unable to move outward) in the earlier study can be overcome by changing the commercial source of agar.     

Vibrio fischeri flagellin A is essential for normal motility and for symbiotic competence during initial squid light organ colonization

The motile bacterium Vibrio fischeri is the specific bacterial symbiont of the Hawaiian squid Euprymna scolopes. Because motility is essential for initiating colonization, we have begun to identify stage-specific motility requirements by creating flagellar mutants that have symbiotic defects. V. fischeri has six flagellin genes that are uniquely arranged in two chromosomal loci, flaABCDE and flaF. With the exception of the flaA product, the predicted gene products are more similar to each other than to flagellins of other Vibrio species. Immunoblot analysis indicated that only five of the six predicted proteins were present in purified flagella, suggesting that one protein, FlaF, is unique with respect to either its regulation or its function. We created mutations in two genes, flaA and flaC. Compared to a flaC mutant, which has wild-type flagellation, a strain having a mutation in the flaA gene has fewer flagella per cell and exhibits a 60% decrease in its rate of migration in soft agar. During induction of light organ symbiosis, colonization by the flaA mutant is impaired, and this mutant is severely outcompeted when it is presented to the animal as a mixed inoculum with the wild-type strain. Furthermore, flaA mutant cells are preferentially expelled from the animal, suggesting either that FlaA plays a role in adhesion or that normal motility is an advantage for retention within the host. Taken together, these results show that the flagellum of V. fischeri is a complex structure consisting of multiple flagellin subunits, including FlaA, which is essential both for normal flagellation and for motility, as well as for effective symbiotic colonization.     

Induction of multiple prophages from a marine bacterium: a genomic approach

Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.     

Swarming motility by Photorhabdus temperata is influenced by environmental conditions and uses the same flagella as that used in swimming motility

Photorhabdus temperata, an insect pathogen and nematode symbiont, is motile in liquid medium by swimming. We found that P.?temperata was capable of surface movement, termed swarming behavior. Several lines of evidence indicate that P. temperata use the same flagella for both swimming and swarming motility. Both motility types required additional NaCl or KCl in the medium and had peritrichous flagella, which were composed of the same flagellin as detected by immunoblotting experiments. Mutants defective in flagellar structural proteins were nonmotile for both motility types. Unlike swimming, we observed swarming behavior to be a social form of movement in which the cells coordinately formed intricate channels covering a surface. The constituents of the swarm media affected motility. Swarming was optimal on low agar concentrations; as agar concentrations increased, swarm ring diameters decreased.     

Transposon mutagenesis in a marine synechococcus strain: isolation of swimming motility mutants

Certain marine unicellular cyanobacteria of the genus Synechococcus exhibit a unique type of swimming motility characterized by the absence of flagella or any other obvious organelles of motility. While the abundant cell surface-associated 130-kDa glycoprotein SwmA is known to be required for the generation of thrust, identification of other components of the motility apparatus has, until recently, been unsuccessful. Here we report on the development of a transposon mutagenesis system for use with marine Synechococcus sp. strain WH8102, a model organism for which the genome has been sequenced. Utilizing this mutagenesis technique, we have isolated 17 independent mutants impaired in swimming motility. These 17 transposon insertions are located in nine open reading frames, which cluster in three separate regions of the genome. Included within these clusters are several multicomponent transport systems as well as a number of glycosyltransferases.     

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective ( sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK ) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.     

Proteus mirabilis mutants defective in swarmer cell differentiation and multicellular behavior

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-microns motile swimmer cell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate by a combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploid swarmer cell is 60 to 80 microns long and possesses hundreds to thousands of surface-induced flagella. These features, combined with multicellular behavior, allow the swarmer cells to move over a surface in a process called swarming. Transposon Tn5 was used to produce P. mirabilis mutants defective in wild-type swarming motility. Two general classes of mutants were found to be defective in swarming. The first class was composed of null mutants that were completely devoid of swarming motility. The majority of nonswarming mutations were the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remaining nonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the second general class of mutants, which made up more than 65% of all defects in swarming were motile but were defective in the control and coordination of multicellular swarming. Analysis of consolidation zones produced by such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation of multicellular behavior. A possible mechanism controlling the cyclic process of differentiation and dediferentiation involved in the swarming behavior of P. mirabilis is discussed.