Immunochemical comparison of phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase among the Enterobacteriaceae.

The bifunctional enzyme of the tryptophan operon, phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthetase (PRAI-InGPS;EC 4.1.1.48), was characterized by an immunochemical study of six representative members of the Enterobacteriaceae: Escherichia coli, Salmonella typhimurium, Enterobacter aerogenes, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris. PRAI-InGPS was purified from E. coli, and antisera were prepared in rabbits. These antisera were utilized in quantitative microcomplement fixation allowing for a comparison of the overall antigenic surface structure of the various homologous enzymes. These data showed E. coli PRAI-InGPS and S. marcescens and E. carotovora PRAI-InGPS (taken as a group) to have an index of dissimilarity of approximately 10, whereas the other organisms had values intermediate. In addition, antiserum to E. coli tryptophan synthetase beta2 subunit was used in microcomplement fixation to extend the previous comparison of this subunit (Rocha, Crawford, and Mills, 1972) to E. carotovora and P. vulgaris. Indexes of dissimilarity for E. coli compared to P. vulgaris of E. carotovora were 1.0 and 1.7, respectively. Agar immunodiffusion using PRAI-Ingps antisera showed significant cross-reaction among E. coli, E. aerogenes, S. typhimurium, and P. vulgaris whereas the enzymes from S. marcescens and E. carotovora cross-reacted to a lesser extent, with the latter reaction being quite weak. Comparative enzyme neutralization using E. coli PRAI-InGPS antisera showed significant cross-reactions among the enzymes in that all were neutralized at least 25%. The data taken together indicate that the trpC gene products in the Enterobacteriaceae are a homologous group of proteins, that the genetic divergene of the trpC gene is basically the same as the trpA gene, and that both are less conserved than the trpB gene. Furthermore, the PRAI-InGPS, enzyme active site appears to represent a more evolutionarily conserved region of the protein. These findings indicate that, with respect to PRAI-InGPS, similarity to E. coli among the organisms examined is in the following order: (E. aerogenes, S. typhimurium, P. vulgaris) greater than (S. marcescens, E. carotovora).     

Aminopeptidase I activities in several microorganisms.

Aminopeptidase I activity which was found to be localized in the same subcellular fraction and to be similarly heat stable was partially purified by a common procedure from Escherichia coli B, Escherichia coli K12, Enterobacter aerogenes, Salmonella typhimurium, Serratia marcescens Pseudomonas aeruginosa, and Proteus vulgaris. The enzyme preparations were shown to contain a single animopeptidase active toward both leucylleucine and methionylalanylserine by mixed-substrate initial-velocity kinetic analysis. The Km value for leucylleucine was virtually identical for the aminopeptidases of all of the organisms, as was the Km value for methionylalanylserine.     

Taurolin, a New Chemotherapeutic Agent

Taurolin, bis-(1,1-dioxo-perhydro-1,2,4-thiadiazinyl-4)-methan, is a novel, broad spectrum, non-systemic chemotherapeutic agent. It is effective in vivo against a wide range of pathogenic organisms including Pseudomonas aeruginosa, Escherichia coli, Proteus vulgaris , and Salmonella typhimurium , and would be of particular use against antibiotic-resistant organisms. It is based on an endogenous substance, taurine, which acts as a non-toxic formaldehyde carrier donating methylol groups to bacterial protein and endotoxin thus causing denaturation and polycondensation of the pathogens and their pyrogens. It is anticipated that Taurolin will be of value in the treatment of faecal peritonitis.     

Escherichia coli molybdoenzymes can be activated by protein FA from several gram-negative bacteria

Six Gram-negative bacteria (Klebsiella pneumoniae, Erwinia chrysanthemi, Proteus vulgaris, Serratia marescens, Salmonella typhimurium, and Pseudomonas aeruginosa) were shown to contain an FA-type protein capable of activating aponitrate reductase, apotrimethylamine N-oxide reductase and apoformate dehydrogenase of Escherichia coli. Protein FA activity was highest in Erwinia chrysanthemi and lowest in Pseudomonas aeruginosa. All the species also contained the low-Mr (less than or equal to 1500) heat-resistant material previously reported to be necessary for the protein-FA-dependent activation of E. coli chlB nitrate reductase.     

PRODUCTION AND WESTERN BLOT CHARACTERIZATION OF MONOCLONAL ANTIBODIES SPECIFIC FOR CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI

Reference strains of the Lior serogroups of Campylobacter jejuni and C. coli most frequently encountered in human infections were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Seven components appeared to be common to all strains. Purified components were obtained from one strain by electrophoretic separation and elution from preparative SDS-PAGE gels. The purified components were used to immunize BALB/c mice for monoclonal antibody production and to screen hybridoma tissue culture supernatants for specific antibody by enzyme immunoassay. Antibodies from selected hybridomas were characterized on western blots of C. jejuni, C. coli, C. fetus, Salmonella typhimurium, Proteus vulgaris, Citrobacter diversus and Escherichia coli whole cell lysates. At least nine of the monoclonal antibodies appeared to be specific for C. jejuni and C. coli and thus may be useful in tests for the rapid detection of these organisms in foods and other specimens.     

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods.

Lectins from Helix pomatia, Canavalia ensiformis, Agaricus bisporus and Triticum vulgaris agglutinated cultures of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. This agglutination was specific as it was inhibited (except with A. bisporus lectin) by the competing sugar substrates. The ability of three of these lectins, immobilized on a variety of supports, to separate these micro-organisms from pure cultures was investigated. Immobilization of the lectins on magnetic microspheres was the most effective method. Immobilized T. vulgaris lectin bound 87-100% of cells from cultures of L. monocytogenes, 80-100% of Staph. aureus, 33-45% of Salmonella spp. and 42-77% of E. coli. The A. bisporus lectin bound 31-63% of cells in cultures of L. monocytogenes, 83% of Staph. aureus but only 3-5% of the salmonella cells. Similarly H. pomatia lectin bound greater than 92% of Staph. aureus and 64% of L. monocytogenes cells but was poor at binding the Gram-negative organisms. This preference for binding Gram-positive organisms was confirmed when mixed cultures were studied. The T. vulgaris lectin was effective in removing L. monocytogenes (43%) and Staph. aureus (26%) from diluted milk and Salmonella (31-54%) from raw egg. Agaricus bisporus lectin removed L. monocytogenes from undiluted milk (10-47%) or ground beef (32-50%).     

Evolution of the ferric enterobactin receptor in gram-negative bacteria.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis of iron-deficient and replete cell envelopes, 59Fe-siderophore uptake studies, and Western immunoblots and cytofluorimetric analyses with monoclonal antibodies (MAbs), we surveyed a panel of gram-negative bacteria to identify outer membrane proteins that are structurally related to the Escherichia coli K-12 ferric enterobactin receptor, FepA. Antibodies within the panel identified FepA epitopes that are conserved among the majority of the bacteria tested, as well as epitopes present in only a few of the strains. In general, epitopes of FepA that are buried in the outer membrane bilayer were more conserved among gram-negative bacteria than epitopes that are exposed on the bacterial cell surface. The surface topology and tertiary structure of FepA are quite similar in E. coli and Shigella flexneri but differ in Salmonella typhimurium. Of the 18 different genera tested, 94% of the bacteria transported ferric enterobactin, including members of the previously unrecognized genera Citrobacter, Edwardsiella, Enterobacter, Haemophilus, Hafnia, Morganella, Neisseria, Proteus, Providencia, Serratia, and Yersinia. The ferric enterobactin receptor contains at least one buried epitope, recognized by MAb 2 (C. K. Murphy, V. I. Kalve, and P. E. Klebba, J. Bacteriol. 172:2736-2746, 1990), that is conserved within the structure of an iron-regulated cell envelope protein in all the bacteria that we have surveyed. With MAb 2, we identified and determined the Mr of cell envelope antigens that are immunologically related to E. coli FepA in all the gram-negative bacteria tested. Collectively, the library of anti-FepA MAbs showed unique patterns of reactivity with the different bacteria, allowing identification and discrimination of species within the following gram-negative genera: Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Escherichia, Haemophilus, Hafnia, Klebsiella, Morganella, Neisseria, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, and Yersinia.     

The use of immobilized lectins in the separation of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. from pure cultures and foods

Lectins from Helix pomatia, Canavalia ensiformis, Agaricus bisporus and Triticum vulgaris agglutinated cultures of Staphylococcus aureus, Escherichia coli, Listeria and Salmonella spp. This agglutination was specific as it was inhibited (except with A. bisporus lectin) by the competing sugar substrates. The ability of three of these lectins, immobilized on a variety of supports, to separate these micro-organisms from pure cultures was investigated. Immobilization of the lectins on magnetic microspheres was the most effective method. Immobilized T. vulgaris lectin bound 87–100% of cells from cultures of L. monocytogenes , 80–100% of Staph. aureus , 33–45% of Salmonella spp. and 42–77% of E. coli. The A. bisporus lectin bound 31–63% of cells in cultures of L. monocytogenes , 83% of Staph. aureus but only 3–5% of the salmonella cells. Similarly H. pomatia lectin bound >92% of Staph. aureus and 64% of L. monocytogenes cells but was poor at binding the Gram-negative organisms. This preference for binding Gram-positive organisms was confirmed when mixed cultures were studied. The T. vulgaris lectin was effective in removing L. monocytogenes (43%) and Staph. aureus (26%) from diluted milk and Salmonella (31–54%) from raw egg. Agaricus bisporus lectin removed L. monocytogenes from undiluted milk (10–47%) or ground beef (32–50%).     

Distribution of the isopropylmalate pathway to leucine among diverse bacteria

alpha-Isopropylmalate synthase and beta-isopropylmalate dehydrogenase activities were detected in extracts of the following organisms: Chromatium D, Rhodopseudomonas spheroides, Hydrogenomonas H16, Pseudomonas aeruginosa, Pseudomonas fluorescens, Vibrio extorquens, Rhizobium japonicum, Alcaligenes viscolactis, Escherichia coli B, Proteus vulgaris, Aerobacter aerogenes, Salmonella typhimurium, Micrococcus sp., Micrococcus lysodeikticus, Bacillus polymyxa, Bacillus subtilis, and Nocardia opaca. The alpha-isopropylmalate synthase activity in these extracts was inhibited by low concentrations of l-leucine. Taken together with other data, these results suggest that the isopropylmalate pathway is widespread among organisms that can synthesize leucine.     

Cross-reactivity of major outer membrane proteins of Enterobacteriaceae, studied by crossed immunoelectrophoresis.

Outer membrane fractions were prepared from 11 bacteria in the family Enterobacteriaceae: Escherichia coli serotypes O1K-, O4K2, O26K60, O75K-, and O111K58, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumonia, Serratia marcescens, Proteus vulgaris, Proteus mirabilis, and Providencia stuartii. All strains studied were found to contain one non-peptidoglycan-bound, heat-modifiable outer membrane protein, and one or two peptidoglycan-associated major outer membrane proteins in the 27,000- to 40,000-dalton range. Crossed immunoelectrophoresis using sodium dodecyl sulfate-polyacarylamide gel electrophoresis for separation of the antigens in the first dimension of the procedure was shown to provide a useful model system for studying the antigenic relationships of the major outer membrane proteins in Enterobacteriaceae species. Peptidoglycan-bound major outer membrane proteins of all bacteria studied reacted with antiserum against the purified peptidogylcan-bound matrix protein I of E. coli O26K60 in this system. Non-peptidoglycan-associated proteins of all strains cross-reacted with protein II of E. coli O26K60 in both their unmodified and their heat-modified forms. These results indicate that the genes coding for the major outer membrane proteins in the family Enterobacteriaceae have been well enough conserved during the course of evolution to allow significant antigenic cross-reactivity between the corresponding proteins in different enterobacterial species.     

Biochemical and genetic analysis of Salmonella typhimurium and Escherichia coli mutants defective in specific incorporation of selenium into formate dehydrogenase and tRNAs

Mutation of a single gene, referred to as selA1 in Salmonella typhimurium and as selD in Escherichia coli, results in the inability of these organisms to insert selenium specifically into the selenopolypeptides of formate dehydrogenase and into the 2-selenouridine residues of tRNAs. The mutation does not involve transport of selenite into the cell or reduction of selenite to selenide since both mutant strains synthesize selenocysteine and selenomethionine from added selenite and incorporate these selenoamino acids non-specifically into numerous proteins of the bacterial cells. Complementation of the mutation in S. typhimurium with the selD gene from E. coli indicates functional identity of the selA1 and selD genes. Although the selA1 gene maps at approximately 21 min on the S. typhimurium chromosome and the selD gene at approximately 38 min on the E. coli chromosome, only a single gene in wild-type S. typhimurium hybridized to the E. coli selD gene probe. Transformation of the mutant Salmonella strain with a plasmid bearing the E. coli selD gene restored formate dehydrogenase activity, 75Se incorporation into formate dehydrogenase seleno-polypeptides and [75Se]seleno-tRNA synthesis. Transformation with an additional plasmid carrying an E. coli formate dehydrogenase selenopolypeptide-lacZ gene fusion showed that the selD gene allowed readthrough of the UGA codon and synthesis of beta-galactosidase in the Salmonella mutant.     

Cloning, heterologous expression, and sequencing of the Proteus vulgaris glnAntrBC operon and implications of nitrogen control on heterologous urease expression

Abstract The glnAntrBC operon of Proteus vulgaris was cloned and heterologously expressed in Escherichia coli . The nucleotide sequence was determined. An open reading frame of 1407 bp was identified as the glnA gene and the deduced amino acid sequence showed 82% identity with the E. coli glutamine synthetase protein. Heterologous expression of the glnA gene in E. coli restored glutamine synthetase (GS) activity in a GS-negative mutant and a 52 kDa protein was detected and addressed as the GS subunit of P. vulgaris . Adjacent to the glnA gene the regulatory genes ntrB and ntrC were identified. Their coding regions comprised 1053 and 1452 bp, respectively, and the deduced gene products NR_II (NtrB) and NR_I (NtrC) shared 72% identity with the corresponding E. coli proteins. Heterologous expression in E. coli revealed only a 54 kDa protein which was shown to be NR_I. NR_II was not detectable using the methods employed.     

Rapid and Sensitive Detection of Bacteria by Gas Chromatography

A gas chromatograph fitted with electron capture and flame ionization detectors was employed for the rapid detection of bacteria by analysis for their metabolic products. The presence of Proteus vulgaris, Streptococcus faecalis, S. liquefaciens, Escherichia coli B, Bacillus cereus, and B. popilliae was detected in 2 to 4 hr in media inoculated with less than 10(4) cells per ml, whereas a 7- to 12-hr growth period was required for the detection of products formed in cultures of Serratia marcescens, Aerobacter aerogenes, E. coli K-12, Staphylococcus aureus, and Salmonella typhimurium. Metabolites elaborated by the equivalent of less than a single cell of B. cereus, S. faecalis, P. vulgaris, or E. coli B were sensed by the electron capture detector. The flame ionization detector was generally not as sensitive. Volatile metabolites were identified, and their concentrations were determined.     

The secreted hemolysins of Proteus mirabilis, Proteus vulgaris, and Morganella morganii are genetically related to each other and to the alpha-hemolysin of Escherichia coli.

Secreted hemolysins were extremely common among clinical isolates of Proteus mirabilis, Proteus vulgaris, and Morganella morganii, and hemolytic activity was either cell associated or cell free. Southern hybridization of total DNA from hemolytic isolates to cloned regions of the Escherichia coli alpha-hemolysin (hly) determinant showed clear but incomplete homology between genes encoding production of hemolysins in the four species. One of the two E. coli secretion genes, hlyD, hybridized only with DNA from P. vulgaris and M. morganii, which produced cell-free hemolysis, but not with that from P. mirabilis, which showed only cell-associated activity. Molecular cloning of the genetic determinants of cell-free hemolytic activity from P. vulgaris and M. morganii chromosomal DNA allowed their functional analysis via inactivation with the transposons Tn1000 and Tn5. Both hemolysin determinants were about 7.5 kilobase pairs and comprised contiguous regions directing regulation, synthesis, and specific secretion out of the cell. Transposon mutations which eliminated secretion of the Proteus and Morganella hemolysins could be complemented specifically by the E. coli hemolysin secretion genes hlyB or hlyD. Alignment of the physically and functionally defined hly determinants from P. vulgaris and M. morganii with that of the E. coli alpha-hemolysin confirmed a close genetic relationship but also indicated extensive evolutionary divergence.     

Glutathione transferase in bacteria: subunit composition and antigenic characterization

The presence of glutathione transferase (GST; EC 2.5.1.18) in Escherichia coli ATCC 25922, E. coli ATCC 25422, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Klebsiella oxytoca CIP 666, K. oxytoca AF 101, Enterobacter cloacae CIP 6085, Serratia marcescens CIP 6755, and Proteus mirabilis AF 2924 was investigated. Using 1-chloro-2,4-dinitrobenzene as substrate, GST activity was found in the glutathione-(GSH-)affinity-purified fraction of all strains tested. SDS-PAGE analysis of GSH-affinity-purified enzyme indicated that the GSTs of all these bacteria are dimers of two identical subunits of Mr about 22,500. Rabbit antiserum directed against the major isoenzyme present in Proteus mirabilis AF 2924, Pm-GST-6.0, was used to investigate the antigenic properties of bacterial GSTs. Western blot analysis indicated that a GST antigenically identical to Pm-GST-6.0 is present in Enterobacter cloacae CIP 6085, Escherichia coli ATCC 25422 and Proteus vulgaris ATCC 8427, but absent in Escherichia coli ATCC 25922, Klebsiella oxytoca CIP 666, K. oxytoca AF 101 and Serratia marcescens CIP 6755. The presence of Pm-GST-6.0, but not mammalian GST, increased the MIC values of amikacin, ampicillin, cefotaxime, cephalothin and nalidixic acid for E. coli ATCC 25922. It is suggested that bacterial GST may represent a defense against the effects of antibiotics.     

Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin.

A physiologically diverse range of Gram-positive and Gram-negative bacteria was found to be susceptible to inhibition and inactivation by lactoferricin B, a peptide produced by gastric pepsin digestion of bovine lactoferrin. The list of susceptible organisms includes Escherichia coli, Salmonella enteritidis, Klebsiella pneumoniae, Proteus vulgaris, Yersinia enterocolitica, Pseudomonas aeruginosa, Campylobacter jejuni, Staphylococcus aureus, Streptococcus mutans, Corynebacterium diphtheriae, Listeria monocytogenes and Clostridium perfringens. Concentrations of lactoferricin B required to cause complete inhibition of growth varied within the range of 0.3 to 150 micrograms/ml, depending on the strain and the culture medium used. The peptide showed activity against E. coli O111 over the range of pH 5.5 to 7.5 and was most effective under slightly alkaline conditions. Its antibacterial effectiveness was reduced in the presence of Na+, K+, Mg2+ or Ca2+ ions, or in the presence of various buffer salts. Lactoferricin B was lethal, causing a rapid loss of colony-forming capability in most of the species tested. Pseudomonas fluorescens, Enterococcus faecalis and Bifidobacterium bifidum strains were highly resistant to this peptide.     

Demonstration of cell division by septation in a variety of gram-negative rods.

Through use of an initial fixative employing a combination of crotonaldehyde and glutaraldehyde, septa were preserved in thin sections of dividing cells of strains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Escherichia coli when grown at 30 C in a dilute basal medium. The same procedures, however, revealed only a constrictive division process in Proteus vulgaris and Erwinia sp. This adds to the evidence that septation, although difficult to demonstrate, is the process of cell division in the enteric gram-negative rods and the pseudomonads and that constriction is a fixation artifact in these organisms.     

Tobramycin,amikacin, sissomicin,and gentamicin resistant Gram-negative rods.

Sensitivities to gentamicin, sissomicin, tobramycin, and amikacin were compared in 196 gentamicin-resistant Gram-negative rods and in 212 similar organisms sensitive to gentamicin, mainly isolated from clinical specimens. Amikacin was the aminoglycoside most active against gentamicin-resistant organisms, Pseudomonas aeruginosa, klebsiella spp, Escherichia coli, Proteus spp, Providencia spp, and Citrobacter spp being particularly susceptible. Most of the gentamicin-resistant organisms were isolated from the urine of patients undergoing surgery. Gentamicin was the most active antibiotic against gentamicin-sensitive E coli, Proteus mirabilis, and Serratia spp. Pseudomonas aeruginosa and other Pseudomonas spp were most susceptible to tobramycin.     

Fishmeal extract bile salt lactose agar--a differential medium for enteric bacteria

Fishmeal extract bile salt lactose agar (FEBLA), a new differential medium for enteric bacteria was developed and evaluated for its ability to grow and differentiate lactose fermenters (LF) from non-lactose fermenters (NLF) in comparison with MacConkeys agar. Performance of FEBLA was at par with the latter. On FEBLA medium, the contrast between LF and NLF colonies was pronounced and Klebsiella pneumoniae produced more mucoid colonies than on MacConkeys agar (Hi Media). Unlike MacConkeys agar, a 24 h culture of K. pneumoniae cells on FEBLA were longer and thicker with abundant capsular material around the bacilli. Escherichia coli produced long and thick cells but only after 48h. No change in cell morphology was evident with regard to Salmonella typhi, S. paratyphi A, Shigella flexneri, Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Citrobacter koseri and Acinetobacter baumannii. Performance of the medium was controlled using E. coli and S. flexneri. FEBLA is simple, cost effective and may be a suitable alternative in the preliminary identification of enteric bacteria.