Kinetic and genetic analyses of D-cycloserine inhibition and resistance in Escherichia coli

Curtiss, Roy, III (Oak Ridge National Laboratory, Oak Ridge, Tenn.), Leigh J. Charamella, Claire M. Berg, and Paula E. Harris. Kinetic and genetic analyses of d-cycloserine inhibition and resistance in Escherichia coli. J. Bacteriol. 90:1238-1250.1965.-Wild-type cells of Escherichia coli growing at 37 C in mineral salts-glucose medium with vigorous aeration were lysed at maximal exponential rates by 10(-4) to 10(-2)md-cycloserine. At concentrations above 2 x 10(-2)m, d-cycloserine was bacteriostatic. Low levels of d-cycloserine (10(-5)m) and pencillin G (10 units per ml) interacted synergistically to cause a rapid exponential rate of lysis. Spontaneous mutations to d-cycloserine resistance occurred in discrete steps at frequencies of 10(-6) to 10(-7) for each step. First-, second-, and third-step d-cycloserine-resistant mutants were lysed at maximal exponential rates by d-cycloserine concentrations of 10(-3), 3 x 10(-3), and 5 x 10(-3)m, respectively. d-Alanine, l-alanine, and dl-alanyl-dl-alanine reversed d-cycloserine-induced lysis, in that order of effectiveness. On the basis of these observations, a d-cycloserine-enrichment cycling technique was developed for isolation of auxotrophic mutants. d-Cycloserine at 2 x 10(-3)m was as efficient as penicillin G (1,000 units per ml) for mutant enrichment in E. coli and should be useful for isolation of mutants in penicillin-resistant microorganisms. Bacterial conjugation experiments indicated that all three mutations conferring d-cycloserine resistance were linked to the met(1) locus. Transduction experiments showed that the mutation conferring first-step resistance was at least 0.5 min away from the mutations conferring second- and third-step resistance. The latter two mutations possibly occurred in the same gene, since they were sometimes carried in the same transducing phage. Studies on expression of d-cycloserine resistance indicated that these mutations were neither dominant nor recessive to each other nor to the d-cycloserine-sensitivity allele. Each allelic state exerted its influence on the phenotype independently of the others. These results are discussed in terms of the known inhibition of alanine racemase and d-alanyl-d-alanine synthetase by d-cycloserine.     

Mechanism of D-cycloserine action: transport systems for D-alanine, D-cycloserine, L-alanine, and glycine

The accumulation of d-alanine, l-alanine, glycine, and d-cycloserine in Escherichia coli was found to be mediated by at least two transport systems. The systems for d-alanine and glycine are related, and are separate from that involved in the accumulation of l-alanine. d-Cycloserine appears to be primarily transported by the d-alanine-glycine system. The accumulation of d-alanine, glycine, and d-cycloserine was characterized by two line segments in the Lineweaver-Burk analysis, whereas the accumulation of l-alanine was characterized by a single line segment. d-Cycloserine was an effective inhibitor of glycine and d-alanine accumulation, and l-cycloserine was an effective inhibitor of l-alanine transport. The systems were further differentiated by effects of azide, enhancement under various growth conditions, and additional inhibitor studies. Since the primary access of d-cycloserine in E. coli is via the d-alanine-glycine system, glycine might be expected to be a better antagonist of d-cycloserine inhibition than l-alanine. Glycine and d-alanine at 10(-5)m antagonized the effect of d-cycloserine in E. coli, whereas this concentration of l-alanine had no effect.     

Presence of an unusual methanogenic bacterium in coal gasification waste

Methanogenic bacteria growing on a pilot-scale, anaerobic filter processing coal gasification waste were enriched in a mineral salts medium containing hydrogen and acetate as potential energy sources. Transfer of the enrichments to methanol medium resulted in the initial growth of a strain of Methanosarcina barkeri, but eventually small cocci became dominant. The cocci growing on methanol produced methane and exhibited the typical fluorescence of methanogenic bacteria. They grew in the presence of the cell wall synthesis-inhibiting antibiotics d-cycloserine, fosfomycin, penicillin G, and vancomycin as well as in the presence of kanamycin, an inhibitor of protein synthesis in eubacteria. The optimal growth temperature was 37 degrees C, and the doubling time was 7.5 h. The strain lysed after reaching stationary phase. The bacterium grew poorly with hydrogen as the energy source and failed to grow on acetate. Morphologically, the coccus shared similarities with Methanosarcina sp. Cells were 1 mum wide, exhibited the typical thick cell wall and cross-wall formation, and formed tetrads. Packets and cysts were not formed.     

Inhibition of staphylococcal enterotoxin B formation by cell wall blocking agents and other compounds

Enterotoxin B formation by Staphylococcus aureus S6 was inhibited by Tween 80, oleic acid, sodium deoxycholate, penicillin, d-cycloserine, or bacitracin. Toxin formation by strain 243 was sensitive to oleic acid, sodium deoxycholate, sodium lauryl sulfate, d-cycloserine, or bacitracin. The effect of d-cycloserine was reversed by d-alanine with strain 243 but not with strain S6. Neither penicillin nor bacitracin inhibited alpha-hemolysin or coagulase activity of strain S6; however, 0.118 mumoles of d-cycloserine per ml increased the alpha-hemolysin titer more than eightfold. Pigmentation of strain 243 was reduced by oleic acid, sodium deoxycholate, or methicillin, and was completely inhibited by d-cycloserine or bacitracin. Glucose was required for the inhibition by spermine of (14)C-valine incorporation into cellular protein of strain S6. These data indicate that the cell surface may contain sites important to the synthesis of enterotoxin B.     

Cell Wall Synthesis by Chlamydia psittaci Growing in L Cells

Biochemical events accompanying changes in structure and behavior of the cell walls of Chlamydia psittaci strain 6BC during its developmental cycle in L cells (mouse fibroblasts) were studied by measuring at short intervals the effect of d-cycloserine and penicillin G on incorporation of labeled intermediates into acid-insoluble fractions of infected L cells in which host incorporation had been inhibited by cycloheximide and into intact chlamydial cells and cell walls separated from the infected L cells. d-Cycloserine enhanced the incorporation of (14)C-l-alanine at all times in the developmental cycle, but the incorporation of (14)C-l-lysine was always inhibited. In parallel experiments, penicillin G had no effect on incorporation of any of these intermediates, but when infected L cells incorporated (14)C-l-alanine in the presence of penicillin G, the labeled alanine was released more rapidly in the subsequent absence of the antibiotic than in its continued presence. When either penicillin G or d-cycloserine was present throughout the developmental cycle, C. psittaci continued to synthesize deoxyribonucleic acid and protein, but at less than normal rates.     

DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.     

D-serine transport system in Escherichia coli K-12

The d-serine transport system in Escherichia coli K-12 was studied by use of a mutant unable to form d-serine deaminase, yet resistant to d-serine. The mutant is greatly impaired in its ability to accumulate d-serine, d-alanine, and glycine. Transport of l-alanine is partially affected but transport of l-serine is unaffected. The mutant is also resistant to d-cycloserine, indicating that d-serine is transported by the system responsible for uptake of d-cycloserine. The d-serine transport system is not inducible, but appears to be formed constitutively, as are the transport systems of most amino acids. The transport mutation appears to be multistep and maps to the right of malB on the E. coli linkage map.     

Über die Bildung von Bakterienhemmstoffen durch Cosmarium impressulum

1. An already published test method for detecting bactericidal substances in paper chromatograms was further improved. 2. In cultures of Cosmarium impressulum free from bacteria, two bactericidal substances were found in the ether extracts from the algae and two others in the extracts of the culture medium. The are active against some or all bacteria testes (Serratia marcescens, Pseudomonas fluorescens; Aerobacter aerogenes or Bacillus pumilus). 3. If the culture medium of Cosmarium or another desmidiale was inoculated with the test bacteria, a clear bactericidal effect was never observed. 4. Because the activity of the bactericidal substances of Cosmarium is only small and the formation is not constant, it is concluded that the water-soluble bactericidal substances of the alga are not the cause that epiphytic bacteria do not grow normally on Cosmarium.     

Mechanism of D-cycloserine action: transport mutants for D-alanine, D-cycloserine, and glycine

The accumulation of d-alanine and the accumulation of glycine in Escherichia coli are related and appear to be separate from the transport of l-alanine. The analysis of four d-cycloserine-resistant mutants provides additional support for this conclusion. The first-step mutant from E. coli K-12 that is resistant to d-cycloserine was characterized by the loss of the high-affinity line segment of the d-alanine-glycine transport system in the Lineweaver-Burk plot. This mutation, which is linked to the met(1) locus, also resulted in the loss of the ability to transport d-cycloserine. The second-step mutation that is located 0.5 min from the first-step mutation resulted in the loss of the low-affinity line segment for the d-alanine-glycine transport system. The transport of l-alanine was decreased only 20 to 30% in each of these mutants. A multistep mutant from E. coli W that is 80-fold resistant to d-cycloserine lost >90% of the transport activity for d-alanine and glycine, whereas 75% of the transport activity for l-alanine was retained. E. coli W could utilize either d- or l-alanine as a carbon source, whereas the multistep mutant could only utilize l-alanine. Thus, a functioning transport system for d-alanine and glycine is required for both d-cycloserine action and growth on d-alanine.     

An L-Form of Staphylococcus aureus Adapted to a Brain Heart Infusion Medium without Osmotic Stabilizers

An L-form derived from halotolerant Staphylococcus aureus Tasaki was adapted to growth in a brain heart infusion medium without any supplemental osmotically protective solutes (360 mOsm/kg). This L-form had no chemically detectable peptidoglycan residues on its surface. Electron microscopic observations confirmed morphologically the absence of the structures and also of other osmotically protective polymers within or exterior to the cytoplasmic membrane. The osmotic stability and susceptibility to bacitracin, d-cycloserine, and vancomycin of the L-form adapted to growth in 360 mOsm osmotically unprotective medium was higher than that of the L-form grown in 1,950 mOsm supplemented with 4.5% NaCl. The adapted L-form tended to be more sensitive to almost all of the antibiotics examined, other than the inhibitors for cell wall-synthesis, than the original L-form strain requiring osmotic protection for growth. Chemical analysis of the membrane of the adapted L-form indicated 16.3% total lipids and 20.6% proteins by dry weight of the membrane, and it contained larger amounts of lipid phosphorus (20.0 μ/mg).     

Effects of Mitomycin C on Macromolecular Synthesis in Escherichia coli

When cells of Escherichia coli B growing in a glucose-synthetic medium were treated with mitomycin C, the effects produced by the antibiotic varied, depending on the concentration. When the concentration was reduced to less than 0.1 mug/ml, the action of the antibiotic was bacteriostatic; cell elongation resulted, but no effect on the synthesis of cellular macromolecules was apparent. At higher levels (more than 5 mug/ml), mitomycin C was highly bactericidal and inhibited deoxyribonucleic acid synthesis almost completely. The exposure of growing cells to a bactericidal level of mitomycin C resulted also in a delayed inhibition of the synthesis of ribonucleic acid (RNA) and protein. The capacity of the treated cells to synthesize beta-galactosidase inducibly in a medium free from a carbon source remained constant for the first 30 min and then was destroyed progressively with time. Prolonged incubation with the bactericidal level of mitomycin C caused a degradation of cellular nucleic acids, particularly RNA. The degraded nucleic acid components were eventually released into the medium.     

Comparative Effects of Anesthetics on the Viability and Integrity of Escherichia coli ML30

Cells of Escherichia coli ML30 in a mineral salts medium were exposed to dichlorodifluoromethane (f-12), cyclopropane, halothane, or Ethrane at concentrations of 1.25, 0.2, 0.04, and 0.008× saturation for times up to 1,200 min, and at temperatures in the range of 2 to 37 C. When any of these anesthetics were applied for 300 min at 1.25× saturation, a substantial decrease in number of survivors occurred. Halothane was most bactericidal, cyclopropane and Ethrane were moderately bactericidal, and f-12 was least bactericidal. At saturation values of less than 1.0, none of the four anesthetics had an appreciable effect on viability of E. coli. Greatest increases in cell permeability occurred when anesthetics were used at saturation values of 1.25, and permeability changes generally decreased as the concentrations of the chemicals were reduced. In many instances, anesthetics in the vapor state caused significant increases in cell permeability but little or no loss of viability. This indicated that a close relationship did not exist between loss of viability and increased permeability. All four anesthetics caused E. coli to lose substantial and similar amounts of compounds absorbing at 260 nm. Release of compounds absorbing at 260 nm generally increased as the saturation value of a given chemical was increased. Halothane, Ethrane, and cyclopropane but not f-12 caused lysis of E. coli ML30. Considering all results, E. coli ML30 was damaged more by halothane or cyclopropane than by f-12 or Ethrane. When f-12 was applied at a saturation value of 1.25, the bactericidal effect on E. coli was much greater at 37 or 22 C than at 12 or 2 C.     

The inhibitory effects of pyrocarbonic acid diethyl ester on lactobacillus casei and its phage Jl – A novel strategy for phage control in technical fermentation processes

The effects of pyrccarbonic acid diethyl ester (PADE) on Lactobacillus casei Sl and its phage Jl was investigated in relation to the control of phages in the dairy industry and other technica fermentation processes. PADE exhibited a bacteriostatic effect at 0.5 to 8 mM and a bactericidal effect at 10 mM or higher. It inhibited the growth of the phage at its bacteriostatic and bactericidal concentrations. The growth inhibition of the phage was reversible at the bacteriostatic concentrations but complete and irreversible at the bactericidal concentrations. PADE inactivated the free phage within several minutes; 10 and 30 mM of PADE inactivated 90 and 100%, respectively, of the phage. It completely decomposed into ineffective components in several minutes. The bacteria grew almost normally when they were inoculated after the complete decomposition of PADE. These four characteristics of PADE – its bactericidal effect, its inhibitory effect on phage growth, its phage-inactivating effect and its decomposition – suggest a novel strategy for phage control in technical fermentation processes, including the dairy industry.     

Lethal Effects of Electric Current on Escherichia coli

An attempt has been made to use low-voltage alternating current to kill microorganisms such as Escherichia coli. The bactericidal effect depends on the energy passing through the suspension and on the time during which the cells are left standing in the medium after the treatment. Most of the toxicity is due to an indirect effect developed with unalterable electrodes in the presence of chlorides in the medium. This method might be applied to eliminate pollution of natural waters.     

Lactoferricin, a new antimicrobial peptide

Lactoferricin B (LF-B) is a peptide derived from acid-pepsin digestion of bovine lactoferrin, which has antimicrobial properties. In order to assess the antimicrobial spectrum of LF-B and its possible in vivo uses, the minimum inhibitory and microbicidal concentrations of pure lactoferricin B were determined for a range of bacterial species and under varying conditions of growth including growth phase and size of the inoculum, pH and ionic strength of the medium. Lactoferricin B was bactericidal against a wide range of bacteria and Candida albicans. Proteus spp., Pseudomonas cepacia and Serratia spp. were resistant. The bactericidal activity of LF-B was inhibited by increasing ionic strength and bacterial inoculum and at acid pH. The activity of lactoferricin B was completely inhibited by the addition of 5% whole cow's milk and was reduced in the presence of increasing concentrations of mucin. These results indicate the potential of LF-B to reduce the numbers of organisms in a simple medium, but raise doubts about its role in vivo because of its sensitivity to changes in physical variables. It may be that lactoferricin exerts a transient antimicrobial effect at mucosal surfaces.     

Genes involved in copper resistance influence survival of Pseudomonas aeruginosa on copper surfaces

Aims:  To evaluate the killing of Pseudomonas aeruginosa PAO1 on copper cast alloys and the influence of genes on survival on copper containing medium and surfaces.
Methods and Results:  Different strains of P. aeruginosa were inoculated on copper containing medium or different copper cast alloys and the survival rate determined. The survival rates were compared with rates on copper-free medium and stainless steel as control. In addition, the effect of temperature on survival was examined.
Conclusions:  Copper cast alloys had been previously shown to be bactericidal to various bacteria, but the mechanism of copper-mediated killing is still not known. In this report, we demonstrate that P. aeruginosa PAO1 is rapidly killed on different copper cast alloys and that genes involved in conferring copper resistance in copper-containing medium also influenced survival on copper cast alloys. We also show that the rate of killing is influenced by temperature.
Significance and Impact of the Study:  To use copper surfaces more widely as bactericidal agents in various settings, it is important to understand how genes influence survival on these surfaces. Here we show that genes shown to be involved in copper resistance in P. aeruginosa PAO1 can have an impact on the length of survival time on copper cast alloys under certain conditions. This is an important first step for evaluation of future use of copper surfaces as bactericidal agents.     

The bactericidal effect of TiO2 photocatalysis involves adsorption onto catalyst and the loss of membrane integrity

The bactericidal effect of photocatalysis with TiO2 is well recognized, although its mode of action is still poorly characterized. It may involve oxidation, as illuminated TiO2 generates reactive oxygen species. Here we analyze the bactericidal effect of illuminated TiO2 in NaCl-KCl or sodium phosphate solutions. We found that adsorption of bacteria on the catalyst occurred immediately in NaCl-KCl solution, whereas it was delayed in the sodium phosphate solution. We also show that the rate of adsorption of cells onto TiO2 is positively correlated with its bactericidal effect. Importantly, adsorption was consistently associated with a reduction or loss of bacterial membrane integrity, as revealed by flow cytometry. Our work suggests that adsorption of cells onto aggregated TiO2, followed by loss of membrane integrity, is key to the bactericidal effect of photocatalysis.     

Effects of various drugs on hemolytic activity of Streptococcus zymogenes

Drugs known to interrupt the chain of protein synthesis (streptomycin, chloramphenicol, ethidium bromide) appeared to affect production of the group D lysin by Streptococcus zymogenes by inhibiting growth. Drugs which block cell wall synthesis (vancomycin, bacitracin, d-cycloserine, and phosphonomycin) inhibited lytic activity by a mechanism independent of growth. Bacitracin appeared not to have a major effect on lysin production but elicited the production of an inhibitor of lytic activity.     

IM-2, a butyrolactone autoregulator,induces production of several nucleoside antibiotics in Streptomyces sp. FRI-5

IM-2 is one of the butyrolactone autoregulators of Streptomyces, which triggers production of a blue pigment in Streptomyces sp. FRI-5 at a concentration of 0.6 ng/ml. In the absence of IM-2, Streptomyces sp. FRI-5 was found to produce d-cycloserine. However, the addition of IM-2 at 5-h cultivation stopped both growth and d-cycloserine production, and instead induced production of several different antibiotics. The IM-2-induced antibiotics were isolated from the culture broth, and assigned as the nucleoside antibiotics, showdomycin and minimycin. Therefore, IM-2 was concluded to be a global regulator of a secondary metabolism, which not only induced the production of nucleoside antibiotics but also suppressed d-cycloserine production.     

Mapping of a D-cycloserine resistance locus in escherichia coli K-12

The mutation conferring first-step resistance to d-cycloserine has been located at min 83 on the Escherichia coli K-12 genetic map.