- Volume 135, Issue 9, 1989

A new bromoperoxidase-catalase was purified from the chloramphenicol-producing actinomy-cete Streptomyces venezuelae ISP 5230. The homogeneous enzyme showed brominating activity, catalase activity and a very low peroxidase activity. The spectral properties and pH dependence of the catalase activity showed similarities to conventional catalases. In contrast to other haem-bromoperoxidases, the bromoperoxidase-catalase was stable when treated with an ethanol/ chloroform mixture. Gel filtration gave an estimated M r of 127000–136000. SDS-PAGE showed a single band corresponding in mobility to a species with an M r of 61000. The pI was estimated to be 4·5. The bromoperoxidase-catalase was not present in active form in a mutant of S. venezuelae ISP 5230, blocked in the chlorination step of chloramphenicol biosynthesis. However, an inactive species of the enzyme was detected in crude extracts of the mutant by using antibodies. From these results it is concluded that this bromoperoxidase participates in the chlorination step during chloramphenicol biosynthesis.

Halobacterium cutirubrum was cultured in a complex medium designed for extreme halophiles containing 4 m-NaCl in the absence or presence of bacitracin (7, 14 or 28 μm) with shaking at 37 °C for 24 h. Bacitracin at 7 or 14 μm inhibited growth (50% and 100%, respectively), phospholipid synthesis (88% and 96%, respectively) and bacterioruberin synthesis (65% and 87%, respectively) relative to a control lacking bacitracin. After 5 h incubation in the presence of 7 or 28 μm-bacitracin, total lipid biosynthesis, as measured by incorporation of 14C from short-term labelling with [14C]glycerol, was inhibited by 26% and 66%, respectively. Bacitracin at 7 or 28μm inhibited biosynthesis of the major phospholipids (9% and 60%), glycolipids (25% and 85%) and polyisoprenyl pyrophosphate (75% at both concentrations). These results support the concept that polyisoprenyl pyrophosphate is involved directly in the biosynthesis of phospholipids, glycolipids and bacterioruberins in the extreme halophile H. cutirubrum.

A new class of Saccharomyces cerevisiae mutants (aat1 - amino acid transport) has been identified. These mutants are unable to grow on rich medium or on minimal medium supplemented with certain amino acids (isoleucine, methionine, phenylalanine, tyrosine or valine). This phenotype is directly linked to the presence of the leu2 allele in these strains: aat1 LEU2 organisms grow normally on all media tested. Leucine uptake through the leucine-specific permease is inhibited to <35% of wild-type levels in aat1 cells preincubated in nonpermissive media, and the activity of the general amino acid permease is also low in these conditions, aat1 cells are therefore unable to grow on rich media because they cannot take up enough leucine to supplement their auxotrophic requirement.

Fourteen lipase low-producing (Lip−) mutants of Yarrowia lipolytica were obtained by UV mutagenesis. Complementation studies showed that ten of these mutants belonged to one complementation group. Two other mutants, Lip− 132 and 223, complemented those of the first group. Lip− 1123 complemented mutants of the first and second groups. Lip− 1351 did not complement Lip− 742, 417, 529 and 1133, which belonged to the first group. Genetic recombination analysis of the Lip− mutants by random ascospore analysis showed that mutants of the first and second groups have lesions in the same gene, which is proposed to be the lipase structural gene. Inter-allelic complementation occurs between certain mutants of these groups. Lip− 1123 has a lesion in a different gene.

A gene coding for endo-l,4-β-glucanase activity has been isolated from Bacteroides ruminicola subsp. brevis by cloning in Escherichia coli. After restriction mapping of a 6·4 kb insert, a 2·2 kb DNA fragment was sub-cloned in pUC19 to produce the enzymically active clone pJW3. Recloning of the gene fragment in the reverse orientation in pUC18 (clone pJW4) indicated that a gene promoter was present in the cloned fragment and was able to function in E. coli. The clone pJW4 displayed increased activity which was attributed to expression from the lac promoter of pUC18. The enzyme encoded by pJW4 was optimally active at pH 5·5–6·0, and in the temperature range 37–42 °C. The preferred substrate was carboxymethylcellulose, but the enzyme displayed 50–60% of maximal activity on both acid-swollen cellulose and soluble xylan. No significant activity was detected on ball-milled filter paper or particulate xylan. Deletion experiments confirmed that both cellulase and xylanase activities were altered to a similar extent by deletion of DNA from the 3′ end of the gene, suggesting that both are a function of the same polypeptide product.

Thirty-seven virulent and 19 temperate bacteriophages of Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were classified in a taxonomic system on the basis of morphology, DNA-DNA hybridization, and protein composition. As judged from electron microscopy and susceptibility to cleavage by restriction endonucleases, the genome of all the bacteriophages investigated is composed of double stranded DNA. Seven virulent phage groups were recognized: types P034 (genome size 18·1 kilobase pairs, kb), P001 (20·2 kb), P008 (29·7 kb), P335 (36·4 kb), P026 (51·5 kb), P107 (51·5 kb), and P087 (54·5 kb). In addition, two temperate phage groups were established: types TP-40-3 (genome size 42·1 kb) and TP-936-1 (37·8 kb). Phages within each group revealed strong DNA homology and similar protein compositions, whereas no significant DNA homology and different proteins were found in phages of different groups. Virulent phages of group P335 exhibited strong DNA homology with the temperate phages of group TP-936-1.

Stable restriction-deficient, modification-proficient galE (JR501) and F′galE + (JR502) strains of Salmonella typhimurium were constructed and the effects of restriction on transformation by plasmid pBR322 were tested. Several factors which affect transformation efficiency were systematically examined to determine optimum transformation conditions and a simplified method is presented.

A library of Treponema pallidum genomic DNA fragments produced by partial Sau3A digestion was established in Escherichia coli K12 using the plasmid vector pAT153. The library was screened using immune syphilitic rabbit serum and six recombinant phenotypes expressing eight treponemal polypeptides were detected. With two exceptions, all the recombinant gene products were the same size as polypeptides detected on Western immunoblots of T. pallidum. The genes encoding three novel gene products, with molecular masses in SDS-PAGE of 42, 17 and 15·5 kDa, which had not been cloned previously from T. pallidum were also identified. Monoclonal antibodies which reacted with four of the eight recombinant polypeptides were generated.

Human epithelial cells and the McCoy cell line were infected with Chlamydia trachomatis, serotype E. The organization of the cytoplasm was then studied with probes which stained cytoskeletal components and membrane compartments. The major actin-containing stress fibre bundles were not associated with inclusions due to the peri-basal and peri-apical location of these bundles within the host cell. The cytokeratin network was distorted by the presence of inclusions so that a common basket of these intermediate filaments surrounded both nucleus and peri-nuclear inclusions. The microtubule network was similarly distorted, but the nucleus and inclusion were surrounded by separate rather than joint baskets of tubules. After reversible depolymerization by nocadazole the microtubules in amniotic epithelial cells began to reassemble at the peri-nuclear microtubule-organizing centre, so that independent microtubule networks were rapidly regenerated around the nucleus and inclusion. Mitochondria of amniotic epithelial cells were vitally stained with the fluorescent probe DiOC6 (3,3′-dihexyloxacarbo-cyanine iodide) after 48 h of infection and found to be widely distributed throughout the host cytoplasm. When the morphology of the Golgi complex was examined with C6-NBD-ceramide (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] aminocaproyl sphingosine) the main cisternae were retained in a juxta-nuclear position, although scattered stained structures were also present close to the cytoplasmic surface of the inclusion. These results demonstrate that the peri-nuclear position of inclusions is determined by the configuration of the cytoskeleton, and that normal host-cell architecture is maintained during infection, albeit in a distorted form.

The aerobactin iron-uptake system of plasmid ColV-K30, genetically isolated from other piasmid determinants by molecular cloning, was sufficient to restore full virulence in a mouse peritonitis model to a clinical Escherichia coli isolate, D551 (O78:H−), whose resident aerobactin-encoding ColV plasmid had been lost by curing. Antiserum was raised in rabbits against live E. coli K12 cells expressing the outer-membrane aerobactin receptor protein and absorbed with an isogenic strain lacking the receptor. This antiserum inhibited binding of aerobactin, cloacin DF13 and bacteriophage B74K to the native protein in whole E. coli K12 bacteria expressing the receptor, or in membranes prepared from such organisms. However, it did not react with the native receptor protein in several wild strains unless lipopolysaccharide was first removed by treatment with trichloroacetic acid, nor did it protect mice in experimental infections with strain D551. Antisera raised in rabbits against partially or fully denatured forms of the aerobactin receptor reacted only in assays involving denatured protein; they showed no inhibition of the biological activities of the native receptor.

By using competition between glucose and its analogue d-glucosamine, we have produced a system in which it is possible to vary the steady-state growth rate of populations of Saccharomyces cerevisiae cells without otherwise altering the composition of the medium or significantly affecting catabolite repression. We demonstrate that d-glucosamine inhibits the accumulation of glucose derived label and the phosphorylation of glucose by hexokinase (EC 2.7.1.1).

Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence-of glucose induced an acidification of the intracellular medium. This acidification appeared to be due to the phosphorylation of the sugar since: (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose-or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification. In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described. Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification. Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar. The apparent K m for glucose in this process was 2 mm. Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux. This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol. Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.

Analyses were made of the fatty-acyl composition of phospholipids from each of two strains of Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically. Residues of C16:0, C16:1 and C18:1 predominated in phospholipids from strains of the first yeast, while phospholipids from Z. bailii contained mainly C16:0, C18:1 and C18:2 residues. S. cerevisiae NCYC 431, grown anaerobically in media supplemented with ergosterol and C14:1, C16:1, C18:1, C18:2, C18:3 or C20:1 fatty acids, contained phospholipids enriched with residues of the exogenously provided acid, to a greater extent with shorter chain than longer chain acids. A plot of the permeability coefficient for sulphite, derived from Woolf-Eadie plots, against the degree of unsaturation in phospholipids (expressed as Δ mol−1 value) showed that the coefficient was greater the lower the degree of unsaturation in the phospholipids. A plot of the permeability coefficient against values for the mean fatty-acyl chain length divided by the Δ mol−1 value, which is an approximation of the cross-section surface area of a phospholipid molecule, showed that the permeability coefficient tended to increase the greater the surface-area value.

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO3 − was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO3 − utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO3 − to N2 and N2O under both anaerobic and aerobic maintenance conditions.

The levels of several enzymes involved in assimilation of different nitrogen compounds were investigated in Streptomyces clavuligems in relation to the nitrogen source supplied to the cultures. Threonine dehydratase, serine dehydratase, proline dehydrogenase, histidase and urocanase were not decreased in the presence of ammonium. The latter two enzymes were induced by histidine in the culture medium, while proline dehydrogenase was induced by proline. Glutamine synthetase, urease and ornithine aminotransferase levels were higher with poor nitrogen sources and were repressed by ammonium. Arginase was induced by arginine and repressed by ammonium. Glutamine synthetase was rapidly inactivated upon addition of ammonium to the culture, and could be reactivated in vitro by treatment with snake venom phosphodiesterase, which suggested that adenylylation is involved in the inactivation. Three previously isolated mutants with abnormal glutamine synthetase activities showed pleiotropic effects on urease formation. All these data point to a mechanism controlling preferential utilization of some nitrogen sources in this species.

Two screening methods for isolation of mutants of Streptomyces clavuligerus with altered control of nitrogen metabolism enzymes are described. Thirty-eight profotrophic mutants with simultaneous deregulation of urease and glutamine synthetase were isolated. Nine mutants were examined in more detail and they also showed deregulated formation of arginase and ornithine aminotransferase. Different patterns of altered control of all four enzymes were observed Inactivation of glutamine synthetase after ammonium shock took place to different extents in these nine strains, and seven of them had a thermosensitive glutamine synthetase activity. It is concluded that a system of nitrogen control, in which glutamine synthetase has a key role, is present in S. clavuligerus. Cephalosporin production was depressed by ammonium in all the mutants, irrespective of the alterations in nitrogen control of primary metabolism.