Facultative Control of Matrix Production Optimizes Competitive Fitness in Pseudomonas aeruginosa PA14 Biofilm Models

As biofilms grow, resident cells inevitably face the challenge of resource limitation. In the opportunistic pathogen Pseudomonas aeruginosa PA14, electron acceptor availability affects matrix production and, as a result, biofilm morphogenesis. The secreted matrix polysaccharide Pel is required for pellicle formation and for colony wrinkling, two activities that promote access to O₂. We examined the exploitability and evolvability of Pel production at the air-liquid interface (during pellicle formation) and on solid surfaces (during colony formation). Although Pel contributes to the developmental response to electron acceptor limitation in both biofilm formation regimes, we found variation in the exploitability of its production and necessity for competitive fitness between the two systems. The wild type showed a competitive advantage against a non-Pel-producing mutant in pellicles but no advantage in colonies. Adaptation to the pellicle environment selected for mutants with a competitive advantage against the wild type in pellicles but also caused a severe disadvantage in colonies, even in wrinkled colony centers. Evolution in the colony center produced divergent phenotypes, while adaptation to the colony edge produced mutants with clear competitive advantages against the wild type in this O₂-replete niche. In general, the structurally heterogeneous colony environment promoted more diversification than the more homogeneous pellicle. These results suggest that the role of Pel in community structure formation in response to electron acceptor limitation is unique to specific biofilm models and that the facultative control of Pel production is required for PA14 to maintain optimum benefit in different types of communities.     

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.     

Genome-wide mutagenesis of Xanthomonas axonopodis pv. citri reveals novel genetic determinants and regulation mechanisms of biofilm formation

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker disease, a major threat to citrus production worldwide. Accumulating evidence suggests that the formation of biofilms on citrus leaves plays an important role in the epiphytic survival of this pathogen prior to the development of canker disease. However, the process of Xac biofilm formation is poorly understood. Here, we report a genome-scale study of Xac biofilm formation in which we identified 92 genes, including 33 novel genes involved in biofilm formation and 7 previously characterized genes, colR, fhaB, fliC, galU, gumD, wxacO, and rbfC, known to be important for Xac biofilm formation. In addition, 52 other genes with defined or putative functions in biofilm formation were identified, even though they had not previously reported been to be associated with biofilm formation. The 92 genes were isolated from 292 biofilm-defective mutants following a screen of a transposon insertion library containing 22,000 Xac strain 306 mutants. Further analyses indicated that 16 of the novel genes are involved in the production of extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS), 7 genes are involved in signaling and regulatory pathways, and 5 genes have unknown roles in biofilm formation. Furthermore, two novel genes, XAC0482, encoding a haloacid dehalogenase-like phosphatase, and XAC0494 (designated as rbfS), encoding a two-component sensor protein, were confirmed to be biofilm-related genes through complementation assays. Our data demonstrate that the formation of mature biofilm requires EPS, LPS, both flagellum-dependent and flagellum-independent cell motility, secreted proteins and extracellular DNA. Additionally, multiple signaling pathways are involved in Xac biofilm formation. This work is the first report on a genome-wide scale of the genetic processes of biofilm formation in plant pathogenic bacteria. The report provides significant new information about the genetic determinants and regulatory mechanism of biofilm formation.     

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.     

Genes involved in matrix formation in Pseudomonas aeruginosa PA14 biofilms

Pseudomonas aeruginosa forms diverse matrix-enclosed surface-associated multicellular assemblages (biofilms) that aid in its survival in a variety of environments. One such biofilm is the pellicle that forms at the air-liquid interface in standing cultures. We screened for transposon insertion mutants of P. aeruginosa PA14 that were unable to form pellicles. Analysis of these mutants led to the identification of seven adjacent genes, named pel genes, the products of which appear to be involved in the formation of the pellicle's extracellular matrix. In addition to being required for pellicle formation, the pel genes are also required for the formation of solid surface-associated biofilms. Sequence analyses predicted that three pel genes encode transmembrane proteins and that five pel genes have functional homologues involved in carbohydrate processing. Microscopic and macroscopic observations revealed that wild-type P. aeruginosa PA14 produces a cellulase-sensitive extracellular matrix able to bind Congo red; no extracellular matrix was produced by the pel mutants. A comparison of the carbohydrates produced by the wild-type strain and pel mutants suggested that glucose was a principal component of the matrix material. Together, these results suggest that the pel genes are responsible for the production of a glucose-rich matrix material required for the formation of biofilms by P. aeruginosa PA14.     

冷激条件下预形成副溶血性弧菌生物被膜的发展变化

【目的】生物被膜可附着在食品或医药生物材料表面引起持续性的感染,而现今极少有关于温度变化对预形成生物被膜影响的研究。本文分析了冷激条件下副溶血性弧菌预形成生物被膜的发展变化。【方法】以改进的结晶紫染色法检测生物被膜总量,以改进的超声法和Lowry法量化胞外多糖和蛋白质,以RNA试剂盒提取并纯化生物被膜形成的相关基因。同时,激光共聚焦扫描显微镜直观显示了冷激条件下预形成生物被膜形态结构的变化,并深入分析了生物被膜结构的变化,以及生物被膜结构参数和基因转录的相关性。【结果】在冷激条件下,副溶血性弧菌生物被膜总量增加,同时,副溶血性弧菌预形成的生物被膜胞外多聚物的主要成分胞外多糖和蛋白质也逐渐增加,被膜形成相关鞭毛基因和毒力基因的转录活跃。生物被膜的平均厚度(MT)、平均扩散距离(ADD)、孔隙率(P)、生物被膜粗糙度(BR)和均匀性(H)在冷激过程中也发生了变化,同时这些参数之间存在显著相关性(P<0.01),而生物被膜结构参数与生物被膜相关基因转录的相关性较弱(P<0.05)。【结论】因此,4°C和10°C冷激不能完全抑制副溶血性弧菌预形成生物被膜的生长,风险评估人员在制定控制食源性感染风险的战略时应考虑到这一因素。     

Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111

Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini-Tn5 insertion mutants of B. cepacia H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three-dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i). genes encoding for surface proteins; (ii). genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii). genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of N-octanoylhomoserine lactone (C8-HSL). This compound serves as the major signal molecule of the cep quorum-sensing system. As this density-dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum-sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the cep system, indicating that the quorum-sensing system of B. cepacia is a major checkpoint for biofilm formation.     

The characterization of functions involved in the establishment and maturation of Klebsiella pneumoniae in vitro biofilm reveals dual roles for surface exopolysaccharides

The ability to form biofilm is seen as an increasingly important colonization strategy among both pathogenic and environmental Klebsiella pneumoniae strains. The aim of the present study was to identify abiotic surface colonization factors of K. pneumoniae using different models at different phases of biofilm development. A 2200 K. pneumoniae mutant library previously obtained by signature-tagged mutagenesis was screened in static and dynamic culture models to detect clones impaired at early and/or mature stages of biofilm formation. A total of 28 mutants were affected during late phases of biofilm formation, whereas 16 mutants displayed early adhesion defect. These mutants corresponded to genes involved in potential cellular and DNA metabolism pathways and to membrane transport functions. Eight mutants were deficient in capsule or LPS production. Gene disruption and microscopic analyses showed that LPS is involved in initial adhesion on both glass and polyvinyl-chloride and the capsule required for the appropriate initial coverage of substratum and the construction of mature biofilm architecture. These results give new insight into the bacterial factors sequentially associated with the ability to colonize an abiotic surface and reveal the dual roles played by surface exopolysaccharides during K. pneumoniae biofilm formation.     

Proteomic analysis of Campylobacter jejuni 11168 biofilms reveals a role for the motility complex in biofilm formation

Campylobacter jejuni remains the leading cause of bacterial gastroenteritis in developed countries, and yet little is known concerning the mechanisms by which this fastidious organism survives within its environment. We have demonstrated that C. jejuni 11168 can form biofilms on a variety of surfaces. Proteomic analyses of planktonic and biofilm-grown cells demonstrated differences in protein expression profiles between the two growth modes. Proteins involved in the motility complex, including the flagellins (FlaA, FlaB), the filament cap (FliD), the basal body (FlgG, FlgG2), and the chemotactic protein (CheA), all exhibited higher levels of expression in biofilms than found in stationary-phase planktonic cells. Additional proteins with enhanced expression included those involved in the general (GroEL, GroES) and oxidative (Tpx, Ahp) stress responses, two known adhesins (Peb1, FlaC), and proteins involved in biosynthesis, energy generation, and catabolic functions. An aflagellate flhA mutant not only lost the ability to attach to a solid matrix and form a biofilm but could no longer form a pellicle at the air-liquid interface of a liquid culture. Insertional inactivation of genes that affect the flagellar filament (fliA, flaA, flaB, flaG) or the expression of the cell adhesin (flaC) also resulted in a delay in pellicle formation. These findings demonstrate that the flagellar motility complex plays a crucial role in the initial attachment of C. jejuni 11168 to solid surfaces during biofilm formation as well as in the cell-to-cell interactions required for pellicle formation. Continued expression of the motility complex in mature biofilms is unusual and suggests a role for the flagellar apparatus in the biofilm phenotype.     

Proteins exported via the PrsD-PrsE type I secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium leguminosarum

The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.     

The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies.     

The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.     

Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis

Populations of surface-attached microorganisms comprising either single or multiple species are commonly referred to as biofilms. Using a simple assay for the initiation of biofilm formation (e.g. attachment to an abiotic surface) by Pseudomonas fluorescens strain WCS365, we have shown that: (i) P . fluorescens can form biofilms on an abiotic surface when grown on a range of nutrients; (ii) protein synthesis is required for the early events of biofilm formation; (iii) one (or more) extracytoplasmic protein plays a role in interactions with an abiotic surface; (iv) the osmolarity of the medium affects the ability of the cell to form biofilms. We have isolated transposon mutants defective for the initiation of biofilm formation, which we term surface attachment defective ( sad ). Molecular analysis of the sad mutants revealed that the ClpP protein (a component of the cytoplasmic Clp protease) participates in biofilm formation in this organism. Our genetic analyses suggest that biofilm formation can proceed via multiple, convergent signalling pathways, which are regulated by various environmental signals. Finally, of the 24 sad mutants analysed in this study, only three had defects in genes of known function. This result suggests that our screen is uncovering novel aspects of bacterial physiology.     

Bap, a Staphylococcus aureus surface protein involved in biofilm formation

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa and Salmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350 S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection model bap was involved in pathogenesis, causing a persistent infection.