vpsA- and luxO-independent biofilms of Vibrio cholerae

The natural life cycle of Vibrio cholerae involves the transitioning of cells between different environmental surfaces such as the chitinous shell of Crustaceae and the epithelial layer of the human intestine. Previous studies using static biofilm systems showed a strict dependence of biofilm formation on the vps and lux genes, which are essential for exopolysaccharide formation and cell-cell signaling, respectively. The authors' report here that in biofilms grown under hydrodynamic conditions, DeltavpsA and DeltaluxO mutants of V. cholerae do form pronounced, three-dimensional biofilms that resemble all aspects of wild-type biofilms. By genetic experiments, it was shown that in hydrodynamically grown biofilms this independence of vpsA is due to the expression of rpoS, which is a negative regulator of vpsA expression. Biofilms also underwent substantial dissolution after 96 h that could be induced by a simple stop of medium flow. The studies indicate that metabolic conditions control the reversible attachment of cells to the biofilm matrix and are key in regulating biofilm cell physiology via RpoS. Furthermore, the results redefine the roles of vps and quorum-sensing in V. cholerae biofilms.     

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.     

The caudal complex of Giardia lamblia and its relation to motility

This paper presents a detailed study of the caudal complex of Giardia lamblia and its relation to movements observed in this region. The caudal complex of Giardia, composed of axonemes from the caudal flagella plus associated microtubular sheets, was investigated by light, electron microscopy, and 3D reconstruction tools. By the use of video-microscopy and digital image processing techniques, we were able to visualize in detail the caudal movements. A non-ionic detergent, Triton X-100, was used to isolate the complex that was afterwards analyzed by video-microscopy and transmission electron microscopy (TEM). We showed for the first time, using video-microscopy, that the intracellular portion of the caudal flagella axonemes presented motility, even after the disrupture of the cell membrane, contrasting with the caudal flagella themselves, that do not show active beating pattern. To check if actin filaments play a role in the above described movements, as previously supposed, we incubated the cells with jasplakinolide, a drug that induces the disruption of actin filaments in living cells. The experiments demonstrated that the drug did not affect the caudal motility. The analysis of the caudal complex by transmission electron microscopy (TEM) revealed that, even after the exposure to higher detergent concentrations, the connections between their components remained intact. The information obtained by TEM and 3D reconstruction tools showed that the region between both nuclei marks the intracellular end of the caudal complex, which proceeds toward the caudal portion of the cell following its longitudinal axis, where the axonemes emerge as the caudal flagella. The results obtained from video-microscopy assays of the isolated beating complex together with the 3D reconstruction data indicated that the internal portion of the caudal flagella is the force-generator of the movements in this region.     

Gene expression by the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough grown on an iron electrode under cathodic protection conditions

The genome sequence of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough was reanalyzed to design unique 70-mer oligonucleotide probes against 2,824 probable protein-coding regions. These included three genes not previously annotated, including one that encodes a c-type cytochrome. Using microarrays printed with these 70-mer probes, we analyzed the gene expression profile of wild-type D. vulgaris grown on cathodic hydrogen, generated at an iron electrode surface with an imposed negative potential of -1.1 V (cathodic protection conditions). The gene expression profile of cells grown on cathodic hydrogen was compared to that of cells grown with gaseous hydrogen bubbling through the culture. Relative to the latter, the electrode-grown cells overexpressed two hydrogenases, the hyn-1 genes for [NiFe] hydrogenase 1 and the hyd genes, encoding [Fe] hydrogenase. The hmc genes for the high-molecular-weight cytochrome complex, which allows electron flow from the hydrogenases across the cytoplasmic membrane, were also overexpressed. In contrast, cells grown on gaseous hydrogen overexpressed the hys genes for [NiFeSe] hydrogenase. Cells growing on the electrode also overexpressed genes encoding proteins which promote biofilm formation. Although the gene expression profiles for these two modes of growth were distinct, they were more closely related to each other than to that for cells grown in a lactate- and sulfate-containing medium. Electrochemically measured corrosion rates were lower for iron electrodes covered with hyn-1, hyd, and hmc mutant biofilms than for wild-type biofilms. This confirms the importance, suggested by the gene expression studies, of the corresponding gene products in D. vulgaris-mediated iron corrosion.     

Salmonella enterica serovar typhimurium swarming mutants with altered biofilm-forming abilities: surfactin inhibits biofilm formation

Swarming motility plays an important role in surface colonization by several flagellated bacteria. Swarmer cells are specially adapted to rapidly translocate over agar surfaces by virtue of their more numerous flagella, longer cell length, and encasement of slime. The external slime provides the milieu for motility and likely harbors swarming signals. We recently reported the isolation of swarming-defective transposon mutants of Salmonella enterica serovar Typhimurium, a large majority of which were defective in lipopolysaccharide (LPS) synthesis. Here, we have examined the biofilm-forming abilities of the swarming mutants using a microtiter plate assay. A whole spectrum of efficiencies were observed, with LPS mutants being generally more proficient than wild-type organisms in biofilm formation. Since we have postulated that O-antigen may serve a surfactant function during swarming, we tested the effect of the biosurfactant surfactin on biofilm formation. We report that surfactin inhibits biofilm formation of wild-type S. enterica grown either in polyvinyl chloride microtiter wells or in urethral catheters. Other bio- and chemical surfactants tested had similar effects.     

Flagellar motility is critical for Listeria monocytogenes biofilm formation

The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. We observed that nonmotile mutants were defective in biofilm formation. To investigate how flagella might function during biofilm formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature biofilm formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent biofilm formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.     

Contribution of mobile genetic elements to Desulfovibrio vulgaris genome plasticity

The genome of Desulfovibrio vulgaris strain DePue, a sulfate-reducing Deltaproteobacterium isolated from heavy metal-impacted lake sediment, was completely sequenced and compared with the type strain D. vulgaris Hildenborough. The two genomes share a high degree of relatedness and synteny, but harbour distinct prophage and signatures of past phage encounters. In addition to a highly variable phage contribution, the genome of strain DePue contains a cluster of open-reading frames not found in strain Hildenborough coding for the production and export of a capsule exopolysaccharide, possibly of relevance to heavy metal resistance. Comparative whole-genome microarray analysis on four additional D. vulgaris strains established greater interstrain variation within regions associated with phage insertion and exopolysaccharide biosynthesis.     

Identification of psl, a locus encoding a potential exopolysaccharide that is essential for Pseudomonas aeruginosa PAO1 biofilm formation

Bacteria inhabiting biofilms usually produce one or more polysaccharides that provide a hydrated scaffolding to stabilize and reinforce the structure of the biofilm, mediate cell-cell and cell-surface interactions, and provide protection from biocides and antimicrobial agents. Historically, alginate has been considered the major exopolysaccharide of the Pseudomonas aeruginosa biofilm matrix, with minimal regard to the different functions polysaccharides execute. Recent chemical and genetic studies have demonstrated that alginate is not involved in the initiation of biofilm formation in P. aeruginosa strains PAO1 and PA14. We hypothesized that there is at least one other polysaccharide gene cluster involved in biofilm development. Two separate clusters of genes with homology to exopolysaccharide biosynthetic functions were identified from the annotated PAO1 genome. Reverse genetics was employed to generate mutations in genes from these clusters. We discovered that one group of genes, designated psl, are important for biofilm initiation. A PAO1 strain with a disruption of the first two genes of the psl cluster (PA2231 and PA2232) was severely compromised in biofilm initiation, as confirmed by static microtiter and continuous culture flow cell and tubing biofilm assays. This impaired biofilm phenotype could be complemented with the wild-type psl sequences and was not due to defects in motility or lipopolysaccharide biosynthesis. These results implicate an as yet unknown exopolysaccharide as being required for the formation of the biofilm matrix. Understanding psl-encoded exopolysaccharide expression and protection in biofilms will provide insight into the pathogenesis of P. aeruginosa in cystic fibrosis and other infections involving biofilms.     

A self-produced trigger for biofilm disassembly that targets exopolysaccharide

Biofilms are structured communities of bacteria that are held together by an extracellular matrix consisting of protein and exopolysaccharide. Biofilms often have a limited lifespan, disassembling as nutrients become exhausted and waste products accumulate. D-amino acids were previously identified as a self-produced factor that mediates biofilm disassembly by causing the release of the protein component of?the matrix in Bacillus subtilis. Here we report that?B.?subtilis produces an additional biofilm-disassembly factor, norspermidine. Dynamic light scattering and scanning electron microscopy experiments indicated that norspermidine interacts directly and specifically with exopolysaccharide. D-amino acids and norspermidine acted together to break down existing biofilms and mutants blocked in the production of both factors formed long-lived biofilms. Norspermidine, but not closely related polyamines, prevented biofilm formation by B.?subtilis, Escherichia coli, and Staphylococcus aureus.     

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.     

Inhibitory effects of D-amino acids on Staphylococcus aureus biofilm development

Biofilms are communities of cells held together by a self-produced extracellular matrix typically consisting of protein, exopolysaccharide, and often DNA. A natural signal for biofilm disassembly in Bacillus subtilis is certain D-amino acids, which are incorporated into the peptidoglycan and trigger the release of the protein component of the matrix. D-amino acids also prevent biofilm formation by the related Gram-positive bacterium Staphylococcus aureus. Here we employed fluorescence microscopy and confocal laser scanning microscopy to investigate how D-amino acids prevent biofilm formation by S. aureus. We report that biofilm formation takes place in two stages, initial attachment to surfaces, resulting in small foci, and the subsequent growth of the foci into large aggregates. D-amino acids did not prevent the initial surface attachment of cells but blocked the subsequent growth of the foci into larger assemblies of cells. Using protein- and polysaccharide-specific stains, we have shown that D-amino acids inhibited the accumulation of the protein component of the matrix but had little effect on exopolysaccharide production and localization within the biofilm. We conclude that D-amino acids act in an analogous manner to prevent biofilm development in B. subtilis and S. aureus. Finally, to investigate the potential utility of D-amino acids in preventing device-related infections, we have shown that surfaces impregnated with D-amino acids were effective in preventing biofilm growth.     

Response of Xylella fastidiosa to Zinc: Decreased Culturability,Increased Exopolysaccharide Production,and Formation of Resilient Biofilms under Flow Conditions

The bacterial plant pathogen Xylella fastidiosa produces biofilm that accumulates in the host xylem vessels, affecting disease development in various crops and bacterial acquisition by insect vectors. Biofilms are sensitive to the chemical composition of the environment, and mineral elements being transported in the xylem are of special interest for this pathosystem. Here, X. fastidiosa liquid cultures were supplemented with zinc and compared with nonamended cultures to determine the effects of Zn on growth, biofilm, and exopolysaccharide (EPS) production under batch and flow culture conditions. The results show that Zn reduces growth and biofilm production under both conditions. However, in microfluidic chambers under liquid flow and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen in the Zn-amended medium. Biofilms formed under these conditions were strongly attached to surfaces and were not removed by medium flow. This phenomenon was correlated with increased EPS production in stationary-phase cells grown under high Zn concentrations. Zn did not cause greater adhesion to surfaces by individual cells. Additionally, viability analyses suggest that X. fastidiosa may be able to enter the viable but nonculturable state in vitro, and Zn can hasten the onset of this state. Together, these findings suggest that Zn can act as a stress factor with pleiotropic effects on X. fastidiosa and indicate that, although Zn could be used as a bactericide treatment, it could trigger the undesired effect of stronger biofilm formation upon reinoculation events.     

鞭毛介导的运动性与细菌生物膜的相互关系

摘要:由于运动缺陷型细菌形成生物膜的能力会下降,长期以来细菌的运动性都被认为与生物膜的形成呈正相关,但这一理论现在证明还有待商榷,而且运动性不是影响膜形成的绝对因素。本文详细介绍了细菌的生物膜和运动性,并重新定义了两者的相互关系。     

Pleiotropic effects of a Yersinia enterocolitica ompR mutation on adherent-invasive abilities and biofilm formation

The OmpR regulator positively influences flagella synthesis and negatively regulates invasin expression in Yersinia enterocolitica. To determine the physiological consequences of this inverse regulation, we analyzed the effect of the ompR mutation on the ability of Y. enterocolitica Ye9 (serotype O9, biotype 2) to adhere to and invade human epithelial HEp-2 cells and to form biofilms. Cell culture assays with ompR, flhDC and inv mutant strains, which vary in their motility and invasin expression, confirmed the important contribution of flagella to the adherent-invasive abilities of Y. enterocolitica Ye9. However, the loss of motility in the ompR strain was apparently not responsible for its low adhesion ability. When the nonmotile phenotype of the ompR mutant was artificially eliminated, an elevated level of invasion, exceeding that of the wild-type strain, was observed. Confocal laser microscopy demonstrated a decrease in the biofilm formation ability of the ompR strain that was only partially correlated with its loss of motility. These data provide evidence that OmpR promotes biofilm formation in this particular strain of Y. enterocolitica, although additional OmpR-dependent factors are also required. In addition, our findings suggest that OmpR-dependent regulation of biofilm formation could be an additional aspect of OmpR regulatory function.     

Steps in the development of a Vibrio cholerae El Tor biofilm

We report that, in a simple, static culture system, wild-type Vibrio cholerae El Tor forms a three-dimensional biofilm with characteristic water channels and pillars of bacteria. Furthermore, we have isolated and characterized transposon insertion mutants of V. cholerae that are defective in biofilm development. The transposons were localized to genes involved in (i) the biosynthesis and secretion of the mannose-sensitive haemagglutinin type IV pilus (MSHA); (ii) the synthesis of exopolysaccharide; and (iii) flagellar motility. The phenotypes of these three groups suggest that the type IV pilus and flagellum accelerate attachment to the abiotic surface, the flagellum mediates spread along the abiotic surface, and exopolysaccharide is involved in the formation of three-dimensional biofilm architecture.     

Engineering a novel c-di-GMP-binding protein for biofilm dispersal

Bacteria prefer to grow attached to themselves or an interface, and it is important for an array of applications to make biofilms disperse. Here we report simultaneously the discovery and protein engineering of BdcA (formerly YjgI) for biofilm dispersal using the universal signal 3,5-cyclic diguanylic acid (c-di-GMP). The bdcA deletion reduced biofilm dispersal, and production of BdcA increased biofilm dispersal to wild-type level. Since BdcA increases motility and extracellular DNA production while decreasing exopolysaccharide, cell length and aggregation, we reasoned that BdcA decreases the concentration of c-di-GMP, the intracellular messenger that controls cell motility through flagellar rotation and biofilm formation through synthesis of curli and cellulose. Consistently, c-di-GMP levels increase upon deleting bdcA, and purified BdcA binds c-di-GMP but does not act as a phosphodiesterase. Additionally, BdcR (formerly YjgJ) is a negative regulator of bdcA. To increase biofilm dispersal, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete removal of biofilms via dispersal without affecting initial biofilm formation.     

Inverse regulation of biofilm formation and swarming motility by Pseudomonas aeruginosa PA14

We previously reported that SadB, a protein of unknown function, is required for an early step in biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa. Here we report that a mutation in sadB also results in increased swarming compared to the wild-type strain. Our data are consistent with a model in which SadB inversely regulates biofilm formation and swarming motility via its ability both to modulate flagellar reversals in a viscosity-dependent fashion and to influence the production of the Pel exopolysaccharide. We also show that SadB is required to properly modulate flagellar reversal rates via chemotaxis cluster IV (CheIV cluster). Mutational analyses of two components of the CheIV cluster, the methyl-accepting chemotaxis protein PilJ and the PilJ demethylase ChpB, support a model wherein this chemotaxis cluster participates in the inverse regulation of biofilm formation and swarming motility. Epistasis analysis indicates that SadB functions upstream of the CheIV cluster. We propose that P. aeruginosa utilizes a SadB-dependent, chemotaxis-like regulatory pathway to inversely regulate two key surface behaviors, biofilm formation and swarming motility.     

Pantoea stewartii subsp. stewartii exhibits surface motility, which is a critical aspect of Stewart's wilt disease development on maize

Pantoea stewartii subsp. stewartii is a plant-pathogenic bacterium that causes Stewart's vascular wilt in maize. The organism is taxonomically described as aflagellated and nonmotile. We recently showed that P. stewartii colonizes the xylem of maize as sessile, cell-wall-adherent biofilms. Biofilm formation is a developmental process that generally involves some form of surface motility. For that reason, we reexamined the motility properties of P. stewartii DC283 based on the assumption that the organism requires some form of surface motility for biofilm development. Here, we show that the organism is highly motile on agar surfaces. This motility is flagella dependent, shown by the fact that a fliC mutant, impaired in flagellin subunit synthesis, is nonmotile. Motility also requires the production of stewartan exopolysaccharide. Moreover, surface motility plays a significant role in the colonization of the plant host.