The Physiological and Molecular Characterization of a Small Colony Variant of Escherichia coli and Its Phenotypic Rescue.

Small colony variants (SCVs) can be defined as a naturally occurring sub-population of bacteria characterized by their reduced colony size and distinct biochemical properties. SCVs of Staphylococcus aureus have been studied extensively over the past two decades due to their role in recurrent human infections. However, little work has been done on SCVs of Escherichia coli, and this work has focused on the physiology and morphology that define these colonies of E.

Rapid approach to identify an unrecognized viral agent.

For epidemic control, rapid identification and characterization of the responsible unknown agent are crucial. To address this critical question, a method was developed for virus discovery based on a flexible nested-PCR subtraction hybridization. As a positive control, we used hepatitis C virus as a hypothetical unrecognized virus and "discover" it in the sample.

Weak organic acids: a panoply of effects on bacteria.

Weak organic acids have been used for centuries to preserve foods, but only recently has the possible mechanism for bacterial growth inhibition been investigated. Although the lowering of internal pH was favored as the cause of growth inhibition, the emphasis has shifted to the anion and its specificity. There are a number of applications of weak organic acids to foods and in the food industry be they pre- or postharvest, However, there is concern that the ability of foodborne pathogens to adapt to these acids may allow longer survival in these commodities and also to better survive transit through the gastric acid barrier of the stomach.

The effect of acid treatment on survival and protein expression of a laboratory K-12 strain Escherichia coli.

Pre-exposure of log phase enteric bacteria to nonlethal acidic pH induces phenotypic changes that protect the organisms against subsequent lethal acidity. Studies have revealed that when Salmonella typhimurium is grown in minimal medium at pH 5.5 and 4.

Detection of extrahepatic hepatitis C virus replication by a novel, highly sensitive, single-tube nested polymerase chain reaction.

We established a cell culture system for the replication of hepatitis C virus (HCV) by using human T and B leukemia cell lines. These 2 cell lines were infected in vitro by using HCV-positive pooled patient serum samples. HCV RNA was extracted from infected cell lines at different times after infection, and a sequence of the virus 5' untranslated region was analyzed.

A superantigen bioassay to detect staphylococcal enterotoxin A.

Current detection methods for enterotoxins of Staphylococcus aureus are labor intensive and limited in sensitivity. Furthermore, these immunochemical protocols fail to adequately detect heat-treated enterotoxins. Staphylococcal enterotoxins cause severe gastrointestinal illness at relatively low concentrations and retain toxigenicity even after heat treatment.

Rapid methods to enumerate Escherichia coli in foods using 4-methylumbelliferyl-beta-D-glucuronide.

Three methods to enumerate Escherichia coli in food were compared. They were based on AOAC methods using lauryl tryptose broth (LST) medium, LST-4-methylumbelliferyl-beta-D-glucuronide (MUG) medium, and a proposed method using regular LST in combination with E. coli (EC)-MUG medium.

Molecular detection of Clostridium botulinum type E neurotoxin gene in smoked fish by polymerase chain reaction and capillary electrophoresis.

The polymerase chain reaction (PCR), a rapid, sensitive technique for amplifying target DNA sequences of pathogenic microorganisms, was used to amplify Clostridium botulinum type E neurotoxin gene fragments in smoked fish. Other botulinal neurotoxin-producing strains, nontoxigenic strains, and food-related microorganisms did not yield nonspecific amplification products with this PCR assay. PCR products were analyzed by capillary electrophoresis (CE) using a low-viscosity entangled polymer system.

The survival benefit of short-chain organic acids and the inducible arginine and lysine decarboxylase genes for Escherichia coli.

The short-chain organic acids (SCOAs), acetic and propionic acids, are used widely as food preservatives. The production of these two acids plus butyric acid in the colon by anaerobes serves as a mechanism for controlling the numbers of enterobacteria (which can be pathogens) in this organ. It has been found in this study that the acid tolerance of cells initially grown at near neutral pH (6.

The occurrence of duplicate lysyl-tRNA synthetase gene homologs in Escherichia coli and other procaryotes.

The lysyl-tRNA synthetase (LysRS) system of Escherichia coli K-12 consists of two genes, lysS, which is constitutive, and lysU, which is inducible. It is of importance to know how extensively the two-gene LysRS system is distributed in procaryotes, in particular, among members of the family Enterobacteriaceae. To this end, the enterics E.

Folding in vivo of bacterial cytoplasmic proteins: role of GroEL.

A general role for chaperonin ring structures in mediating folding of newly translated proteins has been suggested. Here we have directly examined the role of the E. coli chaperonin GroEL in the bacterial cytoplasm by production of temperature-sensitive lethal mutations in this essential gene.

Temperature-dependent induction of an acid-inducible stimulon of Escherichia coli in broth.

The induction of the inducible lysyl-tRNA synthetase, LysU, and the inducible lysine and arginine decarboxylases of Escherichia coli K-12 grown in AC broth to a pH of 5.5 or less is temperature dependent, being distinctly lower at 24 than at 37 degrees C. This induction does not appear to be under HtpR control.

The lrp gene product regulates expression of lysU in Escherichia coli K-12.

In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion.

Partial characterization of a lysU mutant of Escherichia coli K-12.

The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed.

Low-pH-induced effects on patterns of protein synthesis and on internal pH in Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium were grown in a supplemented minimal medium (SMM) at a pH of 7.0 or 5.0 or were shifted from pH 7.

Isolation of Plasmid-Harboring Serratia plymuthica from Facultative Gut Microflora of the Tobacco Hornworm, Manduca sexta.

Aseptic isolation of the facultative gut microflora of the tobacco hornworm, Manduca sexta, yielded four microorganisms. Two were gram-positive Bacillus spp., one was Serratia plymuthica, and another was the yeast Candida guilliermondii.

Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12.

The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis. Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.

Escherichia coli K-12 lysyl-tRNA synthetase mutant with a novel reversion pattern.

Fast-growing revertants have been selected from a slow-growing lysyl-tRNA synthetase mutant. All of the revertants had increased lysyl-tRNA synthetase activity compared with the mutant (5- to 85-fold), and in some revertants this amounted to two to three times the wild-type synthetase activity. Two-dimensional gel electrophoresis of a whole-cell extract of revertant IH2018 (1.

Multiple forms of lysyl-transfer ribonucleic acid synthetase in Escherichia coli.

Lysyl-transfer ribonucleic acid synthetase (EC 6.1.1.

Two modes of metabolic regulation of lysyl-transfer ribonucleic acid synthetase in Escherichia coli K-12.

Lysyl-transfer ribonucleic acid (tRNA) synthetase activity was compared in three independently isolated Escherichia coli K-12 mutants of the enzyme S-adenosyl-L-methionine synthetase (metK mutants) and their isogenic parents. In all three cases the activity of the lysyl-tRNA synthetase was elevated two- to fourfold in the mutant strains. Glycyl-L-leucine (3 mM) usually enhanced lysyl-tRNA synthetase activity two- to threefold in wild-type cells but did not further stimulate the synthetase activity in metK mutants.

An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of the lysyl-tRNA synthetase from Escherichia coli K-12. II. Kinetic evidence.

Wild-type Escherichia coli K-12 was grown in minimal medium alone or with the addition of 20 mM L-alanine or 3 mM glycyl-L-leucine. A lysyl-tRNA synthetase mutant strain was grown in minimal medium containing 20mM L-alanine. The lysyl-tRNA synthetase from these strains was purified to 70-90% of homogeneity.

An in vivo effect of the metabolites L-alanine and glycyl-L-leucine on the properties of lysyl-tRNA synthetase from Escherichia coli K-12. I. Influence on subunit composition and molecular weight distribution.

Lysyl-tRNA synthetase was purified to 70-90% of homogeneity from Escherichia coli K-12. The enzyme was purified from wild-type cells grown in minimal medium, or minimal medium containing either 20 mM L-alanine or 3 mM glycly-L-leucine. The synthetase was similarly purified from a mutant strain grown in minimal medium plus 20 mM L-alanine.

Utilization of selected leucine peptide amides by Escherichia coli.

Studies on the utilization of leucine peptide amides as a source of leucine for a leucine auxotroph showed that in general compounds with the structure leu-chi amide (where chi is any amide) are utilized as well as the free peptide, but that compounds with the structure chi-leu amide (where chi is not leucine) are used less effectively than the free peptide. Growth and enzymological experiments indicated that the lower capacity of Escherichia coli to utilize amides of the structure chi-leu amide is not a result of poor transport of these compounds, but rather the inability to rapidly liberate leucine from the amide when it is supplied to the cell in the form of a peptide. Competition studies indicated that the peptide amides enter the cell via the oligopeptide permease system.