Showing papers in "Fems Microbiology Letters in 1998"
TL;DR: Plasmid reporter vectors have been constructed which respond to activation of LuxR and its homologues LasR and RhlR by N-acyl homoserine lactones by AHLs, allowing a comprehensive evaluation of quorum sensing signals from a test organism.
Abstract: Plasmid reporter vectors have been constructed which respond to activation of LuxR and its homologues LasR and RhlR (VsmR) by N-acyl homoserine lactones (AHLs). The expression of luxCDABE from transcriptional fusions to PluxI, PlasI and PrhlI respectively, occurs in the presence of activating AHLs. A profile of structure/activity relationships is seen where the natural ligand is most potent. The characterisation of individual LuxR homologue/AHL combinations allows a comprehensive evaluation of quorum sensing signals from a test organism.
580 citations
TL;DR: This organism is the first sulfate-reducing bacterium described that can grow with metals or U(VI) as sole electron acceptors.
Abstract: A spore-forming sulfate-reducing bacterium Desulfotomaculum reducens sp. nov. strain MI-1 has been isolated from heavy metal contaminated sediments. Strain MI-1 grows with Cr(VI), Mn(IV), Fe(III), and U(VI), in addition to various sulfur compounds, as electron acceptors. This organism shares physiological properties with both the sulfate-reducing and metal-reducing groups of bacteria and is the first sulfate-reducing bacterium described that can grow with metals or U(VI) as sole electron acceptors.
459 citations
TL;DR: Investigations showed that indolicidin inhibits DNA synthesis in E. coli cells at concentrations at which RNA and protein synthesis are either partially affected or not affected at all, which appears to contribute to the antimicrobial activity of indolicIDin.
Abstract: Indolicidin, a 13-residue antimicrobial peptide isolated from cytoplasmic granules of bovine neutrophils, exhibits activity against Gram-positive and Gram-negative bacteria as well as fungi. Although indolicidin is bactericidal and permeabilizes the bacterial membranes, it does not lyse the bacterial cells. We examined the effect of bactericidal concentrations of indolicidin on the morphology of Escherichia coli cells and found that it induces filamentation. Further investigations showed that indolicidin inhibits DNA synthesis in E. coli cells at concentrations at which RNA and protein synthesis are either partially affected or not affected at all. Since inhibition of DNA synthesis is also known to induce filamentation in E. coli, it appears to contribute to the antimicrobial activity of indolicidin.
404 citations
TL;DR: It is reported here that overexpression of the multidrug efflux pump locus acrAB, or of marA or soxS, both encoding positive regulators of acr AB, decreased susceptibility to triclosan 2-fold.
Abstract: Triclosan (Irgasan) is a broad spectrum antimicrobial agent used in handsoaps, toothpastes, fabrics, and plastics. It inhibits lipid biosynthesis in Escherichia coli, probably by action upon enoyl reductase (FabI) (McMurry L.M., Oethinger M. and Levy S.B. (1988) Nature 394, 531–532). We report here that overexpression of the multidrug efflux pump locus acrAB, or of marA or soxS, both encoding positive regulators of acrAB, decreased susceptibility to triclosan 2-fold. Deletion of the acrAB locus increased the susceptibility to triclosan approximately 10-fold. Four of five clinical E. coli strains which overexpressed marA or soxS also showed enhanced triclosan resistance. The acrAB locus was involved in the effects of triclosan upon both cell growth rate and cell lysis.
343 citations
TL;DR: Western blot analyses utilizing anti-enterotoxin antisera have confirmed the results obtained with the cat reporter system and PCR amplification studies suggest that the sej determinant may be present on all sed-encoding plasmids.
Abstract: Staphylococcus aureus enterotoxin D is one of the serotypes most commonly associated with food poisoning. Further characterization of the enterotoxin D-encoding plasmid revealed the presence of an open reading frame which encodes a previously unidentified enterotoxin, designated staphylococcal enterotoxin J (SEJ). SEJ is a protein of 269 amino acid residues which has substantial sequence similarity to the staphylococcal A, E, D family of enterotoxins. The enterotoxin D and J open reading frames are transcribed in opposite directions and are separated by an 895 nucleotide intergenic region which contains a perfect inverted repeat, with each arm of the repeat having a length of 21 nucleotides. Chloramphenicol acetyl transferase (cat) transcriptional fusions were used to quantify expression from the enterotoxin gene promoters. Both enterotoxin genes are expressed in S. aureus. However, only sed is regulated by the agr virulence gene signal transduction pathway. Western blot analyses utilizing anti-enterotoxin antisera have confirmed the results obtained with the cat reporter system. PCR amplification studies suggest that the sej determinant may be present on all sed-encoding plasmids.
319 citations
TL;DR: The presence and genetic content of integrons was investigated in eight Salmonella enterica Typhimurium DT104 isolates from different pig herds in Denmark and did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.
Abstract: The presence and genetic content of integrons was investigated in eight Salmonella enteritica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sul1 and qacEΔ1 genes characteristic of integrons. The first integron encoded the ant (3″)-Ia gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 β-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.
298 citations
TL;DR: All the strains adhered better to the mucus of adults than to that of infants, and with some of the strains significant differences between the infant age groups were also observed.
Abstract: Human mucus was isolated from faecal samples of newborns, two and six month old infants and adults. The adhesion to this mucus by the bacteria mentioned below was assessed in vitro. Depending on the age group: 44–46% of the applied Lactobacillus GG, 23–30% of Bifidobacterium lactis Bb-12, 9–14% of Lactobacillus johnsonii LJ-1, 3–10% of Lactobacillus salivarius LM2-118, Lactobacillus crispatus M247, Lactobacillus paracasei F19 and 2% of L. crispatus Mu5 adhered. All the strains adhered better to the mucus of adults than to that of infants. With some of the strains significant differences between the infant age groups were also observed. In conclusion, the age of the target group may be worthy of consideration when planning a schedule for probiotic or functional food therapy.
289 citations
TL;DR: Results suggest that cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and whilst short chain (C6) exogenous homoserines can trigger such response in P.fluorescens, its own signal substance is likely to possess a longer (> C8) fatty acyl chain.
Abstract: Pseudomonas fluorescens B52 produces substantial biofilms at the air/liquid/solid interface of glass coverslips clamped vertically and partly submerged in liquid medium at 21 degrees C. Biofilm formation was maximal ca. 20-50 h after inoculation of the liquid medium and as indicated by environmental scanning electron microscopy (ESEM), contained large numbers of bacterial cells that were embedded within an extensive exopolymeric matrix. Incubation beyond 50 h led to reductions in biofilm which ESEM related primarily to losses of exopolymer. Both biofilm formation and the subsequent decline in exopolymer deposition was more rapid, and occurred to greater extents, when supernatants from two-day old cultures of B52 were used as the initial growth media. The addition of N-acyl-hexanoyl homoserine lactone to fresh growth medium had a similar effect upon biofilm formation as using spent culture medium. Homoserine lactones could not be demonstrated in spent culture supernatants by an Agrobacterium tumefaciens bioassay. An exopolysaccharide lyase was detected in spend culture media taken from dense biofilm cultures whose action was specifically directed towards biofilm exopolysaccharide. Results suggest that (i) cell-cell signals such as homoserine lactones are associated with the formation of P. fluorescens biofilms, (ii) the enzymic degradation of exopolymers has a specific role in the detachment of cells under starvation conditions, and (iii) whilst short chain (C6) exogenous homoserines can trigger such response in P. fluorescens, its own signal substance is likely to possess a longer (> C8) fatty acyl chain.
285 citations
TL;DR: The potential of coupling mini-Tn5 luxCDABE promoter probe transposons with automated luminometry and photometry to screen for mutants that exhibit growth phase variation in gene expression is demonstrated.
Abstract: The luxCDABE operon of Photorhabdus luminescens has been cloned and engineered as an easily mobilisable cassette flanked by sites for commonly used restriction enzymes. Constitutive and promoter probe plasmids utilising the P. luminescens luxCDABE have been constructed using a number of compatible replicons and antibiotic markers. Complementary to these plasmids, a range of promoterless and constitutive luxCDABE mini-Tn5 derivatives has been constructed. The potential of coupling mini-Tn5 luxCDABE promoter probe transposons with automated luminometry and photometry to screen for mutants that exhibit growth phase variation in gene expression is demonstrated.
263 citations
TL;DR: Three new primer sets which amplify 1100 bp, 900 bp and 250 bp regions of the nosZ gene were designed and tested and robustly amplified nosZ fragments from samples in which the initial nosZ primers were only marginally successful.
Abstract: Two PCR primer sets for the nitrous oxide reductase gene (nosZ) were developed. The initial primers were based on three sequences in GenBank and used to amplify nosZ from continental shelf sediments and from two denitrifiers in culture, Thiosphaera pantotropha and Pseudomonas denitrificans. Three unique marine sediment nosZ genes were identified and sequenced. The marine nosZ genes were most closely related to the nosZ genes of Paracoccus denitrificans or to Rhizobium meliloti. Alignment of all nosZ sequences currently available (n = 10) facilitated redesign of the PCR primers. Three new primer sets which amplify 1100 bp, 900 bp and 250 bp regions of the nosZ gene were designed and tested. The new primers robustly amplified nosZ fragments from samples in which the initial nosZ primers were only marginally successful.
250 citations
TL;DR: Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed and allowed the same species identification as the DNA/DNA hybridization procedure.
Abstract: A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.
TL;DR: The photodynamic antibacterial properties of a closely related series of phenothiazinium dyes were tested against several pathogenic strains of Staphylococcus aureus, four of which were methicillin-resistant.
Abstract: The photodynamic antibacterial properties of a closely related series of phenothiazinium dyes were tested against several pathogenic strains of Staphylococcus aureus, four of which were methicillin-resistant Illumination of the photosensitisers at a fluence rate of 175 mW cm-2 generally resulted in the enhancement of antibacterial activity in liquid culture and in greater efficacy than the methicillin analogue flucloxacillin For methylene blue, dimethyl methylene blue and new methylene blue illumination led to increases in bactericidal activity or = 05 microM)
TL;DR: The reduced adherence observed with an isogenic SEF14/SEF21-deficient strain implicated the involvement of additional cell surface adherence factors, possibly including SEF21 (type I) fimbriae in the adherence of S. enteritidis to stainless steel or Teflon.
Abstract: Salmonella enteritidis enteropathogens produce a variety of potentially adherent fimbrial types including SEF14, SEF17, SEF18 and SEF21 (type I). In a simplified, pure culture, biofilm generating system the virulent isolate, S. enteritidis 3b, readily adhered to Teflon (polytetrafluoroethylene) and stainless steel forming thick cell aggregates. The inability of an isogenic SEF17-deficient mutant to form thick biofilms suggested a role for SEF17 in stabilizing cell-cell interactions during biofilm formation. Epifluorescent detection of SEF17 in biofilms confirmed the association of these fimbriae with aggregated cells but not with adherent mutants unable to produce SEF17. The reduced adherence observed with an isogenic SEF14/SEF21-deficient strain implicated the involvement of additional cell surface adherence factors, possibly including SEF21 (type I) fimbriae in the adherence of S. enteritidis to stainless steel or Teflon. The role of SEF17 fimbriae in biofilm formation and the contributions of SEF17 to the persistence of Salmonellae on surfaces and in food are discussed.
TL;DR: A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity, and was used in detailed explorations of the structure and function of this membrane protein to high resolution.
Abstract: Combination of an origin repair mutagenesis system with a new mutS host strain increased the efficiency of mutagenesis from 46% to 75% mutant clones. Overexpression with the T7 expression system afforded large quantities of proteins from mutant strains. A series of E. coli BE host strains devoid of major outer membrane proteins was constructed, facilitating the purification of mutant porins to homogeneity. This allowed preparation of 149 porin mutants in E. coli used in detailed explorations of the structure and function of this membrane protein to high resolution.
TL;DR: It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover, and peptidoglycan turnover would cause a turgor on the outer membranes, causing the outer membrane to bulge and finally bleb.
Abstract: It is proposed that the genesis of extracellular membrane vesicles in Gram-negative bacteria is a result of cell wall turnover. Peptidoglycan turnover would cause a turgor on the outer membrane, causing the outer membrane to bulge and finally bleb. Mechanical motion would then shear the blebs into the culture medium.
TL;DR: This represents the first report of quorum-sensing regulation of siderophore production in bacteria and highlights the fact that cell density, while not an essential signal for pyoverdine expression, does enhance production of this siderophile.
Abstract: Cell density-dependent gene expression in Pseudomonas aeruginosa is controlled, in part, by the quorum-sensing regulator LasR. lasR null mutants exhibited a reproducible 2-fold decrease in production of the catecholate-hydroxamate siderophore pyoverdine during grown under iron-limiting conditions. Similarly, lasI mutants defective in the biosynthesis of the autoinducer PAI-1 also exhibited a 2-fold decrease in pyoverdine production which could be largely restored upon addition of exogenous PAI-1. lasR mutants were not altered with respect to expression of the pvdD gene involved in the synthesis of the peptide portion of pyoverdine, indicating that some other pyoverdine biosynthetic gene(s) were affected by the LasRI status of the cell. This represents the first report of quorum-sensing regulation of siderophore production in bacteria and highlights the fact that cell density, while not an essential signal for pyoverdine expression, does enhance production of this siderophore.
TL;DR: A peroxidase oxidizing Mn2+ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds, related to its ability to catalyze Mn(2+)-mediated as well as Mn( 2+)-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.
Abstract: A peroxidase oxidizing Mn2+ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn2+-mediated as well as Mn2+-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.
TL;DR: The physiological behaviour of the fast growing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slow growing pathogenic M. tuberculosis, suggesting that M. smegMatis might be useful as a fast growing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.
Abstract: We report here that the physiological behaviour of the fastgrowing saprophytic Mycobacterium smegmatis under in vitro oxygen-depletion and reactivation conditions is strikingly similar to the characteristics shown by the slowgrowing pathogenic M. tuberculosis. M. smegmatis died rapidly when shifted abruptly from aerobic to anaerobic conditions. In contrast to the lethal shock of abrupt oxygen depletion, the slow depletion through a selfgenerated oxygen gradient permitted an adaptation to a persistent state which showed increased resistance against the bactericidal effects of anaerobiosis. The anaerobic persistent culture did not synthesise DNA and showed synchronised division upon reactivation in oxygen rich medium, indicating that the persistent bacilli are uniformly arrested at a defined stage of the cell cycle. Upon reactivation the persistent culture started synthesising DNA only after the first cell division, suggesting that the persistent cells contain two chromosomes. Furthermore, the persistent culture developed sensitivity to metronidazole and resistance against ofloxacin. These results suggest that M. smegmatis might be useful as a fastgrowing non-pathogenic model for comparative molecular analyses of mycobacterial dormancy.
TL;DR: A proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus is described and surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells.
Abstract: Enteropathogenic Escherichia coli (EPEC) cause a characteristic attaching and effacing (A/E) lesion in intestinal epithelial cells that is associated with the expression and export of specific bacterial proteins via a type III secretion pathway. These effector proteins and components of the type III export apparatus are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE). In this study, we describe a proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus. Whereas an espF deletion mutant does not synthesize or secrete EspF, surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells. Although these results do not indicate an obvious role for EspF in the formation of A/E lesions nor in the invasion of epithelial cells, they do not preclude a role played by EspF in other aspects of EPEC pathogenesis.
TL;DR: The first nucleic sequence report on phytase from a bacterial strain is reported, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution.
Abstract: Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.
TL;DR: On the basis of 16S rRNA sequences, species- and group-specific primers for Bifidobacterium adolescentis, B. bifidum,B.
Abstract: On the basis of 16S rRNA sequences, species- and group-specific primers for Bifidobacterium adolescentis, B. angulatum, B. bifidum, B. breve, the B. catenulatum group (B. catenulatum and B. pseudocatenulatum), and the B. longum group (B. longum and B. infantis), which are species commonly found in human intestinal tracts, were developed. The specificity of these primers was confirmed through the use of DNA extracted from 46 strains of 31 Bifidobacterium species, as well as 14 non-bifidobacterial species that are the predominant bacteria in the human intestinal tract. The present species-specific primers were applied to the identification of 43 isolated strains, consisting of six strains of B. adolescentis, eight of the B. catenulatum group, seven of B. bifidum, nine of B. breve, and 13 of the B. longum group.
TL;DR: High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide, demonstrating that this purification strategy can be used for the mycobacteria.
Abstract: A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.
TL;DR: It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one.
Abstract: The streptomycin/spectinomycin resistance determinant of the 29-kb plasmid pCG4 from Corynebacterium glutamicum was found to be a part of a typical class 1 integron. The sequence analysis revealed that the integron (designated InCg) identified in this Gram-positive bacterium is almost identical to the integron InC present on the plasmid pSA1700 from the Gram-negative bacterium Pseudomonas aeruginosa. Differences in only two base pairs were found in the 3.8-kb sequence. One base substitution (G→C) is present in the streptomycin/spectinomycin resistance determinant which is thus identical to the aadA2a gene from the integron In6 of the broad-host-range plasmid pSa. The other one (C→G) is present in the extended −10 region of the integron promoter involved in expression of the antibiotic resistance gene. It was shown that this novel version of the integron promoter displays five times higher activity in both C. glutamicum and Escherichia coli than the original one.
TL;DR: The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation by the sulfate-reducing bacterium Desulfovibrio desulfuricans were followed by isotope mass spectrometry as discussed by the authors.
Abstract: The fates of the two different sulfur atoms of the thiosulfate molecule during anaerobic disproportionation by the sulfate-reducing bacterium Desulfovibrio desulfuricans were followed by isotope mass spectrometry. During disproportionation, 32S-thiosulfate was preferentially metabolized, and the residual thiosulfate became enriched in 34S. The sulfate formed was isotopically heavier than the inner sulfur of the consumed thiosulfate. Vice versa, the sulfide formed was isotopically lighter than the outer sulfur of the consumed thiosulfate. These results indicate that thiosulfate is cleaved to intermediates that undergo further disproportionation to sulfate and sulfide in a second step. These intermediates are probably elemental sulfur and sulfite. It is concluded that disproportionation of thiosulfate, sulfite and elemental sulfur includes a combined pathway.
TL;DR: The results suggest a novel mechanism for mycobacteria in evading antimicrobial treatment and also indicate that biofilms should be considered possible sites forMycobacterial contamination.
Abstract: Rapidly growing mycobacteria (RGM) are found in soil and diverse aquatic environments. Two species, Mycobacterium fortuitum and Mycobacterium chelonae, are associated with disease and are difficult to eradicate. Biofilm formation may be a contributing factor to their mode of transmission and their resistance to antimicrobial agents. We investigated the ability of the RGM species M. fortuitum to colonise surfaces using a modified Robbins device. M. fortuitum formed dense biofilms within 48 h. The high numbers of sessile organisms recovered and the swiftness of colonisation suggest that M. fortuitum readily forms biofilms. These results suggest a novel mechanism for mycobacteria in evading antimicrobial treatment and also indicate that biofilms should be considered possible sites for mycobacterial contamination.
TL;DR: F fluorimetry indicated that the high-level ethidium bromide resistance was due to improved efflux energised by the proton motive force, and site-directed mutagenesis substituting the Asp-24 residue with Glu-24 had no effect on resistance characteristics.
Abstract: The prevalence of disinfectant-resistant food-related microorganisms is of concern to the food industry. The Staphylococcus saprophyticus strain ST2H6 isolated from a poultry processing plant contained a 2.4-kb plasmid (p2H6) harbouring qacH, which encodes resistance to disinfectants based on quaternary ammonium compounds. The complete p2H6 nucleotide sequence revealed an open reading frame encoding a putative protein of 107 amino acid residues with strong similarity to members of the small multidrug resistance protein family. QacH also conferred high-level ethidium bromide resistance and low-level proflavine resistance and thus differed phenotypically from the similar proteins Smr and QacG. Fluorimetry indicated that the high-level ethidium bromide resistance was due to improved efflux energised by the proton motive force. Site-directed mutagenesis substituting the Asp-24 residue with Glu-24 had no effect on resistance characteristics. An additional open reading frame on p2H6 encoded a putative protein with similarity to rolling circle replication proteins.
TL;DR: Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the polyhydroxyalkanoic acid synthases, in Escherichia coli impaired in fatty acid beta-oxidation results in polyhydroxalkanoi acid accumulation when cells were cultivated on fatty acids is evaluated.
Abstract: Heterologous expression of the phaC1 gene from Pseudomonas aeruginosa, which encodes one of the polyhydroxyalkanoic acid synthases, in Escherichia coli impaired in fatty acid β-oxidation results in polyhydroxyalkanoic acid accumulation when cells were cultivated on fatty acids. We evaluated the application of the fatty acid β-oxidation inhibitor acrylic acid as a tool to channel intermediates of β-oxidation to polyhydroxyalkanoic acid synthesis. Various E. coli strains affected in fatty acid metabolism and the wild-type strain harboring plasmid pBHR71 were analyzed with respect to polyhydroxyalkanoic acid accumulation in the presence of acrylic acid. The E. coli fadR mutant RS3097 revealed the strongest polyhydroxyalkanoic acid accumulation. The optimum inhibitory concentration of acrylic acid was 0.24 mg ml−1 and caused efficient channeling of intermediates of β-oxidation to polyhydroxyalkanoic acid synthesis. Under these conditions and grown on decanoate E. coli RS3097 harboring plasmid pBHR71 revealed a polyhydroxyalkanoic acid accumulation contributing to about 60% of cellular dry weight.
TL;DR: The ACR2 gene of Saccharomyces cerevisiae was disrupted by insertion of a HIS3 gene and cells with the disruption were sensitive to arsenate, and the combination of chimeric MBP-Acr2-6H protein and yeast cytosol from an ACR1-disrupted strain exhibited arsenate reductase activity.
Abstract: The ACR2 gene of Saccharomyces cerevisiae was disrupted by insertion of a HIS3 gene. Cells with the disruption were sensitive to arsenate. This phenotype could be complemented by ACR2 on a plasmid. The ACR2 gene was cloned and expressed in Escherichia coli as a malE gene fusion with a C-terminal histidine tag. The combination of chimeric MBP-Acr2-6H protein and yeast cytosol from an ACR2-disrupted strain exhibited arsenate reductase activity.
TL;DR: Results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.
Abstract: The role of ergosterol in yeast stress tolerance, together with heat shock proteins (hsps) and trehalose, was examined in a sterol auxotrophic mutant of Saccharomyces cerevisiae. Ergosterol levels paralleled viability data, with cells containing higher levels of the sterol exhibiting greater tolerances to heat and ethanol. Although the mutant synthesised hsps and accumulated trehalose upon heat shock to the same levels as the wild-type cells, these parameters did not relate to stress tolerance. These results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.
TL;DR: The data show that phenotypes of Microcystis do not necessarily reflect their phylogeny, and that there is a need to reconstruct the present taxonomy at the species level.
Abstract: Phylogenetic analyses were carried out on 15 strains of Microcystis, with five morphospecies and including two types of phycobilin pigment composition, by sequence determination of the 16S rDNA (16S rRNA gene). All the sequences showed high similarity, in some cases 100% similarity, between different morphospecies and phycobilin types. Derived phylogenetic trees revealed a close relationship between Microcystis strains with and without phycoerythrin. The data show that phenotypes of Microcystis do not necessarily reflect their phylogeny, and that there is a need to reconstruct the present taxonomy at the species level.