Epidemiology of antibiotic and heavy-metal resistance in bacteria: a computer-based data system.
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Biomedical subjects
Publications and source records attributed to F E Young.
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Antibiotics that inhibit the biosynthesis of the cell wall, such as vancomycin, penicillin, d-cycloserine, and bacitracin, stimulate the incorporation of lysine into lipids that are extractable with n-butanol-6 M pyridinium acetate. Approximately 93% of this lysine is in lysylphosphatidylglycerol (LPG). The remaining lysine is incorporated in another as yet uncharacterized lipid. Because the lysine in the latter lipid is released by mild alkaline hydrolysis, it is not the C(55)-isoprenyl-pyrophospho-N-acetyl-muramyl pentapeptide. Vancomycin and penicillin stimulate the incorporation of lysine into both LPG and the minor lipid fractions, whereas treatment with d-cycloserine results in an increase only in LPG. Antibiotics that inhibit protein synthesis do not influence the incorporation of lysine into the lipid fractions. Analysis of the extracted lipids indicate that the incorporation of radioactive lysine into LPG is due to an enhancement in synthesis of LPG from phospholipids in the cytoplasmic membrane.
A stable L-form of Bacillus subtilis 168 (sal-1) has been isolated which grows and divides logarithmically in liquid medium with a generation time of 60 min. This mutant does not synthesize cell wall as evidenced by chemical, biochemical, and morphological analyses. Antibiotics which specifically inhibit cell wall biosynthesis do not affect the growth of the L-form. Significant differences exist between the membrane proteins of the bacillary form and the L-form. The relative profile of membrane proteins varies with the salt concentration of the medium in both the L-form and the bacillary form.
Strains of Bacillus subtilis lysogenic for either temperate bacteriophage phi105 or SPO2 were reduced to less than 1.0% of the level of transformation of the nonlysogenic strains. Strains lysogenic for both phi105 and SPO2 are virtually nontransformable, indicating that the effect of lysogeny is additive. Lysogenic cultures transfected at essentially wild-type levels with deoxyribonucleic acid (DNA) isolated from bacteriophages phi29 and SPO1. The residual transformation and transfection achieved by the lysogenic cultures changed dramatically during growth in SPII medium, whereas nonlysogenic strains remained competent for 5 hr in SPII medium. Despite a marked reduction in transformation, lysogenic cultures initially irreversibly bound as much DNA as nonlysogenic cultures. After 60 min in SPII medium, there was a rapid decrease in the capacity of lysogenic cells to bind DNA irreversibly. These results, as discussed, indicate that the inhibition of transformation is probably due to an alteration of the cell surface or a differential inactivation of bacterial genes after lysogenic conversion.
Genetic analysis of a d-alanine requiring mutant (dal) of Bacillus subtilis reveals that the gene that codes for d,l-alanine racemase is linked to purB. The order of genes in this region of the chromosome is purB, pig, tsi, dal. Thus there are at least two clusters of genes that regulate cell wall biosynthesis in B. subtilis.
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Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis.
Temperature-sensitive mutants of Staphylococcus aureus H which require 1.0 m NaCl for growth at 42 C can be divided into two major classes. Most of the mutants (class A) do not accumulate nucleotide precursors of cell wall biosynthesis in the absence of salt at the nonpermissive temperatures, whereas the class B mutants accumulate these precursors. The most extensively studied mutant RUS 1 (carrying peg-1) is defective in biosynthesis of peptidoglycan at the nonpermissive conditions as evidenced by: (i) reduced incorporation of cell wall precursors into peptidoglycan; (ii) accumulation of the nucleotide, uridine diphosphate (UDP) muramyl-l-alanyl-d-glutamic acid; (iii) reduced specific activity of UDP N-acetylmuramyl (MurNAc)-l-alanyl-d-glutamate: l-lysine ligase (EC 6.3.2.7); and (iv) an increased susceptibility to lysis with sodium dodecyl sulfate. Addition of 1.0 m NaCl reverses these defects with the exception of the specific activity of UDP-MurNAc-l-alanyl-d-glutamate: l-lysine ligase. Nevertheless, the structure of the cell wall is normal at the nonpermissive conditions if 1.0 m NaCl is present. An alteration in the binding of a fluorescent dye, 8-anilino-1-napthalene-4-sulfonic acid at the nonpermissive conditions in the absence of 1.0 m NaCl suggests that there may also be defects in the membrane in this strain.
The effect of various salts on the autolysis of cell wall of a ribitol teichoic acid-deficient mutant of Staphylococcus aureus H (strain 52A5 carrying tar-1) was compared with the parent strain. In the presence of high concentrations of certain salts such as 1.0 m NaCl, the mutant undergoes autolysis with the release of osmotically sensitive spheroplasts. The parent strain is not affected by these conditions. The stimulation of lysis is related to an activation of N-acylmuramyl-l-alanine amidase.
A multiple auxotrophic derivative of Bacillus subtilis 168 (strain BR151 carrying lys-3, trpC2, metB10) was transformed with deoxyribonucleic acid (DNA) isolated from B. subtilis 168, Bacillus amyloliquefaciens H, B. subtilis HSR, Bacillus pumilus, and Bacillus licheniformis. Transformation with heterologous DNA occurred at a very low frequency for the three auxotrophic markers. Heterologous transformation to rifampin resistance was 100 to 1,000 times more efficient than transformation to prototrophy. Transformants from the various heterologous exchanges were used to prepare donor DNA. The fragment of integrated DNA from the heterologous (foreign) species, termed the "intergenote," was capable of transforming BR151 with an efficiency almost equal to that of homologous DNA. When BR151 DNA contained the Rfm(R) (rifampin resistance) intergenote from B. amyloliquefaciens H, the frequency of transformation was frequently greater than that of the homologous DNA. Accompanying this increased efficiency was a marked change in the physiology of the cells. The growth rate of the transformants carrying this intergenote was approximately one-half that of either parental strain. Thus, in a prokaryotic transformation system, adverse side effects can occur after incorporation of a segment of foreign DNA.
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Two linkage groups were established in Bacillus pumilus NRRL B-3275 by bacteriophage PBS1 transduction. Group L contains the auxotrophic markers gly, his, and met. Group M contains the markers argO, met, ura, and cys.
Development of competence in the noncompetent mutant of Bacillus stearothermophilus 4S requires a soluble competence-inducing factor (CF). This study describes several observations that define the mechanism of action of this factor. (i) CF interacts with cells, protoplasts, or cytoplasmic membranes in a temperature-dependent reaction; incubation of CF with cells alters the sedimentation properties of the cells. (ii) Association between CF and the cytoplasmic membrane appears to be the first and rate-limiting step in the process of transfection with deoxyribonucleic acid (DNA) from bacteriophage TP-IC. (iii) The membrane-CF complex is capable of removing infectious DNA from the supernatant fraction in vitro. (iv) Twenty phage-resistant mutants are unable to undergo the above reactions. A model is developed in which CF serves as a dynamic mediator of the reaction between the transfecting DNA and the cell surface.
Polyglycerolteichoic acid:glucosyl transferase (TAG transferase), one of the three enzymes involved in the pathway leading to the glucosylation of teichoic acid in Bacillus subtilis 168, was investigated. During the early stages of the growth of B. subtilis, TAG transferase is predominantly a soluble enzyme found in the cytoplasm. As growth proceeds, the amount of soluble enzyme decreases and the proportion of insoluble, membrane-bound TAG transferase increases, reaching a maximal value at the close of the logarithmic phase. Data are presented which suggest that these are two forms of the same enzyme, or have some common component. The effects of chaotropic agents, such as sodium trichloroacetate and sodium perchlorate, on the cytoplasmic membrane were also studied. These data show that such compounds can effectively remove the TAG transferase from the membrane in a water-soluble form. A study of some of the physical properties of this solubilized enzyme suggests that there is little difference between the two forms of the enzyme. Experiments are described which indicate that the glucosyl transfer by both the membrane-bound and soluble enzymes is not mediated by lipids.
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