- Volume 133, Issue 12, 1987

SUMMARY: Four species of the genus Lysobacter, especially L. brunescens and L. gummosus, produced amylase when grown in liquid medium containing starch. The amylase was purified nearly 1200-fold with a 36% yield from the culture supernatant of L. brunescens by ammonium sulphate precipitation, CM-52 cellulose chromatography and gel filtration. An M r of 47000–49000 was estimated for the enzyme from results of SDS-PAGE and glycerol gradient centrifugation, respectively. Metal ions such as Mg2+, Ca2+, Mn2+ or Zn2+ are not required for activity. The amylase is active from pH 5.0 to 7.5, degrades starch endohydrolytically and has a K m of 2.08 mg starch ml-1. Amylopectin, amylose and glycogen were also hydrolysed. Dextran, maltose, maltotriose, maltotetraose and 4-nitrophenyl α-d-glucoside were hydrolysed very slowly, or not at all. Other enzymes capable of degrading starch, maltose or malto-oligosaccharides were not detected in the culture supernatant of L. brunescens.

SUMMARY: In Bacillus subtilis it was shown that the membrane potential (Δ,ψ) has to reach a threshold value of −180 to −190 mV for efficient uptake of dihydrostreptomycin to occur. The magnitude of Δ,ψ is raised above this threshold, and dihydrostreptomycin uptake greatly enhanced, not only by dihydrostreptomycin itself (autostimulation) and by other miscoding aminoglycoside antibiotics, but also by puromycin, bacitracin and N, N-dicyclohexylcarbodiimide. Stimulation of uptake by dihydrostreptomycin or puromycin was dependent on a specific interference with ongoing protein synthesis. Thus, chloramphenicol prevented the stimulating effect of puromycin by lowering the magnitude of δ+. Although normally severely antagonizing streptomycin accumulation, K+, as well as spermidine and putrescine, which are known to stabilize ribosomes, consequently enhanced autostimulation of dihydrostreptomycin uptake in a K+-retention mutant with impaired protein synthesis. It is suggested that miscoding aminoglycosides and puromycin both enhance dihydrostreptomycin uptake by increasing δ+ due to ion fluxes, which are themselves caused by a dramatic stimulation of intracellular proteolysis of faulty proteins.

SUMMARY: The levels of catalase and superoxide dismutase present in the white-rot fungus Phanerochaete chrysosporium were investigated. The specific activities of both enzymes increased and reached a maximum after 4-6 d growth before falling to a constant low activity. Catalase was purified and found to be the typical eukaryotic enzyme. Activity staining, in the presence and absence of cyanide, revealed that the cytosol of P. chrysosporium contained only the copper/zinc superoxide dismutase commonly found in eukaryotes. These results show that P. chrysosporium meets the challenge of raised levels of activated oxygen by producing enhanced levels of the usual detoxifying enzymes rather than any novel isoenzymes.

SUMMARY: The rate of expression of the cell-associated fructosyltransferase (FTFm) activity of Streptococcus salivarius ATCC 25975 grown in continuous culture was linearly related to the rate of octadecenoic acid (C18:1) incorporation into the membrane lipids irrespective of the presence or absence of Tween 80 in the growth medium. This observation was confirmed with data obtained from cells grown in the presence of a series of n-alkanols. The results suggested that co-synthesis of lipids containing C18:1 residues was necessary for FTFm expression and accounted for the slight stimulation of enzyme expression by Tween 80 at all growth rates. In contrast, addition of Tween 80 to the growth medium resulted in several-fold increases in extracellular glucosyltransferase (GTFe) production irrespective of the growth rate. Following the addition of the surfactant to the growth medium, an exponential relation between the increased rate of GTFe production and the concomitant net increase in the rate of C18:1 incorporation was noted. The results obtained in continuous culture emphasized the underlying effect growth rate had on GTFe production, especially when Tween 80 was added to the growth medium. In the presence of n-alkanols, the rate of GTFe production plotted as a single ‘U’-shaped curve with respect to the rate of C18:1 incorporation irrespective of the chain length of the n-alkanol studied. Rapid analyses of the extracellular proteins by SDS-PAGE suggested that hexan-l-ol and Tween 80 specifically stimulated the synthesis and secretion of GTFe and no other extracellular protein. The combined results emphasized the dissimilarity between amphiphilic modulation of FTFm and GTFe production as well as the apparent unique stimulation of the synthesis and secretion of the latter enzyme(s).

SUMMARY: The topology of tubulin and actin during mating of Saccharomyces cerevisiae was analysed by fluorescence microscopy with the monoclonal anti-tubulin antibody TuO1 and rhodamine-labelled phalloidin. Preconjugatory cells displayed an asymmetric distribution of the microtubule and actin cytoskeleton and an overall polarization of the cells preceding cell fusion. Prior to karyogamy, the haploid spindle pole bodies were associated with abundant cytoplasmic microtubules. Budding zygotes revealed the same tubulin and actin patterns as vegetative cells. Treatment of the mating mixture with the microtubule inhibitor nocodazole (10 μg ml-1) did not prevent polarization and fusion of haploids, zygote formation and emergence of the first zygotic bud. In marked contrast, the migration of the nucleus in preconjugatory cells as well as nuclear migration and fusion within the zygotes was unequivocally blocked by the action of the drug. It is suggested that the problem of the morphogenesis of mating should be approached by considering interactions at the cell periphery.

SUMMARY: R. vannielii swarmer cells were more resistant to the effects of rifampicin than chain or budding cells. RNA polymerase purified from the different cell types showed no difference in susceptibility to rifampicin or in the major protein subunits as visualized by two-dimensional gel electrophoresis. The susceptibility of swarmer and budding cells to sodium deoxycholate gave a similar pattern to that found for rifampicin, with swarmer cells being more resistant than budding cells. This suggests that the changes in susceptibility to the inhibitors as swarmer cells differentiate into budding cells are due to changes in the permeability properties of the cell envelope that relate to changes in the role of cells during the cell cycle.

SUMMARY: The ability of two strains of Rhizobium leguminosarum biovar trifolii (formerly R. trifolii) to compete for nodule sites in soil with indigenous R. l. biovar trifolii was studied using ELISA. In pot experiments with soil containing effective indigenous strains, one of the inoculated strains, 285, was able to form nodules on red clover (Trifolium pratense L.), not only on the tap root but also on later-formed roots. Under field conditions, also in a soil containing effective indigenous R. l. biovar trifolii strains, both inoculated strains were detectable in minor proportions in the nodules of the established red clover. The inoculated strains survived in the soil during the field season. As the indigenous strains were effective, no increase in dry matter production was obtained in this experiment. However, in pot experiments in a soil with ineffective indigenous red clover bacteria, the other inoculated strain, 7612, known to be an effective nitrogen-fixing strain, was less successful and was only detectable incidentally. Although the nodule occupancy of the successful competitor strain, 285, only ranged between 7% and 27% on the upper tap root and was merely occasional on the other parts of the root in the ‘ineffective’ soil, increases in dry matter production were obtained, indicating that the nodules on a specific plant have varying significance for the apparent nitrogen fixation.

SUMMARY: Horizontal movement of Azospirillum brasilense Cd in soil and its vertical movement in the plant rhizosphere were studied. No movement was detected in the absence of living plants. In a controlled environment the bacteria moved horizontally at least 30 cm from the inoculation point to the first growing plant. Once the first root system was colonized, all the neighbouring plants became inhabited. Horizontal movement under field conditions was at least 160 cm, and depended on the presence of live plant roots. Several weeds that grew in the passes between plots acted as efficient vectors. The numbers of A. brasilense Cd decreased with increasing distance from the inoculated plot. Vertical movement in soil columns in a controlled environment was up to 40 cm. Under field conditions, bacteria were detected as deep as 50 cm in the root systems of wheat plants in various types of soil. During the growing season bacteria were mostly found on and in young roots at a depth of 20–50 cm and near the soil surface. A map of depth distribution of A. brasilense Cd showed an uneven colonization pattern within the same root system or between adjacent plants. It was concluded that A. brasilense Cd moved horizontally and vertically in various soil types and that this movement was mainly dependent on the presence of plants.

SUMMARY: A mutant strain of Escherichia coli, strain AK23, is devoid of hydrogenase activity when grown anaerobically on glucose and cannot grow on H2 plus fumarate. From an E. coli chromosomal DNA library, a plasmid, pAK23, was isolated which restored hydrogenase activity in this strain. Two smaller plasmids, pAK23C and pAK23S, containing different parts of the insert DNA fragment of plasmid pAK23, were isolated. The former plasmid restored activity in strain AK23 while the latter did not. The smallest active DNA fragment in plasmid pAK23C was 0.9 kb. This gene is designated hydE. Plasmids pAK23 and pAK23S restored activity in another hydrogenase-negative strain, SE-3-1 (hydB), while plasmid pAK23C did not, suggesting that plasmid pAK23 contains two genes required for hydrogenase expression. Strain AK23 was also devoid of formate hydrogeniyase and formate dehydrogenase activities and these activities were restored by some of the plasmids. Hydrogenase and formate-related activities in strain AK23-were restored by growth of cells in a high concentration of nickel. Plasmid pAK23C led to synthesis of a polypeptide of subunit molecular mass 36 kDa and plasmid pAK23S led to synthesis of polypeptides of subunit molecular masses 30 and 41 kDa.

SUMMARY: A transposon Tn9/7 insertion between gerE and ilvB has identified a new developmental locus, gerM, in Bacillus subtilis. gerM96::Tn917 affects both sporulation and germination. DNA on either side of the transposon has been cloned and includes the previously cloned sdhC and gerE loci. gerE terminates 2.1 kb from the end of the transposon. The gerM96::Tn917 mutant is oligosporogenous, yielding approximately 1% of the number of wild-type heat resistant spores in liquid medium and 10% on solid medium. Six hours after the onset of sporulation alkaline phosphatase and glucose dehydrogenase levels were 90% and 7%, respectively, of those of the wild-type. At this time 50% of the mutant cells were still dividing. The occurrence of multiple polar septa and ‘pygmy’ cells suggested a block at stage II of sporulation. Following addition of germinants, mutant spores prepared on nutrient agar lost heat resistance normally but released slightly less dipicolinic acid than wild-type spores. They also showed only partial loss of optical density, associated with a phase-grey appearance and striations in the cortex suggesting partial degradation. Expression of the gerM gene was monitored by production of β-galactosidase encoded by a promotorless lacZ gene fused to the gerM96:: Tn9/7 insertion. It occurred 1.5–4 h after commencement of sporulation. Transcription was directed from a promoter on the gerE side of gerM and was unaffected by a mutation in the gerE gene.

SUMMARY: An essential region (2.3 kb) for the replication of a low-copy-number plasmid, pBS-2, has been identified and cloned into plasmid pHV60 in Bacillus subtilis. The resultant plasmid, pK W1, and two other plasmids, pC194 (medium copy number) and pTP5 (high copy number), were examined by double radio-labelling and gel electrophoresis to determine which host functions are required for their replication in B. subtilis. Replication of pKW1 requires the functions of most dna genes, in particular dnaB, C, E, F, G and H; pC194 requires only dnaG and H; and pTP5 requires dnaE, F, G and H. Thus dnaG and dnaH are required for the replication of all three plasmids tested, even though each plasmid showed a different spectrum of dependency on other host functions. Because of its greater dependence on host functions and its low copy number, pKW1 should be a useful model with which to investigate the function of host genes in the replication of DNA in B. subtilis. pKW1 should also be a useful shuttle vector for cloning of genes in B. subtilis in cases when high gene dosage might be a problem.

SUMMARY: Two antibiotic-resistant mutants of Enterobacter cloacae (AZT-R and AMA-R), obtained by selection with aztreonam and carumonam, were studied. Both mutants were resistant to β-lactam antibiotics. In addition, AMA-R was also resistant to chloramphenicol, trimethoprim and brodimoprim, whereas AZT-R was hypersensitive to these compounds. Cytoplasmic and outer membranes of these bacteria were separated by sucrose density gradient centrifugation. Analysis of the outer membranes using SDS-PAGE showed marked changes in the bands corresponding to the porins (between 35 and 40 kDa). In the two mutants, the 39 kDa band was reduced to approximately 30% of the wild-type and the 36.5 kDa band was absent. Labelling of the outer membranes with the hydrophobic photolabel 3-(trifluoromethyl)-3-(m-[125I]iodo-phenyl)diazirine ([125I]TID) enabled the above bands as well as a 28.8 kDa band to be identified as integral membrane proteins, thus supporting the suggestion that they correspond to porins and OmpA protein, respectively. Whereas the changes observed in outer-membrane proteins are assumed to be responsible for resistance to β-lactam antibiotics, the basis of hyper-sensitivity of AZT-R to hydrophobic antibiotics remains to be more clearly defined.

SUMMARY: Fifty-six mutants of Bacillus subtilis 168 were selected for resistance to bacteriophages ø29 or ø25. The mutations were all linked to previously described teichoic acid markers gtaA, gtaB or gtaC, for the first and last of which, the gene products have previously been identified. Each linkage group was shown to have two distinct phenotypes with respect to phage resistance and cell-wall galactosamine content. Recombination indexes of 0.35, 0.13 and 0.41 for groups A, B and C respectively were consistent with the presence of two average-sized genes in groups A and C. Correlation between genetic and phenotypic differences supported this conclusion and led to the designation of two new markers, gtaD and gtaE. Two- and three-factor transformation crosses suggested the order hisA-gtaB-gtaD-gtaA-tag-l and gtaC-gtaE-argC. Assays for UDPglucose pyrophosphorylase and phosphoglucomutase activities in soluble extracts of representative mutants revealed that, in contrast to previous findings, the former activity was virtually undetectable in all nine group B mutants examined, suggesting that gtaB is the structural gene of this enzyme. Our results allow us to account for discrepancies with respect to previous reports. The thermosensitive mutation previously designated rodCl was shown to be 90% cotransformable with tag-1. In view of their extremely similar phenotypes the former mutation was renamed tag-3, and the likely order obtained was gtaA-tag-3-tag-l. This suggests that many mutations associated with deformation of cell shape in B. subtilis are located in the region where teichoic acid genes map.

SUMMARY: Rifamycin CGP 4832 is a semisynthetic rifamycin derivative. It is at least 200 times more active than rifampicin against Escherichia coli and related bacteria. This increased activity is shown here to be due to the efficient uptake of CGP 4832 across the E. coli outer membrane via the ferrichrome transport system comprising the outer membrane FhuA (TonA) protein, the ferrichrome receptor, and the inner membrane TonB protein. CGP 4832 competed with ferrichrome and other iron siderophore complexes, and with bacteriophage T5 and colicin M for binding sites on the FhuA protein. Mutations in fhuA or tonB genes reduce CGP 4832 sensitivity to a level comparable to that to rifampicin. There is no evidence that CGP 4832 or rifampicin utilize the inner membrane ferrichrome transport system to gain entry into the cytoplasm.

SUMMARY: Sixty-two ‘crosses’ were attempted using mixed conidial suspensions and a ‘hanging drop’ technique involving 21 auxotrophic mutants obtained from seven isolates of Verticillium lecanil. Frequency of heterokaryon formation varied from 0 to 100%, with apparently complete incompatibility being expressed between some isolates, although this was influenced by the particular auxotrophic marker used. Protoplast fusion between compatible isolates usually resulted in prototrophic colonies, but was often unsuccessful when testing apparently incompatible isolates, suggesting that incompatibility was due mainly to nuclear or cytoplasmic factors, rather than to the wall. Evidence for the formation of prototrophic heterozygous diploids was provided by nutritional studies, and DNA content per conidiospore as measured by Feulgen microdensitometry. Conidiospore volume, determined by Coulter counter analysis, was not a reliable indicator of ploidy when investigating parasexual recombination between isolates of differing conidiospore size. Heterozygous diploids generally tended to be unstable, breaking down to yield recombinant haploids; however, four diploids were isolated which remained stable for 3-4 weeks on artificial media.

SUMMARY: Methylotrophic growth of Eubacterium limosum, a fermentative anaerobe, necessitates the production of butyric acid in quantities proportionate to the methanol dissimilatory flux in order to balance the reducing equivalent status of the cell. Supplementing the medium with low concentrations (<100mM) of butyrate decreased the quantity of methanol dissimilated and brought about a corresponding shift in organic acid production in favour of acetic acid. At higher supplement concentrations the growth rate decreased and synthesis of caproic acid and an unidentified compound, most likely a biopolymer, occurred. In fed-batch cultures similar levels of butyrate were necessary to bring about these biotransformations. The addition of a C2-unit to supplemented organic acids was not restricted to those acids normally involved in the acidogenic metabolism of E. limosum, a finding, which may be of potential biotechnological importance.