Birgit M. Prüß

Global gene regulation in Yersinia enterocolitica: effect of FliA on the expression levels of flagellar and plasmid-encoded virulence genes.

This study describes the involvement of the sigma factor of the flagellar system, FliA, in global gene regulation of Yersinia enterocolitica. In addition to exhibiting a positive effect upon the expression levels of eight class III flagellar operons, FliA also exhibited a negative effect upon the expression levels of four virulence operons that are located on the pYV virulence plasmid. These are yadA, virC, yopQ, and the insertion element ISYen1. While the positive effect on class III flagellar operons by FliA is most likely direct, the negative effect on the virulence operons appears to require the known transcriptional activator of these genes, VirF. This was determined using microarray analysis, quantitative PCR and a search for putative binding sites for FliA. In addition to the FliA regulation of flagellar and plasmid-encoded virulence genes, we studied temperature regulation of these genes. While wild-type cells exhibited increased expression levels of flagellar genes and decreased expression levels of plasmid-encoded virulence genes at 25°C (as compared to 37°C), temperature dependence of gene expression was much reduced in the fliA mutants. We conclude that FliA contributes to the inverse temperature regulation of flagellar and plasmid-encoded virulence genes. We present a network of transcriptional regulation around FlhD/FlhC and FliA.

Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation.

Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria’s transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinations, growth in tryptic soy broth at 37°C supported the most biofilm production. To analyze the complex relationships between the diverse cell-surface organelles, transcriptional regulators, and metabolic enzymes represented by the tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology ‘pyruvate catabolic process,’ which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude that acetate metabolism functions as a metabolic sensor, transmitting changes in environmental conditions to biofilm biomass and structure.

Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity.

This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1-3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

Pleiotropic phenotypes of a Yersinia enterocolitica flhD mutant include reduced lethality in a chicken embryo model

BackgroundThe Yersinia enterocolitica flagellar master regulator FlhD/FlhC affects the expression levels of non-flagellar genes, including 21 genes that are involved in central metabolism. The sigma factor of the flagellar system, FliA, has a negative effect on the expression levels of seven plasmid-encoded virulence genes in addition to its positive effect on the expression levels of eight of the flagellar operons. This study investigates the phenotypes of flhD and fliA mutants that result from the complex gene regulation.ResultsPhenotypes relating to central metabolism were investigated with Phenotype MicroArrays. Compared to the wild-type strain, isogenic flhD and fliA mutants exhibited increased growth on purines and reduced growth on N-acetyl-D-glucosamine and D-mannose, when used as a sole carbon source. Both mutants grew more poorly on pyrimidines and L-histidine as sole nitrogen source. Several intermediates of the tricarboxylic acid and the urea cycle, as well as several dipeptides, provided differential growth conditions for the two mutants. Gene expression was determined for selected genes and correlated with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay. The assay that was previously established for Escherichia coli strains was modified for Y. enterocolitica. The flhD mutant caused reduced chicken embryo lethality when compared to wild-type bacteria. In contrast, the fliA mutant caused wild-type lethality. This indicates that the virulence phenotype of the flhD mutant might be due to genes that are regulated by FlhD/FlhC but not FliA, such as those that encode the flagellar type III secretion system.ConclusionPhenotypes of flhD and fliA mutants are related to central metabolism and virulence and correlate with gene regulation.

Involvement of Two-Component Signaling on Bacterial Motility and Biofilm Development

Two-component signaling is a specialized mechanism that bacteria use to respond to changes in their environment. Nonpathogenic strains of Escherichia coli K-12 harbor 30 histidine kinases and 32 response regulators, which form a network of regulation that integrates many other global regulators that do not follow the two-component signaling mechanism, as well as signals from central metabolism. The output of this network is a multitude of phenotypic changes in response to changes in the environment. Among these phenotypic changes, many two-component systems control motility and/or the formation of biofilm, sessile communities of bacteria that form on surfaces. Motility is the first reversible attachment phase of biofilm development, followed by a so-called swim or stick switch toward surface organelles that aid in the subsequent phases. In the mature biofilm, motility heterogeneity is generated by a combination of evolutionary and gene regulatory events.

Regulation of Cell Division, Biofilm Formation, and Virulence by FlhC in Escherichia coli O157:H7 Grown on Meat

ABSTRACT To understand the continuous problems that Escherichia coli O157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC of E. coli K-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of an E. coli O157:H7 parent strain to that of its isogenic flhC mutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in the flhC mutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity of E. coli O157:H7 on meat by interfering with the signal transduction pathways.

ß-Phenylethylamine as a novel nutrient treatment to reduce bacterial contamination due to Escherichia coli O157:H7 on beef meat.

Bacterial infection by Escherichia coli O157:H7 through the consumption of beef meat or meat products is an ongoing problem, in part because bacteria develop resistances towards chemicals aimed at killing them. In an approach that uses bacterial nutrients to manipulate bacteria into behaviors or cellular phenotypes less harmful to humans, we screened a library of 95 carbon and 95 nitrogen sources for their effect on E. coli growth, cell division, and biofilm formation. In the initial screening experiment using the Phenotype MicroArray(TM) technology from BioLog (Hayward, CA), we narrowed the 190 starting nutrients down to eight which were consecutively tested as supplements in liquid beef broth medium. Acetoacetic acid (AAA) and ß-phenylethylamine (PEA) performed best in this experiment. On beef meat pieces, PEA reduced the bacterial cell count by 90% after incubation of the PEA treated and E. coli contaminated meat pieces at 10°C for one week.

Relating gene expression data on two-component systems to functional annotations in Escherichia coli.

BackgroundObtaining physiological insights from microarray experiments requires computational techniques that relate gene expression data to functional information. Traditionally, this has been done in two consecutive steps. The first step identifies important genes through clustering or statistical techniques, while the second step assigns biological functions to the identified groups. Recently, techniques have been developed that identify such relationships in a single step.ResultsWe have developed an algorithm that relates patterns of gene expression in a set of microarray experiments to functional groups in one step. Our only assumption is that patterns co-occur frequently. The effectiveness of the algorithm is demonstrated as part of a study of regulation by two-component systems in Escherichia coli. The significance of the relationships between expression data and functional annotations is evaluated based on density histograms that are constructed using product similarity among expression vectors. We present a biological analysis of three of the resulting functional groups of proteins, develop hypotheses for further biological studies, and test one of these hypotheses experimentally. A comparison with other algorithms and a different data set is presented.ConclusionOur new algorithm is able to find interesting and biologically meaningful relationships, not found by other algorithms, in previously analyzed data sets. Scaling of the algorithm to large data sets can be achieved based on a theoretical model.

Screening the mechanical stability of Escherichia coli biofilms through exposure to external, hydrodynamic shear forces

The desire to attain a deeper understanding of the fundamental aspects governing the mechanical properties of biofilms has become more prominent in recent years. This has largely been due to the realization that these sessile microbial communities often withstand environments where hydrodynamic turbulence and shearing forces are considerable. In the present study, Escherichia coli K‐12 was used as a model system to develop a laboratory technique that can be used to quickly screen the mechanical integrity or stability of laboratory cultivated bacterial biofilms when exposed to such external, hydrodynamic shear forces. The screening method utilizes a custom‐built, automated water jetting apparatus to generate and precisely apply a pressurized stream of water directly to biofilms cultured in multi‐well plates. An optimized set of water jetting parameters was determined to resolve subtle to moderate differences in the mechanical stability of isogenic strains of E. coli K‐12 as a function of percent biofilm removal. Mutations in both flagella biosynthesis (fliA) and acetate metabolism (ackA and ackA pta) were shown to impair the mechanical integrity of 24‐h biofilms, while a “housekeeping” strain deficient in arginine metabolism (argD) exhibited a mechanical stability profile comparable to the parent strain.

Efficacy of β-phenylethylamine as a novel anti-microbial and application as a liquid catheter flush

Abstract With this study, we introduce a liquid flush for catheters and other tubing‐based applications that consists of a solution of &bgr;‐phenylethylamine (PEA) in tryptic soy broth. The initial experiments in multiwell polystyrene plates were conducted with Escherichia coli K‐12 to assess the effectiveness of PEA at reducing planktonic growth, as well as the biomass and adenosine triphosphate (ATP) content of biofilm; PEA reduced these growth parameters as a function of increasing concentration. This effect was also seen in mutants of PEA catabolism, which leads us to believe that the PEA effect is due to PEA itself and not one of its degradation products. Since PEA reduced planktonic growth and biofilm when added at the time of inoculation, as well as at later time points, we propose PEA as a novel compound for the prevention and treatment of biofilm. PEA reduced planktonic growth and the ATP content of the biofilm for five bacterial pathogens, including an enterohemorrhagic E. coli, two uropathogenic E. coli, Pseudomonas aeruginosa and Staphylococcus aureus. A major finding of this study is the reduction of the ATP content of biofilm that formed in silicone tubing by periodic flushes of PEA. This experiment was performed to model antibiotic‐lock treatment of an intravenous catheter. It was found that 10 mg ml−1 of PEA reduced the ATP content of biofilm of five bacterial strains by 96.3 % or more after 2 weeks of incubation and three treatments with PEA. For P. aeruginosa, the reduction in ATP content was paralleled by an identical percentage reduction in viable cells in the biofilm.