- Volume 129, Issue 6, 1983

Two proteinases have been purified from mycelial extracts of Aspergillus nidulans. Both enzymes have pH optima between 6·5 and 7·5 and are inhibited by phenylmethane sulphonyl fluoride and by di-isopropyl fluorophosphate. The molecular weights and isoelectric points of proteinase I and proteinase II are 30900 and 30000, and 4·6 and 4·3, respectively. Both enzymes have a similar range of substrate specificities. The principal differences in their properties are that proteinase I is sensitive to inhibition by 1 mM mercurials whereas proteinase II is not appreciably inhibited at this concentration, and that proteinase I is much more sensitive to denaturation by urea, guanidine hydrochloride and sodium dodecyl sulphate.

The soluble cytochrome oxidase of Nitrosomonas europaea has been highly purified and shown to be a copper protein devoid of haem, not a cytochrome o as was previously assumed. The native molecular weight was 120000 and the subunit molecular weight 35000. Soluble cytochrome oxidase activity co-purified with nitrite reductase activity; both activities were almost certainly associated with the same protein. The possible physiological role of the nitrite reductase activity is discussed.

The electron transport chain of Escherichia coli grown anaerobically on glycerol with fumarate as terminal electron acceptor, has been studied using electron paramagnetic resonance (EPR) spectroscopy. The analysis did not include cytochromes, but was confined to the lower potential (dehydrogenase) section of the electron transport chain. Several ferredoxin-type centres were detected and partially characterized, and the possible presence of iron-sulphur centres paramagnetic in their oxidized form (‘HiPIP-type’) was also noted. An EPR-detectable signal that may have been due to the presence of molybdenum in the electron transport chain is described and assessed. Most of the centres detected were reducible by substrate in the absence of oxidant and some were found to be wholly or partly reduced during steady state oxidation of substrates in the presence of oxygen.

Cell-free preparations have been made from Thiobacillus A2 that are capable of the complete oxidation of thiosulphate to sulphate and of coupling the associated electron transport to ATP synthesis with a P/O ratio of about one. Spectrophotometric measurements and the effect of electron transport chain inhibitors on cytochrome reduction and oxygen uptake indicate that thiosulphate oxidation is coupled directly to cytochrome c reduction and does not involve cytochrome b. Complete inhibition of phosphorylation by, 2,4-dinitrophenol suggests that ATP synthesis was effected exclusively by electron transport phosphorylation accompanying re-oxidation of cytochrome c. Enzymes for thiosulphate oxidation to sulphate and the reduction of cytochrome c were located in the supernatant fraction after centrifuging at 130000 g, but oxygen uptake required the 130000 g pellet fraction, which provided membrane-bound cytochrome c and cytochrome oxidase.

A crude thiosulphate-oxidizing extract of Thiobacillus A2 has been resolved by ammonium sulphate fractionation and DEAE-Sepharose CL-6B chromatography into fractions containing several components necessary for thiosulphate oxidation and reduction of cytochrome c. These components include two distinct c-type cytochromes, sulphite: cytochrome c oxidoreductase, rhodanese and enzymes probably responsible for thiosulphate cleavage and sulphane-sulphur oxidation. The complete cytochrome c-dependent thiosulphate-oxidizing system could be reconstituted by mixing selected fractions from the column chromatography.

Streptomycin 6-kinase of the streptomycin-producing strain Streptomyces griseus HUT 6037 was purified by fractionation with (NH4)2SO4 and chromatography on DEAE-Sephadex A-25, hydroxyapatite and Sephadex G-100. After PAGE of the final fraction, a protein band corresponding to streptomycin 6-kinase was detected, together with a less intense band having no enzyme activity. Molecular weights determined by SDS-PAGE and by Sephadex G-100 chromatography were about 36000 and 38000, respectively, suggesting that the enzyme was a monomer. The isoelectric point of the enzyme was pH 6.6. Among the nucleoside 5′-triphosphates tested, ATP was the preferred phosphoryl donor. The Km values for streptomycin and ATP were 3.5 mm and 0·4 mm , respectively. The enzyme activity was strongly inhibited by EDTA and AgNO3. It was shown by using an in vitro protein-synthesizing system that purified streptomycin 6-kinase could protect polyphenylalanine synthesis of the streptomycin-susceptible S. griseus strain KSN from inhibition by streptomycin.

55FeCI3 labelling and non-denaturing gel electrophoresis were used to study nitrogenase synthesis in Gloeothece sp. CCAP 1430/3. Nitrogenase synthesis was inhibited by addition of NHX but was unaffected by elevated concentrations of O2. Upon transfer of cultures of Gloeothece from light to darkness, there was initially a slight decrease in the rate of synthesis of nitrogenase but after 4–5 h there was an almost complete cessation of synthesis. This delayed effect of darkness on nitrogenase synthesis could not be related to any change in RNA synthesis, in protein synthesis or in the rate of breakdown of storage glucan.

In cultures of Gloeothece, mRNA, including nif mRNA, was unstable, having a half-life of about 5 min. The synthesis of nif mRNA did not stop immediately upon transfer of cultures to the dark. If darkness exerts its effect on nitrogenase synthesis by inhibiting the synthesis of nif mRNA, it does so only after a lag of about 4 h.

Epoxy fatty acids added to the culture media either with the inoculum or at the end of exponential growth phase stimulated aflatoxin production by toxigenic strains of Aspergillus flavus and Aspergillus parasiticus. This effect did not appear when the unsaturated fatty acids used for the synthesis of the epoxides and the polyhydroxyacids (which can be considered to be derived from the opening of the oxirane ring) replaced the epoxides in the culture media. No significant differences were detected in the lipid fractions (diglycerides, sterols, triglycerides, free fatty acids, sterol esters) extracted from the mycelia grown in the presence of any of the fatty acid derivates.

Details of a relatively inexpensive method for the purification of K99 pili in their native conformation are reported. The method involved sequential precipitation of K99 pili with (NH4)2SO4, followed by precipitation in 12% (w/v) mannose or sorbitol solution. The crude pili preparation was adsorbed with Bio-Gel A-5m and subjected to sequential gel filtration on Bio-Gel A-5m equilibrated with Tris/EDTA/NaN3/NaCl buffer and KSCN/KCl solution, respectively. The K99 containing peak was subjected to sequential ion-exchange chromatography on DEAE-Bio-Gel A equilibrated with 0·02 m-Tris/HCl, pH 8·6 containing 0·05 m-NaCl and DEAE-Sephadex A-50 equilibrated with 0·05 m-phosphate buffer, pH 7·2. The purified pili yielded a single band on SDS-PAGE with an estimated molecular weight of 13000. Attempts to purify pili by other methods evaluated, viz. MgCl2 precipitation and chromatofocusing were unsuccessful. While the amino acid composition of purified K99 pili was similar to that reported previously the N-terminal amino acid was apparently blocked.

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27–30 °C on regeneration R2 medium ( Okanishi et al., 1974 ) containing 0·25 m-sucrose. The addition of phosphate (150–300 mg 1−1) to the medium and incubation at 23 °C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.

A mutation designated spsA1 has been induced in the putrescine (puA2) auxotroph of Aspergillus nidulans which enables this mutant to grow on low concentrations of spermidine in place of putrescine. In addition, the spsA1 mutant, irrespective of putrescine requirement, is abnormally sensitive to high concentrations of spermidine, spermine or the polyamine analogue methylglyoxal bis(guanylhydrazone). When spsA1 strains are grown on medium containing spermidine, uptake of the polyamine continues at a high level for a longer period than in the wild-type and leads to a doubled intracellular spermidine pool. A similar increase in the intracellular spermine pool results from growth on spermine.

A hybrid plasmid. pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a. gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit M r was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.

Previous work has shown that Escherichia coli KH strains carrying the small, high copy number ColE2-P9 plasmid produce large amounts of colicin and then lyse and release colicin when grown in broth culture containing mitomycin C. Strains carrying the larger, low copy number ColIa-CA53 plasmid produced much less colicin and did not lyse or discharge more than 15% of their colicin when grown under the same conditions. Naturally-occurring Col+ strains and E. coli K12 derivatives carrying different Col plasmids could be classified either as ColE2+-like or ColIa+-like according to whether or not they produced large amounts of colicin and lysed and discharged colicin when grown in the presence of mitomycin, and also by the size and presumed copy number of the Col plasmid they carried. Strains carrying multiple copies of the cloned colicin Ia structural gene produced large amounts of colicin but did not lyse or release colicin when grown in the presence of mitomycin. This result rules out the possibility that high level accumulation of colicin is sufficient to cause lysis. Conditions were sought under which colicin Ia could be released from the producing cells. It was found that mitomycin-treated cultures of strains carrying both ColE2 and ColIa plasmids released both colicins when they lysed, although colicin Ia release occurred later than colicin E2 release. It was also noted that colicin Ia-laden cells released their colicin when diluted into fresh culture medium.

Spores produced by a mutant of Bacillus subtilis were slow to develop their resistance properties during sporulation, and were slower to germinate than were wild-type spores. The coat protein composition of the mutant spores, as analysed by SDS-PAGE, was similar to that of the wild-type spores. However, one of the proteins (mol. wt 12000) which is normally present in the outermost layers of mature wild-type spores and which is surface-exposed, was assembled abnormally into the coat of the mutant spores and not surface-exposed. The mutation responsible for this phenotype (spo-520) has been mapped between pheA and leuB on the B. subtilis chromosome, and was 47% cotransformable with leuB16. This mutation, and three others closely linked to it, define a new sporulation locus, spoVIB, which is involved in spore coat assembly. The phenotype of the mutant(s) supports the contention that spore germination and resistance properties may be determined by the assembly of the coat.