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G R Drapeau

Publications and source records attributed to G R Drapeau.

10 recordsLinked to original sources
PubMed1992

The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression.

Wild-type genes which, when overexpressed, are capable of restoring the growth deficiency of the division mutant ftsZ84 of Escherichia coli on L medium containing no added NaCl have been isolated. One of these genes is rcsB, a positive regulator of colanic acid biosynthesis. A direct relationship between rcsB expression and FtsZ activity was observed, suggesting that RcsB specifically increases transcription of ftsZ, thus accounting for the restoration of colony formation by ftsZ84 mutant cells. Analysis of the 5' upstream sequence of rcsB revealed, in addition to the sigma 54 promoter sequence previously reported, a presumptive sigma 70 promoter and LexA-binding site plus an upstream sequence that is found to be essential for the expression of rcsB on a plasmid. The absence of the sigma 54 factor does not have a negative effect on the transcription of rcsB. The RcsB protein is an activator of its own synthesis, particularly in the presence of NaCl. Evidence which suggests that RcsB can be phosphorylated by a presumably modified EnvZ or PhoM sensor protein leading to a suppression of the growth deficiency of ftsZ84 mutant cells and to an increase in colanic acid production was obtained. We also demonstrated that the level of colanic acid is reduced when the cells carry a multicopy rcsC plasmid, suggesting that the RcsC sensor has phosphatase activity.

Amino Acid Sequence↗
PubMed1992

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.

Amino Acid Sequence↗
PubMed1979

Isolation and properties of the protease from the wild-type and mutant strains of Pseudomonas fragi.

A simplified procedure for the purification of the extracellular protease of Pseudomonas fragi was developed. The enzyme was isolated from a derepressed mutant producing 40 times the enzyme level of the parental organism. It was collected from culture filtrates by ammonium sulfate precipitation, and it was obtained in pure form by single chromatography on a column of diethylaminoethyl cellulose. The protease had a molecular weight of 52,000 as estimated by sodium dodecyl sulfate-gel electrophoresis and had properties of a classical neutral endopeptidase with the exception of its substrate specificity. Mutants of P. fragi producing proteases of altered substrate specificities were isolated from plates containing elastin as the sole carbon source. The SP-Sephadex elution patterns of enzymes extracted from each mutant examined were complex, suggesting that either the enzyme was autodigested or several active forms could be generated from a common precursor. The substrate specificities of the mutant enzymes were different from that produced by the parental strain.

Amino Acids↗
PubMed1978

Unusual COOH-terminal structure of staphylococcal protease.

The extracellular enzyme, staphylococcal protease, carries a COOH-terminal tryptic peptide of 43 amino acid residues most of which are aspartic acid, asparagine, and proline. This peptide might have a function equivalent to that of a similar segment previously observed at the NH2-terminal end of the membrane-bound penicillinase precursor of Bacillus licheniformis (Yamamoto, S., and Lampen, J. O. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1457-1461). These observations would suggest that bacterial exoproteins which are secreted in the form of precursors differ from extracellular proteins by the presence of an extra segment at their NH2- and/or COOH-terminal ends.

Amino Acid Sequence↗
PubMed1978

Role of metalloprotease in activation of the precursor of staphylococcal protease.

A metalloprotease was isolated from the culture medium of a mutant of Staphylococcus aureus strain V8. The enzyme had a molecular weight of 38,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an optimum pH of 7.0 and exhibited a specificity for peptide bonds on the N-terminal side of large hydrophobic residues. The protease was fully inactivated by 0-phenanthroline but could be reactivated by zinc ions. Cobalt may be substituted for zinc, producing an activity which corresponds to 160% of that of the native enzyme. All these data indicate that this protease is a typical bacterial neutral metalloprotease. The role of this metalloprotease in the activation of the precursor of another protease secreted by the same organism, staphylococcal protease, has been identified. Mutants which lack the metalloprotease accumulated the precursor, which can be specifically activated by the addition of the purified metalloprotease or the related enzyme thermolysin. The purification of the precursor is also reported.

Amino Acids↗
PubMed1978

The primary structure of staphylococcal protease.

The amino acid sequence of staphylococcal protease has been determined by analysis of tryptic peptides obtained from cyanogen bromide fragments. Selected peptides obtained from digests with staphylococcal protease, thermolysin, and chymotrypsin provided the information necessary to align the tryptic peptides and the cyanogen bromide fragments. The protease is a single polypeptide chain of some 250 amino acids and is devoid of sulfhydryl groups. The COOH-terminal tryptic peptide of of the protease molecule contains some 43 residues, most of which are aspartic acids, asparagines, and prolines. The amino acid sequence of this peptide was not determined. The primary structure near the active serine residue indicates that staphylococcal protease is related to the pancreatic serine proteases. However, it has little or no additional sequence homologies with these enzymes except for the regions near histidine-50 and aspartic acid - 91. These regions have striking similarities with the corresponding regions of protease B and the trypsin-like enzyme of Streptomyces griseus.

Amino Acid Sequence↗
PubMed1976

Purification and properties of the alpha2beta2 complex of tryptophan synthetase of Proteus mirabilis.

A procedure is described for the purification of the tryptophan synthetase alpha2beta2 complex from cell extracts of Proteus mirabilis. A 30-fold purification was achieved with an overall yield of about 23% and a specific activity of 1,600. The complex can be dissociated and the subunits isolated in a pure form. The complex can be reconstituted from the isolated subunits to regain the initial activity. The alpha and beta2 subunits of the tryptophan synthetase complex of P. mirabilis are not significantly different from those of Escherichia coli and other enteric bacteria as to their physical properties, amino acid compositions, and enzymic properties. Complementation studies indicate that the alpha subunit of P. mirabilis hybridizes well with the beta2 subunit from E. coli. Similarly, the beta2 subunit of P. mirabilis readily complexes with the alpha subunits from E. coli, Salmonella typhimurium, and Serratia marcescens. The hybrids formed are all effective in catalyzing the conversion of indoleglycerol phosphate plus serine into tryptophan and glyceraldehyde 3-phosphate. However, these hybrids have reduced or no activity in the other reactions, namely, the condensation of indole and serine to form tryptophan or the aldolytic cleavage of indoleglycerol phosphate.

Escherichia coli↗
PubMed1975

Proton magnetic resonance titration curves of the three histidine residues of staphylococcal protease.

Proton magnetic resonance spectra of staphylococcal protease, a serine protease from Staphylococcus aureus, strain V8, are presented. Initial proton spectra were obtained at 220 MHz, and more detailed studies of the aromatic region were carried out by correlation spectroscopy at 250 MHz. The overall spectrum bears a close resemblance to one calculated from the sum of spectra of the component amino acids. Chemical shifts of the three tyrosine, four phenylalanine, and three histidine residues appear to be equivalent at pH 3.7 and 8.5 indicating that they are all in normal chemical environments in the enzyme. The staphylococcal protease contains a large number of slowly exchanging protons. In fact, interpretable spectra of the aromatic region were obtained only after extensive exchange of N-H groups with deuterium from the D2O solvent. Proton magnetic resonance titration studies of the three histidine residues indicate that these have normal chemical shifts and pK' values. When the data are fitted to single noninteracting titration curves, the histidine pK' values are 7.19 plus or minus 0.02, 6.85 plus or minus 0.03, and 6.69 plus or minus 0.02. The titration curves of two of the histidine residues indicate negative cooperativity. A possible explanation for this is a direct electrostatic interaction between the two histidines. The titration data for these histidines give a significantly better fit to such a mutual interaction model than to noninteracting titration curves. The component microscopic dissociation constants have been calculated. Mutual interaction leads to pK' displacements of 0.31 unit; which indicates a distance of approximately 7 angstrom between the two interacting histidine rings according to the model of Tanford and Roxby. The proton resonances of the two interacting histidines are doubled in the pH region 6.7-7.0 suggesting the presence of two forms of the enzyme having lifetimes in excess of 30 msec.

Amino Acids↗