Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus.

A Lyt- mutant with reduced autolytic activity was isolated after Tn551 mutagenesis of the methicillin-susceptible Staphylococcus aureus laboratory strain RN450. The Lyt- phenotype could be transferred back into the parent and into a variety of other S. aureus strains by transduction of the transposon marker. Southern analysis has located the Tn551 insert to a 3.2-kb HindIII DNA fragment on the SmaI B fragment of the staphylococcal chromosome. The Lyt- phenotype included reduced rates of cell wall turnover and autolysis induced by detergent or methicillin treatment; however, the rate of methicillin-induced killing was not affected. Peptidoglycans prepared from the parental and mutant cells showed identical muropeptide compositions, as resolved by a high-resolution high-pressure liquid chromatography technique. On the other hand, LiCl extracts of the mutant cells contained reduced amounts of total protein and lower specific cell wall-degrading activity compared with those of extracts of parental cells. The profile of bacteriolytic enzymes as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple band differences between mutant and parental cells; a major lytic band with properties characteristic of the staphylococcal endo-beta-N-acetylglucosaminidase was completely absent from the Lyt- cells. The Lyt- phenotype transduced into a series of methicillin-resistant strains of both homogeneous and heterogeneous phenotypes caused only a modest decrease in the level of methicillin resistance, as determined by population analysis.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Isolation and characterization of a bacteriophage T5 mutant deficient in deoxynucleoside 5''-monophosphatase activity.

A bacteriophage T5 mutant has been isolated that is completely deficient in the induction of deoxynucleoside 5'-monophosphatase activity during infection of Escherichia coli F. The mutant bacteriophage has been shown to be deficient in the excretion of the final products of DNA degradation during infection of E. coli F, and about 30% of the host DNA's thymine residues were reinocorporated into phage DNA. During infection with this mutant, host DNA degradation to trichloroacetic acid-soluble products was normal, host DNA synthesis was shut off normally, and second-step transfer was not delayed. However, induction of early phage enzymes and production of DNA and phage were delayed by 5 to 15 min but eventually reached normal levels. The mutant's phenotype strongly suggests that the enzyme's role is to act at the final stage in the T5-induced system of host DNA degradation by hydrolyzing deoxynucleoside 5'-monophosphates to deoxynucleosides and free phosphate; failure to do this may delay expression of the second-step-transfer DNA.     

Isolation of a temperature-sensitive dnaA mutant of Staphylococcus aureus

Of 750 temperature-sensitive mutants of Gram-positive Staphylococcus aureus, one was complemented by the dnaA gene. This mutant had a single base transition in the dnaA gene causing the amino-acid substitution mutation, Ala40Thr. Phage transduction experiments showed that this temperature-sensitive phenotype was linked with a drug-resistant marker inserted near the dnaA gene, suggesting the dnaA mutation is responsible for the phenotype. Flow cytometric analysis revealed that the dnaA mutant was unable to initiate DNA replication at a restrictive temperature and exhibited asynchrony in the replication initiation at a permissive temperature. This is the first report of a temperature-sensitive dnaA mutant in S. aureus, and the results show that DnaA is required for the initiation of chromosomal replication and for the regulation of synchrony in the bacterial cells.     

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity.

A mutant of Neurospora crassa (pm-nbg27) was isolated on the basis of its resistance of p-fluoro-phenylalanine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport activity of all amino acids. Genetic analysis of pm-nbg27 by crossing it to wild-type (74A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types. The above observations confirm the postulated general amino acid permease system as well as a single genetic locus control of that activity.     

Autolysis of microbial cells: salt activation of autolytic enzymes in a mutant of Staphylococcus aureus

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.     

Isolation and characterization of an Escherichia coli mutant deficient in dTMP kinase activity

Escherichia coli LD0181 is sensitive to 15 micrograms of 2',3'-dideoxythymidine per ml. A derivative that was resistant to 40 micrograms of the same chemical per ml at 30 degrees C and that had lost the ability to grow on enriched medium at 42 degrees C was isolated after nitroso-guanidine mutagenesis. This mutant, TD105, produced a dTMP kinase with 25-fold lower specific activity and a 5-fold higher Km for dTMP than the parental strain. The dTMP pool in TD105 was 4.4-fold higher than in the parent. In addition to temperature sensitivity and resistance to 2',3'-dideoxythymidine, the mutant exhibited a hypersensitivity to 5-bromo-2'-deoxyuridine. All three of these phenotypes are cotransducible. The tmk gene was mapped by cotransduction to approximately 30 min on the E. coli map.     

Isolation of a yeast mutant deficient in pyruvate carboxylase activity

To improve our understanding of the catalytic mechanism and regulatory properties of pyruvate carboxylase (EC 6.4.1.1), an important biotin-dependent enzyme, we have sought to isolate mutants in Saccharomyces cerevisiae which are defective in pyruvate carboxylase activity. One mutant was isolated which was unable to grow on glucose minimal medium unless supplemented with aspartate. Although the enzyme had only 25% of the wild type pyruvate carboxylase activity, Western analysis and RNase protection analysis demonstrated that the mutant gene was expressed at approximately 70% of the wild type level. On the basis of genetic crosses and complementation tests, we have attributed the defect to mutations in the PYC gene encoding pyruvate carboxylase.     

Isolation and characterization of lysozyme-sensitive mutants of Staphylococcus aureus.

Four lysozyme-sensitive mutants were isolated after nitrosoguanidine mutagenesis of a lysozyme-insensitive strain of Staphylococcus aureus. One mutant was sufficiently effective for the isolation of macromolecules, such as plasmid deoxyribonucleic acids, from a cell after lysozyme-induced cell lysis.     

Isolation and characterization of a mutant of Salmonella typhimurium deficient in a major deoxyribonucleic acid polymerase activity

A mutant of Salmonella typhimurium strain LT2 that is deficient in a major deoxyribonucleic acid (DNA) polymerase activity has been isolated and characterized. This mutant resembles the pol mutants of E. coli in that it has low DNA polymerase activity and it is sensitive to methyl methane sulfonate as well as ultraviolet irradiation. Revertants selected for methyl methane sulfonate resistance are no longer sensitive to ultraviolet irradiation and contain normal DNA polymerase levels. No direct role in replication can be ascribed to this polymerase activity since cells grow well in its absence. In addition, the LT2 plasmid has been shown to exist in the mutant strain.     

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Isolation of a Saccharomyces cerevisiae mutant strain deficient in deoxycytidylate deaminase activity and partial characterization of the enzyme.

Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.     

Isolation and characterization of a DNA probe for Staphylococcus aureus subspecies aureus biovar

The gene encoding for polynucleotide phosphorylase (pnp) of a new biovar of Staphylococcus aureus subsp. aureus (NBSA) has been isolated from a genomic library of strain M280(0). The coding region consisted of a 1094-bp HindIII-HindIII DNA fragment encoding for a protein of 277 amino acids with a calculated molecular mass of 29.5 kDa. The nucleotide sequence of the structural gene, contained a continuous open reading frame of 836 bp, showed significant homology with the genes of bacterial polynucleotide phosphorylase from Bacillus subtilis (67.7% identity), from Haemophilus influenzae (62.4% identity), from Pseudomonas luminescens (61.6% identity), and from Escherichia coli (59.7% identity). DNA-DNA and DNA-colony slot-blot hybridizations demonstrated that the pnp gene, employed as a molecular probe, is specific for the identification of NBSA strains.     

Isolation and characterization of a mutant of Neurospora crassa deficient in general amino acid permease activity

A mutant of Neurospora crassa (pm-nbg²⁷) was isolated on the basis of its resistance to p-fluoro-phenylalamine on ammonium-deficient Vogel's medium. This mutant was found to be devoid of both conidial and post-conidial (after 180 min of preincubation) transport acitvity of all amino acids.Genetic analysis exhibiting of pm-nbg²⁷ by crossing it to wild-type (74^A) resulted in the predicted segregants exhibiting transport characteristics of pm-n, pm-b, pm-g, pm-nb, pm-ng, pm-bg and parental types.The above observations confirm the postulated general amino acids permease system as well as a single genetic locus control of that activity.     

Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum

In order to facilitate genetic engineering in amino-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.     

Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus.

Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate of Staphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 X 10(6) and 0.75 X 10(6) daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 X 10(6) dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entB- transformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 X 10(6)-dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 X 10(6)-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.     

Lower autolytic activity in a homogeneous methicillin-resistant Staphylococcus aureus strain compared to derived heterogeneous-resistant and susceptible strains

It has been proposed that in addition to production of a penicillin-binding protein with low affinity for beta-lactam antibiotics, control of autolysin activity is involved in the mechanism of staphylococcal methicillin resistance. A homogeneous methicillin-resistant Staphylococcus aureus strain (DU4916) had lower rates of unstimulated, NaCl- and Triton X-100-stimulated autolysis, and daptomycin (LY146032)-induced lysis than a heterogeneous methicillin-resistant strain (DU4916-K7) and a methicillin-susceptible strain (DU4916S) derived from DU4916.