- Volume 130, Issue 5, 1984

The respiratory chains of the related phytopathogenic bacteria Pseudomonas cichorii and Pseudomonas aptata were investigated using the technique of electron paramagnetic resonance which readily detects iron-sulphur centres. In the cytoplasmic membranes of both bacteria three ferredoxin-like centres and a centre detected in the oxidized state (g = 2·015) were resolved. In addition, a Rieske-type iron-sulphur centre (g y = 1·90) was observed in P. cichorii but not in P. aptata. The absence of the Rieske centre in P. aptata parallels the absence of cytochromes c in this bacterium.

Candida boidinii grows well on spermidine as sole nitrogen source, but poorly on spermine. Cells grown on spermidine, cadaverine, putrescine and 1,3-diaminopropane contained a polyamine oxidase which attacks spermine and spermidine at the secondary amino groups, forming putrescine and a product thought to be 3-aminopropionaldehyde. The enzyme was synthesized before growth began when C. boidinii that had been grown in medium containing glucose + ammonium was transferred to medium in which spermidine replaced ammonium. Other enzymes increasing in specific activity during this adaptation were catalase, benzylamine oxidase and N AD-dependent glutamate dehydrogenase. The polyamine oxidase was purified to 50% homogeneity, but was too unstable to obtain completely pure. It had a pH optimum of 10.0, and could be stabilized by addition of inert protein. It oxidized spermine, spermidine, N 1- acetylspermidine, N-n-butylpropylamine, di-n-butylamine and di-n-hexylamine. It did not oxidize di-n-propylamine, diethylamine or N 1,N 8-diacetylspermidine. Apparent Km values were determined for the active substrates. The enzyme was potently inhibited by quinacrine and by divalent cations. The stoicheiometry of the enzyme reaction was established using di-n-butylamine as substrate. The enzyme has a molecular weight in the range 80000 to 110000. Putrescine (the oxidation product of spermidine) was not oxidized by cell-free extracts, but evidence of aminotransferase activity was found. The oxidation/transamination product of putrescine, 4-aminobutyraldehyde (1-pyrroline), was oxidized by extracts and a scheme is presented by which spermidine could be catabolized. Polyamine oxidase was shown to co-sediment with NAD-dependent glycerol 3-phosphate dehydrogenase and catalase in sucrose gradients after mechanical breakage of spheroplasts, and is thus a peroxisomal enzyme. Polyamine oxidase was present in some other yeasts when grown on spermidine, C. nagoyaensis, Hansenula polymorpha and Trichosporon melibiosaceum, but absent from C. steatolytica, Pichia pastoris and Sporopachydermia cereana. These latter yeasts probably contained an enzyme resembling benzylamine/putrescine oxidase which attacks the primary amino groups of spermidine.

Pentacyclic triterpenoids of the hopane family were found in about half of some 100 strains of prokaryotes belonging to diverse taxonomic groups, such a wide distribution indicating the biological significance of these compounds. Hopanoids were found in almost all the cyanobacteria and obligate methylotrophs examined, in all the purple non-sulphur bacteria studied and in many taxonomically diverse Gram-negative or Gram-positive chemohetero-trophs. They were absent in all archaebacteria and purple sulphur bacteria examined as well as in various other Gram-positive or Gram-negative genera. The C30 hopanoids, diploptene and diplopterol, are present in almost all hopanoid-containing prokaryotes. The major compounds are always the C35 bacteriohopanepolyols, which are present at a level of 0.1-2 mg per g dry weight, the most common one being bacteriohopanetetrol. Because of their structural characteristics and their influence on the properties of biological membrane models, these compounds might be the structural equivalents of the sterols found in eukaryotes.

Regenerating spheroplasts of Candida albicans formed organized glucan nets in liquid culture. The nets consisted of interwoven microfibrils about 50 nm wide, but of an undetermined length. Partial acid hydrolysis of the polysaccharide showed the presence of chains of β(1→3)- and β(1→6)-linked glucose residues, but no intrachain β(1→3) andβ(1→6) linkages. Periodate oxidation and GLC of the methylated glucan indicated a highly branched polymer (9·5% branch points). Sequential enzymic degradation of the isolated nets confirmed the presence of chains of β(1→3)- and β(1→6)-linked glucose residues. Degradation by β(1→3) and β(1→6)β-glucanase released 23% (w/w) and 30% (w/w) respectively of the carbohydrate as glucose equivalents. The residual material was degraded by chitinase. Equal amounts of N-acetylglucosamine and glucose equivalents were detected in the chitinase hydrolysate, suggesting a possible linkage between glucan and chitin. Our data indicate that the cell wall of C. albicans contains at least two highly branched glucans with predominantly β(1→3) or β(1→6) linkages.

Exo-(1→3)-β-glucanase, β-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and β-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 °C. However, in situ trehalase, (1 → 3)-β-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. β-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (l→3)-β-glucanase and autolysin activities were optimal at pH 5.6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1 →3)-β-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1→3)-β-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1→3)-β-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1 →3)-β-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1→3)-β-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.

Mutants of Streptomyces griseus blocked in cytodifferentiation regained their capacity to form differentiated mycelia and/or anthracycline pigments in the presence of butyrolactone-type autoregulatory effectors such as trans-2-(6′-methylheptanol-l′yl)-3-hydroxymethyl-4-butanolide. In the pertinent indicator strains, the effect has been correlated with the increase of lipid synthesis, with changes in the composition of lipid fraction and with the restoration of the production of neutral proteinases. The results suggest that autoregulatory butyrolactones from streptomycetes stimulate cytodifferentiation of their producers at an early stage of development.

Pyruvate and ethanol were both effective electron donors for nitrite reduction by Escherichia coli K12. The pyruvate-dependent rate decreased by approximately 50% when either a cysG mutation, which results in loss of NADH-dependent nitrite reductase activity (EC 1.6.6.4), or a chl mutation, which results in loss of the formate-nitrite oxidoreductase activity, was introduced into the prototrophic parental strain CGSC4315. A double mutant deficient in both of these previously described activities retained only 2% of the rate of nitrite reduction of the parental strain after growth on glucose or 5% after growth on pyruvate. We conclude that any third pathway for nitrite reduction contributes little to the in vivo rate of nitrite reduction by wild-type strains.

A strain of Penicillium nigricans, which produces both the antifungal antibiotic griseofulvin and tremorgenic penitrem mycotoxins concurrently in static liquid culture, also elaborated both metabolites in submerged culture when stimulated by calcium chloride to sporulate. Maximum yield of penitrems (60 mg 1−1) occurred within 5 d in a 60 1 stirred fermenter, thus constituting the first significant process for penitrem production in submerged culture.

Several sul-reg mutants of Aspergillus nidulans isolated as constitutive for arylsulphatase were studied with respect to the regulation of enzymes involved in cysteine and homocysteine synthesis and to the pool of sulphur amino acids. All mutants examined showed a decreased concentration of glutathione as compared with the wild type, and all mutants, with one exception, had a decreased total pool of sulphur amino acids. The results suggest that the mutants are leaky in the sulphate assimilation pathway. They show derepression of cysteine synthase, homocysteine synthase, cystathionine β-synthase and γ-cystathionase. In spite of having derepressed homocysteine synthase, the enzyme which constitutes an alternative pathway for homocysteine synthesis, the sul-reg mutations do not suppress lesions in genes required for the main homocysteine-synthesizing pathway. This indicates that the derepression of homocysteine synthase is not in itself sufficient for physiological functioning of this enzyme, but seems to depend also on the effectiveness of cysteine synthesis and sulphide formation.

Mutations in Bacillus subtilis168 have been isolated that confer resistance to arginine hydroxamate in the presence, but not absence, of ornithine. Seven such Ahor mutants have been studied in detail. In common with certain classes of Ahr mutant (resistant to arginine hydroxamate in the absence of arginine precursors) described previously, these Ahor mutants showed little or no inducibility of enzymes of arginine catabolism. Mutants that showed no inducibility were unable to utilize arginine or ornithine as sole nitrogen source. The only biosynthetic enzyme to show any consistent differences in activity from the parent was ornithine carbamoyltransferase, whose level was slightly elevated in cells grown in the presence of ornithine or citrulline. PBS1 transduction crosses showed that two of the ahor mutations map at the ahr A locus, while a third (unique in its resistance to arginine hydroxamate in the presence of citrulline) mapped at a hitherto undescribed locus closely linked to metC, designated ahrD.

We have isolated a mutant of Bacillus subtilis, strain 590, which is blocked at stage III of sporulation. The spo mutation which is carried by this strain is linked to phe A by transformation and defines a previously unidentified locus, spoIIIF. The spoIIIF locus is contiguous with the spoVB locus, in which a mutation causes a block at stage V of sporulation. We also give a detailed genetic map of the phe A region of the chromosome.

The spo0B and spoIVF loci are contiguous on the chromosome of Bacillus subtilis, so that genes in these loci may be parts of a single polycistronic operon. Temperature-sensitive strains having mutations in these loci were isolated, and temperature-shift experiments were carried out to investigate expression of the genes. The temperature-sensitive periods of spo0B mutants extended from the beginning of sporulation until the end of the stage II. The temperature-sensitive periods of spoIVF strains were during stage IV of sporulation. Therefore, although the spo0B and spoIVF loci are contiguous on the chromosome it is unlikely that genes in them are parts of a single polycistronic operon.

The aspartase gene (aspA) of Escherichia coli has been isolated in two plasmids, pGS73 and pGS94, which contain segments of bacterial DNA (12·5 and 2·8 kb, respectively) inserted into the tet gene of the vector pBR322. The plasmids were constructed by sequential sub-cloning from a larger ColEl-frd + hybrid plasmid. The location of the aspA gene confirmed predictions based on a correlation between the genetic and restriction maps of the corresponding region. The aspartase activities of plasmid-containing aspA mutants were amplified four- to sixfold relative to aspA + parental strains. The aspA gene product was tentatively identified as a poly-peptide of M r 55 000, which is somewhat larger than previous estimates (M r 45 000 to 48 000) for aspartase.

The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43000–46000: 43K–46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.

Cells of Pasteurella haemolytica serotypes Al and A6 were extracted with sodium salicylate and the chemical and antigenic composition of both extracts determined. The extracts were concentrated by ultrafiltration and the serotype antigen, measured by the indirect haemagglu-tination test, was estimated to have a molecular weight between 100000 and 300000. The chemical composition of sodium salicylate extracts (SSEs) from both serotypes was similar, having protein, carbohydrate, fatty acid and phosphorus present in the ratio 10:1:0·5:0·1. SDS-PAGE of both SSEs gave similar profiles with at least 48 bands present. These results suggest that sodium salicylate removes the outer membrane of P. haemolytica. Crossed immunoelectrophoresis indicated that a major serotype-specific antigen was present in SSEs of both strains. This antigen was extracted from the SSE with hot phenol/water and analysed by gas chromatography. The sugar composition of Al and A6 phenol/water extract (PWE) was qualitatively identical although some differences in proportions were observed. Al and A6 PWE antigens protected mice against homologous serotype challenge and A6 PWE protected against heterologous (Al) challenge.