- Volume 129, Issue 2, 1983

An autolysin-deficient mutant of Bacillus subtilis was completely tolerant to 5 h incubation with 50–100 µg cycloserine ml−1whereas the wild-type was rapidly lysed and killed by 12 µg ml−1. Lysis also did not occur when low concentrations of β-lactams were added to exponentially growing cultures of the mutant, but over 90% of the bacteria were killed within 90-120 min. Protein, lipid and peptidoglycan synthesis as well as growth were inhibited after about 60 min. At this time, but not earlier, small amounts of these three cell components appeared in culture supernatants. Earlier, at about 20-30 min, the intracellular pools of amino acids started to decline rapidly and there was a temporary apparent increase in the rate of lipid synthesis. Neither of the latter phenomena occurred with cycloserine, with which protein and lipid synthesis declined only slowly and the rate of peptidoglycan synthesis was 80% inhibited within 30 min. Only occasional cells with damaged walls were seen 30–90 min after addition of either β-lactams or cycloserine to the cultures. It thus seems unlikely that wall hydrolysis or penetration by residual autolysins in the mutant are responsible for mass cell death caused by the β-lactams.

Earlier studies have suggested the possible role of host autolytic enzyme in the release of progeny phage from Dp-1 infected pneumococci. Several new experiments described here reinforce this notion. Specifically, the resistance of an autolysis-defective mutant to infection at low phage to cell ratios could be eliminated by prior ‘coating’ of the host bacteria with pneumococcal autolysin isolated from wild-type cells. Similar, productive infection was also possible by lowering the temperature of incubation to 30 °C, a condition that leads to a partial activation of the thermosensitive residual autolysin in the mutant cells. Other experiments, however, clearly indicate the role of the newly discovered phage-associated lysin (PAL), reported in the accompanying communication, in bacteriophage release and culture lysis; specifically, lysis was stimulated by reducing agents and inhibited by cardiolipin. It seems that both the host-related and the PAL activities are involved with Dp-1 induced lysis of pneumococci.

A phage-associated murein hydrolase activity capable of degrading pneumococcal cell walls was isolated and purified to homogeneity from the phage-induced lysate of an autolysis-defective pneumococcal mutant infected with the bacteriophage Dp-1. Some properties of the enzyme resembled those of the wild-type (host) pneumococcal murein hydrolase: cell walls prepared from ethanolamine-grown pneumococci were resistant to the enzyme; the activity was inhibited by the Forssman antigen and was sensitive to proteolytic enzymes. The phage-associated enzyme was not inhibited by antiserum prepared against the purified pneumococcal murein hydrolase; the activity was stimulated by reducing agents and was partially inhibited by cardiolipin. The subunit molecular weight of the phage-associated enzyme was somewhat smaller (31000) than that of the pneumococcal hydrolase (35000). This appears to be the first description of a phage-associated murein hydrolase activity in pneumococci.

The wild-type strain of Streptomyces glaucescens produces hydroxystreptomycin and shows an inherent natural resistance to streptomycin group aminoglycoside antibiotics. Cell free extracts of the wild-type strain were able to inactivate streptomycin, hydroxystreptomycin and dihydrostreptomycin in the presence of ATP. The phosphotransferase did not inactivate other aminoglycoside antibiotics, including spectinomycin.

Mutant strains were isolated, which were highly sensitive to streptomycin group aminoglycosides, had no measurable phosphotransferase activity and were unable to form detectable amounts of hydroxystreptomycin. This suggests either a correlation between phosphotransferase activity, streptomycin resistance and hydroxystreptomycin formation or defects in more than one gene in the strS mutant strains tested.

A new class of temperature-sensitive mutants defective in spore germination (termed spg) was isolated and characterized. The mutants germinate normally at 19 °C but are defective at 27 °C. Analysis of the germination of these mutants at the non-permissive temperature after periods of incubation at 19 °C shows that the temperature-sensitive step in germination occurs somewhere between 60 and 120 min at 19 °C. Activated mutant spores can be deactivated by incubation at 27 °C prior to shift to the permissive temperature. The mutants are not temperature-sensitive for growth or development.

A method is described for the separation of the outer membrane (OM) from the cytoplasmic membrane (CM) of Pseudomonas cepacia grown in nutrient broth and in chemically defined media under different nutrient depletions. The method is particularly valuable since it is effective when applied to stationary phase cells. Enzyme activities indicated that the contamination of the OM with the CM was less than 5%. The OM protein profile of magnesium-depleted cells was much simpler than that of the iron-depleted and nutrient broth grown cells. The apparent molecular weights of the OM proteins of magnesium-depleted cells were: 40000, 36000, 24500 and 14500. Iron depletion induced the synthesis of an OM protein with apparent molecular weight of 66000. The OM proteins with apparent molecular weights of 40000, 36000 and 24500 were heat-modifiable and the 24500 dalton protein was found also to be affected by the presence of 2-mercaptoethanol. The OM consisted of 50% protein and 20% phospholipid and the rest was probably LPS while the CM consisted of 80% phospholipid and 20% protein. The major phospholipid in both membranes was phosphatidylethanolamine with a smaller amount of phosphatidylglycerol and a trace amount of phosphatidylcholine; the OM contained more phosphatidylethanolamine than the CM.

SUMMARY: λgt WES derivatives carrying the citG (fumarase) gene of Bacillus subtilis have been detected by complementation of an Escherichia coli fumarase mutation in lysogen-filled plaques. The gerA gene, mutations in which affect the germination response of spores to l-alanine, is also present on the two citG transducing phages described. The cloned regions in these two phages include EcoRI-generated fragments of 5·0 kb and 1·4 kb; the former fragment carries citG4 +.

SUMMARY: P1Kc-mediated transduction of Escherichia coli trp genes occurred at a frequency of about 10−8 in Salmonella typhimurium trp strains carrying mutations determining sensitivity to P1 and a low level of restriction enzymes. Heterospecific transductants were analysed by using them as donors in second-stage transductions mediated by bacteriophage KB int. One class of heterospecific transductants had the phenotype Trp+ Pro− but were extremely unstable and reverted at high frequency (up to 80%) to the parental phenotype. The Trp+ Pro− phenotype probably represents insertions of the E. coli trp genes in the S. typhimurium pro genes. It was stable in a RecA background.

Mutants of Escherichia coli K12 defective in the gene iex (crr) no longer utilize glucose or N-acetylglucosamine in preference to lactose, but competition between either of these sugars and another that also enters by a phosphotransferase (PT) mechanism is not affected. In this they differ from gsr (tgs) mutants. In gsr mutants, glucose does not exclude any other sugar, though N-acetylglucosamine still does so. In gsr mutants that are also ptsM the phosphoenolpyruvate-dependent phosphorylation of glucose or methyl α-glucoside is reduced by 90%: N-acetylglucosamine phosphorylation is not affected. The iex mutation does not affect the phosphorylation of either of these compounds. The wild-type alleles iex + and gsr + are dominant in λ heterozygotes. Glucose inhibits the lactose permease of wild-type cells, but only when the permease is present in low amounts. The inhibition is also relieved (1) by induction of another transport system that is subject to regulation by the iex system or (2) by an iex mutation. We suggest that the iex gene specifies a protein that, in cells transporting certain sugars by a PT mechanism, acts to inhibit active transport systems. The protein is present in limiting concentration in the cell, sufficient only to inhibit the basal, uninduced, level of the active transport systems. In consequence the inducer (or its precursor) may be excluded from the cell and induction thus prevented.

A lysogen of Escherichia coli K12 with λ cI857 S7 xis6 nin5 b515 b519 integrated into ptsI was induced and the lysates plated on a Pel− host [on which λ strains with less than the wild-type amount of DNA form plaques at low frequency ( Cameron et al., 1977 )]. All of the 40 plaques examined contained phage able to transduce at least two of the genes known from bacteriophage P1 transduction experiments to be closely linked to ptsI. Assuming that each specialized transducing phage arose by a single illegitimate recombination event, the distribution of phage types showed that the gene order is cysA gsr ptsI (ptsH, iex) cysZ lig; both gsr + and iex + were dominant. Analysis of restriction endonuclease digests of the transducing phage confirmed that no unexpected DNA rearrangements had taken place and allowed the construction of a map of the sites of action of the restriction endonucleases EcoRI, HindIII, BamI and Kpn for over 20 kilobases of E. coli DNA.

In an Appendix, we show cysA and cysZ mutants to be deficient in sulphate assimilation.

Methods are described that allow extraction of high molecular weight DNA from germinated conidia of Neurospora crassa. By labelling DNA with ribonucleosides, early conidia were shown to be active in DNA synthesis. These cells when treated with the enzyme Zymolyase became fragile and could be readily lysed with ionic detergents to release high molecular weight DNA.

The DNA extracted from Zymolyase treated cells on to alkaline sucrose gradients sedimented as a heterogeneous species of up to 150 ×106 molecular weight. A minor DNA species (presumably mitochondrial) of 20×106 molecular weight comprised 2-7% of the total. The identity of the DNA was confirmed by sensitivity to DNAase, the diphenylamine assay and TLC. Sedimentation patterns were unaffected by protease digestions and no anomalous high speed rotor effects were evident. Isopycnic gradients suggested that the DNA released was uncomplexed with either protein or carbohydrates. Sepharose chromatography of extracted, RNAase-treated Zymolyase lysate resulted in clearly separate high molecular weight DNA and RNA-protein elution profiles.

UV light preferentially inhibited nuclear DNA synthesis and drastically reduced the size and amount of nascent DNA being synthesized in the excision defective uvs-2 mutant. Sites in parental DNA sensitive to Micrococous luteus UV endonuclease were measured in cells made permeable with Triton X-100.

Streptomyces glaucescens strain GLA0 (= ETH 22794) produces hydroxystreptomycin and has a high natural resistance to hydroxystreptomycin, dihydrostreptomycin and streptomycin. The wild-type strain gives rise spontaneously to streptomycin-sensitive (StrS−) variants at a frequency of 0.2 to 1.4%. These mutants lack streptomycin phosphotransferase activity responsible for the wild-type resistance to streptomycin group antibiotics and are unable to produce detectable amounts of hydroxystreptomycin.

Mapping experiments showed that the strS marker lies between the chromosomal markers lys-2 and ura-3 on the linkage map of S. glaucescens. The molecular basis for instability of this marker is as yet unknown.