How different is the proteome of the extended spectrum ß-lactamase producing Escherichia coli strains from seagulls of the Berlengas natural reserve of Portugal?

UNLABELLED: ß-Lactam antibiotics like cefotaxime are the most commonly used antibacterial agents. Escherichia coli strains 5A, 10A, 12A and 23B isolated from Seagulls feces, are cefotaxime-resistant strains that produces extended-spectrum beta-lactamases. Bacterial resistance to these antibiotics occurs predominantly through structural modification on the penicillin-binding proteins and enzymatic inactivation by extended-spectrum ß-lactamases. Using classical proteomic techniques (2D-GE) coupled to mass spectrometry and bioinformatics extended analysis, in this study, we report several significant differences in cytoplasmic proteins expression when the strains were submitted to antibiotic stress and when the resistant strains were compared with a non-resistant strain. A total of 79 differentially expressed spots were collected for protein identification. Significant level of expression was found in antibiotic resistant proteins like ß-lactamase CTX-M-1 and TEM and also in proteins related with oxidative stress. This approach might help us understand which pathways form barriers for antibiotics, another possible new pathways involved in antibiotic resistance to devise appropriate strategies for their control already recognized by the World Health Organization and the European Commission. BIOLOGICAL SIGNIFICANCE: This study highlights the protein differences when a resistant strain is under antibiotic pressure and how different can be a sensible and resistant strain at the protein level. This survey might help us to understand the specifics barriers for antibiotics and which pathways are involved in its resistance crosswise the wildlife.

Response of Listeria monocytogenes to liquid smoke.

AIMS: To investigate the effect of liquid smoke on growth, survival, proteomic pattern and haemolytic potential of Listeria monocytogenes. METHODS AND RESULTS: Growth and survival curves were recorded in brain-heart infusion broth supplemented with three concentrations of liquid smoke. L. monocytogenes growth was inhibited in the presence of 15 microg ml(-1) phenol while a rapid decrease in cell viability occurred in the presence of 30 microg ml(-1) phenol. The proteome of L. monocytogenes cytosoluble proteins was slightly modified after 2-h incubation with 30 microg ml(-1) phenol but no protein already characterized in response to other known stresses was induced, except the protease ClpP. Liquid smoke inhibited the haemolytic potential without affecting hly gene expression, showing a potential inhibition of protein activity or stability. CONCLUSIONS: The presence of liquid smoke in a rich medium strongly affected growth and survival of L. monocytogenes. Brief smoke stress affected the metabolic pathways and inhibited the haemolytic activity of L. monocytogenes. SIGNIFICANCE AND IMPACT OF STUDY: This study is a first step in the investigation of the influence of a smoked product on L. monocytogenes strains.

Formation of biofilm by Staphylococcus xylosus.

The ability of 12 Staphylococcus xylosus strains to form biofilm was determined through the study of different criteria. Eleven out of the 12 strains were able to form biofilm, 10 preferentially on hydrophilic support (glass) and one, S. xylosus C2a, on both hydrophilic and hydrophobic (polystyrene) supports. The determination of bacterial surface properties showed that all strains were negatively charged with five strains moderately hydrophobic and seven hydrophilic. The bap and icaA genes, important for biofilm formation of some staphylococci, were searched. All strains were bap positive but icaA negative. Furthermore, S. xylosus strain C2a was studied on two supports widely used in the food industry, polytetrafluoroethylene (PTFE, hydrophobic) and stainless steel (hydrophilic) and appeared to adhere preferentially on stainless steel. Addition of 20 g/l of NaCl to Tryptic Soy Broth medium (TSB) did not improve significantly its adhesion but enhanced both bacterial growth and cell survival, which were optimum in this medium. Environmental scanning electron microscopy showed that S. xylosus C2a colonized the surface of stainless steel chips with intercellular spaces. The strain formed cell aggregates embedded in an amorphous polysaccharidic matrix. Indeed, synthesis of polysaccharides increased during growth on stainless steel chips in TSB.

Combined effects of NaCl, NaOH, and biocides (monolaurin or lauric acid) on inactivation of Listeria monocytogenes and Pseudomonas spp.

This study highlighted combinations of chemical stresses that could decrease or eliminate Listeria monocytogenes and Pseudomonas spp. surviving in food processing plants. Strains of L. monocytogenes, Pseudomonas fragi, and Pseudomonas fluorescens isolated from processing environments (meat and milk) were grown at 20 degrees C up to the early stationary phase. The strains were then subjected to 30 min of physicochemical treatments. These treatments included individual or combined acid (acetic acid), alkaline (NaOH), osmotic (NaCl), and biocides (fatty acids) challenges. Survival of the strains was studied after individual or combined acid (acetic acid), alkaline (NaOH), osmotic (NaCl), and biocides (monolaurin, lauric acid) challenges. Individual pH shocks had lower efficiencies than those used in combinations with other parameters. The treatment pH 5.4 followed by pH 10.5 had a low efficiency against L. monocytogenes. The opposite combination, pH 10.5 followed by pH 5.4, led to a 3-log reduction of the L. monocytogenes population. Pseudomonas spp. strains were much more sensitive than L. monocytogenes, and population reductions of 5 and 8 log (total destruction), respectively, were observed after the same treatments. As for L. monocytogenes, the combination pH 10.5 followed by pH 5.4 is more deleterious than the opposite. Whatever the bacterial species, the most efficient treatments were combinations of alkaline, osmotic, and biocide shocks. For instance, the combination pH 10.5 and 10% NaCl plus biocides showed reductions of 5 to 8 log for both bacteria. The origins of the observed lethal effects are discussed.

Cell immobilization induces changes in the protein response of Escherichia coli K-12 to a cold shock.

We have compared the protein expression of gel-entrapped Escherichia coli cells submitted to a cold shock at 4 degrees C with those of exponential- and stationary-phase free-floating counterparts. Autoradiograms of two-dimensional gel electrophoresis patterns of proteins radiolabeled with L-[35S]methionine were compared using computing scanning densitometry. The levels of 203 proteins synthesized during the temperature shift were significantly and reproducibly higher than those corresponding to synthesis at 37 degrees C. A principal component analysis (PCA) was performed on the synthesis levels of these 203 proteins in the different incubation conditions tested. This study showed that the protein response of immobilized cells after the cold shock was significantly different from those of exponential- and stationary-phase free-floating organisms. For instance, protein SSB was specifically overexpressed by shocked immobilized organisms. Such induction of specific molecular mechanisms in immobilized bacteria might explain the high resistance of sessile-like organisms to stresses.

The main cold shock protein of Listeria monocytogenes belongs to the family of ferritin-like proteins.

The transfer of the food-borne pathogen Listeria monocytogenes from 30 to 5 degrees C was characterized by the sharp induction of a low molecular mass protein. This major cold shock protein has an isoelectric point at pH 5.1 and a molecular mass of about 18 kDa, as observed on two-dimensional gel electrophoresis (2-DE) pattern. Its N-terminal sequence, obtained from the 2-DE spot, shared a complete sequence identity with a Listeria innocua non-heme iron-binding ferritin. The purification of these ferritin-like proteins (Flp) revealed a native molecular mass of about 100-110 kDa which indicates a polypeptide composed of six 18 kDa-subunits. Northern analysis indicated the presence of a 0.8-kb monocistronic mRNA in exponential growing cells and an important increase inflp mRNA amount after a downshift but also an upshift in temperature.

Protein synthesis in Escherichia coli at 4 degrees C.

Two-dimensional electrophoresis technology was used to investigate protein synthesis by the mesophilic bacterium Escherichia coli at low temperature. It was confirmed that protein synthesis in E. coli decreased strongly after a temperature downshift from 37 to 4 degrees C. After incubation for 150 min at 4 degrees C, however, the number of synthesized proteins represented 60% of the overall polypeptide number observed at 37 degrees C. Furthermore, the analysis of autoradiograms revealed the overexpression of 69 proteins by shocked bacteria, showing that the number of cold-induced proteins has been significantly underestimated so far.

Protein patterns of gel-entrapped Escherichia coli cells differ from those of free-floating organisms.

The two-dimensional electrophoretic patterns of cellular proteins from gel-entrapped Escherichia coli cells were compared to those of exponential- and stationary-phase free-floating organisms. The amounts of several proteins in immobilized cells were significantly different from those in free bacteria. Immobilized organisms rapidly produced a high level of dipeptide permease and a single-strand binding protein, and progressively accumulated an aldehyde dehydrogenase. Immobilization also induced a decrease in the levels of two proteins, i.e., the YFID protein and a DNA-binding, stationary-phase protein. The possible role of these proteins in the high resistance of immobilized bacteria to stresses is discussed.

Differential protein expression by Pseudomonas fragi submitted to various stresses.

We have analyzed the impact of various stressing conditions on the physiological and molecular responses of the main psychrotrophic spoilage bacterium of refrigerated meat and meat products, Pseudomonas fragi. A survival study using conventional plating was first performed to select the stressing agents and parameters. Some of these mimicked cleaning-disinfection processes but with less drastic conditions in order to keep alive enough bacterial cells for the protein expression characterization. Cultures of P. fragi, at the beginning of the stationary phase of growth, were submitted to individual pH (5.4, 10.5), osmotic (8% Na2SO4, pH 7.0), biocide (fatty amine) shocks or combined treatments (8% Na2SO4, pH 10.5; 8% Na2SO4, pH 10.5 + biocide; pH 5.4 + pH 10.5 and pH 10.5 + pH 5.4) and the molecular responses were investigated by comparing autoradiograms of two-dimensional gel electrophoresis (2-DE) patterns of proteins radiolabeled with L-[35S]methionine. The observation of qualitative and relative quantitative variations in protein expression, determined with Melanie II image analysis software (Bio-Rad), revealed the overexpression of a total of 91 proteins for the eight challenges by comparison with the nonshocked controls. Some proteins appeared to be more or less general stress proteins whereas others were specific for one chemical treatment. The appraisal of the type of molecular response according to the type of treatment was analyzed statistically.

Effect of osmotic, alkaline, acid or thermal stresses on the growth and inhibition of Listeria monocytogenes.

Five strains of Listeria monocytogenes (a, b, c, d and e) isolated from industrial plants have been subjected to different osmotic, alkaline, acid or thermal stresses. The effects of these treatments on lag-phase (L) and growth rate (mu) of cells in mid-log phase have been followed using an automated optical density monitoring system. Increasing the osmotic pressure by the addition of different amounts of NaCl increased the lag phase and decreased the growth rate. The same phenomena were observed after decreasing the pH of the medium to 5.8, 5.6 or 5.4 by addition of acetic, lactic or hydrochloric acids. The inhibitory effect was: acetic acid > lactic acid > hydrochloric acid. The addition of NaOH to attain pH values of 9.5, 10.0, 10.5 or 11.0 in the medium produced a dramatic increase of the lag phase at pH 10.5 and 11. Growth rates were also decreased while the maximal population increased with high pH values. These effects varied according to strains. Strains d and e were the most resistant to acidic and alkaline stresses, and e was the most affected by the addition of NaCl. A cold shock of 30 min at 0 degree C had limited effects on growth parameters. On the other hand, hyperthermal shocks (55 or 63 degrees C, 30 min) led to similar increased lag phases and to significant increases of the maximal population in all five strains.

Cold shock response and low temperature adaptation in psychrotrophic bacteria.

Psychrotrophic bacteria are capable of developing over a wide temperature range and they can grow at temperatures close to or below freezing. This ability requires specific adaptative strategies in order to maintain membrane fluidity, the continuance of their metabolic activities, and protein synthesis at low temperature. A cold-shock response has been described in several psychrotrophic bacteria, which is somewhat different from that in mesophilic microorganisms: (i) the synthesis of housekeeping proteins is not repressed following temperature downshift and they are similarly expressed at optimal and low temperatures (ii) cold-shock proteins or Csps are synthesized, the number of which increases with the severity of the shock (iii) a second group of cold-induced proteins, i.e. the cold acclimation proteins or Caps, comparable with Csps are continuously synthesized during prolonged growth at low temperature. Homologues to CspA, the major cold-shock protein in E. coli, have been described in various psychrotrophs, but unlike their mesophilic counterparts, they belong to the group of Caps. Although they have been poorly studied, Caps are of particular importance since they differentiate psychrotrophs from mesophiles, and they are probably one of the key determinant that allow life at very low temperature.

Effects of pH or a(w) stress on growth of Listeria monocytogenes.

The growth of three strains of Listeria monocytogenes at 20 degrees C in a meat broth of different pH or water activity was investigated. At inoculation or at the beginning of the exponential phase, cells were exposed to stress by the addition of NaOH or NH4+, acetic acid, NaCl or KCl, in order to reach a pH of either 9.0 or 5.6, or an a(w) of 0.950 or 0.965, respectively. The effects of the exposure to stress on the generation and lag times of each strain were analysed by turbidity measurements for cultures in micro-titer plates. Results were confirmed by conducting the same experiments in a fermentor, except for the maximal population reached. The three strains showed similar behaviour. Cells were able to overcome the alkaline stress rapidly whereas acid and osmotic shocks induced important changes of the growth parameters. Cells exposed to acid or osmotic conditions from the time of inoculation were less affected than cells exposed at the beginning of the mid-exponential phase.

The cold shock response of the psychrotrophic bacterium Pseudomonas fragi involves four low-molecular-mass nucleic acid-binding proteins.

The psychrotrophic bacterium Pseudomonas fragi was subjected to cold shocks from 30 or 20 to 5 degrees C. The downshifts were followed by a lag phase before growth resumed at a characteristic 5 degrees C growth rate. The analysis of protein patterns by two-dimentional gel electrophoresis revealed overexpression of 25 or 17 proteins and underexpression of 12 proteins following the 30- or 20-to-5 degrees C shift, respectively. The two downshifts shared similar variations of synthesis of 20 proteins. The kinetic analysis distinguished the induced proteins into cold shock proteins (Csps), which were rapidly but transiently overexpressed, and cold acclimation proteins (Caps), which were more or less rapidly induced but still overexpressed several hours after the downshifts. Among the cold-induced proteins, four low-molecular-mass proteins, two of them previously characterized as Caps (CapA and CapB), and heat acclimation proteins (Haps) as well as heat shock proteins (Hsps) for the two others (TapA and TapB) displayed higher levels of induction. Partial amino acid sequences, obtained by microsequencing, were used to design primers to amplify by PCR the four genes and then determine their nucleotide sequences. A BamHI-EcoRI restriction fragment of 1.9 kb, containing the complete coding sequence for capB, was cloned and sequenced. The four peptides belong to the family of small nucleic acid-binding proteins as CspA, the major Escherichia coli Csp. They are likely to play a major role in the adaptative response of P. fragi to environmental temperature changes.

Purification and characterization of a collagenolytic enzyme produced by Rathayibacter sp. strains isolated from cultures of Clavibacter michiganensis subsp. michiganensis.

We show in this work that collagenolytic Rathayibacter sp. are isolated with phytopathogenic Clavibacter michiganensis subsp. michiganensis strains. The Rathayibacter strains isolated all produced collagenases. One of these collagenases (from the strain 1715) was purified by ammonium sulphate precipitation, DEAE cellulose and Sephadex G 200 chromatography. Characterization of the enzyme showed that it is a true collagenase which is able to degrade both native collagen, gelatin and probably other proteins from plants sharing sequence homologies with collagen.

Effect of Different Temperature Upshifts on Protein Synthesis by the Psychrotrophic Bacterium Pseudomonas fragi

Pseudomonas fragi, a psychrotroph bacterium involved in meat product spoilage, was shifted either from 5degrees to 20degreesC or 30degreesC and from 28degrees to 34degreesC. The heat-shocked cells in the mid-log phase rapidly reached the characteristic growth rate of the postshock temperature. The patterns of synthesized proteins were compared by autoradiography of two-dimensional gel electrophoregrams. The rates of synthesis, after transfer of cells from 5degrees to 30degreesC, 5degrees to 20degreesC, and 28degrees to 34degreesC, changed for 30, 26, and 21 proteins respectively, of which 19, 17, and 12 were increased respectively. Thirteen proteins changed similarly for the three treatments, and two of the seven overexpressed proteins were immunologically related to the Escherichia coli DnaK and GroEL heat shock proteins. From the four low-molecular-mass proteins, belonging to the family of DNA-binding cold shock proteins (CSPs) such as CS7.4, the major E. coli CSP [15], the amounts of C7.0 and C8.0 decreased rapidly after the upshifts, whereas that of E7.0 and E8.0 increased greatly.

Purification and characterization of a dipeptidase from Lactobacillus sake.

A dipeptidase was purified from cell extracts of Lactobacillus sake. This compound was a monomer having a molecular weight of 50,000 and a pI of 4.7 and exhibited broad specificity against all dipeptides except those with proline or glycine at the N terminus. The enzyme was inhibited by EDTA or 1,10-phenanthroline but could be reactivated with CoCl2 and MnCl2.

Effect of growth temperatures on the protein levels in a psychrotrophic bacterium, Pseudomonas fragi.

Pseudomonas fragi has the ability to grow between 0 and 35 degrees C and grows optimally at 30 degrees C. Cellular proteins from mid-log-phase cells growing from 4 to 34 degrees C were labeled with L-[35S]methionine during 1 generation time and analyzed by two-dimensional gel electrophoresis. The electrophoretic patterns revealed differences in the patterns of protein synthesis over this temperature span. A qualitative comparison of cellular proteins led to their separation into five thermal classes. The first class contained proteins whose relative rates of synthesis were unaffected by the growth temperature. Three other classes included proteins with optimal expression at 4 to 10, 15 to 20, and 25 to 30 degrees C. A fifth class contained proteins which were more specifically synthesized at a supraoptimal growth temperature (34 degrees C). Two low-molecular-mass proteins, designated C7.0 and C8.0, were highly concentrated at 4 to 10 degrees C, and their relative rates of synthesis steadily increased with decreasing temperature. Polyclonal antibodies were separately raised against these two proteins. Immunological analyses revealed cross-reaction between these two proteins and between two additional low-molecular-mass proteins which were maximally produced at elevated temperatures. Antisera directed against C8.0 recognized the major cold shock protein of Escherichia coli, CspA, indicating the presence of similarities between these proteins.

Molecular cloning of genes from the rumen anaerobic fungus Neocallimastix frontalis: expression during hydrolase induction.

Glycoside and polysaccharide hydrolase production by the rumen anaerobic fungus, Neocalimastix frontalis is induced by the presence of crystalline cellulose. A differential screening of a cDNA library was used to isolate DNA sequences transcribed at high levels under growth conditions which induce enzyme production. Seven clones were isolated that preferentially hybridized to the induced cDNA probe versus the non-induced cDNA probe. Southern analysis showed that a cDNA clone (118) hybridized to a DNA probe encoding part of the exo-cellobiohydrolase I (CBH I) gene of Trichoderma reesei. Northern analysis demonstrated that the cDNA 118 was transcribed to yield a 2.1 kb RNA. This transcript was induced in the presence of cellulose.

Purification and Characterization of an Aspecific Glycoside Hydrolase from the Anaerobic Ruminal Fungus Neocallimastix frontalis.

A glycoside hydrolase characterized by beta-fucosidase (EC 3.2.1.38) and beta-glucosidase (EC 3.2.1.21) activities was purified from the culture medium of the anaerobic ruminal phycomycete Neocallimastix frontalis grown on 0.5% Avicel. The enzyme had a molecular mass of 120 kilodaltons and a pI of 3.85. Optimal activity against p-nitrophenyl-beta-d-fucoside and p-nitrophenyl-beta-D-glucoside occurred at pH 6.0 and 50 degrees C. The beta-fucosidase and beta-glucosidase activities were stable from pH 6.0 to pH 7.8 and up to 40 degrees C. They were both inhibited by gluconolactone, sodium dodecyl sulfate, p-chloromercuribenzoate, and Hg cation. The enzyme had K(m)s of 0.26 mg/ml for p-nitrophenyl-beta-d-fucoside and 0.08 mg/ml for p-nitrophenyl-beta-d-glucoside. The purified protein also had low beta-galactosidase activity.

Purification and characterization of an extracellular beta-xylosidase from the rumen anaerobic fungus Neocallimastix frontalis.

The purification of beta-xylosidase (beta-D-xyloside xylohydrolase, EC 3.2.1.37) from Neocallimastix frontalis was performed by ammonium sulphate precipitation, ion exchange chromatography, gel filtration and preparative isoelectric focusing. The enzyme had a molecular mass of 180,000 Da, an isoelectric point at pH 4.35 and catalysed the hydrolysis of p-nitrophenyl-beta-D-xylopyranoside optimally at pH 6.5 and 35 degrees C with a Km of 0.33 mg ml-1. The enzymatic activity was strongly increased by the presence of Ca2+, Mn2+, Zn2+, Co2+ or Mg2+ and completely inhibited by Hg2+ and p-chloromercuribenzoate. The purified protein also had a low level of xylanase activity.