Possible intermediates in the biosynthesis of mycoside B by Mycobacterium microti.

Two lipids were isolated from Mycobacterium microti which became labelled when the cells were grown in the presence of [2-14C]propionate. They were purified by thin-layer chromatography and studied by chemical degradation and mass spectrometry. The lipids were identified as phenolphthiocerol dimycocerosate and phenolphthiodiolone dimycocerosate, the aglycosyl derivatives of mycoside B, the phenolic glycolipid produced by M. microti. Cell-free extracts of the organism were able to glycosylate the lipids to form mycoside B in vitro. It is probable that the lipids are intermediates in the biosynthesis of phenolic glycolipids by mycobacteria.

The bactericidal action of beta-lactam antibiotics on an autolysin-deficient strain of Bacillus subtilis.

An autolysin-deficient mutant of Bacillus subtilis was completely tolerant to 5 h incubation with 50-100 micrograms cycloserine ml-1 whereas the wild-type was rapidly lysed and killed by 12 micrograms ml-1. Lysis also did not occur when low concentrations of beta-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 beta-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 beta-lactams.

Double mutants of Bacillus subtilis growing as helices.

Double mutants of Bacillus subtilis, deficient in autolysin and rod in genotype, grow as helical filaments of unseparated cells when changed from the cocci to rods.

Temperature-sensitive nature of the rodB maturation in Bacillus subtilis.

The Mg2+ requirement of a morphological mutant of Bacillus subtlis, rodB strain 104 was highly temperature sensitive in the presence of halide or nitrate anions. Likewise the morphological change from rod shapes to spheres was dependent upon temperature, the same anions, and the Mg2+ concentration. The three factors interacted. Other rodB mutants behaved similarly. If the rodB strain 104 in its rod form was treated at high temperatures in the absence of either protein or peptidoglycan synthesis and restored to lower temperatures with the syntheses restarted, a partial temporary change toward cocci occurred. In the absence of halides or in the presence of Cl- but not Br-, the cells increased in volume when they changed from rods to cocci.

Magnesium and anion requirements of rodB mutants of Bacillus subtilis.

rodB mutants of Bacillus subtilis have been found to require several hundred-fold more Mg2+ in a minimal growth medium than the wild type to achieve rapid growth. In the presence of all concentrations of Cl-, the organisms grow as deformed cocci, but with 10 mM Mg2+ and Br-, I-, or NO3- present they grow as rods. The morphology is then directly under the control of the concentration of both Mg2+ and anion. Originally, it was found that L-glutamic acid in the medium brought about the change from deformed spheres to rods. This amino acid will similarly function at a much lower concentration when the higher concentrations of Mg2+ and Cl- are also present. At a constant concentration of L-glutamate, the morphology can be controlled by varying the Mg2+ concentration. In the presence of Mg2+ and I-, the morphological change is temperature sensitive. At 30 C rods are formed and at 42 C deformed cocci are formed. The requirement of a rodB mutant for a high concentration of magnesium and the round morphology have been shown to be most probably due to a single mutation.

Estimates of the porosity of Bacillus licheniformis and Bacillus subtilis cell walls.

The maximum porosity of Bacillus subtilis and Bacillus licheniformis cell walls was estimated by two independent and relatively simple methods. Peptidoglycan was isolated from Bacillus subtilis cell wall preparations and used as an insoluble support for exclusion chromatography of dextrans of known average molecular size. In an alternative approach the leakage of radioactively labelled proteins from Bacillus licheniformis cells incubated in butanol-saline mixtures was measured and their size estimated by exclusion chromatography on Sephadex G-100. Under these conditions the permeability barrier of the cytoplasmic membrane was destroyed with preservation of the structural integrity of the outer cell wall. The apparent exclusion threshold of the cell wall of either organism as determined by these means corresponded to molecules with a diffusional radius of not more than 2.5 nm.

Some structural features of the teichuronic acid of Bacillus licheniformis N.C.T.C. 6346 cell walls.

A teichuronic acid, containing glucuronic acid and N-acetylgalactosamine, was purified from acid extracts of Bacillus licheniformis 6346 cell walls as described by Janczura, Perkins & Rogers (1961). After reduction of the carboxyl function of glucuronic acid residues in the polysaccharide the reduced polymer contains equimolar amounts of N-acetylgalactosamine and glucose. Methylation of the reduced polysaccharide by the Hakamori (1964) technique showed the glucose residues to be substituted on C-4. A disaccharide, 3-O-glucuronosylgalactosamine, was isolated from partial acid hydrolysates of teichuronic acid. After N-acetylation the disaccharide produces chromogen readily on heating at pH7, in agreement with C-3 substitution of the reducing N-acetylamino sugar. Teichuronic acid also produces chromogen under the same conditions, with concurrent elimination of a modified polysaccharide from C-3 of reducing terminal N-acetylgalactosamine residues of the teichuronic acid chains. The number-average chain lengths of several preparations of teichuronic acid were estimated from the amounts of chromogen produced in comparison with the N-acetylated disaccharide. The values obtained are in good agreement with the weight-average molecular weight determined by ultracentrifugal analysis. The reducing terminals of teichuronic acid are shown to be exclusively N-acetylgalactosamine by reduction with sodium boro[(3)H]hydride. The number-average chain lengths of the teichuronic acid preparations were estimated by the extent of in corporation of tritium and are in agreement with values obtained by the other methods.