Two type IV pili of Vibrio parahaemolyticus play different roles in biofilm formation

Thirty-eight Shiga toxin-producing Escherichia coli (STEC) O157:H7/H(-) strains isolated from human infections, cattle and foods in Brazil and in some other Latin American countries were compared with regard to several phenotypic and genotypic characteristics. The genetic relatedness of the strains was also determined by pulsed-field gel electrophoresis (PFGE). Similar biochemical behaviour was identified, regardless of the origin and country of the strains. Most (89.5%) strains were sensitive to the antimicrobial agents tested, but resistance to at least one drug was observed among bovine strains. Although a diversity of stx genotypes was identified, most (77.8%) of the human strains harboured stx(2) or stx(2)stx(2c(2vha)), whereas stx(2c(2vha)) prevailed (64.2%) among strains isolated from cattle. stx(1) and stx(1)stx(2c(2vha)) were the genotypes identified less frequently, and occurred exclusively among strains isolated from food and cattle, respectively. Despite differences in the stx genotypes, all strains carried eae-gamma, efa1, ehx, iha, lpf(O157) and toxB sequences. Many closely related subgroups (more than 80% of similarity) were identified by PFGE, and the presence of a particular O157:H7 STEC clone more related to human infections in Brazil, as well as a common origin for some strains isolated from different sources and countries in Latin America can be suggested.     

Biofilm formation by Pseudomonas aeruginosa wild type,flagella and type IV pili mutants

Biofilm formation by Gfp-tagged Pseudomonas aeruginosa PAO1 wild type, flagella and type IV pili mutants in flow chambers irrigated with citrate minimal medium was characterized by the use of confocal laser scanning microscopy and comstat image analysis. Flagella and type IV pili were not necessary for P. aeruginosa initial attachment or biofilm formation, but the cell appendages had roles in biofilm development, as wild type, flagella and type IV pili mutants formed biofilms with different structures. Dynamics and selection during biofilm formation were investigated by tagging the wild type and flagella/type IV mutants with Yfp and Cfp and performing time-lapse confocal laser scanning microscopy in mixed colour biofilms. The initial microcolony formation occurred by clonal growth, after which wild-type P. aeruginosa bacteria spread over the substratum by means of twitching motility. The wild-type biofilms were dynamic compositions with extensive motility, competition and selection occurring during development. Bacterial migration prevented the formation of larger microcolonial structures in the wild-type biofilms. The results are discussed in relation to the current model for P. aeruginosa biofilm development.     

Developmental pathway for biofilm formation in curli-producing Escherichia coli strains: role of flagella, curli and colanic acid

This work was performed to establish a model describing bacterial surface structures involved in biofilm development, in curli-overproducing Escherichia coli K-12 strains, at 30°C, and in minimal growth medium. Using a genetic approach, in association with observations of sessile communities by light and electron microscopic techniques, the role of protein surface structures, such as flagella and curli, and saccharidic surface components, such as the E. coli exopolysaccharide, colanic acid, was determined. We show that, in the context of adherent ompR234 strains, (i) flagellar motility is not required for initial adhesion and biofilm development; (ii) both primary adhesion to inert surfaces and development of multilayered cell clusters require curli synthesis; (iii) curli display direct interactions with the substratum and form interbacterial bundles, allowing a cohesive and stable association of cells; and (iv) colanic acid does not appear critical for bacterial adhesion and further biofilm development but contributes to the biofilm architecture and allows for the formation of voluminous biofilms.     

Pseudomonas aeruginosa exhibits sliding motility in the absence of type IV pili and flagella

Pseudomonas aeruginosa exhibits swarming motility on 0.5 to 1% agar plates in the presence of specific carbon and nitrogen sources. We have found that PAO1 double mutants expressing neither flagella nor type IV pili (fliC pilA) display sliding motility under the same conditions. Sliding motility was inhibited when type IV pilus expression was restored; like swarming motility, it also decreased in the absence of rhamnolipid surfactant production. Transposon insertions in gacA and gacS increased sliding motility and restored tendril formation to spreading colonies, while transposon insertions in retS abolished motility. These changes in motility were not accompanied by detectable changes in rhamnolipid surfactant production or by the appearance of bacterial surface structures that might power sliding motility. We propose that P. aeruginosa requires flagella during swarming to overcome adhesive interactions mediated by type IV pili. The apparent dependence of sliding motility on environmental cues and regulatory pathways that also affect swarming motility suggests that both forms of motility are influenced by similar cohesive factors that restrict translocation, as well as by dispersive factors that facilitate spreading. Studies of sliding motility may be particularly well-suited for identifying factors other than pili and flagella that affect community behaviors of P. aeruginosa.     

GGDEF proteins YeaI, YedQ, and YfiN reduce early biofilm formation and swimming motility in Escherichia coli

The second messenger 3′–5′-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.     

Expression of type I pili is abolished in verotoxin-producing Escherichia coli O157

Verotoxin-producing Escherichia coli (VTEC) were examined for production of type I pili. None of 34 strains of VTEC serogroup O157 examined expressed any pili, whereas 26 strains of 27 VTEC serogroup O26 and seven strains of nine non-VTEC O157 produced type I pili. These VTEC strains were collected from sporadic human cases and cattle in Okinawa in 1997. The genes encoding the major structural component (FimA) and the adhesin (FimH) of type I pili were detected in all 70 strains examined. The inability to express type I pili could be a unique character of VTEC O157 and this trait could be a new candidate to identify the organisms.     

Archaeal flagella, bacterial flagella and type IV pili: a comparison of genes and posttranslational modifications

The archaeal flagellum is a unique motility organelle. While superficially similar to the bacterial flagellum, several similarities have been reported between the archaeal flagellum and the bacterial type IV pilus system. These include the multiflagellin nature of the flagellar filament, N-terminal sequence similarities between archaeal flagellins and bacterial type IV pilins, as well as the presence of homologous proteins in the two systems. Recent advances in archaeal flagella research add to the growing list of similarities. First, the preflagellin peptidase that is responsible for processing the N-terminal signal peptide in preflagellins has been identified. The preflagellin peptidase is a membrane-bound enzyme topologically similar to its counterpart in the type IV pilus system (prepilin peptidase); the two enzymes are demonstrated to utilize the same catalytic mechanism. Second, it has been suggested that the archaeal flagellum and the bacterial type IV pilus share a similar mode of assembly. While bacterial flagellins and type IV pilins can be modified with O-linked glycans, N-linked glycans have recently been reported on archaeal flagellins. This mode of glycosylation, as well as the observation that the archaeal flagellum lacks a central channel, are both consistent with the proposed assembly model. On the other hand, the failure to identify other genes involved in archaeal flagellation by homology searches likely implies a novel aspect of the archaeal flagellar system. These interesting features remain to be deciphered through continued research. Such knowledge would be invaluable to motility and protein export studies in the Archaea.     

Novel roles for the AIDA adhesin from diarrheagenic Escherichia coli: cell aggregation and biofilm formation

Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact with antigen 43 (Ag43)-expressing cells, which is indicative of an intercellular AIDA-Ag43 interaction. Additionally, AIDA expression dramatically enhances biofilm formation by E. coli on abiotic surfaces in flow chambers.     

Effect of temperature on motility and chemotaxis of Escherichia coli.

The swimming velocity of Escherichia coli at various constant temperatures was found to increase with increasing temperature. The frequency of tumbling had a peak at 34 degrees C and was very low both at 20 and at 39 degrees C. The swimming tracks near the surface of a slide glass showed curves, and the curvature increased the temperature. When the temperature of a bacterial suspension was suddenly changed, a transient change of the tumbling frequency was observed. A temperature drop induced a temporary increase in the tumbling frequency, and a quick rise of temperature, on the other hand, resulted in a temporary suppression of the tumbling. These dynamic responses to sudden changes of temperature was not observed in the smoothly swimming nonchemotactic strains bearing the mutations cheA and cheC and also in a mutant with the metF mutation under a smooth swimming condition.     

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.     

Localization of proteins controlling motility and chemotaxis in Escherichia coli.

Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.     

Catabolite repression of Escherichia coli biofilm formation

Biofilm formation was repressed by glucose in several species of Enterobacteriaceae. In Escherichia coli, this effect was mediated at least in part by cyclic AMP (cAMP)-cAMP receptor protein. A temporal role for cAMP in biofilm development was indicated by the finding that glucose addition after approximately 24 h failed to repress and generally activated biofilm formation.     

Dissociation and reassembly of Escherichia coli type 1 pili.

Escherichia coli type 1 pili, which mediate the mannose-sensitive adherence of the bacterium to eucaryotic cells, are comprised of very stable arrays of pilin protein subunits (molecular weight, approximately 17,000). Previous methods for the dissociation of pili caused their irreversible denaturation. We have found that incubation of pili in saturated guanidine hydrochloride at 37 degrees C led to their complete dissociation, as evidenced by nephelometry and electron microscopy. Gel chromatography of the dissociated pili on a Sepharose CL-6B column in the presence of saturated guanidine hydrochloride yielded a single protein peak with a molecular weight corresponding to that of pilin. Dialysis of this peak against 5 mM tris(hydroxymethyl)aminomethane hydrochloride (pH 8.0) and rechromatography in the same buffer afforded a major protein peak, probably consisting of pilin dimers. About 25% of the protein in this peak bound to a mannan-sepharose column and could be eluted with methyl alpha-D-mannoside. The pilin dimer gave a single protein band upon polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (molecular weight, 16,600) or 10 M urea and penetrated completely into 7% gels in the absence of denaturants. Reassembly of the pilin dimers into pili was achieved upon dialysis against the tris(hydroxymethyl)aminomethane buffer containing 5 mM MgCl2, as observed by electron microscopy. Thus, the conditions used allow renaturation of the dissociated subunits and may aid in further studies of the structure-function relationship of pili.     

Cell envelopes of chemotaxis mutants of Escherichia coli rotate their flagella counterclockwise.

Flagella rotated exclusively counterclockwise in Escherichia coli cell envelopes prepared from wild-type cells, whose flagella rotated both clockwise and counterclockwise, from mutants rotating their flagella counterclockwise only, and even from mutants rotating their flagella primarily clockwise. Some factor needed for clockwise flagellar rotation appeared to be missing or defective in the cell envelopes.     

Mechanical removal of F pili, type I pili, and flagella from Hfr and RTF donor cells and the kinetics of their reappearance

The effect of mechanical agitation (blending) on the removal of F pili, type I pili, and flagella from Hfr (high-frequency recombinant) and resistance transfer factor (RTF) fi(+)Escherichia coli cells was studied by electron microscopy. The reduction in number and length of appendages was measured as a function of blendor speed under standard conditions of temperature, medium, cell density, and blendor configuration. F pili and flagella were removed within the same narrow range of blendor speeds. Type I pili were removed within a higher and broader range of speeds. The speed which reduced the average length of type I pili to 50% was 3.5 times the speed which reduced the average length of F pili to 50%. None of the speeds employed inhibited cell growth, viability, or the ability to produce cell appendages. The kinetics of reappearance of F pili and type I pili after removal by blending were also different. F pili grew out very rapidly, reaching 50% of their full length in 30 sec and their full length in 4 to 5 min. The number of attached F pili per cell also increased rapidly, reaching a constant value in 4 to 5 min. After 5 min, F pilus lengths were distributed around a modal value of about 1.2 mum, and the numbers of F pili per cell were distributed according to a Poisson distribution, with an average of 1.0 per cell. These reappearance kinetics, length distributions, and number distributions are consistent with a model of F-pilus outgrowth in which new F pili appear at random locations on the cell surface at an average rate of about once every 4 min, grow to their characteristic length in about 4 min, and then separate from the cell. F pili which had separated could absorb to the cells, leading to the presence of two classes of F pili on cells: those in the process of natural out-growth and those attached by absorption. Type I pili increased in length much more slowly than did F pili, although the fraction of cells having visible type I pili increased very rapidly after blending because of the large number of type I pili per cell. The fraction of flagellated cells increased even more slowly, reaching only 30% of the unblended fraction in 30 min. The application of blending spectra and reappearance kinetics to the identification of cell functions with surface structures is discussed.