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S E West

Publications and source records attributed to S E West.

At least 37 records · Page 2Linked to original sources
PubMed1994

Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa.

The nucleotide sequence of the 1.9-kb PstI fragment from pRO1614, that allows stable maintenance of pMB1 (ColE1)-based cloning vectors in Pseudomonas, was determined. This fragment encodes a putative origin of replication (ori), a replication-controlling protein, and the C terminus of the Tn3 beta-lactamase-encoding gene. Improved versions of the broad-host-range plasmid vectors, pUCP18 and pUCP19, were constructed by deletion of nonessential DNA or replacement of nonessential DNA with an antibiotic-resistance cassette.

Amino Acid Sequence↗
PubMed1994

Characterization of Pseudomonas aeruginosa mutants that are deficient in exotoxin A synthesis and are altered in expression of regA, a positive regulator of exotoxin A.

In Pseudomonas aeruginosa, production of exotoxin A, an ADP-ribosyltransferase, is a complex and highly regulated process. Two positively acting regulatory genes, regA and regB, have been cloned and characterized. To identify additional exotoxin A regulatory genes, we have characterized four N-methyl-N'-nitro-N-nitrosoguanidine-generated mutants of P. aeruginosa PA103 which are deficient in exotoxin A production. These mutants (PA103-8, PA103-15, PA103-16, and PA103-19) do not accumulate intracellular exotoxin A and are not complemented by the cloned toxA or regAB genes. This observation indicates that the lesion(s) in the mutants is probably in an exotoxin A regulatory gene(s) and is not in the genes for secretion of exotoxin A or in the toxA or regAB genes. To assess the effect of the putative regulatory mutations on the toxA and regAB genes, we compared the activity of the toxA and regAB promoters in the mutant and parental strains using plasmids containing the genes for beta-galactosidase or chloramphenicol acetyltransferase under the control of either the toxA or the regAB promoter. The toxA promoter-beta-galactosidase fusion plasmid could not be maintained in PA103-8. beta-Galactosidase expression driven by the toxA promoter was absent in the mutant PA103-19 and occurred at a low level, which was not repressed by iron in mutants PA103-15 and PA103-16. The regAB genes are temporally controlled by two promoters, P1 and P2. In all four mutants, regAB P1 promoter activity was reduced; however, expression under the control of the regAB P2 promoter was normal. These observations suggest the existence of one or more regulatory genes which directly affect expression of both the toxA and the regAB P1 promoters.

ADP Ribose Transferases↗
PubMed1994

The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family.

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.

ADP Ribose Transferases↗
PubMed1992

Isolation and characterization of toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1.

We have isolated and characterized four toxin A excretion-deficient mutants of Pseudomonas aeruginosa PAO1. Similar to previously described mutants (B. Wretlind and O. R. Pavlovskis, J. Bacteriol. 158:801-808, 1984), the mutants appear to have a pleiotropic defect in the excretion of several extracellular products, including toxin A, elastase, alkaline phosphatase, and phospholipase C. However, the mutants are not defective in the excretion of either alkaline protease or exoenzyme S. We also examined the localization and processing of toxin A in these mutants by using pulse-labeling experiments. Mature toxin A was found to be localized to the membranes only. Our results suggest that toxin A is localized to the outer membrane but is not exposed to the extracellular surfaces of the outer membranes. The results also suggest that toxin A obtained from the excretion-deficient mutants has intact disulfide bonds.

ADP Ribose Transferases↗
PubMed1988

Codon usage in Pseudomonas aeruginosa.

We have generated a codon usage table for Pseudomonas aeruginosa. Codon usage in P. aeruginosa is extremely biased. In contrast to E. coli and yeast, P. aeruginosa preferentially uses those codons within a synonymous codon group with the strongest predicted codon-anticodon interaction. We were unable to correlate a particular codon usage pattern with predicted levels of mRNA expressivity. The choice of a third base reflects the high guanine plus cytosine content of the P. aeruginosa genome (67.2%) and cytosine is the preferred nucleotide for the third codon position.

Bacteriophages↗
PubMed1987

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.

Bacterial Outer Membrane Proteins↗
PubMed1985

Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production.

For acquisition of iron, an essential nutrient, most microorganisms produce siderophores (low-molecular-weight iron-chelating compounds) and membrane proteins to serve as receptors for the iron-siderophore complexes. The gonococcus does not appear to produce a siderophore, since the quantity of siderophore detected by bioassays of culture supernatants from strains F62 and FA19 was never greater than the amount present in the uninoculated medium. Iron limitation of the laboratory strains F62 and FA19 and 12 recent clinical isolates resulted in the expression of several iron-repressible membrane proteins. The expression of proteins in the apparent molecular weight range of 70,000 to 100,000 was strain dependent. All strains expressed 36,000-dalton (36K) and 19.5K proteins. FA19 and F62 were also grown in medium containing iron sources commonly encountered in vivo (i.e., transferrin, lactoferrin, hemoglobin, or hemin). Comparison of growth rates indicates that transferrin and lactoferrin were more readily utilized as iron sources than hemin and hemoglobin were. Expression of the iron-repressible proteins varied depending upon the iron source. Fewer iron-repressible proteins were observed when cells were supplied with transferrin or lactoferrin than when the cultures were grown with either hemin or hemoglobin. The 36K protein was expressed with all four iron sources.

Bacterial Proteins↗
PubMed1983

Therapy of anaerobic infections.

Anaerobic bacteria commonly cause soft tissue infections in humans, usually as a result of trauma. Although the susceptibility of some species to antibiotics can be predicted reasonably well, many clinical laboratories can do susceptibility tests more easily than they can correctly identify anaerobes. The broth-disk test is the simplest test for clinical laboratories, and a new standardized MIC method is now available for research laboratories. Several types of experimental anaerobic infections can be initiated in animals, and these infections have been used to test the efficacy of therapeutic regimens. Metronidazole was the most effective, and clindamycin was also very active. Elimination of the anaerobic component of abscesses may be sufficient therapy; it may be possible to ignore the facultative organisms.

Abscess↗
PubMed1980

In vitro production of human fecal mutagen.

The feces of some normal humans were previously shown to be mutagenic by the Salmonella mutagenicity assay with strain TA100. In the present study, the mutagenicity of feces of certain donors can be increased by anaerobic incubation for 96 h. The increase in mutagenicity did not occur upon incubation in the cold or in air, in the presence of antimicrobial agents or if the feces were sterilized by heat. On thin-layer chromatographs, the relative mobility of fecal mutagen for all donors after incubation was the same in any one of 4 different solvent systems. The major mutagenicity appears to be due to a single type of compound which may be produced by anaerobic bacteria.

Adult↗
PubMed1977

Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon.

Ten Bacteroides species found in the human colon were surveyed for their ability to ferment mucins and plant polysaccharides ("dietary fiber"). A number of strains fermented mucopolysaccharides (heparin, hyaluronate, and chondroitin sulfate) and ovomucoid. Only 3 of the 188 strains tested fermented beef submaxillary mucin, and none fermented porcine gastric mucin. Many of the Bacteroides strains tested were also able to ferment a variety of plant polysaccharides, including amylose, dextran, pectin, gum tragacanth, gum guar, larch arabinogalactan, alginate, and laminarin. Some plant polysaccharides such as gum arabic, gum karaya, gum ghatti and fucoidan, were not utilized by any of the strains tested. The ability to utilize mucins and plant polysaccharides varied considerably among the Bacteroides species tested.

Bacteroides↗
PubMed1977

Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon.

A total of 154 strains from 22 species of Bifidobacterium, Peptostreptococcus, Lactobacillus, Ruminococcus, Coprococcus, Eubacterium, and Fusobacterium, which are present in high concentrations in the human colon, were surveyed for their ability to ferment 21 different complex carbohydrates. Plant polysaccharides, including amylose, amylopectin, pectin, polygalacturonate, xylan, laminarin, guar gum, locust bean gum, gum ghatti, gum arabic, and gum tragacanth, were fermented by some strains from Bifidobacterium, Peptostreptococcus, Ruminococcus, and Eubacterium species. Porcine gastric mucin, which was fermented by some strains of Ruminococcus torques and Bifidobacterium bifidum, was the only mucin utilized by any of the strains tested.

Anaerobiosis↗