- Volume 129, Issue 8, 1983

Benzylpenicillin-resistant mutants of Clostridium perfringens have been isolated by in vitro selection. The sole mechanism of resistance was a decreased affinity of the highest molecular weight penicillin-binding protein (PBP1) for the antibiotic.

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0·2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1·9 and a s 0·5 of 0·6 mm, whereas in cell extracts, it had a Hill coefficient of 1·9 and a s 0·5 of 1·0 mm. The K m for ATP was 1·6 mm in cell extracts and 1·8 mm in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (s 0·5 of 2·3 mm, Hill coefficient of 4·0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.

Using [3H]corticosterone as a probe, corticosteroid-binding protein (CBP) was detected in eight out of eight isolates of Candida albicans, of both A and B serotypes. The apparent dissociation constant (K d) in the various isolates ranged between 8 and 19 nm; the binding capacity varied from 122 to over 2400 fmol (mg cytosol protein)−1. There was no correlation between the amount or affinity of CBP and isolate virulence for murine hosts. Further analysis revealed demonstrable CBP in six out of six Candida species other than C. albicans. One isolate of C. tropicalis has been identified which fails to bind [3H]corticosterone. Saccharomyces cerevisiae, Neurospora crassa and Paracoccidioides brasiliensis also failed to bind [3H]corticosterone. Preliminary attempts were made to determine functions mediated by CBP in Candida, but in vitro growth, phase conversion and glucose oxidation by Candida were unaffected by the addition of a variety of steroid hormones. These data indicate that the presence of CBP in Candida does not correlate with either virulence or serotype. The physiological significance of CBP remains to be determined.

Rhodopseudomonas palustris was grown on benzoate, cyclohexanecarboxylate or succinate under anaerobic or aerobic conditions. Studies of oxygen uptake by intact bacteria indicated that cyclohexanecarboxylate was metabolized aerobically by a β-oxidation sequence and that cultures grown anaerobically on benzoate also possessed this capacity. Bacteria grown on succinate were able to oxidize octanoate but not alicyclic acids. The enzymes necessary for the β-oxidation of cyclohexanecarboxylate appeared to be constitutive in both anaerobic and aerobic bacteria, the only exception being an acyl-CoA synthetase which used benzoate and some alicyclic acids as substrates. This acyl-CoA synthetase differed from the constitutive short-chain fatty acyl-CoA synthetase in that it was induced by anaerobic growth on benzoate or by aerobic growth on cyclohexanecarboxylate.

Taurine has been reported to be a component of the capsular polysaccharide of the encapsulated M strain of Staphylococcus aureus. This led to a study of the uptake and metabolism of [1,2-14C]taurine in a variety of encapsulated and unencapsulated S. aureus strains. Taurine was taken up by all strains studied. A discrepancy between uptake measured as depletion of radioactivity from growth medium and as cell-associated radioactivity suggested that taurine may be catabolized to CO2 in some strains. In most strains, cell-associated radioactivity was located mainly in cold TCA-soluble (pool metabolites) fractions. About 90% of the cell-associated radioactivity was present in the pool metabolites fraction in the M strain, and about 10% in hot TCA-soluble (nucleic acid-teichoic acid-capsular polysaccharide) fraction. Radioactivity in spent medium and the capsular polysaccharide-containing fraction appeared to be present as taurine in this strain. Radioactivity in the pool metabolites fraction of three of the strains examined did not chromatograph as taurine, indicating that taurine was converted into other cell metabolites. One strain incorporated radioactivity from taurine into cellular macromolecules, thus revealing a heterogeneity of staphylococcal taurine metabolism.

The presence of a plasmid conferring resistance to penicillin (PC plasmid, e.g. pI258 blaI−) in Staphylococcus aureus NCTC 8325 increases the sensitivity of such a bacterium to the growth inhibitory effects of linoleic acid, whereas a plasmid conferring resistance to tetracycline does not affect linoleic acid sensitivity. The increased linoleic acid sensitivity of bacteria containing a PC plasmid may be related to the penicillinase protein itself since (i) strains having inducible penicillinase show increased sensitivity only after induction, (ii) strains in which penicillinase is directed from chromosomal or plasmid-borne genes show similar increased linoleic acid sensitivity and (iii) notwithstanding the above, the linoleic acid inhibitory effect is enhanced in a strain in which penicillinase activity is greatly reduced by a point mutation in the structural gene for penicillinase. The enhanced linoleic acid sensitivity seems to require the membrane-bound penicillinase since added extracellular penicillinase does not confer this sensitivity, and there appears to be a specific interaction between the membrane-bound penicillinase activity and linoleic acid.

Forespores of Bacillus subtilis 60015 were isolated from sporulating cells at t 5 and were incubated with an amino acid mixture containing [14C]phenylalanine. Three species of 14C-labelled cytoplasmic proteins synthesized in the forespores were purified to homogeneity by gel filtration in the presence of detergents and by ion-exchange chromatography. The molecular weights of the purified proteins, called A, B and C, were estimated by SDS-PAGE as 24200, 11500 and 12700, respectively. Protein B contained remarkably high amounts of tyrosine (23·8%) and alanine (22·8%), and proteins A and C contained relatively high amounts of glutamate + glutamine, glycine, and alanine (10·4 to 13·3%). The synthesis of these proteins can provide markers for the control events in the forespore compartment during sporulation.

Density labelling with 2H2O has been used in association with isopycnic centrifugation to isolate proteins of known age from cultures of Escherichia coli. The physical properties of protein samples of known age have been examined to detect a correlation between specific properties and susceptibility to degradation. No evidence of a correlation between size, charge, thermodynamic properties or amino acid composition, on one hand, and susceptibility to degradation, on the other hand, was observed following step-down conditions of growth. However, the SH content of proteins in E. coli does appear to be correlated with their susceptibility to degradation.

Extracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exolarge penicillinase (mol. wt, 30500; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.

The biosynthesis of wax esters by the psychrophilic bacterium Micrococcus cryophilus has been investigated using radio-gas chromatographic analysis of the constituent fatty acid and fatty alcohol moieties. Degradation studies, using a;-oxidation, and the kinetics of labelling from radioactive precursors show that wax ester fatty alcohols are derived from fatty acids. Based on different labelling patterns in wax esters and phospholipids formed from acetate or a saturated fatty acid supplied exogenously, two pathways of wax ester biosynthesis are proposed. It is suggested that wax esters can be derived in the main from either a cytoplasmic or a membrane-bound pool of fatty acyl-coenzyme A thioesters and alcohols, depending on whether the fatty acid is synthesized endogenously or is supplied exogenously.

A strain of Pseudomonas fluorescens that had been isolated from a freshwater source on a plastic substratum was grown in continuous culture in minimal medium. The ‘adsubble’ process (adsorptive bubble separation process) was found to foam-fractionate wild-type cells from the fermenter during flow conditions. This selection pressure favoured the enrichment of two major classes of mutant, both having cell surface characteristics fundamentally different from the wild-type. The wild-type produced very little extracellular polysaccharide, whereas a ‘mucoid’ mutant, found predominantly in the aqueous-phase, produced an alginate exopolymer. The second class of mutant was isolated from the walls of the fermenter and, like the wild-type, produced little exopolymer. This mutant, with crenated colony morphology, showed increased attachment to solid surfaces compared to the wild-type and mucoid cells when assayed for attachment to polystyrene surfaces for 2 h. Outer-membrane protein, lipopolysaccharides and exopolysaccharides of the wild-type and both mutants were analysed. The results demonstrate the role of cell surface characteristics in the adaptability of the organism to micro-environments such as a solid/liquid or air/liquid interface or the aqueous phase.

A mutant of Deinococcus (formerly Micrococcus) radiodurans (strain 302, mutant in mtcA) sensitive to both the lethal effect of mitomycin C and the mutagenic effect of simple alkylating agents, but having wild-type resistance to UV light, was treated with the mutagen N-methyl-N′-nitro-N-nitrosoguanidine in an attempt to isolate strains deficient in the ability to excise UV-induced pyrimidine dimers. Three strains were isolated that were UV-sensitive, but had wild-type resistance to the lethal effect of methyl methanesulphonate and all were shown to be unable to excise pyrimidine dimers. The three strains UVS9, UVS25 and UVS78 had, in addition to the mutation in mtcA, mutations in loci designated uvsC, uvsD and uvsE, respectively. When the mutant mtcA gene was replaced by its wild-type allele in all three strains they became UV- and mitomycin C-resistant. On incubating the double mutants UVS9, UVS25 and UVS78 with wild-type DNA about 50% of the transformants selected for UV resistance were mitomycin C-sensitive and about 50% resistant depending on whether the mutant mtcA or the uvsC, D or E genes had been replaced by their wild-type alleles. Although strains mutant singly in uvsC, D or E were UV-resistant the rates of excision of pyrimidine dimers differed between them and was slower in all of them than in the wild-type and strain 302. The results indicate that wild-type D. radiodurans possesses two pathways for the excision of pyrimidine dimers and that mutational blocks in both must exist for the excisionless phenotype to be expressed.

Hybridization experiments were carried out between strains of the myxomycete Physarum polycephalum which differed in their sensitivities to the cations Na+, K+, Mg2+ and Ca2+ as determined by the threshold for changes in membrane potential on application of these ions. The sensitivities were explicable on the basis of control by three genes (mon, mag and cal) for monovalent cations, magnesium and calcium, respectively, with low sensitivity dominant. Departures from the expected ratios for three unlinked loci are explicable by high sensitivity for divalent cations reducing viability, but the additional possibility that high divalent cation sensitivity cannot be expressed in the presence of low monovalent cation sensitivity has not been excluded. Chemotactic thresholds corresponded with membrane potential thresholds.

Triploid and aneuploid strains of the xylose-fermenting yeast Pachysolen tannophilus were constructed. This species is a strongly homothallic organism in which the haploid phase normally predominates. The technique for producing polyploids involved prototrophic selection and interruption of the normal sequence of events leading from nuclear fusion to meiosis. Confirmation of triploidy was obtained by tetrad analysis. The haploid chromosome number of P. tannophilus was estimated to be between 5 and 7.

Increasing chromosome number of Pachysolen tannophilus above the haploid level increased the yield of ethanol from d-xylose. There was a large increment on going from the haplophase to the diplophase, and the highest yields were obtained with either a triploid or a probable tetraploid, depending on the concentration of d-xylose. In addition, the rate of ethanol production from d-xylose and d-glucose increased, with the increment being larger on d-xylose. On d-galactose, the amount of ethanol produced within a given time also increased. The level of a by-product from d-xylose, xylitol, decreased on going from a haploid to higher ploidy, but there was no discernible trend for two other by-products, acetic acid and arabinitol. Increasing ploidy increased growth rate on d-galactose, but not appreciably on d-xylose. The altered properties on D-xylose of the strains tested were not due to increased levels of either d-xylose reductase or xylitol dehydrogenase, nor probably of alcohol dehydrogenase.

Transformation of Rhizobium japonicum CB1809 was studied using DNA from the cyanobacterium Nostoc muscorum ATCC 27893. A spontaneous nitrate reductase deficient (Nar−) mutant (NR-6) of R. japonicum CB1809 was isolated with a frequency of 8·4 × 10−7. Streptomycin (Sm) and Neomycin (Neo) resistance markers were introduced into strain NR-6, and the resulting strain was designated NR-6 SmR NeoR. Experiments with cyanobacterial DNA and live cells of strain NR-6 SmR NeoR indicated transformation of nitrate reductase (nar) genes of N. muscorum into this strain. This conclusion was supported by the reversion frequency of strain NR-6 SmR NeoR to Nar+ and the transformation frequency when recipient cells were exposed to N. muscorum DNA (with heat-treated DNA as control). Comparisons of growth, nitrate uptake, assimilatory nitrate reductase activity and nodulation of parent CB1809, NR-6 SmR NeoR and five transformant clones (Nar+) suggest that there may be considerable homology between the nar genes of R. japonicum CB1809 and N. muscorum.

Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pIDl, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix− phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.