- Volume 135, Issue 10, 1989

A soluble dimeric haemoprotein, structurally and functionally similar to plant and animal haemoglobins, is found in the Gram-negative aerobic bacterium Vitreoscilla sp., strain C1. Vitreoscilla haemoglobin (VtHb) increases in concentration when the cells are exposed to hypoxic conditions. The globin part of VtHb is encoded by a single gene (vgb). An RNA transcript, approximately 500 bases long, specific for vgb was detected after Northern hybridization. The relative amount of this mRNA increased in cells grown at low levels of oxygen. Two enzymes important for haemoglobin function are δ-aminolaevulinic acid synthase (ALAS), which is necessary for haem biosynthesis, and NADH-methaemoglobin reductase, which is necessary to keep VtHb in the physiologically functional ferrous state. An increase in ALAS specific activity under hypoxic conditions preceded the increased haem production. Cellular reductase content also increased when the VtHb increased in cells grown under hypoxic conditions. The ratio of cellular reductase activity to VtHb content remained relatively constant in cells grown under a variety of conditions. The data suggest that in Vitreoscilla the transcription of the globin gene and the biosynthesis of two enzymes important for VtHb function are regulated by oxygen.

Haloferax gibbonsii strain Ma2.39 produces an inhibitor substance designated halocin H6. This halocin was isolated from culture supernatants of the producer strain. It appeared to be a non-inducible bacteriocin with a bactericidal mode of cation and typical ‘single-hit’ kinetics. The protein was purified by a combination of hydroxylapatite affinity chromatography, gel filtration and HPLC. Halocin H6 is a protein of M r 32000; it is heat resistant, non-salt-dependent (it retains its activity after exposure to distilled water) and sensitive to pronase but not to trypsin.

The disproportionate difficulty in obtaining compelling experimental evidence from 14C-radio-labelling that the indole moiety of the otherwise isoprenoid penitrem A is biosynthesized by Penicillium crustosum directly from tryptophan has been explored, [benzene ring-14C]Tryptophan added to the broth beneath the mycelial mat of stationary liquid cultures labelled penitrem A with 1·4% incorporation, only threefold more than that determined for [methylene-14C]tryptophan or [U-14C]tyrosine, incorporation of which could only have been indirect. In contrast, the substituted tryptophan-histidine diketopiperazine roquefortine, biosynthesized concurrently with penitrems by this organism, was labelled with compelling efficiency (23·4% incorporation of [benzene ring-14C]tryptophan). In submerged culture, Claviceps paspali concurrently biosynthesized an analogous pair of metabolites, 3-hydroxy-3-methylbutenyl paspalinine and lysergic acid α-hydroxyethylamide. This feature enabled experimental demonstratation of [benzene ring-14C]tryptophan incorporation to an extent more consistent with direct contribution of the indole moiety of the indole-diterpenoid paspalinine derivative. The same precursor applied to the sporing surface of P. crustosum stationary cultures also provided stronger evidence for a direct biosynthetic role in the formation of penitrem A. In the absence of competition from any other indolic secondary metabolite, a submerged culture of Penicillium paxilli incorporated 5% of the [benzene ring-14C]tryptophan given during growth into the indole-diterpenoid paxilline. A double-labelling time-course experiment indicated temporal separation of steps in the biosynthesis of roquefortine. The inadequacy of classical precursor techniques for studying biosynthesis of indole-diterpenoids in P. crustosum is discussed. The more homogeneous submerged culture fermentation system is preferred for experimentation.

Lipids from the autotrophic thermophilic archaeobacterium Desulfwolobus ambivalens grown under aerobic and anaerobic conditions were analysed and compared with those of Sulfolobus solfataricus, a related micro-organism. The ether lipids of aerobically and anaerobically grown D. ambivalens, as well as those of S. solfataricus, had the same general features except for the degree of cyclization of the C40 isopranic chains. The quinone content of D. ambivalens was strongly affected by growth conditions. Aerobically grown cells contained caldariellaquinone, 6-(3,7,11,15,19,23-hexamethyltetracosyl)-5-methylthiobenzo[b]thiophen-4,7-quinone (83% of the quinone pool), sulfolobusquinone, 6-(3,7,11,15,19,23-hexamethyltetracosyl)-5-methyl-benzo[b]thiophen-4,7-quinone (16%) and the tricyclic quinone benzo[1,2-b; 4,5-b′]dithiophen-4,8-quinone (trace amounts). In anaerobically grown D. ambivalens sulfolobusquinone was the only quinone present.

Streptococcus salivarius HB and four adhesion deficient mutants, HB-7, HB-V5, HB-V51 and HB-B, were grown in continuous culture in a defined medium under glucose limitation over a range of growth rates from 0·1 to 1·1 h−1. The ability to coaggregate with Veillonella parvula V1 cells and the ability to adhere to buccal epithelial cells did not alter with increasing growth rate. Cell surface hydrophobicity decreased markedly with increasing growth rate for the non-fibrillar non-adhesive mutant HB-B but not for the other four strains which all carry different combinations of fibril classes. The thickness of the ruthenium red staining layer (RRL) also varied with growth rate for strain HB-B, ranging from 19·5 ± 3·8 nm at high growth rate to a minimum of 12·3 ± 4·8 nm at low growth rate. Low cell surface hydrophobicity correlated with a thicker RRL for strain HB-B. Strains HB-V5 and HB-7 also showed a significant increase in RRL thickness at high growth rates although to a lesser degree than HB-B. SDS-PAGE revealed a large number of protein bands common to all strains at all growth rates, with the major common protein occurring at 15·6 kDa. Protein bands at 70, 56, 40·5 and 39 kDa appeared stronger at high growth rates than at low. A protein band at 82 kDa showed strongly only at low growth rates. Therefore, adhesion and coaggregation are not phenotypically variable with increasing growth rate but RRL thickness, hydrophobicity and cell surface proteins may be phenotypically variable depending on the strain.

The surface hydrophobicity of 12 strains of Bacillus spp. was examined in a hexadecane-aqueous partition system. Mature and germinated spores of Bacillus megaterium QM B1551 transferred to the hexadecane layer, while vegetative and sporulating cells did not. Wild-type spores were more hydrophobic than spores of an exosporium-deficient mutant of B. megaterium QM B1551, although the mutant spores were shown to be hydrophobic to some extent by using increased volumes of hexadecane. This result suggests that the exosporium is more hydrophobic than the spore coat and that the surface hydrophobicity of spores depends mainly on components of the exosporium. The surface hydrophobicity of spores of nine other species of Bacillus was also examined, and spores having an exosporium were more hydrophobic than those lacking an exosporium. Thus measurement of the hydrophobicity of spores by the hexadecane partition method may provide a simple and rapid preliminary means of determining the presence or absence of an exosporium.

The acetyl-CoA synthase (acuA) gene from Ustilago maydis has been isolated using a fragment of the acu5 gene from Neurospora crassa as a heterologous probe. The U. maydis acuA gene transformed Acu− mutants of U. maydis to acetate utilization. Transformation was accomplished by integration of the vector sequences into the chromosomal DNA.

Growth of Escherichia coli on glutamate as sole carbon source only occurs in strains carrying mutations that increase the expression of genes encoding glutamate transport systems. From an analysis of mutants able to grow on glutamate we have identified a genetic locus that when mutated elevates the expression of the GltII glutamate/aspartate transport system. The mutants exhibit increased sensitivity to the toxic aspartate analogues cysteate and dl-threo-β-hydroxyaspartate. Data from the analysis of mutants that are impaired in this transport activity are consistent with the presence of the structural gene for the transport system at the same genetic locus. The locus was mapped by P1 transduction to a region of the E. coli chromosome lying at approximately. 92·5 min on the E. coli genetic map.

Haemophilus influenzae type b strains isolated from children with meningitis, septicaemia and pharyngitis were studied for their ability to undergo genetic transformation by two chromosomal markers, streptomycin resistance and nalidixic acid resistance. Fifty-eight percent of the strains were non-transformable while the remaining 42% showed considerable strain variation with regard to their transformation frequencies, which ranged from 8 × 10−4 to 1 × 10−6. The effect of type b capsule on competence development and transformation activity was studied by comparing encapsulated strains with their non-encapsulated variants. Type b capsule did not inhibit either competence development or transforming efficiency. The lack of transformability in the majority of strains was not due to the presence of a capsule.

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3′ end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.

Treatment of female BALB/c mice with oestradiol rendered them susceptible to vaginal colonization by three of four different strains of Mycoplasma hominis. Overall, the organisms were recovered persistently from the vagina of 68 (87%) of 78 of these mice. Strain TO mice given one of the strains were at least as susceptible, all of ten becoming colonized and larger numbers of organisms being recovered. The hormone arrested the reproductive cycle in the oestrous phase, characterized by non-nucleated, cornified vaginal epithelial cells. In contrast, M. hominis organisms were isolated transiently from only seven (10·5%) of 66 BALB/c mice not treated with oestradiol, after intravaginal inoculation; treatment with progesterone, which induced the dioestrous phase of the cycle, did not render any of 10 BALB/c mice susceptible to vaginal colonization. The minimum number of organisms (2·5 × 105) of one strain of M. hominis and the minimum dose of oestradiol (0·05 mg) required to induce persistent colonization were established. Vaginal colonization persisted for more than 200 d in some mice, the numbers of organisms recovered ranging between 101 and 108. At autopsy there was evidence of spread to the uterine horns and ovaries, and also to the oropharynx, of some animals but not to other organs. Infection was not associated with a polymorphonuclear leucocyte response in the vagina or elsewhere, but a fourfold serum antibody response to M. hominis, measured by the metabolism-inhibition technique, was detected in almost half of the mice tested.

Summary: Five distinct glycolipids were readily detected in isolates of Mycobacterium tuberculosis. Spectroscopic methods and chemical degradation techniques allowed the structural identification of four of these glycolipids. The specific phenolic glycolipid antigen previously characterized from the Canetti strain was found in all the strains examined, with identical structural features (triglycosyl phenol phthiocerol dimycocerosate). The other three glycolipids identified were acylated trehaloses: penta-acyl trehalose (containing phthienoyl substituents), tetra-acyl trehalose 2'-sulphate (with C40-C50 hydroxyphthioceranoyl substituents) and diacyl trehalose 2'-sulphate (with C16 and C18 substituents). The two latter glycolipids as well as the phenolic glycolipid immunoreacted with whole-cell antiserum, indicating their surface location. The occurrence of these glycolipid antigens in recent clinical isolates suggests their possible utilization in the serodiagnosis of tuberculosis and the rapid identification of M. tuberculosis with specific antisera.

The involvement of glutamine aminotransferase activity in glutamine catabolism by Saccharomyces cerevisiae under microaerophilic conditions was studied. We were able to show that there are at least two different glutamine aminotransferase activities that are differentiated genetically, by their substrate specificity (pyruvate and glyoxylate dependence), and their different modes of regulation. The pyruvate-dependent glutamine aminotransferase activity plays a major role in glutamine catabolism under microaerophilic conditions since the wild-type strain S288C showed a 10-fold higher activity in static cultures than in agitated ones. The same strain also had 3-fold higher glutaminase B activity in agitated cultures than in static ones. Pyruvate-dependent glutamine aminotransferase activity is not regulated directly by O2 itself since a ρ − strain showed a high activity regardless of the extent of aeration of cultures. Finally, we were able to isolate a mutant, strain CN20, derived from the ρ − strain and unable to utilize glutamine as the sole nitrogen source, which was severely affected in pyruvate-dependent but not in glyoxylate-dependent aminotransferase activity.

Neurospora crassa wild-type is almost unable to grow on glutamine as sole nitrogen and carbon source but a GDH−; GS± double mutant strain, lacking NADP-dependent glutamate dehydrogenase and partially lacking glutamine synthetase did grow. Under these conditions, the double mutant had a higher chemical energy content than the wild-type. Enzyme assays and labelling experiments with glutamine indicated that in the double mutant glutamine was degraded to ammonium and to carbon skeletons by glutamate synthase, the catabolic (NADH-dependent) glutamate dehydrogenase and the glutamine transaminase-ω-amidase pathway.

Validamycin A decreased the biomass and extracellular protein produced by cultures of Rhizoctonia solani A79 grown on medium containing cellulose as the carbon source. In the presence of the antibiotic, the production of inducible enzymes active on filter paper (FPase), carboxymethylcellulose (CMCase) and (cellobiase) was decreased, but not the production of constitutive enzymes involved in hemicellulose (xylanase) and pectin (polygalacturonase) degradation. When incorporated in the assay mixtures, validamycin A did not affect the activity of the above enzymes, but the antibiotic did act as a substrate for cellobiase. Validamycin A did not affect the distribution of cellulase enzymes in stationary phase cultures of R. solani, and incorporation of inositol in the culture medium did not reverse the effect of the antibiotic on cellulase production. Addition of inositol to the medium decreased the activities of FPase and CMCase in the culture supernatant but had no effect on cellobiase activity. The results are discussed in relation to the way validamycin A controls rice sheath blight.