Biofilm formation of Pseudomonas putida IsoF: the role of quorum sensing as assessed by proteomics

Pseudomonas putida strains are frequently isolated from the rhizosphere of plants and many strains promote plant-growth, exhibit antagonistic activities against plant pathogens and have the capacity to degrade pollutants. Factors that appear to contribute to the rhizosphere fitness are the ability of the organism to form biofilms and the utilization of cell-to-cell-communication systems (quorum sensing, QS) to co-ordinate the expression of certain phenotypes in a cell density dependent manner. Recently, the ppu QS locus of the tomato rhizosphere isolate P. putida Iso F was characterized and an isogenic QS-negative ppuI mutant P. putida F117 was generated. In the present study we investigated the impact of QS and biofilm formation on the protein profile of surface-associated proteins of P. putida IsoF. This was accomplished by comparative proteome analyses of the P. putida wild type IsoF and the QS-deficient mutant F117 grown either in planktonic cultures or in 60 h old mature biofilms. Differentially expressed proteins were identified by peptide mass fingerprinting and database search in the completed P. putida KT2440 genome sequence. The sessile life style affected 129 out of 496 surface proteins, suggesting that a significant fraction of the bacterial genome is involved in biofilm physiology. In surface-attached cells 53 out of 484 protein spots were controlled by the QS system, emphasizing its importance as global regulator of gene expression in P. putida IsoF. Most interestingly, the impact of QS was dependent on whether cells were grown on a surface or in suspension; about 50% of the QS-controlled proteins identified in planktonic cultures were found to be oppositely regulated when the cells were grown as biofilms. Fifty-seven percent of all identified surface-controlled proteins were also regulated by the ppu QS system. In conclusion, our data provide strong evidence that the set of QS-regulated proteins overlaps substantially with the set of proteins differentially expressed in sessile cells.     

Quorum sensing-dependent biofilms enhance colonization in Vibrio cholerae

Vibrio cholerae is the causative agent of the diarrheal disease cholera. By an incompletely understood developmental process, V. cholerae forms complex surface-associated communities called biofilms. Here we show that quorum sensing-deficient mutants of V. cholerae produce thicker biofilms than those formed by wild-type bacteria. Microarray analysis of biofilm-associated bacteria shows that expression of the Vibrio polysaccharide synthesis (vps) operons is enhanced in hapR mutants. CqsA, one of two known autoinducer synthases in V. cholerae, acts through HapR to repress vps gene expression. Vibrio biofilms are more acid resistant than planktonic cells. However, quorum sensing-deficient biofilms have lower colonization capacities than those of wild-type biofilms, suggesting that quorum sensing may promote cellular exit from the biofilm once the organisms have traversed the gastric acid barrier of the stomach. These results shed light on the relationships among biofilm development, quorum sensing, infectivity, and pathogenesis in V. cholerae.     

Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.     

Survival strategies of infectious biofilms

Modern medicine is facing the spread of biofilm-related infections. Bacterial biofilms are difficult to detect in routine diagnostics and are inherently tolerant to host defenses and antibiotic therapies. In addition, biofilms facilitate the spread of antibiotic resistance by promoting horizontal gene transfer. We review current concepts of biofilm tolerance with special emphasis on the role of the biofilm matrix and the physiology of biofilm-embedded cells. The heterogeneity in metabolic and reproductive activity within a biofilm correlates with a non-uniform susceptibility of enclosed bacteria. Recent studies have documented similar heterogeneity in planktonic cultures. Nutritional starvation and high cell density, two key characteristics of biofilm physiology, also mediate antimicrobial tolerance in stationary-phase planktonic cultures. Advances in characterizing the role of stress response genes, quorum sensing and phase variation in stationary-phase planktonic cultures have shed new light on tolerance mechanisms within biofilm communities.     

The Small Molecule DAM Inhibitor,Pyrimidinedione, Disrupts Streptococcus pneumoniae Biofilm Growth In Vitro

Streptococcus pneumoniae persist in the human nasopharynx within organized biofilms. However, expansion to other tissues may cause severe infections such as pneumonia, otitis media, bacteremia, and meningitis, especially in children and the elderly. Bacteria within biofilms possess increased tolerance to antibiotics and are able to resist host defense systems. Bacteria within biofilms exhibit different physiology, metabolism, and gene expression profiles than planktonic cells. These differences underscore the need to identify alternative therapeutic targets and novel antimicrobial compounds that are effective against pneumococcal biofilms. In bacteria, DNA adenine methyltransferase (Dam) alters pathogenic gene expression and catalyzes the methylation of adenine in the DNA duplex and of macromolecules during the activated methyl cycle (AMC). In pneumococci, AMC is involved in the biosynthesis of quorum sensing molecules that regulate competence and biofilm formation. In this study, we examine the effect of a small molecule Dam inhibitor, pyrimidinedione, on Streptococcus pneumoniae biofilm formation and evaluate the changes in global gene expression within biofilms via microarray analysis. The effects of pyrimidinedione on in vitro biofilms were studied using a static microtiter plate assay, and the architecture of the biofilms was viewed using confocal and scanning electron microscopy. The cytotoxicity of pyrimidinedione was tested on a human middle ear epithelium cell line by CCK-8. In situ oligonucleotide microarray was used to compare the global gene expression of Streptococcus pneumoniae D39 within biofilms grown in the presence and absence of pyrimidinedione. Real-time RT-PCR was used to study gene expression. Pyrimidinedione inhibits pneumococcal biofilm growth in vitro in a concentration-dependent manner, but it does not inhibit planktonic cell growth. Confocal microscopy analysis revealed the absence of organized biofilms, where cell-clumps were scattered and attached to the bottom of the plate when cells were grown in the presence of pyrimidinedione. Scanning electron microscopy analysis demonstrated the absence of an extracellular polysaccharide matrix in pyrimidinedione-grown biofilms compared to control-biofilms. Pyrimidinedione also significantly inhibited MRSA, MSSA, and Staphylococcus epidermidis biofilm growth in vitro. Furthermore, pyrimidinedione does not exhibit eukaryotic cell toxicity. In a microarray analysis, 56 genes were significantly up-regulated and 204 genes were significantly down-regulated. Genes involved in galactose metabolism were exclusively up-regulated in pyrimidinedione-grown biofilms. Genes related to DNA replication, cell division and the cell cycle, pathogenesis, phosphate-specific transport, signal transduction, fatty acid biosynthesis, protein folding, homeostasis, competence, and biofilm formation were down regulated in pyrimidinedione-grown biofilms. This study demonstrated that the small molecule Dam inhibitor, pyrimidinedione, inhibits pneumococcal biofilm growth in vitro at concentrations that do not inhibit planktonic cell growth and down regulates important metabolic-, virulence-, competence-, and biofilm-related genes. The identification of a small molecule (pyrimidinedione) with S. pneumoniae biofilm-inhibiting capabilities has potential for the development of new compounds that prevent biofilm formation.     

The Influence of Fluid Flow on Modeling Quorum Sensing in Bacterial Biofilms

In this paper, we study quorum sensing in Pseudomonas aeruginosa biofilms. Quorum sensing is a process where bacteria monitor their population density through the release of extra-cellular signalling molecules. The presence of these molecules affects gene modulation leading to changes in behaviour such as the release of virulence factors. Here, we use numerical methods to approximate a 2-D model of quorum sensing. It is observed that the shape of the biofilm can have a profound effect on the onset of quorum sensing. This has serious repercussions for experimental observations since biofilms of the same biomass but different shapes can produce quite different results.     

Antibodies against the majority subunit of type IV pili disperse nontypeable Haemophilus influenzae biofilms in a LuxS‐dependent manner and confer therapeutic resolution of experimental otitis media

Despite resulting in a similar overall outcome, unlike antibodies directed against the DNABII protein, integration host factor (IHF), which induce catastrophic structural collapse of biofilms formed by nontypeable Haemophilus influenzae (NTHI), those directed against a recombinant soluble form of PilA [the majority subunit of Type IV pili (Tfp) produced by NTHI], mediated gradual ‘top‐down’ dispersal of NTHI from biofilms. This dispersal occurred via a mechanism that was dependent upon expression of both PilA (and by inference, Tfp) and production of AI‐2 quorum signaling molecules by LuxS. The addition of rsPilA to a biofilm‐targeted therapeutic vaccine formulation comprised of IHF plus the powerful adjuvant dmLT and delivered via a noninvasive transcutaneous immunization route induced an immune response that targeted two important determinants essential for biofilm formation by NTHI. This resulted in significantly earlier eradication of NTHI from both planktonic and adherent populations in the middle ear, disruption of mucosal biofilms already resident within middle ears prior to immunization and rapid resolution of signs of disease in an animal model of experimental otitis media. These data support continued development of this novel combinatorial immunization approach for resolution and/or prevention of multiple diseases of the respiratory tract caused by NTHI.     

Characterization of starvation-induced dispersion in Pseudomonas putida biofilms

The biofilm lifestyle, where microbial cells are aggregated because of expression of cell-to-cell interconnecting compounds, is believed to be of paramount importance to microbes in the environment. Because microbes must be able to alternate between sessile and planktonic states, it is anticipated that they must be able to regulate their ability to form biofilm and to dissolve biofilm. We present an investigation of a biofilm dissolution process occurring in flow-chamber-grown Pseudomonas putida biofilms. Local starvation-induced biofilm dissolution appears to be an integrated part of P. putida biofilm development that causes characteristic structural rearrangements. Rapid global dissolution of entire P. putida biofilms was shown to occur in response to carbon starvation. Genetic analysis suggested that the adjacent P. putida genes PP0164 and PP0165 play a role in P. putida biofilm formation and dissolution. PP0164 encodes a putative periplasmic protein of previously unknown function, and PP0164 mutant bacteria are sticky, and unable to reduce their adhesiveness and dissolve their biofilm in response to carbon starvation. PP0165 encodes a putative transmembrane protein containing GGDEF and EAL domains, and PP0165 mutant bacteria are unable to increase their adhesiveness and form biofilm. We suggest that the PP0164 and PP0165 proteins are involved in the regulation of the adhesiveness of the bacteria; the PP0165 protein through c-di-GMP signalling, and the PP0164 protein as a transducer of the signal.     

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.     

Biological and Physiochemical Methods of Biofilm Adhesion Resistance Control of Medical-Context Surface

The formation of biofilms on medical-context surfaces gives the EPS embedded bacterial community protection and additional advantages that planktonic cells would not have such as increased antibiotic resistance and horizontal gene transfer. Bacterial cells tend to attach to a conditioning layer after overcoming possible electrical barriers and go through two phases of attachments: reversible and irreversible. In the first, bacterial attachment to the surface is reversible and occurs quickly whilst the latter is permanent and takes place over a longer period of time. Upon reaching a certain density in the bacterial community, quorum sensing causes phenotypical changes leading to a loss in motility and the production of EPS. This position paper seeks to address the problem of bacterial adhesion and biofilm formation for the medical surfaces by comparing inhabiting physicochemical interactions and biological mechanisms. Several physiochemical methodologies (e.g. ultrasonication, alternating magnetic field and chemical surface coating) and utilizing biological mechanisms (e.g. quorum quenching and EPS degrading enzymes) were suggested. The possible strategical applications of each category were suggested and evaluated to a balanced position to possibly eliminate the adhesion and formation of biofilms on medical-context surfaces.     

Fitness landscape of antibiotic tolerance in Pseudomonas aeruginosa biofilms

Bacteria in biofilms have higher antibiotic tolerance than their planktonic counterparts. A major outstanding question is the degree to which the biofilm-specific cellular state and its constituent genetic determinants contribute to this hyper-tolerant phenotype. Here, we used genome-wide functional profiling of a complex, heterogeneous mutant population of Pseudomonas aeruginosa MPAO1 in biofilm and planktonic growth conditions with and without tobramycin to systematically quantify the contribution of each locus to antibiotic tolerance under these two states. We identified large sets of mutations that contribute to antibiotic tolerance predominantly in the biofilm or planktonic setting only, offering global insights into the differences and similarities between biofilm and planktonic antibiotic tolerance. Our mixed population-based experimental design recapitulated the complexity of natural biofilms and, unlike previous studies, revealed clinically observed behaviors including the emergence of quorum sensing-deficient mutants. Our study revealed a substantial contribution of the cellular state to the antibiotic tolerance of biofilms, providing a rational foundation for the development of novel therapeutics against P. aeruginosa biofilm-associated infections.     

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.     

The use of glass wool as an attachment surface for studying phenotypic changes in Pseudomonas aeruginosa biofilms by two-dimensional gel electrophoresis

Two-dimensional polyacrylamide gel electrophoresis was used to demonstrate phenotypic differences between Pseudomonas aeruginosa biofilm cells and the planktonic counterpart cells under defined culture conditions. Glass wool was used as a substratum for cell attachment as it affords a large surface-to-volume ratio (1 g with a mean diameter of 15 microns = 1300 cm2), supports the growth of biofilms, allows for free movement of cells between the inter-strand spaces, and it facilitates the exchange of nutrients and oxygen. It also allows for the separation of the biofilm biomass from the surrounding surface influenced planktonic (SIP) cells for further characterization. Comparative analysis of the respective proteomes indicated striking differences in the protein patterns of planktonic, biofilm and SIP cells. We selected 41 proteins, the levels of which varied in a significant and reproducible way in the respective protein profiles. In the biofilm cells, a general up-regulation of the spots was seen, but in SIP cells expression of these spots were generally down-regulated. Altogether six unique proteins were seen in the planktonic cells, while the biofilm and SIP cells contained five and two unique proteins, respectively. Glass wool, therefore, appears to be an ideal attachment surface for the study of biofilm development.     

Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation

Using flow chamber‐grown Pseudomonas aeruginosa biofilms as model system, we show in the present study that formation of heterogeneous biofilms may occur through mechanisms that involve complex subpopulation interactions. One example of this phenomenon is expression of the iron‐siderophore pyoverdine in one subpopulation being necessary for development of another subpopulation that does not itself express the pyoverdine synthesis genes. Another example is quorum sensing‐controlled DNA release in one subpopulation being necessary for development of another subpopulation that does not itself express the quorum‐sensing genes.     

Signs of biofilm formation in the genome of Labrenzia sp. PO1

Labrenzia sp. are important components of marine ecology which play a key role in biochemical cycling. In this study, we isolated the Labrenzia sp. PO1 strain capable of forming biofilm, from the A. sanguinea culture. Growth analysis revealed that strain reached a logarithmic growth period at 24 hours. The whole genome of 6.21813 Mb of Labrezia sp. PO1 was sequenced and assembled into 15 scaffolds and 16 contigs, each with minimum and maximum lengths of 644 and 1,744,114 Mb. A total of 3,566 genes were classified into five pathways and 31 pathway groups. Of them, 521 genes encoded biofilm formation proteins, quorum sensing (QS) proteins, and ABC transporters. Gene Ontology annotation identified 49,272 genes that were involved in biological processes (33,425 genes), cellular components (7,031genes), and molecular function (7,816 genes). We recognised genes involved in bacterial quorum sensing, attachment, motility, and chemotaxis to investigate bacteria's ability to interact with the diatom phycosphere. As revealed by KEGG pathway analysis, several genes encoding ABC transporters exhibited a significant role during the growth and development of Labrenzia sp. PO1, indicating that ABC transporters may be involved in signalling pathways that enhance growth and biofilm formation.     

铜绿假单胞菌中一个新的吩嗪合成调节基因

[目的]在次抑制浓度四环素条件下,研究铜绿假单胞菌phzAl操纵子的调节基因及调节途径.[方法]对转座突变库中phaAl操纵子表达发生变化的突变体,进行随机PCR、基因测序及比对,确定突变位点.并以发光杆菌的荧光素酶基因操纵子luxCDABE为报道基因,研究基因调节作用及调节路径.[结果]在两株突变体PAM0487和PAM0487R中phzAl操纵子的表达降低,这两株突变体的突变基因确定为假定钼元素转运蛋白调节子PA0487基因.[结论]PA0487是phzAl操纵子表达的一个新的正向调节子,并对密度感应系统相关基因的表达有凋节作用.