Search PubMed⌕ Search

Biomedical subjects

R Kolter

Publications and source records attributed to R Kolter.

At least 55 records · Page 3Linked to original sources
PubMed1997

Green fluorescent protein as a marker for Pseudomonas spp.

The development of sensitive methods for observing individual bacterial cells in a population in experimental models and natural environments, such as in biofilms or on plant roots, is of great importance for studying these systems. We report the construction of plasmids which constitutively express a bright mutant of the green fluorescent protein of the jellyfish Aequorea victoria and are stably maintained in Pseudomonas spp. We demonstrate the utility of these plasmids to detect individual cells in two experimental laboratory systems: (i) the examination of a mixed bacterial population of Pseudomonas aeruginosa and Burkholderia cepacia attached to an abiotic surface and (ii) the association of Pseudomonas fluorescens WCS365 with tomato seedling roots. We also show that two plasmids, pSMC2 and pGB5, are particularly useful, because they are stable in the absence of antibiotic selection, they place an undetectable metabolic burden on cells that carry the plasmids, and cells carrying these constructs continue to fluoresce even after 7 days in culture without the addition of fresh nutrients. The construction of improved Escherichia coli-Pseudomonas shuttle vectors which carry multiple drug resistance markers also is described.

Biomarkers↗
PubMed1997

Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps.

Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents. Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli. We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks. dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide. In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles. In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps. The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.

Bacterial Proteins↗
PubMed1996

From peptide precursors to oxazole and thiazole-containing peptide antibiotics: microcin B17 synthase.

Esherichia coli microcin B17 is a posttranslationally modified peptide that inhibits bacterial DNA gyrase. It contains four oxazole and four thiazole rings and is representative of a broad class of pharmaceutically important natural products with five-membered heterocycles derived from peptide precursors. An in vitro assay was developed to detect heterocycle formation, and an enzyme complex, microcin B17 synthase, was purified and found to contain three proteins, McbB, McbC, and McbD, that convert 14 residues into the eight mono- and bisheterocyclic moieties in vitro that confer antibiotic activity on mature microcin B17. These enzymatic reactions alter the peptide backbone connectivity. The propeptide region of premicrocin is the major recognition determinant for binding and downstream heterocycle formation by microcin B17 synthase. A general pathway for the enzymatic biosynthesis of these heterocycles is formulated.

Adenosine Triphosphate↗
PubMed1996

Processing of colicin V-1, a secretable marker protein of a bacterial ATP binding cassette export system, requires membrane integrity, energy, and cytosolic factors.

Extracellular secretion of the peptide antibiotic colicin V (ColV) in Escherichia coli is mediated by a dedicated exporter system consisting of host TolC protein and the products of two specific genes, cvaA and cvaB, the latter being a member of the ATP binding cassette (ABC) superfamily. An amino-terminal export signal of ColV is specific for the CvaA-CvaB-TolC exporter and is processed concomitant with secretion. In this study, we attempt to characterize this processing with a secretable marker protein, ColV-1, using a newly developed in vitro assay. Processing is found to be dependent on both CvaA-CvaB transporters and the TolC protein and to require membrane integrity. An additional cytoplasmic soluble factor(s) is also necessary for the processing. Although the sequence of the cleavage site suggests it could be a substrate, ColV-1 cannot be processed in vitro by the purified leader peptidase I. Moreover, ColV-1 processing is inhibited by antipain and N-ethylmaleimide. Furthermore, the processing requires energy in the form of nucleotide hydrolysis. These results indicate that the processing of ColV-1 is specific and more complex than expected, requiring the CvaA-CvaB-TolC transporter intact in the membrane, energy, and cytosolic factors for rapid cleavage.

ATP-Binding Cassette Transporters↗
PubMed1996

SurA assists the folding of Escherichia coli outer membrane proteins.

Many proteins require enzymatic assistance in order to achieve a functional conformation. One rate-limiting step in protein folding is the cis-trans isomerization of prolyl residues, a reaction catalyzed by prolyl isomerases. SurA, a periplasmic protein of Escherichia coli, has sequence similarity with the prolyl isomerase parvulin. We tested whether SurA was involved in folding periplasmic and outer membrane proteins by using trypsin sensitivity as an assay for protein conformation. We determined that the efficient folding of three outer membrane proteins (OmpA, OmpF, and LamB) requires SurA in vivo, while the folding of four periplasmic proteins was independent of SurA. We conclude that SurA assists in the folding of certain secreted proteins.

Amino Acid Sequence↗
PubMed1995

Glutathionylspermidine metabolism in Escherichia coli. Purification, cloning, overproduction, and characterization of a bifunctional glutathionylspermidine synthetase/amidase.

Glutathionylspermidine (GSP) synthetases of Trypanosomatidae and Escherichia coli couple hydrolysis of ATP (to ADP and Pi) with formation of an amide bond between spermidine (N-(3-aminopropyl)-1,4-diaminobutane) and the glycine carboxylate of glutathione (gamma-Glu-Cys-Gly). In the pathogenic trypanosomatids, this reaction is the penultimate step in the biosynthesis of the antioxidant metabolite, trypanothione (N1,N8-bis-(glutathionyl)spermidine), and is a target for drug design. In this study, GSP synthetase was purified to near homogeneity from E. coli B, the gene encoding it was isolated and sequenced, the enzyme was overexpressed and purified in quantity, and the recombinant enzyme was characterized. The 70-kDa protein was found to have an unexpected second catalytic activity, glutathionylspermidine amide bond hydrolysis. Thus, the bifunctional GSP synthetase/amidase catalyzes opposing amide bond-forming and -cleaving reactions, with net hydrolysis of ATP. The synthetase activity is selectively abrogated by proteolytic cleavage 81 residues from the C terminus, suggesting that the two activities reside in distinct domains (N-terminal amidase and C-terminal synthetase). Proteolysis at this site is facile in the absence of substrates, but is inhibited in the presence of ATP, glutathione, and Mg2+. A series of analogs was used to probe the spermidine-binding site of the synthetase activity. The activity of diaminopropane as a substrate, inactivity of the C4-C8 diaminoalkanes, and greater loss of specificity for analogs modified in the 3-aminopropyl moiety than for those modified in the 4-aminobutyl moiety indicate that the enzyme recognizes predominantly the diaminopropane portion of spermidine and corroborate N-1 (the aminopropyl N) as the site of glutathione linkage (Tabor, H. and Tabor, C. W. (1975) J. Biol. Chem. 250, 2648-2654). Trends in Km and kcat for a set of difluorosubstituted spermidine derivatives suggest that the enzyme may bind the minor, deprotonated form of the amine nucleophile.

Amide Synthases↗
PubMed1995

A functionally diverse enzyme superfamily that abstracts the alpha protons of carboxylic acids.

Mandelate racemase and muconate lactonizing enzyme are structurally homologous but catalyze different reactions, each initiated by proton abstraction from carbon. The structural similarity to mandelate racemase of a previously unidentified gene product was used to deduce its function as a galactonate dehydratase. In this enzyme superfamily that has evolved to catalyze proton abstraction from carbon, three variations of homologous active site architectures are now represented: lysine and histidine bases in the active site of mandelate racemase, only a lysine base in the active site of muconate lactonizing enzyme, and only a histidine base in the active site of galactonate dehydratase. This discovery supports the hypothesis that new enzymatic activities evolve by recruitment of a protein catalyzing the same type of chemical reaction.

Amino Acid Sequence↗
PubMed1995

Microcins.

Explore the source record for details and available documents.

Amino Acid Sequence↗
PubMed1995

Genetic analysis of the colicin V secretion pathway.

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.

Bacterial Outer Membrane Proteins↗
PubMed1995

MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli.

The Escherichia coli gene murZ was recently shown to encode UDP-N-acetylglucosamine enolpyruvyl transferase, which catalyzes the first committed step of peptidoglycan biosynthesis (J. L. Marquardt, D. A. Siegele, R. Kolter, and C. T. Walsh, J. Bacteriol. 174:5748-5752, 1992). The map position of murZ (69.3 min) differed from that determined for murA (90 min), a gene which had been previously proposed to encode the same activity (P.S. Venkateswaran and H. C. Wu, J. Bacteriol. 110:935-944, 1972). Here we describe the construction of a chromosomal deletion of murZ and a plasmid containing murZ under arabinose control. Growth of cells containing the murZ deletion was dependent on the expression of murZ from the plasmid. We conclude that murZ is an essential gene and encodes the sole UDP-N-acetylglucosamine enolpyruvyl transferase of E. coli. To simplify the nomenclature, we recommend that murA be used to designate the gene at 69.3 min that encodes this activity and that the designation murZ be abandoned.

Alkyl and Aryl Transferases↗
PubMed1994

Sensing starvation: a homoserine lactone--dependent signaling pathway in Escherichia coli.

When nutrients become limiting, many bacteria differentiate and become resistant to environmental stresses. For Escherichia coli, this process is mediated by the sigma s subunit of RNA polymerase. Expression of sigma s was induced by homoserine lactone, a metabolite synthesized from intermediates in threonine biosynthesis. Homoserine lactone-dependent synthesis of sigma s was prevented by overexpression of a newly identified protein, RspA. The function of homoserine lactone derivatives in many cell density-dependent phenomena and the similarity of RspA to a Streptomyces ambofaciens protein suggest that synthesis of homoserine lactone may be a general signal of starvation.

4-Butyrolactone↗
PubMed1994

Purification and characterization of colicin V from Escherichia coli culture supernatants.

The peptide antibiotic, colicin V (ColV), has been purified and characterized from Escherichia coli culture supernatants by precipitation with trichloroacetic acid (TCA) and high-performance liquid chromatography (HPLC). Polyacrylamide gel electrophoresis (PAGE) and Western analysis identifies ColV as a polypeptide with an apparent molecular mass of 5.8 kDa. The protein identified remains biologically active after purification and SDS-PAGE. A mutant form of ColV, ColV-1, removes the carboxy-terminal 21 amino acids and replaces them with eight heterologous residues. The ColV-1 mutant is also secreted into the extracellular medium, demonstrating that the carboxy-terminal 21 amino acids are not required for secretion by the dedicated ColV export system, CvaAB/TolC. N-Terminal amino acid sequencing shows that the primary translation product of cvaC, the ColV structural gene, is processed to remove the N-terminal 15 amino acids. The cleavage site is preceded by the sequence Ser-Gly-Gly, making it a potential substrate for leader peptidase. The ColV leader sequence has many characteristics in common with the amino-terminal leader sequences of the lactococcins, lactacins, and pediocins from Gram-positive bacteria. Mass spectroscopy of purified ColV shows that it has a mass of 8741.0 amu, consistent with the mass of the unmodified 88 amino acid polypeptide. The purification scheme provides a rapid and simple way to obtain ColV for further biochemical analysis.

Amino Acid Sequence↗
PubMed1994

Posttranslational modifications in microcin B17 define an additional class of DNA gyrase inhibitor.

Drugs that inhibit the activity of DNA gyrase fall almost exclusively into two structural classes, the quinolones and the coumarins. A third class of DNA gyrase inhibitor is defined by the ribosomally synthesized peptide antibiotic microcin B17 (MccB17). MccB17 contains 43 amino acid residues, but 14 of these are posttranslationally modified. Here we describe the characterization of the structure of these modifications. We propose that four cysteine and four serine side chains undergo condensation with the carbonyl group of the preceding residue, followed by alpha/beta dehydrogenation to yield four thiazole and four oxazole rings, respectively. The three proteins implicated in catalyzing these modifications (McbBCD) would constitute the only thiazole/oxazole biosynthetic enzymes identified. These results open up possibilities for the design of DNA gyrase inhibitors and add to the repertoire of posttranslational modifications with potential for protein engineering. Escherichia coli sbmA mutants, which lack the inner membrane protein (SbmA) involved in MccB17 uptake, were found to be resistant to bleomycin. Bleomycin is structurally unrelated to MccB17 except for the fact that it contains two thiazole rings. This suggests that thiazole rings are part of the MccB17 structure recognized by SbmA. This observation and the finding that SbmA homologs are widely conserved and can play developmental roles [Glazebrook, J., Ichige, A. & Walker, G. C. (1993) Genes Dev. 7, 1485-1497] suggest that thiazole- and oxazole-containing compounds may serve as signaling molecules for a wide variety of bacteria in diverse environments, including pathogen interactions with plant and animal hosts.

Amino Acid Sequence↗
PubMed1994

Role of leuX in Escherichia coli colonization of the streptomycin-treated mouse large intestine.

Escherichia coli F-18, a normal human fecal isolate, is an excellent colonizer of the streptomycin-treated mouse large intestine. E. coli F-18 Col-, a derivative of E. coli F-18 that no longer makes the E. coli F-18 colicin, colonizes the mouse large intestine as well as E. coli F-18 when fed alone, but is eliminated when fed together with E. coli F-18. Recently, a random bank of E. coli F-18 DNA was transformed into E. coli F-18 Col-, the resultant population was fed to streptomycin-treated mice, and the intestine was used to select the best colonizer. In this fashion, a 6.5 kb E. coli F-18 DNA fragment was isolated. This fragment was shown to enhance E. coli F-18 Col- mouse large intestinal colonizing ability and survival during stationary phase in intestinal mucus in vitro, as well as stimulate the synthesis of type-1 fimbriae. Here, we present evidence that the gene responsible for the enhanced E. coli F-18 Col- colonizing ability and survival during stationary phase in vitro is leuX. This gene encodes a rare leucine tRNA specific for the UUG codon. In addition, we show that the presence of a functional leuX gene is necessary for E. coli K-12 intestinal colonization and for survival in stationary phase.

Animals↗
PubMed1994

The dps promoter is activated by OxyR during growth and by IHF and sigma S in stationary phase.

Dps is a non-specific DNA-binding protein abundant in starved Escherichia coli cells and is important for the defence against hydrogen peroxide. We found that dps mRNA levels are controlled by rpoS-encoded sigma S, the transcriptional activator OxyR and the histone-like IHF protein. In exponentially growing cells, dps is induced by treatment with hydrogen peroxide in an OxyR-dependent manner. This OxyR-dependent induction occurs only during log phase, although the OxyR protein is present in stationary phase. In the stationary phase cells, dps is expressed in a sigma S- and IHF-dependent manner. The purified OxyR and IHF proteins are also shown to bind upstream of the dps promoter. Our results suggest that the dps promoter is recognized by both sigma 70-holoenzyme and sigma S-holoenzyme, since OxyR acts through sigma 70 and the starts of the OxyR- and sigma S-dependent transcripts are identical.

Amino Acid Sequence↗
PubMed1993

Microbial competition: Escherichia coli mutants that take over stationary phase cultures.

Many microorganisms, including Escherichia coli, can survive extended periods of starvation. The properties of cells that survived prolonged incubation in stationary phase were studied by mixture of 10-day-old (aged) cultures with 1-day-old (young) cultures of the same strain of Escherichia coli. Mutants from the aged cultures that could grow eventually took over the population, which resulted in the death of the cells from the young cultures. This phenotype was conferred by mutations in rpoS, which encodes a putative stationary phase-specific sigma factor. These rapid population shifts have implications for the studies of microbial evolution and ecology.

Acridine Orange↗