RecA-independent resistance to irradiation with u.v. light in acid-habituated Escherichia coli

Growth of Escherichia coli 1829 ColV, I-K94 at pH 5.0 led to an increase in u.v. resistance compared with cells grown at pH 7.0. This was due to a phenotypic change, since organisms grown at pH 7.0 showed increased resistance after only 2.5-5.0 min incubation at the mildly acid pH. Other E. coli K12 derivatives became more u.v.-resistant at pH 5.0 including uvrA, recA and polA1 mutants. Organisms grown at pH 5.0 also showed increased Weigle reactivation of u.v.-irradiated lambda phage and this applied to the repair-deficient mutants as well as the parent strains. Both the increased u.v. resistance of acid-habituated cells and their increased ability to bring about Weigle reactivation appear to involve RecA-independent processes and are presumably, therefore, independent of the SOS response.     

Effect of Lysogeny on Serum Sensitivity

When Escherichia coli K-12 was infected with lambda phage and mutants of lambda characterized by the production of temperature-sensitive repressors, the lysogenic bacteria were significantly more resistant to normal serum than the uninfected organisms. Infection of E. coli K-12 with a lambdoid phage, phi80, whose prophage attachment site is different from that of lambda, did not result in a detectable change in serum resistance. Similarly, infection with certain Pseudomonas and Shigella phages caused no detectable differences in serum resistance. Finally, the well-known conversion of the Salmonella anatum serotype to S. newington by E(15) phage indicated that, despite the relatively greater roughness of S. anatum, S. newington was more sensitive to normal serum than S. anatum. Thus, the effects of lysogeny on the sensitivity of bacteria to the bactericidal action of serum mediated by the complement system may be quite variable.     

A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.     

Isolation of the Bacteriophage Lambda Receptor from Escherichia coli

A factor which inactivates the phage lambda can be extracted from Escherichia coli. This factor is a protein and is located in the outer membrane of the bacterial envelope. It is found in extracts of strains which are sensitive to phage lambda, but not in extracts of strains specifically resistant to this phage. We conclude that this factor is the lambda receptor, responsible for the specific adsorption of the phage lambda to E. coli cells. A partial purification of the lambda receptor is described. Inactivation of the phage by purified receptor is shown to be accompanied by the release of deoxyribonucleic acid from the phage.     

Pseudouridylate Synthetase of Escherichia coli: a Catabolite-Repressible Enzyme

The growth on pseudouridine of two pyrimidine auxotrophs of Escherichia coli (Bu(-) and W63-86) was markedly enhanced when glycerol replaced glucose as a carbon source or when adenosine 3':5'-cyclic monophosphoric acid was added to medium containing glucose. These results indicated that an enzyme catalyzing a reaction in the pathway of pseudouridine conversion to uracil was sensitive to catabolite repression. The following pathway is proposed for pseudouridine utilization: [Formula: see text] [Formula: see text] Pseudouridylate synthetase was sensitive to catabolite repression in strains Bu(-) and W63-86. In contrast, strains B5RU and W5RU, mutants of Bu(-) and W63-86 which were selected for their ability to grow rapidly on pseudouridine in the presence of glucose, had high levels of pseudouridylate synthetase in the presence of glucose. In the case of B5RU but not W5RU, synthetase activity was greater in cells grown on glycerol or on glucose plus adenosine 3':5-cyclic monophosphoric acid than on glucose.     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Catabolite repression of tryptophanase in Escherichia coli

Catabolite repression of tryptophanase was studied in detail under various conditions in several strains of Escherichia coli and was compared with catabolite repression of beta-glactosidase. Induction of tryptophanase and beta-galactosidase in cultures grown with various carbon sources including succinate, glycerol, pyruvate, glucose, gluconate, and arabinose is affected differently by the various carbon sources. The extent of induction does not seem to be related to the growth rate of the culture permitted by the carbon source during the course of the experiment. In cultures grown with glycerol as carbon source, preinduced for beta-galactosidase or tryptophanase and made permeable by ethylenediaminetetraacetic acid (EDTA) treatment, catabolite repression of tryptophanase was not affected markedly by the addition of cAMP (3',5'-cyclic adenosine monophosphate). Catabolite repression by glucose was only partially relieved by the addition of cAMP. In contrast, under the same conditions, cAMP completely relieved catabolite repression of beta-galactosidase by either pyruvate or glucose. Under conditions of limited oxygen, induction of tryptophanase is sensitive to catabolite repression; under the same conditions, beta-galactosidase induction is not sensitive to catabolite repression. Induction of tryptophanase in cells grown with succinate as carbon source is sensitive to catabolite repression by glycerol and pyruvate as well as by glucose. Studies with a glycerol kinaseless mutant indicate that glycerol must be metabolized before it can cause catabolite repression. The EDTA treatment used to make the cells permeable to cAMP was found to affect subsequent growth and induction of either beta-galactosidase or tryptophanase much more adversely in E. coli strain BB than in E. coli strain K-12. Inducation of tryptophanase was reduced by the EDTA treatment significantly more than induction of beta-galactosidase in both strains. Addition of 2.5 x 10(-3)m cAMP appeared partially to reverse the inhibitory effect of the EDTA treatment on enzyme induction but did not restore normal growth.     

Antibacterial activity of surfactants against Escherichia coli cells is influenced by carbon source and anaerobiosis

AIMS: In order to clarify the involvement of an energy-yielding system in the antibacterial action of surfactants, the effects of carbon source and anaerobiosis during the growth period on the surfactant sensitivity of Escherichia coli cells were investigated. METHODS AND RESULTS: Cetyltrimethylammonium bromide (CTAB) and N-dodecyl-N,N-dimethylglycine, at relatively low concentrations, caused a delay in growth of E. coli cells. Cells grown in M9 medium supplemented with glycerol, succinate or acetate as a carbon source were more sensitive to surfactants and had a higher respiratory activity than those grown with glucose. Cultivation under anaerobiosis made cells resistant to CTAB. CONCLUSIONS: Bacterial sensitivity to surfactants was affected by carbon source and anaerobiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained should be helpful in determining suitable conditions of treatment in the practical use of surfactants for bacterial decontamination.     

Temperature Sensitivity of Maltose Utilization and Lambda Resistance in Escherichia coli B

Escherichia coli B strains that have acquired the malB region from E. coli K-12 are able to utilize maltose and to adsorb phage lambda when grown at 30 C, but when grown at 40 C they do not absorb phage lambda and are devoid of amylomaltase activity. These Mal(ts) Lam(ts) cells can be mutated or transduced to become able to grow on maltose at 40 C, but they still have no detectable amylomaltase activity nor functional lambda receptors at that temperature. This Mal(40) phenotype is governed by a gene located near or at malA. It is suggested that the temperature sensitivity of both characters results from a defect in malT. However, transduction of malA from E. coli B to E. coli K-12 results in a wild-type phenotype, whereas E. coli B cells that have acquired malA from E. coli K-12 donors are still temperature sensitive for both amylomaltase and lambda-receptor production.     

Sensitivity of Escherichia coli cells to seawater closely depends on their growth stage.

Sensitivity of Escherichia coli cells in seawater, considered in terms of culturability loss, was examined after different growth periods in a mineral medium supplemented with glucose (M9) at 37 degrees C under aerobic or anaerobic conditions. Their sensitivity varied considerably during the different growth phases and differed when cells were grown under aerobic or anaerobic conditions. Sensitivity of aerobic cells rapidly increased during the lag phase, then decreased during the exponential phase and became minimal during the stationary phase. Coliforms isolated from human faeces showed a similar sensitivity after incubation in wastewater at 37 degrees C for 3 h. The sensitivity phase was completely eliminated when cells were incubated with chloramphenicol. Variation of sensitivity in anaerobic cells according to their growth phase was comparable with that found for aerobic cells which had been left in seawater for a long period (6 d). However, for shorter periods in this medium (1-2 d), cells grown until the mid-exponential phase remained resistant to seawater. During the second half of the growth phase, they were as sensitive as aerobic cells at lag phase. Escherichia coli cells grown under anaerobic conditions, such as found in the intestine, progressively adapt to aerobic conditions after their transfer into aerated seawater and their sensitivity to seawater increases. On a practical level, these observations show that it is necessary to control accurately the age of cells before inoculation in seawater microcosms to conserve a comparative value in results. The importance of this factor is vital as all variations in sensitivity of cells to seawater according to their prior growth phase proved to be logarithmic functions of time.     

Inactivation and mutagenesis of lambda phage by O-methylhydroxylamine and O-delta-aminooxybutylhydroxylamine

The inactivation and the mutagenesis of lambda phage Cl 857 virR by O-methylhydroxylamine (OMHA) and O-delta-aminooxybuthylhydroxylamine (delta-HA) were studied. The inactivation of OMHA-treated phage was shown to be stronger in E. coli polA cells defective in DNA-polymerase I as compared to wild-type host E. coli W3350. In contrast delta-HA caused similar phage inactivation in these two strains. Wave-type kinetics of the inactivation and the mutagenesis of phage by OMHA and delta-HA was observed. delta-HA appeared to be a more effective mutagen than OMHA: it induced higher mutant yield at a given level of inactivation.     

Isolation and characterization of respiratory-deficient mutants of Escherichia coli K-12.

Several mutants of Escherichia coli K-12 defective in aerobic metabolism were isolated. One such mutant was found to be deficient in cytochromes, heme, and catalase. Aerobically grown cells did not consume oxygen and could grow only on fermentable carbon sources. Supplementation of the growth medium with delta-aminolevulonic acid, protoporphyrin IX, or hemin did not restore aerobic metabolism. The lack of heme and catalase in mutant cells grown on glucose was not due to catabolite repression, since the addition of exogenous cyclic AMP did not restore the normal phenotype. When grown aerobically on complex medium containing glucose, the mutant produced lactic acid as the principal fermentation product. This pleotropic mutation was attributed to an inability of the cells to synthesize heme, and preliminary data mapped the mutation to between 8 and 13 min on the E. coli genome.     

New nalidixic acid resistance mutations related to deoxyribonucleic acid gyrase activity.

In Escherichia coli K-12 mutants which had a new nalidixic acid resistance mutation at about 82 min on the chromosome map, cell growth was resistant to or hypersusceptible to nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, and novobiocin. Deoxyribonucleic acid gyrase activity as tested by supercoiling of lambda phage deoxyribonucleic acid inside the mutants was similarly resistant or hypersusceptible to the compounds. The drug concentrations required for gyrase inhibition were much higher than those for cell growth inhibition but similar to those for inhibition of lambda phage multiplication. Transduction analysis with lambda phages carrying the chromosomal fragment of the tnaA-gyrB region suggested that one of the mutations, nal-31, was located on the gyrB gene.     

RecA-independent resistance to irradiation with u.v. light in acid-habituated Escherichia coli

Growth of Escherichia coli 1829 ColV, I-K94 at pH 5.0 led to an increase in u.v. resistance compared with cells grown at pH 7.0. This was due to a phenotypic change, since organisms grown at pH 7.0 showed increased resistance after only 2.5–5.0 min incubation at the mildly acid pH. Other E. coli K12 derivatives became more u.v.-resistant at pH 5.0 including uvrA, recA and polA1 mutants. Organisms grown at pH 5.0 also showed increased Weigle reactivation of u.v.-irradiated Λ phage and this applied to the repair-deficient mutants as well as the parent strains. Both the increased u.v. resistance of acid-habituated cells and their increased ability to bring about Weigle reactivation appear to involve RecA-independent processes and are presumably, therefore, independent of the SOS response.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Changes in the intracellular concentration of acetyl-CoA and malonyl-CoA in relation to the carbon and energy metabolism of Escherichia coli K12

Intracellular concentrations of acetyl-CoA and malonyl-CoA in Escherichia coli K12 were determined by a malonyl-CoA: acetyl-CoA cycling technique. Under aerobic growth conditions with glucose the acetyl-CoA and malonyl-CoA concentrations varied over a range of 0.05-1.5 nmol (mg dry wt)-1 (20-600 microM) and 0.01-0.23 nmol (mg dry wt)-1 (4-90 microM), respectively. The intracellular concentration of acetyl-CoA was highest in exponentially growing cells and it fell rapidly to less than 5% of the maximum level when the organism entered stationary phase after exhaustion of glucose. A linear relationship was observed between the intracellular concentration of total acyl-CoA and the logarithm of the concentration of glucose in the medium. Consequently, the acetyl-CoA/malonyl-CoA ratios also varied drastically, in a range of 0.6-41.7, under different conditions. Of several carbon sources tested, glucose was the most effective for promoting the synthesis of cellular acetyl-CoA. For cells grown on glycerol or acetate the maximum concentrations of total acyl-CoA were significantly lower. In cells incubated with citrate (not used as a carbon source by E. coli), the level was consistent with that in cells starved for exogenous carbon sources.     

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.     

Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli

Izaki, Kazuo (University of Tokyo, Tokyo, Japan), Kan Kiuchi, and Kei Arima. Specificity and mechanism of tetracycline resistance in a multiple drug resistant strain of Escherichia coli. J. Bacteriol. 91:628-633. 1966.-A decrease in the uptake of tetracycline occurred concurrently with a rise in the level of resistance of a multiple drug resistant strain of Escherichia coli grown in the presence of tetracycline. Although the strain was also resistant to streptomycin and chloramphenicol, growth in the presence of these two antibiotics did not influence the uptake of tetracycline. The induction of resistance, or decreased uptake of tetracycline, was dependent on growth of the organism in the presence of the drug. Decreased uptake of tetracycline could not be induced in a sensitive strain of the same organism under conditions suitable for induction of the resistant strain. The decrease in accumulating power of the resistant organism cultured in the presence of tetracycline does not appear to be due to selection of a resistant strain from cultures containing both resistant and sensitive strains.     

Expression of pyruvate formate-lyase of Escherichia coli from the cloned structural gene

It is shown here that a plasmid (p29) derived from the transducing phage aspC2 (Christiansen and Pedersen 1981) codes for pyruvate formate-lyase. The identity of the 80 kilodaltons (kd) gene product of plasmid p29 with the pyruvate formate-lyase polypeptide was proven (i) by comigration of the gene product expressed in the maxicell system with purified enzyme on O'Farrell gels, and (ii) by comparison of the peptide maps obtained from limited proteolysis. In vivo the 80 kd form of the enzyme was proteolytically converted to a 78 kd polypeptide. The two polypeptides (80 kd and 78 kd) and their charge isomers present in purified enzyme preparations are therefore products of a single gene.Aerobically grown cells of Escherichia coli contained a basal level of pyruvate formate-lyase which was derepressed 5-to 10-fold under anaerobiosis. Derepression also occurred during anaerobic growth on glycerol plus fumarate. Presence of plasmid p29 caused overproduction of pyruvate formatelyase, 11-fold upon anaerobic growth on glucose, 14-fold upon aerobic growth on glucose and 33-fold upon aerobic growth at the expense of D-lactate.Non-Standard Abbreviation MOPS 4-morpholine-propane sulfonic     

Glycerol metabolism in superoxide dismutase-deficient Escherichia coli.

Escherichia coli, which lacks cytoplasmic superoxide dismutases, exhibits various phenotypic deficits if grown aerobically. Here we report that sodAsodB E. coli cannot use glycerol under aerobic conditions. The reason is low activity of glycerol kinase (GK), the rate-limiting enzyme in glycerol metabolism. Superoxide does not inactivate GK, but makes it susceptible to inactivation by a heat-labile factor present in the cell-free extracts. This factor seems to be part of a proteolytic system, which recognizes and degrades oxidatively modified proteins.