- Volume 132, Issue 2, 1986

Summary: The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since (i) inactivation did not occur during carbon starvation, (ii) when a fermentable sugar was added to starved cells, inactivation began, (iii) when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, (iv) when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.

Summary: Phenylalanineaminopeptidase was isolated and purified from the culture filtrate of Legionella pneumophila by affinity chromatography on O-tert-butyl-l-threonyl-l-phenylalanyl-l-prolyl-glycyl-aminosilochrom and by gel-filtration; a 401-fold purification with a yield of 18% was achieved. The enzyme was a metalloenzyme with a molecular weight of 35000 and a pI of 5.8. It was stable at pH 7–9 and had an activity optimum in the range of pH 8–9.5 with l-phenylalanine p-nitroanilide as substrate. Enzyme activity was highest towards the latter compound, substantially lower towards l-leucine p-nitroanilide and only marginal towards other p-nitroanilides. Besides phenylalanineaminopeptidase, a metalloproteinase and a serine proteinase were also detected in L. pneumophila culture filtrate.

Summary: Plasmid-encoded symbiotic determinants from the Rhizobium leguminosarum strain MA1 (817) with uptake hydrogenase activity (Hup+) and from the Hup− strain MC1 (18a) were mobilized by recombination with the self-transmissible plasmid pVW5JI. The symbiotic determinants were transferred by conjugation from strain MA1 to strain MC1 and to a derivative of MC1 without the symbiotic plasmid, and vice versa, thus constructing four types of transconjugants. The determinants for a total recycling hydrogenase in strain MA1 were found to be encoded on the symbiotic plasmid.

Strain MC1 fixed 60% more N2 in pea root nodules, determined as mg nitrogen per plant, than strain MA1. This difference was not increased in the MC1 derivative that obtained hydrogenase activity. Plants inoculated with a derivative of strain MA1, however, where the symbiotic plasmid was replaced by that of strain MC1 had a high percentage nitrogen content. It was concluded that the symbiotic plasmid and the genetic background were more important for plant nitrogen accumulation than uptake hydrogenase.

Summary: The DNA of Rhodomicrobium vannielii was analysed for the presence of inverted repeat sequences (IR DNA) by S1 nuclease digestion. Approximately 7% of chromosomal DNA was found to be IR DNA which comprised two size classes. The large IR DNA was heterogeneous and contained species in the size range 100–700 bp. The smaller size class contained species of 17 and 27 bp. Both size classes of IR DNA hybridized to many chromosomal restriction fragments, suggesting that these IR DNA sequences are dispersed throughout the genome. Hybridization studies also indicated sequence homology between the two classes of IR DNA and suggested that the 17 and 27 bp IR DNA sequences may exist in clusters.

Summary: Genes for lactose catabolism and proteinase production in Streptococcus lactis 712 are encoded by a 56.5 kb metabolic plasmid, pLP712. A lactose mini-plasmid of only 23.7 kb, pMG820, was constructed by introducing two deletions into pLP712, and was cloned as two segments of DNA into the Escherichia coli vector pAT153 using restriction endonuclease PstI. The lactose genetic region of pLP712, which has been defined by deletion and restriction mapping, was cut into two parts by this process. When the smaller 10.8 kb segment of pMG820 DNA was present, the key lactic streptococcal lactose splitting enzyme, phospho-β-d-galactosidase, was expressed in E. coli. The gene for phospho-β-d-galactosidase was more precisly located by introducing a series of deletions into cloned DNA by in vitro manipulations and then assaying for enzyme activity. The presence of this phospho-β-d-galactosidase activity was correlated with the production of a 58kDa 35S-labelled protein both by E. coli minicells and after coupled transcription and translation of cloned DNA. The product of a second gene, a 37 kDa protein (“protein X”), and a possible truncated phospho-β-d-galactosidase protein of 16 kDa were also detected in minicells.

Summary: The locus spoIID, involved in the sporulation of Bacillus subtilis, was cloned into derivatives of the temperate phage ϕ105. Two recombinant phages were obtained which contain chromosomal DNA covering 1∙6 kbp. They are both able to complement mutations spo-68 and spo-298. These mutations, which were believed to be in different loci, spoIID and spoIIC respectively, were shown to be closely linked, and both map at the position assigned to spoIID on the genetic map of B. subtilis. The sequence of 1656 bp carrying the spoIID locus was determined. Only one open reading frame was found; this codes for a protein of 343 amino acids. It is preceded by a ribosome binding site and possible recognition sequences for σ32- and σ29-RNA polymerases. Studies of the locus by means of integrational plasmid vectors defined the outer limits of the transcriptional unit. These results are completely compatible with the sequence data. The combination of sequence and mapping and the information obtained by the use of integrational plasmids confirm that the spoIID locus functions as a monocistronic operon.

Summary: Microcin B17 is a novel peptide antibiotic of low M r (about 4000) produced by Escherichia coli strains carrying plasmid pMccB17. The action of this microcin in sensitive cells is essentially irreversible, follows single-hit kinetics, and leads to an abrupt arrest of DNA replication and, consequently, to the induction of the SOS response. RecA− and RecBC− strains are hypersensitive to microcin B17. Strains producing a non-cleavable SOS repressor (lexA1 mutant) are also more sensitive than wild-type, whereas strains carrying a mutation which causes constitutive expression of the SOS response (spr-55) are less sensitive to microcin. Microcin B17 does not induce the SOS response in cells which do not have an active replication fork. The results suggest that the mode of action of this microcin is different from all other well-characterized microcins and colicins, and from other antibiotics which inhibit DNA replication.

Summary: When microcin B17-immune cells are treated with microcin B17 they show many of the physiological effects displayed by microcin B17-sensitive cells treated in the same way. DNA replication stops immediately and several SOS functions are subsequently induced. In sensitive cells these effects are irreversible and lead to cell death, whereas in immune cells they are reversible and there is no loss of viability. This is an unusual mechanism of immunity because it does not prevent the primary action of the microcin. The implications of this mechanism concerning the mode of action of microcin B17 and the induction of the SOS system are discussed.

Summary: A mutant of Bacillus subtilis 168 was isolated which resists infection by all the group III temperate bacteriophages except SPR, while allowing full infection by phages of the other groups (I, II and IV). The mutation conferring this phenotype, pha-3, shows 52–54% PBS1-mediated cotransduction with the hisA1 marker, mapping therefore in the gtaA and gtaB region of the B. subtilis chromosome. Nevertheless, it does not affect the infection by phages sensitive to gta mutations.

Summary: Among secreted proteins of Bacillus subtilis 168 four polypeptides, separated by electrophoresis in polyacrylamide gradient gels under denaturing conditions, were shown to have lichenanhydrolysing enzyme activity; one of them, a 22 kDa protein, represents the main β-glucanase activity. A number of N-methyl-N-nitro-N-nitrosoguanidine-induced mutants defective in β-glucanase activity at an elevated temperature (44°C) were isolated and characterized. All mutants analysed by PAGE had no active 22 kDa β-glucanase in their supernatants and showed no cross-reactivity with antibodies raised against purified 22 kDa β-glucanase. However, formation of other exoenzymes, including the other minor β-glucanases, was not affected in these mutant strains. The mutations (bgl-1, bgl-12, bgl-35, bgl-101) were mapped by phage PBS1-mediated transduction and found to be located between purA and sacA, close to the hutH marker of the B. subtilis chromosome. A 3.8 kb EcoRI fragment of the B. subtilis chromosome which directs β-glucanase synthesis in Escherichia coli as well as complementing a β-glucanase deficient mutant, bgl-35, of B. subtilis to synthesize active 22 kDa β-glucanase was mapped after homologous recombination by means of an integratable plasmid. The cointegrated chloramphenicol-resistance marker of the plasmid was found at the same map position as the mutations bgl-1, bgl-12, bgl-35 and bgl-101, suggesting that the gene affected encodes for the 22 kDa β-glucanase and lies within the order sacA–sacA–bgl–purA.

Summary: A clinical isolate of Candida albicans, a member of the Fungi Imperfecti, was polyploid as shown by the fact that it contained two kinds of nuclei, one of diploid and one of tetraploid DNA content. These determinations were made by fluorescence microscopy-photometry. The nucleus-associated organelles (NAOs), or spindle pole bodies, of yeast cells in this isolate were classified into two groups, one diploid and the other tetraploid, according to their dimensions as determined by serial thin-sectioning electron microscopy. A ploidy shift from diploid to tetraploid was found in individual cells of a culture of this isolate undergoing diphasic growth in minimal salts medium. A process of shift-down or reduction of ploidy from tetraploid to diploid was also observed by electron microscopy during these growth conditions: this appeared to occur in large cells which showed multiple spindle formation during nuclear division, a phenomenon apparently similar to the process of meiosis II during sporogenesis of Saccharomyces cerevisiae, but differing in that it produces diploid daughter nuclei by the vegetative process.

Summary: Chromosomal and plasmid DNA have been extracted from six isolates of Coxiella burnetii, the aetiological agent of Q fever. Restriction fragment length polymorphisms detected after HaeIII digestions of chromosomal DNA revealed four different patterns that distinguished the American from the European isolates, and the Nine Mile phase I prototype strain from a spontaneously derived, isogenic phase II nonrevertant variant. At least one of the HaeIII fragments visible in the pattern from Nine Mile phase I and not in that from Nine Mile phase II could not be detected by DNA–DNA hybridization, and thus may have been deleted during the phase transition. Comparison of Nine Mile phase II, which does not survive animal passage, with Grita M44 phase II, which does, indicated that the HaeIII fragment was present in the Grita strain. These results suggest that this HaeIII fragment may be concerned with functions necessary to survive the cellular immune response in vivo. Isolates from two human endocarditis cases showed the greatest divergence from all the other isolates, having at least five fragments of unique mobility in the HaeIII digestion pattern of their chromosomal DNA. Also, a plasmid obtained from these two isolates was 2 to 3 kb larger than the plasmid present in the other five isolates, and its restriction pattern could be distinguished from that of the other plasmids by several endonucleases. Detection of chromosomal and plasmid restriction fragment length polymorphisms among strains of phase I or phase II C. burnetii from various geographical locations and environmental sources will facilitate Q fever diagnosis and strain identification.

Summary: Expression of the mercury-resistance (mer) genes of the transposon Tn501 is positively and negatively controlled by the product of the merR gene. DNA sequence analysis has identified three open reading frames as potential candidates for this gene, one of which is oriented divergently with respect to the mercury-resistance genes. We have demonstrated that although RNA polymerase will bind to fragments containing the potential control regions for all three reading frames, only the control region for this divergent reading frame shows detectable promoter activity in vivo. Transcription of this reading frame is required for repression and induction of mer transcription. We have also shown that the Tn501 merR gene product negatively regulates its own synthesis, and have identified the start point of the transcript for this reading frame and for the mercury-inducible transcript of the mercury-resistance genes.

Summary: Mutations which allow tolerance to 5-bromo-2′-deoxyuridine (BUdR) in a thymidine (TdR)-requiring strain of Bacillus subtilis have been examined. Differences in sensitivity to BUdR existed between isogenic strains harbouring the mutations. Those mutations originally isolated as BUdR-tolerant also bestowed tolerance to 5-bromouracil and vice versa. The strain exhibiting the greatest tolerance to BUdR maintained a normal rate of replication in the presence of BUdR whereas the parent strain did not, but the tolerant strain incorporated less analogue into DNA than the parent strain. The basis of the tolerance mutation appeared to lie at the point of uptake of the analogue into the cell as the tolerant mutant preferentially took up TdR over BUdR into whole cells. DNA polymerase activity measured in vitro did not distinguish between TdR and BUdR in either the parent or the mutant strain and although TdR kinase activity showed a preference for TdR over BUdR as a substrate, the extent of discrimination was similar in both strains.

Summary: A 5-bromo-2ʹ-deoxyuridine (BUdR)-tolerant derivative of a thymidine (TdR)-requiring strain of Bacillus subtilis was used to examine the effect of BUdR, an analogue of TdR, on sporulation. At a TdR: BUdR ratio which had little effect on growth, sporulation was inhibited if cells were exposed to BUdR during the period of DNA synthesis at the onset of the process. Cells recovered from BUdR inhibition of sporulation if the analogue was removed and DNA replication allowed to continue with TdR alone. BUdR prolonged the period of DNA synthesis during sporulation and experiments with chloramphenicol suggested that this was due in part to unscheduled initiation of new rounds of replication.