- Volume 129, Issue 11, 1983

Menaquinone-6 (2-methyl-3-farnesyl-farnesyl-1,4-naphthoquinone) and a methyl-substituted menaquinone-6 (2,[5 or 8]-dimethyl-3-farnesyl-faniesyl-1,4-naphthoquinone) were the major isoprenoid quinones found in membrane preparations of Campylobacter jejuni and Campylobacter fetus subsp. fetus. By reverse-phase high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) the faster-eluting menaquinone-6 co-chromatographed with a menaquinone-6 standard. The identity of menaquinone-6 was confirmed by UV spectrophotometry, mass spectrometry and nuclear magnetic resonance (NMR) analysis. The slower-eluting methyl-substituted menaquinone-6 co-chromatographed with a menaquinone-7 standard by reverse-phase TLC but eluted between menaquinone-6 and menaquinone-7 standards by HPLC. The UV spectrum of the rnethyl-substituted menaquinone-6 did not correlate with either authentic menaquinone or demethylmenaquinone. Mass spectra showed an increase of 14 mass units when compared to menaquinone-6, and indicated that a methyl substituent was on the naphthoquinone nucleus. NMR spectra confirmed the presence of a methyl substituent at a peri position (carbon-5 or -8) on the benzenoid ring.

Purified adenosine triphosphatase (F1-ATPase) (EC 3.6.1.3) from Micrococcus lysodeikticus membranes required divalent metal ions for its reattachment to membranes depleted of the enzyme. This requirement showed specificity: Mg2+ ions were able to reconstitute a physiologically significant F1-ATPase-membrane complex, whereas Zn2+ ions produced one differing from the native and the Mg2+-reconstituted ATPase-membrane complexes. Micrococcus lysodeikticus membranes contained a limited number of specific binding sites, which did not seem to be modified after ATPase release and membrane manipulation. The binding properties of F1-ATPase preparations correlated well with their content of δ-subunit. The results suggested that F1-ATPase molecules able to rebind to M. lysodeikticus membranes must contain at least two copies of the δ-subunit. The isolated subunits (particularly α- and β-subunits) showed a certain capacity for rebinding to the membranes, but an enzyme form consisting only of α- and β-subunits was unable to reattach to membranes. These results prove unambiguously that δ-, but not α-or any other polypeptide, was involved in the attachment of F1-ATPase to M. lysodeikticus membranes, unlike other bacterial F1-ATPases. These results were consistent with a subunit stoichiometry and arrangement: α3 β3 γ(1-2) δ2-Mg2+- membrane.

The specific activity of X-prolyl-dipeptidyl aminopeptidase in Saccharomyces cerevisiae grown on glucose-containing medium remains constant during exponential growth and increases less than twofold when cells reach the stationary phase. In cells harvested from exponential growth on glucose-containing medium the specific activity of the enzyme is found to be 20-30% lower than the specific activity observed in media without glucose, containing acetate or ethanol as the carbon source. X-Prolyl-dipeptidyl aminopeptidase is not inactivated after the addition of glucose to stationary phase cells. Growth of the yeast on poor nitrogen sources or under nitrogen-starvation results in a three- to fourfold increase in the level of the enzyme.

A mutant of Aspergillus nidulans, isolated for inability to form asexual spores (conidia) on complete medium, was found to regain the ability to conidiate if the medium was supplemented with arginine On minimal medium the mutant required arginine for growth but at a much lower concentration than that required for conidiation. This mutant, designated argB12, thus defines a phase-critical gene, i.e. a gene whose function is in greater demand for development than for growth. In addition to its aconidial phenotype, the mutant also exhibited (depending on the medium) aberrant sexual development and a low efficiency of conidial germination. In crosses, each of these developmental phenotypes segregated with arginine auxotrophy. Genetic and biochemical analyses showed the argB12 mutation to be an allele of the previously described argB locus, mutants of which lack ornithine transcarbamylase. Arginine-requiring mutants at at least two other loci were also found to be defective in asexual sporulation, but the germination defect appears to be specific to argB mutants.

Gene fusions between the lac structural genes and various genes of the hexuronate system of Escherichia coli K12 were isolated by the technique of Casadaban. Mud(Apr lac) and λplac-Mu insertion mutants were constructed in which the lac genes were fused to the regulatory region of the uxu operon. In all the uxu-lac fusion strains, β-galactosidase expression was shown to be inducible by the natural inducers of the uxu operon (glucuronate and fructuronate) and sensitive to catabolite repression by glucose. In addition we isolated a Mud(Apr lac) fusion where the lac genes were fused to the uxuR regulatory gene. In this fusion the synthesis of β-galactosidase reflects the regulation of the uxuR gene. In the presence of a wild-type uxuR allele, partial repression of β-galactosidase expression was found; the repression was removed when inducer was added. This result indicates that while the uxuR gene is subject to autogenous control, the uxuR repressor may have only a low affinity for its own operator.

Escherichia coli K12 growing anaerobically on l-fucose excretes l-1,2-propanediol as a fermentation product whose formation is catalysed by an inducible NAD-linked oxidoreductase. The activity of this enzyme is highly induced only during anaerobic growth. Three bacterial strains bearing a hybrid operon with the structural genes for lactose utilization (lacZYA) fused to the promoter of the propanediol oxidoreductase gene (fucO) were constructed to test whether or not transcriptional control was involved. In contrast to propanediol oxidoreductase of wild-type cells, β-galactosidase in the ɸ(fuc-lac) strains was induced by fucose to high levels both aerobically and anaerobically. Data from this work are in accord with the previous report that the enzyme protein (assayed by specific antibodies) was induced both aerobically and anaerobically, but that only in anaerobically grown cells was the oxidoreductase catalytically active. In the present study, we found that the oxidoreductase induced anaerobically in wild-type cells remained enzymically active during aerobic growth in the absence of fucose. On the other hand, wild-type cells grown aerobically in the presence of fucose and then allowed limited anaerobic growth on glucose did not gain any oxidoreductase activity. The mechanism of this post-transcriptional control remains to be discovered.

Mutations of the dye gene on the E. coli chromosome result in sensitivity to the dye toluidine blue and, in male cells, cause loss of F pili, producing sterility in conjugation. Compared with its dye + parent, a strain deleted for dye (∆dye) showed an altered sensitivity to a wide range of dyes and antibiotics which affect different intracellular processes, and hence it appeared likely that the barrier properties of the cell envelope were impaired. Unlike mutants known to be defective in LPS structure, there appeared to be no correlation between the hydrophobicity of the compounds and the sensitivity of the ∆dye strain. Moreover there was no difference between dye + and ∆dye strains in their sensitivity to LPS-specific phages, and chemical and GLC analysis of LPS components revealed no difference between the two strains. Examination of outer and inner membrane proteins from isogenic strains having the ∆dye deletion and the dye + gene cloned into the plasmid pACYC184, with or without insertional inactivation of dye by the transposon γδ, was performed by SDS-PAGE. This revealed a number of differences in the profile of proteins from both inner and outer membranes, correlated with mutation in the dye gene. The dye gene appears to be identical to the sfrA gene, which has been shown to be required for efficient transcription of the sex factor F. It is therefore proposed that the dye (sfrA) gene product may also control the expression of chromosomal genes coding for envelope proteins.

Phage j2, a PI-like phage in Salmonella typhi, was heteroimmune to phage PI and existed in the lysogenic state as a plasmid of molecular size 58.6 MDal. The phage j2 plasmid was incompatible with the P1 plasmid (IncY group). A j2-sensitive mutant of Salmonella typhimurium LT2 was isolated by transduction of j2Ap phage into LT2 followed by curing of the prophage. The mutant was used to demonstrate transduction between S. typhi and S. typhimurium by phage j2.

Mutants of Bacillus subtilis 168 were isolated which allow adsorption but not infection by bacteriophage SPP1. The adsorbed phages can be subsequently recovered in active form. From ten other bacteriophages tested, only the SPP1-related phages 41c, 22a, ρ15 and SF6 failed to plate on the mutant cells. The mutation responsible for this behaviour (pha-2) was mapped by PBS1 transduction, showing 95% cotransduction with ald-1.

The uptake and retention of purine bases and of uracil requires both a protonmotive force and intracellular conversion to nucleotides. Inhibition of uptake by arsenate does not imply that ATP is required for the transport process because arsenate caused a rapid fall in the level of 5-phosphoribosyl 1-pyrophosphate. A mutation defective in high-affinity adenine transport has been identified and is designated purP. This mutation has been found to lie in the neighbourhood of mtl. Competition experiments indicate that at least two other systems are used to transport guanine, xanthine and hypoxanthine.

The nuclear (n) and mitochondrial (mt) genomes of the take-all fungus, Gaeumannomyces graminis var. tritici, were examined by reassociation kinetics with Escherichia coli DNA as internal standard. Only one kinetic component was detected in each DNA, with second-order rate constants of 0·022 m −1 s −1 for nDNA and 10·9 m −1 s −1 for mtDNA, corresponding to sequence complexities of 29 × 106 base pairs and 60 × 103 base pairs respectively.

Hybrids were produced by protoplast fusion between strains of Aspergillus rugulosus and mitotic master strains of Aspergillus nidulans with a genetic marker on each linkage group. Analysis of segregants induced by growth on benomyl revealed recombination between every pair of unlinked markers. Parental combinations of markers were often recovered at significantly higher frequencies than expected. This aberrant segregation was not correlated with any particular pair of linkage-groups and was attributed to inter-species incompatibility. The segregation of genetic markers of A. rugulosus from the hybrids suggested that A. nidulans and A. rugulosus may differ in haploid chromosome number and chromosome size. In sexual crosses between A. nidulans and strains containing chromosomes of mixed parental origin recombinants were recovered. The results support the classification of A. nidulans and A. rugulosus as separate species.

A simple, fast, and in situ method of detecting the inapparent infection of cultured cells with mycoplasmas is reported. Animal cells grown on Formvar-coated electron microscopic grids were directly fixed with glutaraldehyde, negatively stained with phosphotungstic acid and examined by transmission electron microscopy. Cells contaminated with mycoplasmas could be discriminated from uncontaminated cells. The micro-organisms in the negatively stained preparations corresponded with those revealed by thin sectioning, and the distribution of mycoplasmas in cultured cells coincided with those revealed by the Hoechst staining method. Most of the highly resolved mycoplasmas were polymorphic, and closely associated with host cells; often more than 500 organisms per host cell were seen.

In contrast to most yeasts, which ferment glucose more rapidly than fructose, Saccharomyces bailii ferments fructose first, then glucose. Thus, in a medium containing fructose and glucose, diauxic growth results. Cells of S. bailii that were grown on fructose were unable to ferment glucose when suspended in a glucose-containing buffer solution. Fructose-grown cells were cryptic for glucose fermentation but contained the enzymes for glucose metabolism. When suspended for 2 h in a growth medium containing glucose, fructose-grown cells acquired the ability to ferment glucose, due to the synthesis of a carrier protein. This induction was prevented by cycloheximide. In S. bailii, fructose was transported into the cells by a constitutive carrier system that was insensitive to uranyl ions. The inducible glucose carrier system was completely inhibited by 10−4 M-uranyl ions. If subsequent metabolism of hexoses was inhibited by iodoacetic acid, the uptake of hexoses could be measured by the increase in their intracellular concentrations. Fructose-grown cells took up only fructose whereas glucose-grown cells possessed an inducible glucose carrier and uptake of both glucose and fructose was observed. A model is proposed to explain the sequential fermentation of fructose and glucose in S. bailii.