- Volume 131, Issue 12, 1985

Summary: Pseudomonas BB1, grown on alcohols, contained a quinoprotein alcohol dehydrogenase, whose substrate specificity was between those of typical methanol dehydrogenases and ethanol dehydrogenases, and which had a higher turnover number and a different amino acid composition. The enzyme occurred in a dimeric as well as in a monomeric form, and the ratio in which the two forms were found depended on the culture conditions. The mechanism of action and the substrate specificity and affinity of the two forms were identical, while the turnover number of the dimer was twice that of the monomer. This indicates that the catalytic activity of the monomer does not change on dimerization. On inactivating alcohol oxidation of whole cells with cyclopropanol, monomeric and dimeric enzyme became fully inactivated. It is tentatively concluded that both forms participate in alcohol oxidation in vivo.

Summary: The accumulation of amino acids and the excretion of ammonium have been studied in various Neurospora crassa ammonium assimilation mutants, together with the labelling of glutamine in the presence of [U-14C]sucrose when N. crassa grows on glutamine as the nitrogen source. Ammonium coming from glutamine degradation by the ω-amidase pathway is assimilated by NADP-dependent glutamate dehydrogenase and glutamine synthetase. The operation of these enzymes results in a cycling of glutamine that seems to be essential for cell growth. In addition, glutamine is also converted to glutamate by glutamate synthase.

Summary: The influence of the nitrogen source on pyoverdine production by Pseudomonas fluorescens grown in chemostat culture in synthetic media at constant pH was studied. Pigment synthesis was highest in a medium with proline as the nitrogen source; other amino acids were less effective in promoting pigment synthesis. The pH and Fe3+ content of the media affected pyoverdine production. The ratio of pigment to growth was maximal at pH 8. When the Fe3+ concentration was increased from 10 to 200 μg 1−1 in media with the same nitrogen source, pyoverdine synthesis decreased. Since the Fe3+ contents of the synthetic media were different, the amount present was adjusted to a standard concentration to permit comparisons between the various nitrogen sources.

Summary: The intracellular ATP content of Mycobacterium leprae isolated from armadillo tissue was approximately 1.5 x 10−16 g per bacillus. During in vitro incubation of bacilli at 4 °C, 33 °C or 37 °C there was an exponential decrease in ATP content, the rate depending on the medium and the temperature. M. leprae incorporated phosphate into ATP and into other nucleotide materials during in vitro incubation.

Summary: An alkalophilic Bacillus sp., no. 1139, was isolated from soil and this bacterium produced a carboxymethyl cellulase (CMCase). The production of enzyme was strongly inhibited by the addition of glucose. The CMCase was purified on a DEAE-Toyopearl 650 ion-exchange column followed by Toyopearl HW-55F gel filtration and passage through a DEAE-Toyopearl 650 ion-exchange column. The purified enzyme gave a single band of protein on PAGE. The enzyme hydrolysed carboxymethylcellulose with an optimum at pH 9.0 and a K m of 0.48 mg ml−1; no activity was observed at pH 6.0. The enzyme had a molecular weight (SDS-PAGE) of 92000 and an isoelectric point of 3.1. The maximum degree of hydrolysis of carboxymethylcellulose was about 30% and trans-glucosidase activity was also observed.

Summary: An extracellular glucosyltransferase (sucrose: 1,6-, 1,3-α-D-glucan 3-α- and 6-α-D-glucosyltransferase, EC 2.4.1.-) of Streptococcus mutans HS6 (serotype a) was purified from culture supernatant by DEAE-Sepharose chromatography and preparative isoelectric focusing. The molecular weight measured by SDS-PAGE was 159000 and the isoelectric point was pH 4.9. The specific activity was 89.7 i.u. (mg protein)−1 and the optimum pH was 6.0. The K m value for sucrose was 4.9 mM and the enzyme activity was not stimulated by exogenous dextran T10. Glucan was synthesized de novo from sucrose by the purified enzyme and consisted of 49.1 mol% 1,6-α-linked glucose and 33.9 mol% 1,3-α-linked glucose, with 13.6 mol% terminal glucose and 3.3 mol% 1,3,6-α-branched glucose.

Summary: Anomalous nodulation of Trifolium subterraneum (subterranean clover) roots by Rhizobium leguminosarum 1020 was examined as a model of modified host-specificity in a Rhizobium-legume symbiosis. Consistent with previous reports, these nodules (i) appeared most often at sites of secondary root emergence, (ii) were ineffective in nitrogen fixation and (iii) were as numerous as nodules formed by an effective Rhizobium trifolii strain. R. leguminosarum 1020, grown on agar plates or in the clover root environment, did not bind the white clover lectin, trifoliin A. This strain did not attach in high numbers, and did not induce shepherd's crooks or infection threads, in subterranean clover root hairs. However, R. leguminosarum 1020 did cause branching, moderate curling and other deformations of root hairs. The bacteria probably entered the clover root through breaks in the epidermis at sites of lateral root emergence. The anomalous nodulation was inhibited by nitrate. Only trace amounts of leghaemoglobin were detected in the nodules by Western blot analysis. The nodules were of the meristematic type and initially contained well-developed infection, bacteroid and senescent zones. Infection threads were readily found in the infection zone of the nodule. However, the bacteroid-containing tissue senesced more rapidly than in the effective symbiosis between subterranean clover and R. trifolii 0403. This anomalous nodulation of subterranean clover by R. leguminosarum 1020 suggests a naturally-occurring alternative route of infection that allows Rhizobium to enlarge its host range.

Summary: Protoplasts were formed from aged germlings and mycelia of Conidiobolus lamprauges, using a combination of chitinase and β-glucanase. Methanol, ethanol and propanol stimulated protoplast formation, apparently by a mechanism different from that of mercaptoethanol. Regeneration of protoplasts in soft agar was detectable by light microscopy 2 h after incubation and colonies were observable by the naked eye within 40 h; the regeneration rate was 65%.

Summary: We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by Dictyostelium discoideum amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into D. discoideum amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.

Summary: The effects of elevated temperature and of digestion with a variety of proteinases on the floc-forming ability of flocculent strains of Saccharomyces cerevisiae, both genetically defined (FLO1 and FLO5) laboratory and genetically undefined brewing strains, have been determined. This has permitted classification of the flocculent phenotypes of these strains according to criteria other than quantitative grading of flocculence. The flocculent phenotypes conferred by both the FLO1 and the FLO5 gene were irreversibly lost upon treatment with pronase, proteinase K, trypsin or 2-mercaptoethanol treatments. However, the floc-forming ability of cells of the FLO1 strain ABXL-1D was destroyed by chymotrypsin digestion and was stable to incubation at 70°C, whereas the floc-forming ability of cells of the FLO5 strain ABXR-11A was resistant to the action of chymotrypsin and was heat labile. Tetrad analysis of a cross of these FLO1 and FLO5 strains indicated that the chymotrypsin and heat sensitivity phenotypes were FLO-gene determined. It appears that expression of the FLO1 and FLO5 genes leads to the production of different and characteristic cell-wall proteins underlying their respective flocculent phenotypes.

Summary: Sixteen wild-type isolates of Verticillium lecanii from different insects and non-insect hosts, soils and locations exhibited a wide range in spore volumes, from 2.18 μm3 to 18.25 μm3, as recorded by Coulter counter analysis. A cold hydrolysis technique for Feulgen DNA microdensitometry indicated that all the isolates were haploid. Two wild-type strains of V. dahliae, from rape and sugarbeet from Sweden, were shown to be stable diploids by the criteria of conidial volume and relative DNA content. After treatment with p-fluorophenylalanine, both V. dahliae strains produced haploid segregants with half the conidial volume and relative DNA content of the original parents. Both diploid strains are regarded as new records for V. dahliae var. longisporum (Stark).

Summary: Two spontaneous mutant plasmids of TOL plasmid pWW0 are described. pWW0-660 contains an 18 kbp deletion of the entire xylCAB operon and pWW0-673 has a 3.4 kbp insert in the operator-promoter (OP1) region of xylCAB. In Pseudomonas putida hosts containing either plasmid, m-xylene was unable to act as a growth substrate or as an inducer of enzymes of the xylCAB or of the xylDLEGF operons. Complementation of these strains with a recombinant plasmid pWW0-3006 carrying the HindIII region D (HD) of pWW0 restored the Mxy+ phenotype and the ability of m-xylene to induce the xylDLEGF operon. The cloned HD region had no regulatory function but carried the OP1 region, the entire xylC structural gene and sufficient of xylA to support growth on m-xylene. XylC was more precisely located on a 1.9 kbp Sa/I subclone of the HD region. Comparison of the roles of m-xylene and m-methylbenzyl alcohol as inducers shows they are not equivalent and that whereas m-xylene can induce only xylCAB, m-methylbenzyl alcohol can induce both xylCAB and xylDLEGF. The restoration of the induction of xylDLEGF by m-xylene is due to the presence of xylA on the recombinant pWW0-3006, converting m-xylene to m-methylbenzyl alcohol.

Summary: A gene for the β-lactamase from alkalophilic Bacillus sp. strain 170 was cloned in a functional state on a 1-0 kb DNA fragment and its nucleotide sequence was determined. The coding sequence showed an open reading frame of 257 amino acids, which represents the β-lactamase precursor protein. It is considered that the signal peptide consisted of 30 amino acids including 12 hydrophobic amino acids.

Summary: Bacterial DNA was incubated with xanthine plus xanthine oxidase plus excess iron as an oxygen-species-generating system, and DNA injury was measured by agarose gel electrophoresis and by the ability of the DNA to transform competent bacteria. After 5 to 10 min incubation, the covalently closed circular form of plasmid DNA was converted into the open circular form, and after 30 min, to some extent into the linear form. Biological activity, measured as the number of transformed bacteria, decreased rapidly after 10 min incubation. Incubation of chromosomal DNA with the enzymic oxygen-species-generating system resulted in the degradation of DNA to small fragments within about 1 h. Excess iron was essential for the damaging effect of xanthine plus xanthine oxidase. Damage to DNA could be prevented by oxygen scavengers such as superoxide dismutase, catalase, mannitol and thiourea. Our results suggest that hydroxyl radical is the injurious oxidant for bacterial DNA, and that it can mediate physicochemical as well as biological alterations in DNA.

Summary: The non-conjugative plasmid pAV5 specifies resistance to kanamycin/neomycin (KmR) and tetracycline (TcR). Physical evidence is presented to show that pAV5 gives rise to two plasmids, pAV51 (KmR) and pAV52 (TcR), which are formed by deletion of apparently non-overlapping segments of pAV5. Expression of TcR has been obtained in Escherichia coli and is associated with a 1.9 kb HindIII fragment found in pAV5 and in pAV52. Expression of KmR has been obtained in E. coli and is associated with a 1.3 kb PstI fragment found in pAV5 and pAV51. Evidence is presented that the KmR gene is flanked by inverted repeat sequences and is therefore tentatively identified as a transposon, designated Tn4411. The KmR gene specifies an aminoglycoside 3'-phosphotransferase-type I (APH(3')-I) enzyme.

Summary: The existence of the plasmid incompatibility group D was reaffirmed as a result of compatibility experiments done on plasmids R687, R711b, R778b and R840 which were previously tentatively accepted as constituting the group. The group was further delineated by the isolation of a phage, phage D, which adsorbed specifically to IncD plasmid- encoded pili produced by Escherichia coli K12 strains and strains of Salmonella typhimurium, Proteus morganii and Klebsiella oxytoca harbouring one of these plasmids. Plaque formation, like that of phage pilHα, was temperature sensitive in that plaques formed at 26 °C but not at 37 °C. Plaques were fairly clear, regular in outline and varied from pinpoint to about 1.5 mm in diameter on E. coli hosts where plaques were detected, but on the other hosts the plaques were more turbid and often irregular in outline. The phage did not plate (or propagate) on IncD plasmid-carrying strains of Providencia alcalifaciens. Providencia stuartii or Serratia marcescens. The phage had an isometric hexagonal outline with a diameter of about 27 nm. It contained RNA and resembled two other RNA-containing phages, M and pilHα, by being sensitive to chloroform. It adsorbed to the sides of the very distal ends of the shafts of IncD plasmid-coded pili.