- Volume 131, Issue 09, 1985

Summary: O2 affinities of the bioluminescence systems have been measured in five species of luminous bacteria. Photobacterium phosphoreum He-1a, recently isolated from a fish light-organ, showed halfmaximal bioluminescence at 0·015 µM-O2 whereas another symbiotic strain (PJ-1a) gave a 33-fold higher value. Vibrio fischeri MJ-1 gave a value of 0·60 µM-O2, and the symbiotic Photobacterium leiognathi LN-la gave half maximal emission at 0·7 µM-O2. These results indicate the importance of strain selection for sensitive O2 measurements.

Summary: The nitro radical-anion of the anti-trichomonal drug metronidazole has been detected by electron spin resonance spectrometry under anaerobic conditions in suspensions of intact hydrogenosomes isolated from the parasitic protozoon Trichomonas vaginalis. Metronidazole reduction was driven by pyruvate, but progressive damage to the radical generating system was observed. Quenching of signals due to metronidazole radicals by chromium oxalate suggests that the radicals generated within the organelle can cross the hydrogenosomal membrane into the external medium. Even if a similar process of radical migration occurs in vivo, it seems likely that intrahydrogenosomal damage may explain drug action.

Summary: Lysine was found in the vegetative peptidoglycan of Bacillus sphaericus (23 strains), and of the related species Bacillus pasteurii and Bacillus globisporus. None of these organisms formed diaminopimelate epimerase, but all contained diaminopimelate dehydrogenase. All other species of Bacillus studied had diaminopimelate in their walls, and produced diaminopimelate epimerase and N-acetyldiaminopimelate deacetylase, but not diaminopimelate dehydrogenase. One exception, Bacillus macerans, contained all three of these enzymes; diaminopimelate (and no lysine) was found in its walls. A new spectrophotometric assay for diaminopimelate epimerase was developed, which had advantages over the previous methods.

Summary: Organism 4025 contained only b-type and c-type cytochromes. In the absence of any a-type cytochrome oxidase it was concluded that respiration in these bacteria is catalysed entirely by way of the o-type cytochrome oxidase. After growth on methanol, the soluble protein contained cytochromes c H and c L, and a single blue copper protein of unknown function, ‘azurin’. After growth on methylamine the soluble proteins were strikingly different. The total concentration of soluble cytochrome c was about 70% lower; cytochrome c L was about 65%, and cytochrome c H about 16% of the concentrations present after growth on methanol. Although some ‘azurin’ was still present, a second copper protein, amicyanin (94% of the total), was induced during growth on methylamine. The concentration of this protein was considerably higher than that of blue copper proteins measured in other bacteria and 35 times higher than the concentration in the facultative methylotroph Pseudomonas AMI during growth on methylamine. It is the very high concentration of amicyanin that gave suspensions of methylamine-grown organism 4025 their blue-green colour. All of the soluble cytochrome c, and all of the methanol dehydrogenase, methylamine dehydrogenase and blue copper proteins were located in the periplasmic fraction. All these results are consistent with the conclusion that the main electron acceptor for methylamine dehydrogenase in organism 4025 is amicyanin.

Pure methanol dehydrogenase of Pseudomonas AMI oxidizes propanediol in a transitory fashion when measured in a dye-linked assay with phenazine ethosulphate (PES) as electron acceptor. It was shown that the transitory nature of this oxidation is not because the product (lactaldehyde) is an inhibitor but because the dehydrogenase is inactivated by PES. Substrates having low affinities for the enzyme, such as propanediol, are unable to protect against this inactivation. A ‘stimulatory factor’, previously shown to facilitate the continuous oxidation by methanol dehydrogenase of propanediol to lactate, was shown to consist of two proteins, a modifier protein (M-protein) and an aldehyde dehydrogenase. The M-protein increased the affinity of methanol dehydrogenase for propanediol; this enabled the substrate to protect the enzyme against inactivation by PES, and it facilitated the continuous oxidation of low concentrations of propanediol to lactaldehyde. The M-protein was purified almost to homogeneity (more than 95% pure) and shown to be an acidic, dimeric protein having subunits of molecular weight 64000. The second protein component of the ‘stimulatory factor’ was a dye-linked aldehyde dehydrogenase which oxidized lactaldehyde to lactate but which had a greater affinity for longer-chain normal aliphatic aldehydes and the following characteristics: pH optimum, 7-0; isoelectric point, 4 0; molecular weight, 115000; and K m for lactaldehyde, 16 mm.

Summary: Wall mannoproteins of the two (yeast and mycelial) cellular forms of Candida albicans were solubilized by different agents. Boiling in 2% (w/v) SDS was the best method, as more than70% of the total mannoprotein was extracted. Over 40 different bands (from 15 to 80 kDal) were detected on SDS-polyacrylamide gel electrophoresis of this material. The residual wall mannoproteins were releasedafter enzymic (Zymolyase and endogenous wall β-glucanases) degradation of wall glucan, suggesting that theyare covalently linked to this structural polymer. Four bands (of 160 kDal, 205 kDal and higher molecular mass) were observed in the material released from yeast walls but only the two smaller components weredetected in the material obtained from mycelial walls. Moreover, the mannoproteins of high molecular mass, which are covalently linked in walls of normal cells, were not incorporated into walls of regeneratingprotoplasts, but non-covalently linked mannoproteins were retained from the beginning of the process.

Summary: When budding cells of Candida albicans are starved for 20 min and then diluted into fresh nutrient medium at 37 °C, pH 6.7, they form mycelia by two alternative modes. For cells with small buds, the bud expands apically, resulting in a transiently tapered daughter cell. With continued growth, the daughter cell tapers into an elongated mycelium. For cells with large buds, the bud completes expansion in the budding form, the mother cell and then the daughter bud evaginate, and the evaginations grow as mycelia. The present study investigates whether the temporal and spatial changes in the zones of wall expansion during bud growth are involved in the two modes of mycelium formation. Data are presented which demonstrate that the transition circumference which determines the two modes of mycelium formation and the transition circumference at which the active apical expansion zone shuts down are both 7 µm. This exact correlation suggests that starved cells with buds with a circumference of less than 7 µm form mycelia in the tapering mode due to the reactivation of the still present apical expansion zone, and that starved cells with buds with a circumference greater than 7 µm complete bud growth by general expansion due to the absence of the apical expansion zone at the time of starvation.

Summary: A new mutation, nalD, determining resistance of Escherichia coli to nalidixic acid (NAL) is reported. The nalD mutant described is resistant to NAL at 37°C but sensitive at 30 C. It is defective in penetration of NAL and glycerol through the outer membrane at 37°C. The nalD mutation is located half-way between 89 and 89.5 min on the E. coli genetic map.

Summary: Plasmids were isolated from E colicinogenic strains and transformed into prototrophic Escherichia coli K12 strain DB364. Screening of E colicinogenic transformants for growth on defined medium revealed an apparent amino acid auxotrophy mediated by E4 and, to a lesser extent, E colicin plasmids. The auxotrophy was further investigated in E4 colicinogenic strains. From such auxotrophic transformants, denoted Pmi+ (plasmid-mediated inhibition of growth). Pmi− variants were obtained at a frequency of 3 x 10−4 per bacterium. Plasmid loss was not detected among Pmi− clones. Isolation of E4 colicin plasmids from Pmi− clones and re-transformation of strain DB364 with these plasmids showed that 40% of the plasmids were unable to inhibit growth of DB364 and were inferred to have alterations in an E4 colicin plasmid gene termed pmi. All such plasmids were indistinguishable from native E4 colicin plasmids, with respect to colicin immunity, colicin production and excretion, and sensitivity to lysis by mitom C. Experiments examining the nutritional basis of the plasmid-mediated auxotrophy indicated that at least seven amino acids, isoleucine, leucine, valine, arginine, methionine, serine and glycine, were involved in the auxotrophy. However, supplementation with only these seven amino acids did not completely restore growth. Assays of the activities of enzymes involved in amino acid biosynthesis in colicinogenic and non-colicinogenic strains under repressing and derepressing growth conditions suggested that E4 colicin plasmids did not repress synthesis of the implicated amino acids.

Summary: RNA polymerase was purified from both vegetative and symbiotic forms of Rhizobium japonicum strain 3I 1b 110 and the physical and transcriptional properties of the enzyme were compared. The subunit composition of the enzyme from both sources appeared similar when analysed by SDS-polyacrylamide gel electrophoresis. Removal of an 82 kDal protein from the purified enzyme from either source, using procedures developed for isolation of the sigma factor from Escherichia coli RNA polymerase, resulted in a decreased ability of the enzyme to utilize phage T4 DNA as a template in vitro. These results indicate that the 82 kDal protein may act as a sigma-like factor for the RNA polymerase of R. japonicum. The transcriptional specificity of the enzyme from both sources was similar using a variety of exogenous DNA templates. There were no apparent differences in the kinetic properties of the enzyme from both sources, or in the specificity of promoter utilization when phage T7 DNA was used as a template. This would indicate that a novel form of RNA polymerase may not be responsible for the transcription of nif genes in the symbiotic state.

Summary: Isolates of Rhizobium meliloti and of R. leguminosarum biovars viceae, trifolii and phaseoli were obtained from a single site in Norfolk, England, and examined by enzyme electrophoresis of glucose-6-phosphate dehydrogenase, superoxide dismutase, β-galactosidase and esterases. All the enzymes had mobility variants, but the variation of the different enzymes was highly correlated, so that only a restricted number of combinations (electrophoretic types, ETs) were found. Some ETs were confined to a single biovar, but others were shared amongst R. leguminosarum biovars viceae, trifolii and phaseoli. Most R. meliloti isolates were quite distinct from those of R. leguminosarum. Electrophoresis of total soluble protein in the presence of sodium dodecyl sulphate revealed variation that correlated with the ET rather than with the host range. It is suggested that the Rhizobium isolates from this site comprise a number of genetically distinct lineages, some of which may carry any of several different host-range determinants, and which therefore appear in more than one biovar.

Summary: A mutation near cysB on the Bacillus subtilis chromosome marks a new sporulation locus, spoVIC. It causes spores to germinate more slowly than those of the wild-type under all conditions and, from indirect evidence, it does not appear to alter the affinity for the germinant l-alanine. The mutant spores have some deficiency of coat proteins (particularly the alkalisoluble coat protein, M r = 12000) and the spore coat layers are disorganized. The mutant strain grows normally and sporulates normally until stage II, after which its sporulation is delayed by about 2 h compared to that of the wild-type. This delay results in the prolonged synthesis of some coat proteins and the late synthesis of others. The abnormal coat may be the cause of the germination deficiency. A double mutant strain carrying the spoVIC610 mutation together with gerE36 sporulates slowly. Its spores have very little coat protein, are sensitive to heat, lysozyme and organic solvents, but germinate as well as the strain carrying the spoVIC mutation alone. The role of the spore coat in germination is discussed in the light of these findings.

Summary: Early in sporulation, cells of wild-type Bacillus subtilis produce three proteases (b, c and d) with monomeric M r values of about 65000, 53000 and 43500, anda further protease, e (M r about 30000) at the time of coat assembly. An additional protease, f (M r about 15000) appears transiently in sporangia at about the time of spore release. Three strains with defective spore coats were examined for alterations in sporulation proteases. A strain carrying the gerE36 mutation produces b, c and d normally, fails to produce e and accumulates f on or inits spores. A strain carrying the spoVIC610 mutation produces normal quantities of proteases b, c and d, but has a reduced amount of proteases e and f. A strain carrying both the gerE36 and the spoVIC610 mutations accumulates neither protease e nor f. The wild-type allele of the gerE gene was cloned in the vector, phage ϕ105J9. Complementation tests with the cloned gene showed that the gerE36 mutation is recessive to the wild-type allele.

Summary: Transposon mutagenesis in a tomato isolate of Pseudomonas solanacearum (strain Kourou) is reported, using Tn7 and Tn5 inserted in suicide conjugative plasmids. Whereas Tn7 integrates at high frequency in a particular site of the the genome, Tn5 appears to transpose much more randomly, allowing isolation of auxotrophic mutants with a frequency of 0.35%. The mutants showed a wide range of nutritional requirements. Following Tn5 mutagenesis, screening of 8250 clones on axenic tomato seedlings led to the isolation of 12 avirulent mutants. Southern blot analysis revealed that, for avirulent mutants, insertion of Tn5 occurred in at least 10 different EcoRI restriction fragments. Additional independent insertions of IS50 were also detected in four of these mutants. For each mutant, transformation experiments demonstrated that the Tn5-encoded kanamycin resistance and the avirulent phenotype are linked. Based on their ability or inability to induce a collapse of tobacco leaf parenchyma, and on the timing of reaction of the plant, avirulent mutants have been divided in to two and possibly three groups.

Summary: Examination of approximately 700 soil samples yielded about 100 actinophages. Restriction analysis of phage DNA indicated that 57 are unique, and of these, 20 produce turbid plaques on one or more of the streptomycetes tested. Five phages are shown to insert into the genome of Streptomyces avermitilis. None of the phages was able to perform generalized transduction of S. avermitilis.

Summary: Two bacteriophages, designated GW6210 and JW2040, were isolated from decaying apples using Gluconobacter oxydans ATC 621 and G. oxydans VPI 204JW, respectively. Electron microscopy showed that phage GW6210 belonged to group A and phage JW2040 to group C of Bradley's morphological classification. Phage GW6210 was unusually large, with a head diameter of 170 nm. Both phages contained double stranded DNA. The G + C content of the DNA of phage GW6210 was 293 mol % (T m), and the size of the genome was approximately 250-300 kb. The size of the DNA of phage JW2040 was found to be 37 kb and the G + C content was 565 mol% (T m). The host ranges of both phages were determined using 54 Gluconobacter, 52 bacter and three Pseudomonas strains. Only the Gluconobacter strains were hosts for these phages.