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PubMed2004

Proteome analysis of intraclonal diversity of two Pseudomonas aeruginosa TB clone isolates.

Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients. Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not. Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins. About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity. This percentage reflects a relatively high degree of intraclonal variability. Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors. It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

Clone Cells↗

PubMed2006

Impact of the accessory gene regulatory system (Agr) on extracellular proteins, codY expression and amino acid metabolism in Staphylococcus epidermidis.

The quorum-sensing system Agr is part of a complex regulatory network of gene expression in staphylococci. This study presents the effect of an agr mutation on a biofilm-forming Staphylococcus epidermidis isolate by employing proteome and transcriptome analysis. The agr mutant exhibited a significantly lowered amount of extracellular proteins: amongst others SspA, AtlE, GehD and the phenol soluble modulins PSM1/2. Cytoplasmic proteome analysis and expression profiling indicated that the agr inactivation led to a strongly altered regulation of metabolism and virulence. Most strikingly, expression of CodY, a global regulator of virulence and stationary phase gene expression, was decreased in the agr mutant. In this respect, homologous genes known to be controlled by CodY in Bacillus subtilis and Lactococcus lactis were found to be up-regulated in the S. epidermidis agr mutant. The combined data show that wild-type and agr mutant differ with respect to amino acid biosynthesis and oligopeptide transport, carbohydrate utilization, as well as GMP and IMP interconversion. Due to the varying physiological properties S. epidermidis agr mutants, which often occur spontaneously, might be capable of colonizing alternative ecological niches in the human host and could, therefore, have an advantage in adapting to changing environmental conditions.

Amino Acids↗

PubMed2001

Cell death in Tetrahymena thermophila: new observations on culture conditions.

We previously suggested that the cell fate of the protozoan ciliate, Tetrahymena thermophila, effectively relates to a quorum-sensing mechanism where cell-released factors support cell survival and proliferation. The cells have to be present above a critical initial density in a chemically defined nutrient medium in order to release a sufficient level of these factors to allow a new colony to flourish. At a relatively high rate of metabolism and/or macromolecular synthesis and below this critical density, cells began to die abruptly within 30 min of inoculation, and this death took the form of an explosive disintegration lasting less than 50 milliseconds. The cells died at any location in the culture, and the frequency of cell death was always lower in well-filled vials than those with medium/air interface. Cell death was inhibited by the addition of Actinomycin D or through modifications of the culture conditions either by reducing the oxygen tension or by decreasing the temperature of the growth medium. In addition, plastic caps in well-filled vials release substances, which promote cell survival. The fate of low-density cultures is related to certain 'physical' conditions, in addition to the availability of oxygen within closed culture systems.

Animals↗

PubMed2000

Luminescence control in the marine bacterium Vibrio fischeri: An analysis of the dynamics of lux regulation.

A mathematical model has been developed based on the fundamental properties of the control system formed by the lux genes and their products in Vibrio fischeri. The model clearly demonstrates how the components of this system work together to create two, stable metabolic states corresponding to the expression of the luminescent and non-luminescent phenotypes. It is demonstrated how the cell can "switch" between these steady states due to changes in parameters describing metabolic processes and the extracellular concentration of the signal molecule N-3-oxohexanoyl-l-homoserine lactone. In addition, it is shown how these parameters influence how sensitive the switch mechanism is to cellular LuxR and N-3-oxohexanoyl-l-homoserine lactone and complex concentration. While these properties could lead to the collective phenomenon known as quorum sensing, the model also predicts that under certain metabolic circumstances, basal expression of the lux genes could cause a cell to luminesce in the absence of extracellular signal molecule. Finally, the model developed in this study provides a basis for analysing the impact of other levels of control upon lux regulation.

4-Butyrolactone↗

PubMed2006

Furanones.

The red alga Delisea pulchra has been a model organism for understanding the ecological role of secondary metabolites as natural antifoulants. Furanones are produced by the plant and delivered to the surface at a concentration where they regulate bacterial colonisation and the settlement of epibiota. This biological understanding has led to the application of furanones as inhibitors of bacterial- and macro-fouling. Furanones inhibit bacterial colonisation and biofilm development through interference with a key bacterial quorum-sensing pathway, the acylated homoserine lactone regulatory system in Gram-negative bacteria. They also interfere with the alternative AI-2 signalling system in Gram-negative and Gram-positive bacteria. Synthetic programs have developed a library of more than 200 furanone and furanone-analogues including surface attached-furanones. These furanone analogues are potent anti-infectives and inhibit pathogenic phenotypes in Gram-negative and Gram-positive bacteria as demonstrated in-vitro using gene microarrays, and in-vivo using mouse models. Additionally, furanones inhibit the expression of bacterial exo-enzymes that actively degrade components of the immune system thereby enhancing the immune response. Surface-attached furanones immobilised on catheters also inhibit bacterial attachment and retain activity for extended periods. Furanones are strong deterrents of the settlement and growth of macrofouling organisms and as such have potential application as a marine antifouling technology. Laboratory antifouling assays have been used to identify effective and safe furanone-analogues while field trials of furanones incorporated into coatings and polymers demonstrate efficacies similar to commercial biocides. Further development is required to control the release of compounds from suitable carriers to extend coating/polymer lifespans. This review summarises the extensive work on furanones focusing on their natural and applied antifouling activities.

Anti-Infective Agents↗

PubMed2006

Biofilms.

Biofilms of bacteria, frequently in association with algae, protozoa and fungi, are found on all submerged structures in the marine environment. Although it is likely that for the majority of organisms a biofilmed surface is not a pre-requisite for settlement, in practice, colonization by spores and larvae of fouling organisms almost always takes place via a biofilmed surface. Therefore, the properties of the latter may be expected to influence colonization, positively or negatively. Biofilms are responsible for a range of surface-associated and diffusible signals, which may moderate the settling behaviour of cells, spores and larvae. However, there is no consensus view regarding either cause and effect or the mechanism(s) by which biofilms moderate settlement. Studies with mixed biofilms, especially field experiments, are difficult to interpret because of the conflicting signals produced by different members of the biofilm community as well as their spatial organisation. Molecular techniques highlight the deficiencies of culture methods in identifying biofilm bacteria; hence, the strains with the most impact on settlement of spores and larvae may not yet have been isolated and cultured. Furthermore, secondary products isolated from cultured organisms may not reflect the situation that pertains in nature. The evidence that bacterial quorum sensing signal molecules stimulate settlement of spores of the green macroalga, Ulva, is discussed in some detail. New molecular and analytical tools should provide the opportunity to improve our fundamental understanding of the interactions between fouling organisms and biofilms, which in turn may inform novel strategies to control biofouling.

4-Butyrolactone↗

PubMed1995

Modification by surface association of antimicrobial susceptibility of bacterial populations.

In the majority of natural situations in which bacteria are found, they are associated with and attached to surfaces. In the presence of moisture and nutrients, they grow to form extensive bacterial films which are often enveloped within copius exopolymeric matrices. Biofilms are ubiquitous to many different situations in industry, the environment and medicine. Their presence can be either beneficial or more commonly detrimental to such systems. In this respect, biofilm populations possess physiological properties distinct from those of unattached, planktonic bacteria. Moreover, it is generally accepted that bacteria growing within a biofilm are more resistant to antimicrobial agents than their planktonic counterparts. However, although the consequences of attachment to antimicrobial resistance have been known for many years, the mechanistic bases for such effects have still to be fully elucidated. In this article the nature of different resistance mechanisms, including those of the exopolymeric matrix, environmental modulation, attachment-specific physiologies and quorum sensing are reviewed.

Bacteria↗

PubMedDate not supplied

Identification of a negative regulator for the pathogenicity island of enterohemorrhagic Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue. The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island. The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons, LEE1, LEE2, LEE3, LEE4, and tir(LEE5). The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing. Current knowledge is that regulation is hierarchical: a pivotal positive regulator, ler, is first stimulated, which in turn activates the expression of other operons. Herein, we report on the presence of a negative regulation protein located within the LEE. L0044 is 372 bp in length and is located outside of the 5 major operons. An isogenic L0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins. Reciprocally, trans expression of L0044 suppressed the expression of the LEE. Furthermore, mRNA of ler increased as a result of deleting L0044, and disrupting ler in a L0044-deleted background reversed the loss of the repression phenotype. Thus, L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation of ler.

Bacterial Proteins↗

PubMedDate not supplied

The role of RsmA in the regulation of swarming motility in Serratia marcescens.

Swarming motility is a multicellular phenomenon comprising population migration across surfaces by specially differentiated cells. In Serratia marcescens, a network exists in which the flhDC flagellar regulatory master operon, temperature, nutrient status, and quorum sensing all contribute to the regulation of swarming motility. In this study, the rsmA (repressor of secondary metabolites) gene (hereafter rsmA(Sm)) was cloned from S. marcescens. The presence of multicopy, plasmid-encoded rsmA(Sm) expressed from its native promoter in S. marcescens inhibits swarming. Synthesis of N-acylhomoserine lactones, presumably by the product of smaI (a luxI homolog isolated from S. marcescens), was also inhibited. Knockout of rsmA(Sm) on the S. marcescens chromosome shortens the time before swarming motility begins after inoculation to an agar surface. A single copy of the chromosomal PrsmA(Sm)::luxAB reporter of rsmA(Sm) transcription was constructed. Using this reporter, the roles of the flhDC flagellar regulatory master operon, temperature and autoregulation in the control of rsmA(Sm) expression were determined. Our findings indicate that RsmA(Sm) is a component of the complex regulatory network that controls swarming.

Amino Acid Sequence↗

PubMed2001

Stress-induced evolution and the biosafety of genetically modified microorganisms released into the environment.

This article is focused on the problems of reduction of the risk associated with the deliberate release of genetically modified microorganisms (GMMs) into the environment. Special attention is given to overview the most probable physiological and genetic processes which could be induced in the released GMMs by adverse environmental conditions, namely: (i) activation of quorum sensing and the functions associated with it, (ii) entering into a state of general resistance, (iii) activation of adaptive mutagenesis, adaptive amplifications and transpositions and (iv) stimulation of inter-species gene transfer. To reduce the risks associated with GMMs, the inactivation of their key genes responsible for stress-stimulated increase of viability and evolvability is proposed.

Adaptation, Physiological↗

PubMed2003

On the origin of differentiation.

Following the origin of multicellularity in many groups of primitive organisms there evolved more than one cell type. It has been assumed that this early differentiation is related to size the larger the organism the more cell types. Here two very different kinds of organisms are considered: the volvocine algae that become multicellular by growth, and the cellular slime moulds that become multicellular by aggregation. In both cases there are species that have only one cell type and others that have two. It has been possible to show that there is a perfect correlation with size: the forms with two cell types are significantly larger than those with one. Also in both groups there are forms of intermediate size that will vary from one to two cell types depending on the size of the individuals, suggesting a form of quorum sensing. These observations reinforce the view that size plays a critical role in influencing the degree of differentiation.

Animals↗

PubMed2003

Production of N-acylhomoserine lactone signal molecules by gram-negative soil-borne and plant-associated bacteria.

Quorum-sensing control mediated by N-acylhomoserine lactone (AHL) signal molecules has been established as a key feature in the regulation of various metabolic traits in many bacteria. Approximately 300 strains representing 6 genera and 18 species of soil-borne and plant-associated Gram-negative bacteria isolated in various regions of the former USSR using two reporter systems were screened for AHL production. The production was observed in 17.5% of the screened bacterial strains. Positive response was detected in all of the 14 tested strains of Erwinia herbicola, in 41 of the 239 strains of Pseudomonas species; in all 5 strains of Xanthomonas ampelina, X. campestris pv. malvacearum, pv. translucens, pv. vesicatoria and in one strain of Pantoea stewartii. AHL assay of 41 strains of X. maltophilia (syn. Stenotrophomonas maltophilia) isolated from soils with Chromobacterium violaceum reporter has revealed no strains synthesizing these signal molecules; 26 strains analyzed with Agrobacterium tumefaciens reporter showed the same result.

4-Butyrolactone↗

PubMed1998

Identification of the DNA-binding sites for two response regulators involved in control of bacteriocin synthesis in Lactobacillus plantarum C11.

In Lactobacillus plantarum C11, bacteriocin production has previously been shown to be an inducible process, in which a secreted peptide, produced by the host itself, is involved. The inducing factor, designated plantaricin A (PlnA), is a bacteriocin-like peptide encoded by a gene (plnA) located on the same operon as the genes for a two-component regulatory system (plnBCD). This system consists of a histidine kinase (PlnB) and two response regulators (PlnC,D), and belongs to a recently defined subfamily of two-component regulatory systems, which are activated by secreted peptide pheromones through a quorum-sensing mechanism. We show here that the two response regulators PlnC and PlnD bind specifically to imperfect direct repeats found within the adjacent promoter of the plnABCD operon, and to similar sequences found within the promoter regions of two nearby operons containing bacteriocin structural genes (plnEFI and plnJKLR). Binding of PlnC and PlnD was increased two to three fold in the presence of acetyl phosphate. The results suggest that bacteriocin synthesis in L. plantarum C11 is regulated by the DNA-binding activity of the two response regulators PlnC and PlnD.

Bacteriocins↗

PubMed2003

[Microbiological aspects and antibiotic therapy of diabetic foot infections].

IMMUNOLOGICAL AND MICROBIOLOGICAL ASPECTS OF DIABETIC FOOT INFECTIONS: Diabetic patients are at increased risk of severe skin and bone infections. Immunological disturbances are reasonable and due to altered specific and unspecific cellular immune responses. Analysis of epidemiology and microbial pathogenicity shows that staphylococci seem to be predestined to induce such infections. Staphylococcus aureus and coagulase-negative staphylococci are able to adhere to the wound ground by a sequela of mechanisms. Initial bacterial adherence is due to hydrophobicity, ion exchanges, and specific binding of bacterial adhesion molecules to cellular receptors. Moreover, staphylococci secrete polysaccharides which form a biofilm together with multilayer cell clusters. The highly structured communities within a biofilm are resistant to distinct immunoeffectors and have a decreased susceptibility to antibiotics in vivo. ASPECTS OF ANTIBIOTIC THERAPY: Assessing the severity of an infection is essential to selecting an antibiotic regimen, the mode of drug administration, and the duration of therapy. Regimens for severe and chronic infections are broader spectrum and often intravenously to obtain high drug concentrations immediately. Infections of the bone often require an antibiotic therapy for > 4 weeks, while a 1- to 2-week therapy for mild to moderate infections has been found to be effective. CONCLUSIONS: Because of the tremendous progress in diagnostics and therapy of diabetic foot infections, infectious complications can be successfully treated by appropriate wound care, metabolic control, and early surgical and antibiotic intervention. Bacterial biofilms involved into chronic infections are new aspects currently not visualized by clinical therapy. Besides the classic antimicrobial therapy, new concepts of an enzymatic therapy or the inhibition of bacterial "communication" (quorum sensing) are in progress and the hope for the future.

Anti-Bacterial Agents↗

PubMed2002

Identification of Salmonella enterica serovar Typhimurium genes associated with growth suppression in stationary-phase nutrient broth cultures and in the chicken intestine.

Over 2,800 Tn 5 insertion mutants of Salmonella enterica sv. Typhimurium were screened for the loss of ability to suppress the multiplication of a spectinomycin-resistant (Spc(r)) but otherwise isogenic S. enterica sv. Typhimurium strain, when the Spc(r) mutant was added to 24-h LB broth cultures of the mutants. Selected "growth non-suppressive" (GNS) mutants were defective in respiration (insertions in arcA and fnr), amino acid biosynthesis (aroA and aroD), nutrient uptake and its regulation (tdcC and crp), and chemotaxis (fliD). In the last GNS mutant, the transposon inactivated yhjH, an ORF with unknown function which shows sequence similarity to di-guanylate cyclase and to novel two-component signal transduction proteins. In newly hatched chickens, all of the mutants, with the exception of the fliDmutant, were also unable to suppress colonization of the alimentary tract by the parent strain inoculated 1 day later. Defined mutations in luxS or sdiA,genes which contribute to quorum sensing in S. enterica sv. Typhimurium, had no effect on the stationary-phase growth suppression. Analysis of a transcriptional fusion construct indicated that yhjH was moderately expressed in the exponential phase of growth and up-regulated upon entry into stationary phase. Expression of yhjH was also considerably suppressed by the addition of supernatant from a 24-h stationary-phase S. enterica sv. Typhimurium culture, suggesting that the gene belongs to a new sensing and signaling regulatory pathway in S. enterica sv. Typhimurium.

Amino Acid Sequence↗

PubMed2003

New ubiquitous translocators: amino acid export by Corynebacterium glutamicum and Escherichia coli.

Molecular access to amino acid excretion by Corynebacterium glutamicum and Escherichia coli led to the identification of structurally novel carriers and novel carrier functions. The exporters LysE, RhtB, ThrE and BrnFE each represent the protoype of new transporter families, which are in part distributed throughout all of the kingdoms of life. LysE of C. glutamicum catalytes the export of basic amino acids. The expression of the carrier gene is regulated by the cell-internal concentration of basic amino acids. This serves, for example, to maintain homoeostasis if an excess of l-lysine or l-arginine inside the cell should arise during growth on complex media. RhtB is one of five paralogous systems in E. coli, of which at least two are relevant for l-threonine production. A third system is relevant for l-cysteine production. It is speculated that the physiological function of these paralogues is related to quorum sensing. ThrE of C. glutamicum exports l-threonine and l-serine. However, a ThrE domain with a putative hydrolytic function points to an as yet unknown role of this exporter. BrnFE in C. glutamicum is a two-component permease exporting branched-chained amino acids from the cell, and an orthologue in B. subtilis exports 4-azaleucine.

Amino Acids↗

PubMed2006

Distinct mechanisms regulate expression of the two major groEL homologues in Rhizobium leguminosarum.

We investigated the regulation of the two of the three groE operons (cpn.1 and cpn.2) of the root-nodulating bacterium R. leguminosarum strain A34. Both are heat inducible, and both have a CIRCE sequence in their upstream regions, suggesting regulation by an HrcA repressor. Mutagenesis of the CIRCE sequence upstream of cpn.1 led to an increase in the levels of cpn.1 mRNA, and knock-out of the hrcA gene increased the level of Cpn60.1 protein (the GroEL homologue encoded by the cpn.1 operon). Inactivation of the hrcA gene also caused increased expression of a 29 kDa protein that was identified as RhiA, a component of a quorum-sensing system. However, neither loss of the upstream CIRCE sequence, nor loss of HrcA function, had any effect on expression from the cpn.2 promoter. Further analysis of the cpn.2 upstream region suggested regulation could be mediated by an RpoH system, and this was confirmed by deleting the rpoH gene from the chromosome, which led to a decreased level of Cpn60.2 expression. Inactivation of RpoH led to a reduction in growth rate which could be partly compensated for by inactivation of HrcA, indicating an overlap in the in vivo function of the proteins regulated by these two systems.

Chaperonin 60↗

PubMed2006

Identification of bacterial N-acylhomoserine lactones (AHLs) with a combination of ultra-performance liquid chromatography (UPLC), ultra-high-resolution mass spectrometry, and in-situ biosensors.

N-Acylated homoserine lactones (AHLs) are produced by Gram-negative bacteria as communication signals and are frequently studied as mediators of the "quorum sensing" response of bacterial communities. Several reports have recently been published on the identification of AHLs from different species and attempts have been made to study their role in natural habitats, for example the surface of plant roots in the rhizosphere. In this article, different analytical methods, including bacterial biosensors and chromatographic techniques, are reviewed. A concept for assignment of the structures of AHLs is also presented. The retention behaviour of derivatives of AHLs containing beta-keto or hydroxyl groups and/or double bonds has been evaluated in relation to the separation behaviour of AHLs with saturated and unsubstituted alkanoyl chains. Samples have also been analysed by high resolution mass spectrometry (Fourier-transform ion-cyclotron-resonance mass spectrometry, FTICR-MS), nano liquid chromatography-electrospray ionization ion trap mass spectrometry (nano-LC-MS) and by the aid of a biosensor. The results obtained from ultra performance liquid chromatography (UPLC), FTICR-MS, nano-LC-MS, and bioassays have been compared to attempt structural characterisation of AHL without chemical synthesis of analytical standards. The method was used to identify the major AHL compound produced by the rhizosphere bacterium Acidovorax sp. N35 as N-(3-hydroxydecanoyl)homoserine lactone.

4-Butyrolactone↗