Multiple Streptococcus mutans Genes Are Involved in Biofilm Formation

Streptococcus mutans has been strongly implicated as the principal etiological agent in dental caries. One of the important virulence properties of these organisms is their ability to form biofilms known as dental plaque on tooth surfaces. Since the roles of sucrose and glucosyltransferases in S. mutans biofilm formation have been well documented, we focused our attention on sucrose-independent factors. We have initially identified several mutants that appear to be defective in biofilm formation on abiotic surfaces by an insertional inactivation mutagenesis strategy applied to S. mutans. A total of 27 biofilm-defective mutants were isolated and analyzed in this study. From these mutants, three genes were identified. One of the mutants was defective in the Bacillus subtilis lytR homologue. Another of the biofilm-defective mutants isolated was a yulF homologue, which encodes a hypothetical protein of B. subtilis whose function in biofilm formation is unknown. The vast majority of the mutants were defective in the comB gene required for competence. We therefore have constructed and examined comACDE null mutants. These mutants were also found to be attenuated in biofilm formation. Biofilm formation by several other regulatory gene mutants were also characterized using an in vitro biofilm-forming assay. These results suggest that competence genes as well as the sgp and dgk genes may play important roles in S. mutans biofilm formation.     

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.     

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。     

Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili

We have used Escherichia coli as a model system to investigate the initiation of biofilm formation. Here, we demonstrate that E. coli forms biofilms on multiple abiotic surfaces in a nutrient-dependent fashion. In addition, we have isolated insertion mutations that render this organism defective in biofilm formation. One-half of these mutations was found to perturb normal flagellar function. Using defined fli , flh , mot and che alleles, we show that motility, but not chemotaxis, is critical for normal biofilm formation. Microscopic analyses of these mutants suggest that motility is important for both initial interaction with the surface and for movement along the surface. In addition, we present evidence that type I pili (harbouring the mannose-specific adhesin, FimH) are required for initial surface attachment and that mannose inhibits normal attachment. In light of the observations presented here, a working model is discussed that describes the roles of both motility and type I pili in biofilm development.     

The spatial architecture of Bacillus subtilis biofilms deciphered using a surface-associated model and in situ imaging

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding "beanstalk-like" structures with certain strains. Indeed, these structures can reach a height of more than 300 μm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities.     

Evidence for Cyclic Di-GMP-Mediated Signaling in Bacillus subtilis

Cyclic di-GMP (c-di-GMP) is a second messenger that regulates diverse cellular processes in bacteria, including motility, biofilm formation, cell-cell signaling, and host colonization. Studies of c-di-GMP signaling have chiefly focused on Gram-negative bacteria. Here, we investigated c-di-GMP signaling in the Gram-positive bacterium Bacillus subtilis by constructing deletion mutations in genes predicted to be involved in the synthesis, breakdown, or response to the second messenger. We found that a putative c-di-GMP-degrading phosphodiesterase, YuxH, and a putative c-di-GMP receptor, YpfA, had strong influences on motility and that these effects depended on sequences similar to canonical EAL and RxxxR-D/NxSxxG motifs, respectively. Evidence indicates that YpfA inhibits motility by interacting with the flagellar motor protein MotA and that yuxH is under the negative control of the master regulator Spo0A～P. Based on these findings, we propose that YpfA inhibits motility in response to rising levels of c-di-GMP during entry into stationary phase due to the downregulation of yuxH by Spo0A～P. We also present evidence that YpfA has a mild influence on biofilm formation. In toto, our results demonstrate the existence of a functional c-di-GMP signaling system in B. subtilis that directly inhibits motility and directly or indirectly influences biofilm formation.     

MotPS is the stator-force generator for motility of alkaliphilic Bacillus, and its homologue is a second functional Mot in Bacillus subtilis

The stator-force generator that drives Na+-dependent motility in alkaliphilic Bacillus pseudofirmus OF4 is identified here as MotPS, MotAB-like proteins with genes that are downstream of the ccpA gene, which encodes a major regulator of carbon metabolism. B. pseudofirmus OF4 was only motile at pH values above 8. Disruption of motPS resulted in a non-motile phenotype, and motility was restored by transformation with a multicopy plasmid containing the motPS genes. Purified and reconstituted MotPS from B. pseudofirmus OF4 catalysed amiloride analogue-sensitive Na+ translocation. In contrast to B. pseudofirmus, Bacillus subtilis contains both MotAB and MotPS systems. The role of the motPS genes from B. subtilis in several motility-based behaviours was tested in isogenic strains with intact motAB and motPS loci, only one of the two mot systems or neither mot system. B. subtilis MotPS (BsMotPS) supported Na+-stimulated motility, chemotaxis on soft agar surfaces and biofilm formation, especially after selection of an up-motile variant. BsMotPS also supported motility in agar soft plugs immersed in liquid; motility was completely inhibited by an amiloride analogue. BsMotPS did not support surfactin-dependent swarming on higher concentration agar surfaces. These results indicate that BsMotPS contributes to biofilm formation and motility on soft agar, but not to swarming, in laboratory strains of B. subtilis in which MotAB is the dominant stator-force generator. BsMotPS could potentially be dominant for motility in B. subtilis variants that arise in particular niches.     

Initial Phases of biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.     

The effect of mutations in the synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by Azospirillum brasilense

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

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.     

Effect of Different Nutritional Conditions on the Synthesis of Tricarboxylic Acid Cycle Enzymes

The effect of various nutritional conditions on the levels of Krebs cycle enzymes in Bacillus subtilis, B. licheniformis, and Escherichia coli was determined. The addition of glutamate, alpha-ketoglutarate, or compounds capable of being catabolized to glutamate, to a minimal glucose medium resulted in complete repression of aconitase in B. subtilis and B. licheniformis. The synthesis of fumarase, succinic dehydrogenase, malic dehydrogenase, and isocitric dehydrogenase was not repressed by these compounds. It is postulated that glutamate or alpha-ketoglutarate is the true corepressor for the repression of aconitase. A rapidly catabolizable carbon source and alpha-ketoglutarate or glutamate must be simultaneously present for complete repression of the formation of aconitase. Conditions which repress the synthesis of aconitase in B. subtilis restrict the flow of carbon in the sequence of reactions leading to alpha-ketoglutarate but do not prevent glutamate oxidation in vivo. The data indicate that separate and independent mechanisms regulate the activity of the anabolic and catabolic reactions of the Krebs cycle in B. subtilis and B. licheniformis. The addition of glutamate to the minimal glucose medium results in the repression of aconitase, isocitric dehydrogenase, and fumarase, but not malic dehydrogenase in E. coli K-38.     

Rapid surface motility in Bacillus subtilis is dependent on extracellular surfactin and potassium ion

Motility on surfaces is an important mechanism for bacterial colonization of new environments. In this report, we describe detection of rapid surface motility in the wild-type Bacillus subtilis Marburg strain, but not in several B. subtilis 168 derivatives. Motility involved formation of rapidly spreading dendritic structures, followed by profuse surface colonies if sufficient potassium ion was present. Potassium ion stimulated surfactin secretion, and the role of surfactin in surface motility was confirmed by deletion of a surfactin synthase gene. Significantly, this motility was independent of flagella. These results demonstrate that wild-type B. subtilis strains can use both swimming and sliding-type mechanisms to move across surfaces.     

The EpsE flagellar clutch is bifunctional and synergizes with EPS biosynthesis to promote Bacillus subtilis biofilm formation

Many bacteria inhibit motility concomitant with the synthesis of an extracellular polysaccharide matrix and the formation of biofilm aggregates. In Bacillus subtilis biofilms, motility is inhibited by EpsE, which acts as a clutch on the flagella rotor to inhibit motility, and which is encoded within the 15 gene eps operon required for EPS production. EpsE shows sequence similarity to the glycosyltransferase family of enzymes, and we demonstrate that the conserved active site motif is required for EPS biosynthesis. We also screen for residues specifically required for either clutch or enzymatic activity and demonstrate that the two functions are genetically separable. Finally, we show that, whereas EPS synthesis activity is dominant for biofilm formation, both functions of EpsE synergize to stabilize cell aggregates and relieve selective pressure to abolish motility by genetic mutation. Thus, the transition from motility to biofilm formation may be governed by a single bifunctional enzyme.     

Characterization of Bacillus subtilis mutants with carbon source-independent glutamate biosynthesis

Bacillus subtilis synthesizes glutamate from 2-oxoglutarate and glutamine using the glutamate synthase, encoded by the gltAB operon. Glutamate degradation involves the catabolic glutamate dehydrogenase (GDH) RocG. Expression of both gltAB and rocG is controlled by the carbon and nitrogen sources. In the absence of glucose or other well-metabolizable carbon sources, B. subtilis is unable to grow unless provided with external glutamate. In this work, we isolated mutations that suppressed this growth defect of B. subtilis on minimal media (sgd mutants). All mutations enabled the cells to express the gltAB operon even in the absence of glucose. The mutations were all identified in the rocG gene suggesting that the catabolic GDH is essential for controlling gltAB expression in response to the availability of sugars.     

Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.     

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.     

The effect of mutations affecting synthesis of lipopolysaccharides and calcofluor-binding polysaccharides on biofilm formation by <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

The thickness and antigenic properties of biofilms produced by Azospirillum brasilense Sp245 and its mutants deficient in the synthesis of lipopolysaccharides (Lps) and calcofluor-binding polysaccharides (CBPS) at the interface between water and hydrophilic or hydrophobic solid surfaces were compared. The mutants deficient in acidic LpsI synthesis produce thicker biofilms on hydrophilic surfaces. Biofilms produced on hydrophobic surfaces by bacteria that are unable to synthesize CBPS are less pronounced. Defects in CBPS production in Azospirillum mutants with impaired flagellar motility can cause adverse effects on the cell ability to attach to hydrophobic and hydrophilic surfaces. The loss of the neutral LpsII antigen by the mutants capable of producing CBPS does not affect their behavior on hydrophobic surfaces, which is probably due to the compensatory increase in the total polysaccharide production. The fundamental change in the Lps structure correlates with the activation of biofilm formation by the relevant mutants on hydrophilic and hydrophobic surfaces.     

Genes involved in formation of structured multicellular communities by Bacillus subtilis

The spore-forming bacterium Bacillus subtilis is capable of assembling multicellular communities (biofilms) that display a high degree of spatiotemporal organization. Wild strains that have not undergone domestication in the laboratory produce particularly robust biofilms with complex architectural features, such as fruiting-body-like aerial projections whose tips serve as preferential sites for sporulation. To discover genes involved in this multicellular behavior and to do so on a genome-wide basis, we took advantage of a large collection of mutants which have disruptions of most of the uncharacterized genes in the B. subtilis genome. This collection, which was generated with a laboratory strain, was screened for mutants that were impaired in biofilm formation. This subset of mutated genes was then introduced into the wild strain NCIB 3610 to study their effects on biofilm formation in liquid and solid media. In this way we identified six genes that are involved in the development of multicellular communities. These are yhxB (encoding a putative phosphohexomutase that may mediate exopolysaccharide synthesis), sipW (encoding a signal peptidase), ecsB (encoding an ABC transporter subunit), yqeK (encoding a putative phosphatase), ylbF (encoding a regulatory protein), and ymcA (a gene of unknown function). Further analysis revealed that these six genes play different roles in B. subtilis community development.