- Volume 113, Issue 1, 1979

The growth inhibition of Salmonella typhimurium aziA mutants by sodium azide is reversed by cystine and related compounds. NADPH-sulphite reductase (hydrogen-sulphide: NADP+ oxidoreductase; EC 1.8.1.2), an enzyme of cysteine biosynthesis, is inhibited in cell extracts by sodium azide. AziB mutants which are able to grow in the presence of the inhibitor without cystine were isolated. About half of them were mapped in the cysK gene and have only residual activity of its product, O-acetylserine sulphydrylase A [O-acetyl-l-serine acetate-lyase (adding hydrogen-sulphide); EC 4.2.99.8]. Sensitivity of wild type and aziA mutants to azide was also reversed by a constitutive mutation in cysB, the regulatory gene of cysteine biosynthesis. CysK and cysB mutants showed cross-resistance to azide and 1,2,4-triazole. It is suggested that the resistance of these mutants to azide is due to an increased activity of NADPH-sulphite reductase.

Gel electrophoresis of DNA from 95 clinical isolates of Shigella sonnei and Shigella flexneri resistant to antibiotics revealed a heterogeneous plasmid population. Most of the plasmids were smaller than 6 megadaltons (Mdal). Six S. sonnei isolates with the most common antibiotic resistance pattern were characterized. They had two plasmids in common: one was a self-transmissible Fi+ plasmid of 46 Mdal encoding tetracycline resistance, while the other was a 5·5 Mdal non-conjugative plasmid encoding resistance to streptomycin and sulphafurazole. In addition, several cryptic plasmids ranging in size from 1·0 to 24·5 Mdal were present. Mobilization of the 5·5 Mdal SuSm plasmid and a 1·0 Mdal cryptic plasmid was demonstrated with all six S. sonnei isolates during conjugation. This mobilization was mediated by the 46 Mdal self-transmissible Fi+ R plasmid and also by a 24·5 Mdal Fi− plasmid carrying no known drug resistance determinants.

Twenty-five mutants unable to utilize nitrate as sole nitrogen source were isolated from Rhizobium meliloti 41. These mutations mapped at four different sites, narA, narB, narC and narD; narB, C and D were located between trp-15 and ade-15 on the chromosome. NarA mutants were affected in assimilatory nitrate reduction but not in ‘respiratory’ nitrate reduction and had methyl viologen-coupled nitrate reductase activity. NarB mutants were affected in both assimilatory and ‘respiratory’ nitrate reduction and lacked methyl viologen-coupled nitrate reductase activity. NarC and narD mutants were impaired not only in assimilatory and ‘respiratory’ nitrate reduction but lacked xanthine dehydrogenase activity as well. Acid-treated crude extracts of these two mutant classes were unable to restore NADPH-coupled nitrate reductase activity to the nit-1 mutant of Neurospora crassa, indicating the lack of active molybdenum cofactor. All mutants tested were effective in symbiotic plant tests and had normal nitrogenase activity, indicating that nitrogenase and nitrate reductase do not share the same molybdenum cofactor.

Three spore colour, two mycelial colour and 12 auxotrophic mutants were isolated from an aflatoxigenic strain of Aspergillus parasiticus. These mutants and heterozygous diploids formed by pairwise combinations of auxotrophs were assayed for aflatoxin production; norsolorinic acid and versicolorin A production were also assayed in the diploids. In general, introduction of an auxotrophic marker lowered aflatoxin production in haploids. The green-spored, prototrophic diploids resembled haploid wild-type strains in that they produced high levels of aflatoxin, low levels of versicolorin A, and no detectable norsolorinic acid. Parasexual analysis of segregants from four heterozygous diploids was hampered by the uniform conidiospore diameter of haploids and diploids and by the non-random recovery of genotypes among somatic segregants with and without treatment with p-fluorophenylalanine. Nevertheless, this technique is useful for recombinational analysis of mutants blocked in aflatoxin synthesis. The fortuitous association of mycelial pigmentation with certain blocked aflatoxin mutants should prove useful in future analyses of the genetics and biosynthesis of these economically important secondary metabolites.

Fifty-five haemin-requiring mutants were isolated from haemin-permeable mutants. According to their growth responses to haem precursors and their patterns of porphyrin accumulation, the 55 mutants fell into three groups which were judged to have defects in 5-aminolaevulinate dehydratase, ferrochelatase, and uroporphyrinogen III cosynthase or uroporphyrinogen decarboxylase. In mutants of the group deficient in 5-aminolaevulinate dehydratase, the mutations were adjacent to lac, and evidence is presented that the mutations were in hem B and were commonly deletions extending into proC.

Gonococci adapted to growth in guinea pig chambers [strain BS4 (agar)] were predominantly smooth organisms and produced a type-specific antigen. A vaccine prepared by treating these gonococci with formalin, protected guinea pig chambers against homologous challenge in contrast to a similarly treated laboratory strain (BSDH) which had been selected in vitro from the same parent strain and which did not produce the type-specific antigen. Surface washes of BS4 (agar) contained the type-specific antigen but attempts to immunize guinea pigs with complexes of rabbit antibody with this antigen excised from gels failed. However, good immunity could be produced by combining such complexes with formalin-killed rough gonococci (strain BS4R), lacking the type-specific antigen, which were found in some chambers of challenged guinea pigs that had been immunized with the complexes. Hence, at least two antigens - one the type-specific antigen and the other(s) possessed by both BS4 (agar) and BS4R - are needed for immunogenicity. Surface washes of BS4 (agar) and BS4R contained three antigens, distinct from the type specific antigen, which might complement it in producing immunity. Similar antigens were present in surface washes of five fresh isolates from human urethral pus, but only a few organisms from these isolates possessed antigens similar to the type-specific antigen. The variability of gonococci in antigenicity, immunogenicity and probably virulence is discussed.

The common brewery contaminant Citrobacter freundii has been used to investigate how oxygen, nitrate and glucose concentrations determine the growth rates, cell yields, the flux of metabolites and the rates of synthesis of tricarboxylic acid cycle enzymes and terminal electron transfer pathways. The growth rate was inhibited for 4 or 8 h when anaerobic batch cultures were either supplemented with nitrate or aerated, respectively. The subsequent exponential growth rates and yield coefficients were greater than in unsupplemented anaerobic cultures, but glucose was only partially oxidized to organic acids even in the most vigorously aerated cultures. The least active enzyme of the tricarboxylic acid cycle was succinate dehydrogenase, and although there were large differences in individual cytochrome concentrations and NADH oxidase activity between aerated and anaerobic cultures, only small differences in 2-oxoglutarate dehydrogenase activity were detected. Similar results were obtained with sulphate-limited continuous cultures, but in a glucose-limited continuous culture, succinate dehydrogenase activity was derepressed 12-fold and the yield coefficient increased 7·5-fold during aerobic growth.

It is concluded that the major determinant of the potential tricarboxylic acid cycle activity is catabolite repression of succinate dehydrogenase synthesis rather than oxygen induction of 2-oxoglutarate dehydrogenase. Synthesis of 2-oxoglutarate dehydrogenase was derepressed, possibly by glutamate or by traces of oxygen in the gas used to sparge the culture during anaerobic growth.

Four species of Balansia (clavicipitaceous systemic grass pathogens) isolated from pastures where cattle showed signs of ergot toxicity were grown in culture. Balansia epichloë, one isolate of B. claviceps, B. henningsiana and two isolates of B. strangulans produced conidia in submerged culture during the first stage of a two-stage fermentation procedure. When transferred to a glucose/sorbitol/inorganic salts medium during the second stage, these four species produced ergot alkaloids in stationary cultures. The transfer of fungi cultured in the first medium to the second medium was necessary for alkaloid biosynthesis. One isolate of B. claviceps did not produce alkaloids. Balansia epichlo produced chanoclavine (I), agroclavine, penniclavine, elymoclavine, ergonovine and ergonovinine. Balansia claviceps produced chanoclavine (I), ergonovine and ergonovinine. This is the first report of isolating ergonovine and ergonovinine, two lysergic acid derivatives, from fungi outside the genus Claviceps. Only chanoclavine (I) was identified from extracts of B. strangulans and B. henningsiana. Chanoclavine (I) and ergonovine were identified from smut grass (Sporobolus poiretii) parasitized by B. epichlo, indicating that this endophyte produces alkaloids both in vivo and in vitro.

Cytophaga sp. NCMB 1314 produces, especially under Mg2+-limited growth conditions, a factor that liberates a cholinesterase from plaice muscle. Just after the onset of Mg2+ limitation in a batch culture, there is a period of rapid production of the factor. Changes also occur in the morphology, ATP content and gross chemical composition of the bacteria. The protein and carbohydrate contents are raised by 60 % and 100 %, respectively, while the RNA and ATP contents are reduced to 70 % and 40 % of the original values. Increased amounts of carbohydrate found outside the cells after Mg2+ limitation at least partly correlated with extracellular slime observed in electron micrographs.

There was no correlation between glutamate dehydrogenase activity and the ability to excrete glutamic acid in the wild type and two auxotrophic mutants of Citrobacter intermedius. Differences in the ability to excrete glutamic acid were attributed to mechanisms which regulated the availability of 2-oxoglutarate, as this intermediate induced the synthesis of glutamate synthase.

Mutants of Schizosaccharomyces pombe with single and multiple requirements for aromatic amino acids were isolated and allocated to 15 loci. Difficulties in cultivation of many of these auxotrophs were largely overcome by using a minimal medium with l-glutamic acid as sole nitrogen source. The locus-typical growth factor requirements of mutants allowed a preliminary allocation of genes to reaction sequences in aromatic amino acid biosynthesis. Several explanations for the occurrence of mutants with locus-atypical growth factor requirements are discussed.

l-Arabinose is metabolized by different pathways in fast- and slow-growing rhizobia.

Although bacteriophage Mu cannot infect Erwinia stewartii, lysogens were constructed by conjugal transfer of an RK2::Mu cts 62 hybrid plasmid, pRK210, into this species from Escherichia coli. The pRK210 transconjugants were stable and produced infectious Mu virions. The Mu cts 62 prophage was thermally induced at 42°C and zygotically induced following transfer of the plasmid to E. stewartii recipients. Induction was accompanied by an increased frequency of Gal− and Thy− mutants among the survivors. Mu promises to be an important tool for studying the genetics of pathogenicity in E. stewartii.