envM genes of Salmonella typhimurium and Escherichia coli.

Conjugation and bacteriophage P1 transduction experiments in Escherichia coli showed that resistance to the antibacterial compound diazaborine is caused by an allelic form of the envM gene. The envM gene from Salmonella typhimurium was cloned and sequenced. It codes for a 27,765-dalton protein. The plasmids carrying this DNA complemented a conditionally lethal envM mutant of E. coli. Recombinant plasmids containing gene envM from a diazaborine-resistant S. typhimurium strain conferred the drug resistance phenotype to susceptible E. coli cells. A guanine-to-adenine exchange in the envM gene changing a Gly codon to a Ser codon was shown to be responsible for the resistance character. Upstream of envM a small gene coding for a 10,445-dalton protein was identified. Incubating a temperature-sensitive E. coli envM mutant at the nonpermissive temperature caused effects on the cells similar to those caused by treatment with diazaborine, i.e., inhibition of fatty acid, phospholipid, and lipopolysaccharide biosynthesis, induction of a 28,000-dalton inner membrane protein, and change in the ratio of the porins OmpC and OmpF.     

Periplasmic space in Salmonella typhimurium and Escherichia coli.

The volume of the periplasmic space in Escherichia coli and Salmonella typhimurium cells was measured. This space, in cells grown and collected under conditions routinely used in work with these bacteria, was shown to comprise from 20 to 40% of the total cell volume. Further studies were conducted to determine the osmotic relationships between the periplasm, the external milieu, and the cytoplasm. Results showed that there is a Donnan equilibrium between the periplasm and the extracellular fluid, and that the periplasm and cytoplasm are isoosmotic. In minimal salts medium, the osmotic strength of the cell interior was estimated to be approximately 300 mosM, with a net pressure of approximately 3.5 atm being applied to the cell wall. A corollary of these findings was that an electrical potential exists across the outer membrane. This potential was measured by determining the distributions of Na+ and Cl- between the periplasm and the cell exterior. The potential varied with the ionic strength of the medium; for cells in minimal salts medium it was approximately 30 mV, negative inside.     

Studies on the UDP-sugar hydrolases from Escherichia coli and Salmonella typhimurium

A simple procedure for purification of the UDP-sugar hydrolase from Escherichia coli is described. The enzyme has an apparent molecular weight of 61,000. The UDP-sugar hydrolase from Salmonella typhimurium has been solubilized and partially purified from total cell membranes. According to several criteria, antibodies raised against the purified E. coli enzyme do not seem to react with partially purified Salmonella enzyme.     

Polymorphic transition of the flagellar polyhook from Escherichia coli and Salmonella typhimurium

Bacterial flagellar polyhook fibers were reversibly transformed into a set of helical forms depending on pH, ionic strength and temperature. Electron microscopy with formalin fixation and freeze-drying was useful for observing three-dimensional shapes of various polyhook helices and determining their helical handedness. A Cartesian plot of curvature against twist for these polyhook helices gave a sinusoidal curve as in the case of the polymorphic forms of flagellar filament. In the study on the polymorphism of flagellar filaments. Calladine (1976, 1978) and Kamiya et al. (1979) pointed out that such a relation in the polymorphic forms could be derived from the assumption that the subunits on the near-longitudinal (11-start) helical lines should work as elastic fibers (protofilaments) having two distinct states of conformation. In contrast, the observed twist for the polyhook helices is too large to be explained by the same assumption. Instead, we must assume that subunits on the strongly twisted, 16-start helical line should work as the co-operative protofilament.     

Characterization of recombinant diaminopropionate ammonia-lyase from Escherichia coli and Salmonella typhimurium

Diaminopropionate ammonia-lyase gene from Escherichia coli and Salmonella typhimurium was cloned and the overexpressed enzymes were purified to homogeneity. The k(cat) values, determined for the recombinant enzymes with DL-DAP, D-serine, and L-serine as substrates, showed that the enzyme from S. typhimurium was more active than that from E. coli and the K(m) values were found to be similar. The purified enzymes had an absorption maximum (lambda(max)) at 412 nm, typical of PLP dependent enzymes. A red shift in lambda(max) was observed immediately after the addition of 10mM DL-DAP, which returned to the original lambda(max) of 412 nm in about 4 min. This red shift might reflect the formation of an external aldimine and/or other transient intermediates of the reaction. The apoenzyme of E. coli and S. typhimurium prepared by treatment with L-cysteine could be partially (60%) reconstituted by the addition of PLP. The holo, apo, and the reconstituted enzymes were shown to be present as homo dimers by size exclusion chromatography.     

Intergeneric conjugational crossing of Escherichia coli with Salmonella typhimurium. II. Transfer of a polA1 mutation from Escherichia coli to Salmonella typhimurium and its phenotypic expression in the salmonella genome]

The Escherichia coli structural gene for DNA polymerase I was inserted into Salmonella typhimurium chromosome by conjugal transfer. The genetic analysis of P1-mediated transduction of obtained hybrid showed that polA gene is located in it between metE and rha loci and is cotransduced with metE (about 50%) and rha (12%). The phenotypic properties of polA1 hybrid E. coliXS. typhimurium concerning UV-MMS-NG and gamma-ray sensitivity are similar to the polA1 mutants of E. coli.     

Glycerol elicits energy taxis of Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium show positive chemotaxis to glycerol, a chemical previously reported to be a repellent for E. coli. The threshold of the attractant response in both species was 10(-6) M glycerol. Glycerol chemotaxis was energy dependent and coincident with an increase in membrane potential. Metabolism of glycerol was required for chemotaxis, and when lactate was present to maintain energy production in the absence of glycerol, the increases in membrane potential and chemotactic response upon addition of glycerol were abolished. Methylation of a chemotaxis receptor was not required for positive glycerol chemotaxis in E. coli or S. typhimurium but is involved in the negative chemotaxis of E. coli to high concentrations of glycerol. We propose that positive chemotaxis to glycerol in E. coli and S. typhimurium is an example of energy taxis mediated via a signal transduction pathway that responds to changes in the cellular energy level.     

Signal transduction in chemotaxis to oxygen in Escherichia coli and Salmonella typhimurium.

Pathways previously proposed for sensory transduction in chemotaxis to oxygen (aerotaxis) involved either (i) cytochrome o, the electron transport system, and proton motive force or (ii) enzyme IIGlucose and the phosphoenolpyruvate:carbohydrate phosphotransferase system for active transport. This investigation distinguished between these possibilities. Aerotaxis was absent in a cyo cyd strain of Escherichia coli that lacked both cytochrome o and cytochrome d, which are the terminal oxidases for the branched electron transport system in E. coli. Aerotaxis, measured by either a spatial or temporal assay, was normal in E. coli strains that had a cyo+ or cyd+ gene or both. The membrane potential of all oxidase-positive strains was approximately -170 mV in aerated medium at pH 7.5. Behavioral responses to changes in oxygen concentration correlated with changes in proton motive force. Aerotaxis was normal in ptsG and ptsI strains that lack enzyme IIGlucose and enzyme I, respectively, and are deficient in the phosphotransferase system. A cya strain that is deficient in adenylate cyclase also had normal aerotaxis. We concluded that aerotaxis was mediated by the electron transport system and that either the cytochrome d or the cytochrome o branch of the pathway could mediate aerotaxis.     

Intracellular activation of albomycin in Escherichia coli and Salmonella typhimurium.

The antibiotic albomycin is actively taken up by Escherichia coli via the transport system for the structurally similar iron complex ferrichrome. Albomycin is cleaved, and the antibiotically active moiety is released into the cytoplasm, whereas the iron carrier moiety appears in the medium. Besides transport-negative mutants, additional albomycin-resistant mutants were isolated. The mutations were mapped outside the transport genes close to the pyrD gene at 21 min. The mutants were devoid of peptidase N activity. The molecular weight, sensitivity to inhibitors, and cytoplasmic location of the enzyme hydrolyzing albomycin in vitro corresponded to the known properties of peptidase N. The aminoacyl thioribosyl pyrimidine moiety of albomycin apparently has to be cleaved off the iron chelate transport vehicle to inhibit growth. Peptidase N is the major hydrolyzing enzyme. In Salmonella typhimurium peptidase N and peptidase A were equally active in hydrolyzing and activating albomycin.     

Peptidases and proteases of Escherichia coli and Salmonella typhimurium

A number of peptidases and proteases have been identified in Escherichia coli. Although their specific physiological roles are often not known, some of them have been shown to be involved in: the maturation of nascent polypeptide chains; the maturation of protein precursors; the signal peptide processing of exported proteins; the degradation of abnormal proteins; the use of small peptides as nutrients; the degradation of colicins; viral morphogenesis; the inactivation of some regulatory proteins for which a limited lifetime is a physiological necessity. Some of these enzymes act in concert to carry out specific functions. At present, twelve peptidases and seventeen proteases have been characterized. The specificity for only a few of them is known. The possible roles and the properties of these enzymes are discussed in this review.     

Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties.

The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.     

Uptake of cell wall peptides by Salmonella typhimurium and Escherichia coli.

During bacterial growth, cell wall peptides are released from the murein and reused for the synthesis of new cell wall material. Mutants defective in peptide transport were unable to reutilize cell wall peptides, demonstrating that these peptides are taken up intact into the cytoplasm prior to reincorporation into murein. Furthermore, cell wall peptide recycling was shown to play an important physiological role; peptide transport mutants which were unable to recycle these peptides showed growth defects under appropriate conditions. Using mutants specifically defective in each of the three peptide transport systems, we showed that the uptake of cell wall peptides was mediated solely by the oligopeptide permease (Opp) and that neither the dipeptide permease (Dpp) nor the tripeptide permease (Tpp) played a significant role in this process. Our data indicate that the periplasmic oligopeptide-binding protein has more than one substrate-binding site, each with different though overlapping specificities.