- Volume 124, Issue 1, 1981

Summary: The response of six amino acid synthetic enzymes, including four concerned with arginine synthesis, one with histidine synthesis and one with lysine synthesis, to conditions of histidine and arginine limitation and to exogenously provided amino acids is described in Neurospora crassa. The activities of all these enzymes increased in response to lowered levels of histidine or arginine, but showed little or no repression in wild-type cultures supplemented with casein hydrolysate. The activity of glucose-6-phosphate dehydrogenase (an enzyme not concerned with amino acid synthesis) remained unaffected by any of these conditions. In the case of the four arginine synthetic enzymes evidence is presented that suggests the ‘general’ system implicated in the ‘cross-pathway’ response to histidine is also entirely responsible for the derepression occurring under conditions of arginine limitation. Further investigations into the control of one enzyme, ornithine carbamoyltransferase, are also reported. These show that the enzyme is stable, and suggest that its derepression in response to histidine limitation entails new protein synthesis and involves control at a stage prior to translation.

Summary: Gloeocapsa sp. 1430/3 fixed N2 in the dark, but at a lower rate than in the light. Following the transfer of exponentially growing cultures to the dark, the rate of N2 fixation increased for between 2 and 5 h and then decreased, becoming negligible after 12 h. No substantial increase in activity occurred for about 10 h following re-illumination. It is suggested that the decrease in nitrogenase activity which occurred about 5 h after transfer to the dark was not caused by exhaustion of carbon reserves but by a cessation of nitrogenase synthesis coupled with an irreversible inactivation of the enzyme, probably by O2. Subsequent recovery of activity apparently depended upon resynthesis of nitrogenase.

Wall-deficient forms of Mycobacterium aurum were prepared by agitating the cells during exponential growth with d-cycloserine, glycine, lysozyme, EDTA and LiCl for approximately the time of three cell divisions (18 h). Wall-deficient forms were then converted to spheroplasts by gentle stirring with lysozyme and EDTA in a Tris/HCl buffer containing sucrose until all the cells appeared spherical by phase contrast microscopy. Subsequent lysis by nucleases followed by osmotic shock produced membrane vesicles. Ultrastructural and chemical properties of the spheroplasts and membrane vesicles are described. The spheroplasts were susceptible to lysis by 0·25% (w/v) sodium dodecyl sulphate and were permeable to certain enzyme substrates.

A regular array exhibiting hexagonal periodicity with a centre-to-centre spacing of about 6 nm was found on the wall surface of freeze-etched Lactobacillus buchneri. The regular array was composed of a subunit protein with a molecular weight of about 55000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Subunits isolated from the wall by extraction with guanidine hydrochloride reassembled into the original regular array after dialysis against distilled water. The subunits could reattach to wall fragments from which most of the teichoic acid had been extracted with cold trichloroacetic acid, but not to wall fragments from which most of the neutral polysaccharide had been removed by treatment with hot formamide. Heterologous reattachment of the subunits took place on to Lactobacillus casei subsp. casei wall fragments which had been partially hydrolysed under mild acid conditions. These observations suggest that the subunit protein binds to a neutral polysaccharide moiety in the underlying wall layer but not to peptidoglycan or teichoic acid.

Summary: Previous work had shown that haploid strains of Aspergillus nidulans with a duplicate chromosome segment (one in normal position, one translocated to another chromosome) were unstable at mitosis; genome balance was restored by spontaneous deletion of either duplicate segment. Diploids with an extra, translocated segment showed high instability which was confined to the excess segments; loss of one of these, usually that in translocated position, gave balanced diploid nuclei and the loss was assumed to be by deletion. This led to the proposal that high-frequency deletion was provoked by, and confined to, the excess segment. In the present work it has been shown that elimination of the translocated segment in such diploids occurs more frequently by mitotic crossing over than by deletion. Accordingly, in a more rigorous test of the possible association of excess segments and deletions, a diploid homozygous for an extra, translocated segment has been studied as mitotic crossing over in this strain could not give a balanced genome. The strain was extremely unstable and gave variants of which most had a balanced, or near-balanced, diploid genome. Some variants arose by simultaneous deletions involving both non-translocated segments; almost all variants had deletions with breakpoints different from those most frequent in the corresponding, duplication haploid. The results have shown the diversity of mechanisms available for the correction of genome imbalance and that, at least in the case of Dp(I, II), the degree and modalities of mitotic instability are functions of the balance of chromosome segments and of ploidy.

Summary: The Clarke-Carbon colony bank containing ColEl-Escherichia coli hybrid plasmids was screened by conjugation for complementation of the citrate synthase lesion of a gltA mutant. Three ColEl-gltA + plasmids were identified: pLC26-17 (16·3 kilobase pairs), pLC27-18 (16·35 kb) and pLC31-28 (26·0 kb). The citrate synthase activities of strains containing the hybrid plasmids were amplified 3- to 10-fold. Genetic studies indicated that the smaller plasmids may contain at least part of the succinate dehydrogenase gene (sdh). A physical map of a 19·4 kb region of the E. coli chromosome containing the citrate synthase gene (gltA) was constructed by restriction analysis with the isolated plasmids. The relative positions of 20 restriction sites were defined and a region (3·1 kb) containing the gltA + gene was identified in the segment common to all three plasmids.

Southern hybridization experiments carried out between pSC101:: TN951 DNA and λdlac DNA allowed the location and orientation of the lac operon within the transposon to be deduced. The same method was used to detect Tn951 on Lac plasmids from 11 independent isolates from three continents. None of these plasmids was found to carry an entire Tn951 sequence but they all contained lac genes homologous to the lac genes of Tn951. The lac operon of one of these plasmids was bordered by a sequence homologous to that found at the left-hand side of Tn951. It is concluded that the lac determinants of the Lac plasmids analysed and of Tn951 have evolved from a common ancestor but that the distribution of these determinants cannot be attributed to a spread of the transposon Tn951.

Summary: Thirty-four epidermolytic toxin-producing strains of Staphylococcus aureus obtained from a variety of sources were screened for the presence of plasmid DNA. All serotype ii toxin producers harboured a large 42 kilobase pairs (kb) plasmid. Elimination of these plasmids resulted in the simultaneous loss of a bacteriocin determinant (Bac+) and type ii toxin production (Tox+ ii). Some strains producing serotype i toxin (Tox+ i) contained similar 42 kb plasmids. Elimination of these plasmids resulted in the loss of only bacteriocin production. Strains producing both toxin serotypes readily lost Tox+ ii and Bac+, which were carried on the same plasmid, but Tox+ i could not be eliminated. Thus Tox+ i was probably chromosomally determined, while Tox+ ii was a plasmid-encoded marker. In some strains cadmium resistance was also linked to the 42 kb plasmid. The 42 kb plasmids from seven strains with different phenotypes were analysed with restriction endonucleases EcoRI and HindIII. The plasmids shared 19 of 22 HindIII fragments indicating that they are closely related to each other.

Summary: Caulobacter strains which contain plasmid RP1 are resistant to carbenicillin, kanamycin and tetracycline and can serve as donors of the plasmid, but such R+ strains are not susceptible to the RP1-specific phage PRR1. Since both conjugation and phage PRR1 infection are mediated by the plasmid-specified pilus, the lack of phage binding suggests some functional alteration of the pilus. In this report we demonstrate that RP1 pili are produced by Caulobacter R+ cells, but unlike the RP1 pili produced by PRR1-susceptible strains, the Caulobacter R+ pili do not inactivate phage PRR1. Antibody produced against Caulobacter R+ pili prevents phage PRR1 binding to RP1 pili from PRR1-susceptible strains of Pseudomonas aeruginosa and Escherichia coli, and also inhibits plasmid transfer by those strains. Antibody produced against RP1 pili from P. aeruginosa and E. coli inhibits plasmid transfer both by those strains and by Caulobacter R+ donors. Phage PRR1 rendered non-infectious by RNAase treatment prevents plasmid transfer by P. aeruginosa and E. coli but not by Caulobacter.

Summary: The effects of u.v. irradiation on the survival of Staphylococcus aureus and its phage 𝜙11 were studied. The recA and uvr mutations affected their survival in a similar way to synonymous mutations in Escherichia coli. Weigle reactivation (W-reactivation) of 𝜙11 occurred in wild-type S. aureus and in a uvr mutant but to a lesser extent than has been found for phage λ in E. coli. Reactivation was recA-dependent and was accompanied by u.v.-induced mutagenesis in a temperature-sensitive mutant of 𝜙11. Bacterial mutation to streptomycin resistance was induced by u.v. and was also recA-dependent. In S. aureus, as in E. coli, u.v. was a more effective mutagen in the uvr genetic background. However, a dose-squared response for u.v.-induced mutation of wild-type and uvr strains of S. aureus to streptomycin resistance, and of a trp auxotroph to tryptophan independence, was found only with u.v. doses below 1 J m−2. We suggest that, in relation to the Uvr mechanism of DNA repair, u.v. mutagenesis in S. aureus involves both repairable and non-repairable lesions. As in E. coli, the uvr genetic background reduced the u.v. dose required for maximal W-reactivation of u.v.-irradiated phage. However, there was no enhancement of W-reactivation by post-irradiation broth incubation of S. aureus. Our results are compatible with a non-inducible mechanism for this phenomenon.

Summary: Incubation of a strain of Escherichia coli K12 with 25 mm-methyl methanesulphonate (MMS) for 1 h changed the sedimentation coefficient of the nucleoids from 1600S to 850S. When isolated nucleoids were treated with MMS under identical conditions in vitro there was no change in the sedimentation coefficient. Alkaline sucrose-gradient centrifugation of DNA from cells treated with 25 mm-MMS for 1 h indicated that there were approximately 100 breaks plus apurinic sites per chromosome. Titration with ethidium bromide of nucleoids from MMS-treated cells showed that almost all supercoiling had been lost, suggesting that the breaks plus apurinic sites consisted mostly of breaks. Further experiments showed that the apurinic sites were probably created by non-enzymic depurination and that little non-enzymic strand breakage had occurred. The depurinated sites thus created could then serve as substrates for the apurinic-specific endonucleases of the cell, with the result that strand breakage occurred. MMS treatment did not cause any changes in the DNA: RNA ratio of the nucleoids. Removal of MMS followed by a period of incubation resulted in a decrease in the number of breaks plus apurinic sites and an increase in the sedimentation coefficient of the nucleoids. After 2 h incubation in MMS-free medium the sedimentation coefficient of the nucleoids from MMS-treated cells was the same as that of the control; the supercoiling was also partially restored.

The effect of MMS on two MMS-sensitive mutants of E. coli, one a polA and the other a recA mutant, was also studied. In both cases MMS caused complete collapse of the nucleoid structure.

SUMMARY: The susceptibility of Klebsiella aerogenes to cycloserine varied according to the growth conditions. In batch culture, cells were less susceptible to the antibiotic when glycine was present in the medium, presumably due to competition between glycine and cycloserine for the uptake system by which glycine, d-alanine and cycloserine are transported into the cell. In the chemostat at average dilution rates, ammonia-limited cultures were more susceptible to the antibiotic than were glucose-limited cultures. Under phosphate-limiting conditions cultures were at least ten times less susceptible. Under ammonia and phosphate limitation the susceptibility increased with increasing growth rate. The sensitivity of glucose-limited cells was independent of the growth rate. A high-affinity uptake system for cycloserine (as measured by d-alanine transport) was present in ammonia- and glucose-limited cells, but not in phosphate-limited cells. Thus, the phenotypically defined alterations in the susceptibility of the bacterium to cycloserine could be correlated with variations in its uptake system for the antibiotic.

SUMMARY: A strain of Escherichia coli with a mutation in the ana gene was shown to lack acetaldehyde dehydrogenase and alcohol dehydrogenase. The requirement of this strain for an external oxidant to grow anaerobically on glucose shows that the reduction of acetyl-CoA is the principal means of reoxidation of NADH produced during glycolysis in E. coli. Further mutants derived from the ana strain were shown to be affected in the enzymes involved in the fermentation of pyruvate (pyruvate formate-lyase, phosphotransacetylase, acetate kinase). A gene controlling acetate kinase (ackB) activity has been located at 39 min on the chromosomal map. Evidence is presented that anaerobic nitrite reduction with pyruvate involves at least the dehydrogenase subunit of the pyruvate dehydrogenase complex.

A theory is developed to predict the growth rates of photosynthetic microbes in a photobiological reactor with light-sufficient zones and dark zones. The theory also predicts that in light-limited cultures the maintenance energy will increase in proportion to the duration of the dark period.

The theory was tested and quantitatively confirmed by means of a loop reactor which permitted light and dark periods to be varied with a total cycle time of 40 s. An axenic Chlorella culture grown at the maximum rate under light-sufficient conditions could continue growth at the maximum rate in the dark for 9·2 s. It was found that the maintenance energy was zero in the light, but endogenous metabolism of resting cells in the dark corresponded to a large maintenance energy of 8·8 kJ (g dry wt)−1 h−1. These results have much significance for the design of photobiological reactors.