Antigen 43 from Escherichia coli induces inter- and intraspecies cell aggregation and changes in colony morphology of Pseudomonas fluorescens

Antigen 43 (Ag43) is a surface-displayed autotransporter protein of Escherichia coli. By virtue of its self-association characteristics, this protein is able to mediate autoaggregation and flocculation of E. coli cells in static cultures. Additionally, surface display of Ag43 is associated with a distinct frizzy colony morphology in E. coli. Here we show that Ag43 can be expressed in a functional form on the surface of the environmentally important Pseudomonas fluorescens strain SBW25 with ensuing cell aggregation and frizzy colony types. Using green fluorescence protein-tagged cells, we demonstrate that Ag43 can be used as a tool to provide interspecies cell aggregation between E. coli and P. fluorescens. Furthermore, Ag43 expression enhances biofilm formation in P. fluorescens to glass surfaces. The versatility of this protein was also reflected in Ag43 surface display in a variety of other gram-negative bacteria. Display of heterologous Ag43 in selected bacteria might offer opportunities for rational design of multispecies consortia where the concerted action of several bacterial species is required, e.g., waste treatment and degradation of pollutants.     

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

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

Role of bacterial ribosomes in barotolerance.

The effects of high hydrostatic pressures on protein synthesis by whole cells and cell free preparations of Escherichia coli, Pseudomonas fluorescens, and Pseudomonas bathycetes were determined. Actively growing cells of P. bathycetes and P. fluorescens were less sensitive than were E. coli cells. Protein synthesis by cell free preparations of E. coli and P. fluorescens showed the same extent of inhibition as their respective whole cell preparations, whereas cell free preparations of P. bathycetes showed a marked increase in pressure sensitivity over whole cells. Protein synthesis by hybrid protein synthesizing cell free preparations (the ribosomes from one organism and the S-100 supernatant fraction from another) demonstrated that response to high pressure is dependent on the source of the ribosome employed. A hybrid system containing E. coli ribosomes and P. fluorescens S-100 shows the same sensitivity to pressure as a homologous E. coli system, whereas a hybrid containing P. fluorescens ribosomes and E. coli S-100 shows the greater pressure tolerance characteristic of the P. fluorescens homologous system.     

The Escherichia coli biofilm-promoting protein Antigen 43 does not contribute to intestinal colonization

Abstract Escherichia coli is a versatile organism capable of causing a variety of intestinal and extraintestinal diseases, as well as existing as part of the commensal flora. A variety of factors permit specific attachment to host receptors including fimbrial adhesins and outer membrane proteins such as autotransporters. One of the better characterized autotransporters is Antigen 43 (Ag43), the major phase-variable surface protein of E. coli. Ag43 is associated with bacterial cell-cell aggregation and biofilm formation. Nevertheless, the precise biological significance and contribution to intestinal colonization remain to be elucidated. Here we investigated the contribution of Ag43 to E. coli adherence to intestinal epithelial cells and colonization of the mouse intestine. These investigations revealed that Ag43 increased in vitro adherence of E. coli to epithelial cells by promoting bacterial cell-cell aggregation but that Ag43 did not promote specific interactions with the mammalian cells. Furthermore, Ag43 did not contribute significantly to colonization of the mouse intestine and expression of Ag43 was lost a few days after colonization of the mouse was established. Unexpectedly, considering its similarity to other adhesins, our findings suggest that Ag43 does not act as a direct colonization factor by binding to mammalian cells.     

Growth of Escherichia coli in model distribution system biofilms exposed to hypochlorous acid or monochloramine

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH(2)Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH(2)Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila, E. coli, and beta and delta proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.     

Differential expression of the Escherichia coli autoaggregation factor antigen 43

Antigen 43 (Ag43) is a self-recognizing surface adhesin found in most Escherichia coli strains. Due to its excellent cell-to-cell aggregation characteristics, Ag43 expression confers clumping and fluffing of cells and promotes biofilm formation. Ag43 expression is repressed by the cellular redox sensor OxyR. Here we used mutant versions of OxyR that are locked in either the reduced or the oxidized form as well as the addition of a simple redox-changing chemical to show that the redox state of OxyR influences Ag43 expression. Furthermore, the redox state of OxyR influences the biofilm-forming potential of E. coli. Finally, we demonstrated that Ag43-mediated cell aggregation confers significant protection against hydrogen peroxide killing.     

Antigen-43-Mediated Autoaggregation of Escherichia coli Is Blocked by Fimbriation

Antigen 43 (Ag43), the product of the flu gene, is a surface-displayed autotransporter protein of Escherichia coli. Ag43 is responsible for the autoaggregation and flocculation of static liquid cultures of many E. coli strains. The expression of Ag43 has been reported to be phase variable and controlled by the product of the oxyR gene. Type 1 fimbriae are thin adhesive thread-like surface organelles responsible for bacterial receptor recognition and tissue colonization. Like that of Ag43, the expression of type 1 fimbriae is phase variable. Interestingly, previous results have suggested that the expression of type 1 fimbriae and the expression of Ag43 are mutually exclusive. In the present report, we show, by use of well-defined mutants, that fimbriation abolishes Ag43-mediated autoaggregation but does not affect Ag43 expression. Autoaggregation is shown to require an intercellular Ag43-Ag43 interaction, and the physical presence of fimbriae on the cells seems to abrogate this interaction. The Ag43 or OxyR status does not appear to influence fimbria expression, and our results suggest that the expression of Ag43 and the expression of fimbriae are independent processes.     

The effect of hydrodynamic conditions on the phenotype of Pseudomonas fluorescens biofilms

This study investigated the phenotypic characteristics of monoculture P. fluorescens biofilms grown under turbulent and laminar flow, using flow cells reactors with stainless steel substrata. The cellular physiology and the overall biofilm activity, structure and composition were characterized, and compared, within hydrodynamically distinct conditions. The results indicate that turbulent flow-generated biofilm cells were significantly less extensive, with decreased metabolic activity and a lower protein and polysaccharides composition per cell than those from laminar flow-generated biofilms. The effect of flow regime did not cause significantly different outer membrane protein expression. From the analysis of biofilm activity, structure and composition, turbulent flow-generated biofilms were metabolically more active, had twice more mass per cm(2), and higher cellular density and protein content (mainly cellular) than laminar flow-generated biofilms. Conversely, laminar flow-generated biofilms presented higher total and matrix polysaccharide contents. Direct visualisation and scanning electron microscopy analysis showed that these different flows generate structurally different biofilms, corroborating the quantitative results. The combination of applied methods provided useful information regarding a broad spectrum of biofilm parameters, which can contribute to control and model biofilm processes.     

Coordinate gene regulation by fimbriae-induced signal transduction.

Fimbriae are thread-like polymers displayed in large amounts on the bacterial surface and used by many pathogens to attach to receptors on host tissue surfaces. Fimbriae contain disulfide bridges, contrary to many Escherichia coli surface proteins produced in bulk amounts. Here we investigate whether fimbriae expression can affect expression of other genes. Analysis of gene expression in two E.coli strains, differing in the fim locus, indicated the flu gene to be affected. The flu gene encodes the antigen 43 (Ag43) surface protein, specifically involved in bacterial aggregation, and microcolony and biofilm formation. Ag43 production is repressed by the global regulator OxyR, which monitors the cell's thiol-disulfide status. Only the thiol form of OxyR represses Ag43 production. We demonstrate that production of several different disulfide-containing fimbriae results in the abolition of Ag43 production. No effect was observed in an oxyR mutant. We conclude that fimbriae expression per se constitutes a signal transduction mechanism that affects a number of unrelated genes via the thiol-disulfide status of OxyR. Thus, phase variation in fimbrial expression is coordinated with the expression of other disease- and colonization-related genes.     

Expression of Pseudomonas fluorescens D-galactose dehydrogenase in E. coli

To investigate the heterologous expression of Pseudomonas genes in Escherichia coli we have cloned P. fluorescens DNA in an E. coli [cosmid] system. A colony bank representing the whole P. fluorescens chromosome was screened immunologically using a modification of the method described by Broome and Gilbert (1978). Radioactive labelling of the antibodies was replaced by conjugation with horseradish peroxidase. Among 523 E. coli colonies one was D-galactose dehydrogenase-positive. The expression of this enzyme in primary clones was lower than in the uninduced Pseudomonas. Subcloning of the D-galactose dehydrogenase gene, in vitro mutagenesis of the DNA, and coupling to a strong E. coli promoter yielded an E. coli strain that produces 90 times more of the enzyme than the induced P. fluorescens.     

Nucleotide sequence of the Pseudomonas fluorescens signal peptidase II gene (lsp) and flanking genes.

The lsp gene encoding prolipoprotein signal peptidase (signal peptidase II) is organized into an operon consisting of ileS and three open reading frames, designated genes x, orf149, and orf316 in both Escherichia coli and Enterobacter aerogenes. A plasmid, pBROC128, containing a 5.8-kb fragment of Pseudomonas fluorescens DNA was found to confer pseudomonic acid resistance on E. coli host cells and to contain the structural gene of ileS from P. fluorescens. In addition, E. coli strains carrying pBROC128 exhibited increased globomycin resistance. This indicated that the P. fluorescens lsp gene was present on the plasmid. The nucleotide sequences of the P. fluorescens lsp gene and of its flanking regions were determined. Comparison of the nucleotide sequences of the lsp genes in E. coli and P. fluorescens revealed two highly conserved domains in this enzyme. Furthermore, the five genes which constitute an operon in E. coli and Enterobacter aerogenes were found in P. fluorescens in the same order as in the first two species.     

Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins

In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm formation are studied. We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with site-directed insertion of a repression and expression (RExBAD) cassette which places a functional pBAD promoter upstream of a target gene. We showed that this method permits both inactivation and modulation of most Escherichia coli gene expression, including expression of toxin and essential genes. We used this strategy to study adhesion and bacterial biofilms by placing the RExBAD cassette in front of the tra operon, encoding the DNA transfer and pilus genes on the F conjugative plasmid, and in front of flu, the antigen 43 (Ag43) autotransporter adhesin-encoding gene. In silico analysis revealed the existence of 10 genes with homology to the Ag43 gene that were good candidates for genes that encode putative new adhesins in E. coli. We used the RExBAD strategy to study these genes and demonstrated that induction of expression of four of them is associated with adhesion of E. coli to abiotic surfaces. The potential use of the RExBAD approach to study the function of cryptic or uncharacterized genes in large-scale postgenomic functional analyses is discussed.     

Effects of low temperature on in vivo and in vitro protein synthesis in Escherichia coli and Pseudomonas fluorescens.

The effects of temperature on protein synthesis by Escherichia coli, a mesophile, and Pseudomonas fluorescens, a psychotroph, were investigated by using whole-cell and cell extract preparations. After shifts to 5 degrees C, protein was synthesized at a slowly decreasing rate for 1 h by both organisms, after which P. fluorescens synthesized protein at a new rate corresponding to its 5 degrees growth rate, in contrast to E. coli which did not synthesize protein at a measurable rate. In vitro protein-synthesizing systems using MS-2 RNA, endogenous mRNA, and purified polysomes were utilized to investigate initiation of translation at 5 degrees C. In these systems, P. fluorescens cell extracts synthesized protein at linear rates for up to 2 h at 5 degrees C, whereas E. coli cell extracts synthesized protein for only 25 min at 5 degrees C. The rates of polypeptide elongation, as tested by the incorporation of phenylalanine into polyphenylalanine by cell extract protein-synthesizing systems from both organisms, were identical over the range of 25 to 0 degrees C. The polysome profiles of E. coli whole cells shifted from 37 to 5 degrees C showed accumulation of 70S ribosomal particles and ribosomal subunits at the expense of polysomes. Similar experiements done with P. fluorescens resulted in polysome reformation at 5 degrees C. In vitro experiments demonstrated that the 70S ribosomal particles, which accumulated in E. coli at 5 degrees C, were capable of synthesizing protein in vitro in the absence of added mRNA. These in vivo and in vitro results suggest that incubation of E. coli at subminimal temperatures results in a block in initiation of translation causing polysomal runoff and the accumulation of 70S particles, some of which are 70S monosomes.     

Integration and Proliferation of Pseudomonas aeruginosa PA01 in Multispecies Biofilms

Despite an increased awareness of biofilm formation by pathogens and the role of biofilms in human infections, the potential role of environmental biofilms as an intermediate stage in the host-to-host cycle is poorly described. To initiate infection, pathogens in biofilms on inanimate environmental surfaces must detach from the biofilm and be transmitted to a susceptible individual in numbers large enough to constitute an infectious dose. Additionally, while detachment has been recognized as a discrete event in the biofilm lifestyle, it has not been studied to the same extent as biofilm development or biofilm physiology. Successful integration of Pseudomonas aeruginosa strain PA01 expressing green fluorescent protein (PA01GFP), employed here as a surrogate pathogen, into multispecies biofilm communities isolated and enriched from sink drains in public washrooms and a hospital intensive care unit is described. Confocal laser scanning microscopy indicated that PA01GFP cells were most frequently located in the deeper layers of the biofilm, near the attachment surface, when introduced into continuous flow cells before or at the same time as the multispecies drain communities. A more random integration pattern was observed when PA01GFP was introduced into established multispecies biofilms. Significant numbers of single PA01GFP cells were continuously released from the biofilms to the bulk liquid environment, regardless of the order of introduction into the flow cell. Challenging the multispecies biofilms containing PA01GFP with sub-lethal concentrations of an antibiotic, chelating agent and shear forces that typically prevail at distances away from the point of treatment showed that environmental biofilms provide a suitable habitat where pathogens are maintained and protected, and from where they are continuously released.     

Antagonism between Bacillus cereus and Pseudomonas fluorescens in planktonic systems and in biofilms

In the environment, many microorganisms coexist in communities competing for resources, and they are often associated as biofilms. The investigation of bacterial ecology and interactions may help to improve understanding of the ability of biofilms to persist. In this study, the behaviour of Bacillus cereus and Pseudomonas fluorescens in the planktonic and sessile states was compared. Planktonic tests were performed with single and dual species cultures in growth medium with and without supplemental FeCl3. B. cereus and P. fluorescens single cultures had equivalent growth behaviours. Also, when in co-culture under Fe-supplemented conditions, the bacteria coexisted and showed similar growth profiles. Under Fe limitation, 8 h after co-culture and over time, the number of viable B. cereus cells decreased compared with P. fluorescens. Spores were detected during the course of the experiment, but were not correlated with the decrease in the number of viable cells. This growth inhibitory effect was correlated with the release of metabolite molecules by P. fluorescens through Fe-dependent mechanisms. Biofilm studies were carried out with single and dual species using a continuous flow bioreactor rotating system with stainless steel (SS) substrata. Steady-state biofilms were exposed to a series of increasing shear stress forces. Analysis of the removal of dual species biofilms revealed that the outer layer was colonised mainly by B. cereus. This bacterium was able to grow in the outermost layers of the biofilm due to the inhibitory effect of P. fluorescens being decreased by the exposure of the cells to fresh culture medium. B. cereus also constituted the surface primary coloniser due to its favourable adhesion to SS. P. fluorescens was the main coloniser of the middle layers of the biofilm. Single and dual species biofilm removal data also revealed that B. cereus biofilms had the highest physical stability, followed by P. fluorescens biofilms. This study highlights the inadequacy of planktonic systems to mimic the behaviour of bacteria in biofilms and shows how the culturing system affects the action of antagonist metabolite molecules because dilution and consequent loss of activity occurred in continuously operating systems. Furthermore, the data demonstrate the biocontrol potential of P. fluorescens on the planktonic growth of B. cereus and the ability of the two species to coexist in a stratified biofilm structure.     

New alkane-responsive expression vectors for Escherichia coli and pseudomonas

We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E. coli. XylE expression levels of more than 10% of total cell protein were obtained for E. coli as well as for Pseudomonas strains.     

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.