- Volume 131, Issue 9, 1985

Summary: Yeast strains resistant to the L-asparagine analogue β-L-aspartylhydroxamate have been shown to have mutations in any of at least three unlinked genes. Mutation in one of these genes, ahrl, is dominantly expressed and affects a function that involves nitrogen metabolism in the cells, including the exclusion of amino acids at the level of transport. The general amino acid permease is rendered sensitive to ammonium ion in strains carrying the dominantly expressed mutations in ahrl, but other functions related to nitrogen metabolism probably are involved as well.

Summary: From a Bacillus subtilis gene bank constructed in Escherichia coli and based on a low copy number cloning vector we have isolated a hybrid plasmid, pSH1047, containing an 8.0 kb segment of B. subtilis DNA coding for the sdhA. B and C genes, which code for the component polypeptides of succinate dehydrogenase, and the gerE gene, which may code for or regulate a protease involved in producing spores which germinate normally. We report the restriction map of this segment and the analysis of deletion derivatives which allow us to correlate the physical and genetic maps of these chromosomal segments.

Summary: Nifurzide is a nitrofuran with antibacterial activity. As nitrofurans have been reported to interact with DNA, we tested the ability of nifurzide to inhibit plasmid transfer. Inhibition of plasmid transfer between Escherichia coli strains was obtained for ten plasmids belonging to nine incompatibility groups. The same effect was observed when plasmid RP4 was harboured in six different members of the Enterobacteriaceae. Inhibition depended on the reduction of the −NO2 group of nifurzide and was obtained with four other nitrofuran derivatives.

Summary: Production of the red antibiotic, undecylprodigiosin, by Streptomyces coelicolor A3(2) was studied by DNA cloning and biochemical analysis. Over 21 kb of genomic DNA were cloned, in several segments, into plasmid vectors. The cloned DNA ‘complemented’ several specific mutations in the red gene cluster. Four red genes (redA, B, E, and F) were mapped to different regions within the cloned DNA. Screening with redE probes for DNA homologies among various streptomycetes revealed hybridizing DNA in three strains, one of them not known to synthesize prodigiosin pigments. Biochemical studies using protoplasted cells revised our interpretation of the nature of redE and redF mutations. Two forms of undecylnorprodigiosin:S-adenosylmethionine O-methyltransferase activity on gel filtration columns were detected: a very high molecular mass peak (>5 MDal) and a 49 kDal peak. Analyses of extracts from red mutants suggested that these two forms are related, and that at least the redE and redF gene products are necessary for O-methyltransferase activity in vivo. Lack of activity of the redE gene in a heterologous host, S. glaucescens, is consistent with the necessity for a biosynthetic complex involving several red gene products for efficient expression. Experiments in liquid antibiotic production medium indicated that prodigiosin compounds in S. coelicolor are examples of ‘secondary metabolites’ whose synthesis lags behind that of cell mass. The peak of specific activity of O-methyltransferase coincided with the ‘late exponential’ phase of growth. Thus, understanding the genetic regulation of undecylprodigiosin biosynthesis in S. coelicolor may be relevant to other antibiotic production pathways, and perhaps to ‘secondary’ metabolism in general.

Summary: The tetracycline resistance region of the multi-resistance plasmid pBP16 is flanked by direct repeats of the insertion sequence IS160. The tetracycline resistance region plus the flanking IS elements can transpose as a discrete unit. The composite transposon, designated Tn2440, has a size of 4.0 kb.

Summary: The molecular weight of Gluconobacter bacteriophage A-1 DNA was determined by restriction analysis and electron microscopic measurements to be about 30 × 106. The DNA has cohesive ends and forms ring-like monomers. A circular map with the relative positions of the cleavage sites for the restriction enzymes EcoRI, SmaI, XhoI and BamHI is proposed.

Summary: Mutants of Bacteroides fragilis sensitive to mitomycin C were isolated after mutagenesis with ethyl methane sulphonate. One mutant (MTC25) was markedly sensitive to mitomycin C but was unaffected as regards UV sensitivity; another mutant (UVS9) was sensitive to UV radiation but was only moderately sensitive to mitomycin C. Caffeine decreased the survival after UV-irradiation of the wild-type, MTC25 and UVS9 strains by the same relative amount. Aerobic liquid holding recovery occurred in each of the three strains. The MTC25 and UVS9 mutants showed reduced host cell phage reactivation. The wild-type, MTC25 and UVS9 strains all showed UV-and H2O2-induced phage reactivation. The physiological characterization of the MTC25 and UVS9 mutants indicates that it is possible to differentiate between mechanisms for the repair of mitomycin C-and UV-induced DNA damage in B. fragilis.

Summary: The photosensitizing activity of haematoporphyrin (HP) on Mycoplasma hominisand Acholeplasma laidlawii was studied as a function of the phase of growth and the amount of sterols in the cell membrane. Less HP was bound to cells when the membrane had a high sterol content. Both strains in the exponential but not in the stationary phase of growth were sensitive to HP treatment (above µg ml−1) in the dark. Visible light irradiation of HP-loaded cells caused in all cases a decrease of cell survival, with concomitant changes in the pattern of membrane proteins that suggested protein-protein cross-linking, and the appearance of ultrastructural alterations (rounded andlysed cells); the photosensitivity was indirectly related to the sterol content of the cell membrane. On the whole, our findings suggest that the cell membrane is a major target for HP photosensitization of mycoplasma cells.

Summary: Cultures ofMicrococcus cryophilus continue to grow without a lag following a sudden increase in temperature from O ºC to 20 °C (shift-up) or a reciprocal decrease (shift-down); the growth rate changes gradually to that typical of cultures grown isothermally at the final temperature. After a shift-down the phospholipid acyl chain length begins to change immediately, whereas there is a delay following a shift-up. However, the final fatty acid composition is attained within the same number of cell division times after a shift-up or shift-down. The changes in chain length after a shift-up or shift-down can be partially prevented by adding streptomycin or erythromycin at the time of the temperature shift. There is little or no turnover of the head-group or acyl chains of phospholipids during isothermal growth, at O °C or 20 °C, nor after a shift-down. In contrast, after a shift-up approximately 40% of the phosp_holipid turns over during the first 12 h after the shift, but none during the subsequent 24 h; phosphatidylglycerol turns over slightly faster than phospholipids during isothermal growth at O °C or 20 °C, nor after a shift-down. In contrast, and acyl chains of each phospholipid. There is evidence for the conversion of phosphatidylglycerol to cardiolipin. It is argued that as a psychrophilic organism, well adapted to growth at low temperatures,M.cryophilus is more stressed by sudden increases of growth temperature than by a sudden decrease.

Summary: True cellulase activity (i.e. degradation of crystalline cellulose) was markedly derepressed when cellobiose-grown cells were transferred to fructose or sorbitol, especially while the cells were adapting over many hours to these carbon sources. On the other hand, the long lag phase on glucose was not accompanied by derepression. Transfer to cellobiose resulted in low cellulase production. Growth on crystalline cellulose derepressed cellulase production. Addition of cellobiose (the preferred carbon source for growth) to cells adapting to fructose caused a rapid burst of growth and cessation of cellulase synthesis. After cells had adapted to growth on fructose or sorbitol, repression set in and, after a number of transfers, growth on these carbon sources yielded cellulase at a level even lower than with cellobiose or glucose. It appears that rapid growth on a soluble sugar such as cellobiose causes carbon source repression which is relieved during slow growth on crystalline cellulose or during the growth lag on fructose or sorbitol. The reason for the lack of cellulase derepression during the growth lag on glucose is unexplained.

Microbial death was studied under starvation conditions. The kinetics of cell death were described by dN/dt = κN α+1, where N is the number of viable cells at time t and κ and α are constants concerning growth or death of bacteria. Death in the decline phase ofthe culture also fitted the above equation, except for irregular oscillations with relatively short periods. This suggested that bacterial death in the decline phase was mainly caused by the same factor(s) as death under starvation conditions.

Summary: A study was made of Streptornyces rimosusand mutant strains to compare the phenotype of high and low oxytetracycline producers. Strains were identified using a probabilistic identification matrix for the genusStreptomyces.Mutant strains separated into two groups: high-titre strains and blocked mutants. The former identified with theS. rimosuscluster whereas the latter were not identified. Two further oxytetracycline producers identified with theStreptornyces lydicuscluster.