- Volume 136, Issue 4, 1990

Three species-specific, temperate actinophages of Streptomyces coelicolor Müller, ϕSC623, ϕSC347 and ϕSC681, were compared with respect to host range, virion structure, antiserum cross-inactivation, DNA-restriction pattern, DNA hybridization, and DNA base composition. The restriction map of ϕSC623 (57 kb) was established with eight restriction enzymes; the homologies of this phage with ϕSC347 and ϕSC681 suggested that it might be a hybrid phage composed of approximately equal parts homologous to one of the other two phages. No homology was detected between ϕSC623 and R4, a temperate, wide-host-range phage which can also lysogenize S. coelicolor Müller.

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hybridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.

Time-lapse cinematography and immunofluorescence microscopy were used to study cellular events during amoebal fusions and sexual plasmodium development in Physarum polycephalum. Amoebal fusions occurred frequently in mixtures of strains heteroallelic or homoallelic for the mating-type locus matA, but plasmodia developed only in the matA-heteroallelic cultures. These observations confirmed that matA controls development of fusion cells rather than cell fusion. Analysis of cell pedigrees showed that, in both types of culture, amoebae fused at any stage of the cell cycle except mitosis. In matA-heteroallelic fusion cells, nuclear fusion occurred in interphase about 2 h after cell fusion; interphase nuclear fusion did not occur in matA-homoallelic fusion cells. The diploid zygote, formed by nuclear fusion in matA-heteroallelic fusion cells, entered an extended period of cell growth which ended in the formation of a binucleate plasmodium by mitosis without cytokinesis. In contrast, no extension to the cell cycle was observed in matA-homoallelic fusion cells and mitosis was always accompanied by cytokinesis. In matA-homoallelic cultures, many of the binucleate fusion cells split apart without mitosis, regenerating pairs of uninucleate amoebae; in the remaining fusion cells, the nuclei entered mitosis synchronously and spindle fusion sometimes occurred, giving rise to a variety of products. Immunofluorescence microscopy showed that matA-heteroallelic fusion cells possessed two amoebal microtubule organizing centres, and that most zygotes possessed only one; amoebal microtubule organization was lost gradually over several cell cycles. In matA-homoallelic cultures, all the cells retained amoebal microtubule organization.

d(−)-β-hydroxybutyrate dehydrogenase (BOHB-DH) (EC 1.1.1.30) was purified 991-fold from Azospirillum brasilense Cd. Its specific activity was 5650 units (mg protein)−1 min−1. The enzyme is a tetramer, with identical subunits and a total molecular mass of 100 kDa. BOHB-DH is not a glycoprotein. It is acidic and contains six disulphide bonds without free -SH groups. Under the assay conditions used, BOHB-DH activity was maximal at pH 8·0 and at 36 °C. The enzyme is an NAD+ oxidoreductase, and is inhibited by NADPH and NADH. It has high affinity for β-hydroxybutyrate: the K m value for the β-hydroxybutyrate substrate is 1 mm. Adenosine phosphates, pyruvate, acetyl-coenzyme A, oxaloacetate and 2-oxoglutarate inhibited purified BOHB-DH.

A new family, the Halomonadaceae, has recently been proposed for members of the genera Deleya and Halomonas. The three strains investigated, Deleya halophila, Halomonas elongata and Flavobacterium halmephilum (reclassified as H. halmophila), are aerobic heterotrophic micro-organisms exhibiting an extreme salt tolerance. The major organic osmoregulatory solutes of these organisms were examined using 13C-nuclear magnetic resonance spectroscopy. The relative proportions of the solutes varied with respect to salt concentration, temperature and carbon source. The recently described amino acid ectoine was found to be a dominant solute. For the first time it could be shown for halophilic eubacteria that the intracellular concentration of solutes is sufficient to balance the osmotic pressure of the medium. Thus, there is no need to postulate a hypo-osmotic cytoplasm.

A transitory cessation of growth was recorded in Streptomyces coelicolor A3(2) at the end of vegetative mycelium formation on solid medium. In the same phase a striking reduction in protein and nucleic acid synthesis was detected. Growth and macromolecular synthesis resumed, nearly reaching the original values, when morphological differentiation occurred. It is concluded that a physiological stress occurs within the bacterial population just before the onset of the morphological differentiation.

The extremely thermophilic organism Thermus aquaticus possesses high activities of enzymes catalysing the degradation of xylans and metabolizing d-xylose via the pentose phosphate pathway. The d-xylose isomerase (d-xylose ketol-isomerase, EC 5.3.1.5), an important enzyme of this process, is efficiently induced by its substrate d-xylose, and, to a lesser extent, by related pentoses and some derivatives of d-xylose. The d-xylose isomerase from T. aquaticus has been purified by anion-exchange chromatography, chromatography on d-xylose agarose and gel filtration. A single band migrating according to an M r of 50000 was obtained by SDS-PAGE. An M r of 196000 for the native enzyme, determined by gel filtration and ultracentrifugation in a glycerol gradient, suggested that the d-xylose isomerase is a homomeric tetramer. Arrhenius plots of the enzyme activity of the d-xylose isomerase were linear up to a temperature of 85 °C. At 70 °C the enzyme was inactivated in the absence of divalent cations, with a half-life of 4 d, while in the presence of Mn2+ or Co2+ it remained fully active for at least 1 month. The enzyme had an isoelectric point at 4·4 and showed a broad optimum in the pH range from 5·5 to 8·5. No significant differences in the pH and temperature behaviour could be observed when d-xylose was compared with d-glucose as substrate. Different methods of immobilization of the enzyme to solid supports as well as inclusion into nylon beads were studied. Attachment of the enzyme to epoxy-activated agarose and its co-aggregation with bovine serum albumin gave immobilized preparations with the same stability as free enzyme supplemented with Mn2+ or Co2+.

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.

Mixtures of d-glucosamine and glucose were used to slow the growth of wild-type and whi2 mutant strains of Saccharomyces cereuisiae without affecting the level of catabolite repression. The following observations were made. Firstly, mutant cells were found to be partially resistant to the inhibitory effect of glucosamine. Secondly, slow growth induced by glucosamine resulted in cells becoming larger, in direct contrast to the effect of slowing growth by glucose limitation in a chemostat or by carbon source substitution. It is concluded that the level of repression/derepression, rather than absolute growth rate, is responsible for controlling cell size. Thirdly, when WHI2 transcript levels were measured it was found that expression was correlated with growth rate rather than the level of repression. These results are interpreted in terms of a model which envisages that the WHI2 product acts as a negative regulator of of catabolite repression. A test of this model is reported: it is shown that mutant cells respired more actively in the presence of glucose and grew more rapidly on glycerol, whereas overexpression of WHI2 from multicopy plasmids prevented growth on glycerol and depressed respiration.

An ornithine-amide lipid is present in Mycobacterium tuberculosis. Its structure was established by a combination of chemical analysis and mass spectrometry. 3-Hydroxyoctadecanoic and 3-hydroxyeicosanoic acids (and homologues) were found to be linked through an amide bond to the α-amino group of l-ornithine, the hydroxyl group of the fatty acid being esterified mainly by tuberculostearic acid (10-methyloctadecanoic acid). This ornithine-amide lipid was detected in several other slow-growing pathogenic mycobacteria by thin layer chromatography, but not in an avirulent strain (H37 Ra) of M. tuberculosis. In each case mass spectrometry showed that all the structures were identical, thus revising an earlier reported structure for the lipid from M. bovis.

Certain isolates of Escherichia coli from humans and animals with enteric disease attach to enterocytes and cause ‘attaching and effacing’ (AE) lesions. E. coli strain S22–1, serotype O103:H2, isolated from a child with diarrhoea, contained two plasmids; one of these (pDEP12) hybridized with the CVD419 DNA probe derived from a plasmid found in E. coli 0157:H7 and associated with expression of fimbriae and ability to adhere to Intestine 407 cells. Strain S102–9, serotype O5:H−, isolated from a calf with dysentery, contained six plasmids, one of which also hybridized with the CVD419 probe. Loss of pDEP12 coincided with reduced adhesion to HEp-2 or Intestine 407 cells cultured in vitro; reintroduction of this plasmid restored adhesiveness. Loss of the plasmid in strain S102–9 that hybridized with the CVD419 probe did not cause a decrease in adhesion. Accumulations of actin were seen in vitro in the fluorescence actin staining (FAS) test of strains S22–1, S102–9 and their derivatives, irrespective of the plasmid content of these strains or the prevalence of attached bacteria. Strain S22–1 and its plasmidless derivative caused AE lesions of equal severity in experimentally infected gnotobiotic piglets; piglets inoculated with an isolate from a healthy human or pig did not develop these lesions. These results indicate that the CVD419 probe is not specific for genes conferring the ability to adhere to HEp-2 or Intestine 407 cells by these E. coli and that the adhesins detected in vitro, or plasmid-encoded properties, are not required for strain S22–1 to cause AE lesions in gnotobiotic pigs or to cause the accumulation of actin in cells in vitro.

The type IIIb dihydrofolate reductase, a novel plasmid-encoded enzyme recently identified in Shigella sonnei, has been shown to have some similar biochemical properties to the type IIIa dihydrofolate reductase which was first identified in New Zealand in 1979. However, the type IIIb enzyme has a K i for trimethoprim of 0·4 μm, and a pI of 5·35 (as compared to 19 nm and 6·1 for the type IIIa); both these results suggest that it is a different enzyme from the prototype type IIIa. The type IIIb dihydrofolate reductase was purified by methotrexate agarose affinity chromatography, yielding a pure protein as determined by HPLC. Automatic amino acid analysis of the purified enzyme showed it to be distinct from all other known plasmid-encoded dihydrofolate reductases and quite different from the type IIIa enzyme. The purified enzyme was examined by SDS-PAGE, which revealed that the type IIIb dihydrofolate reductase was a monomeric protein of M r 17200.

Isopenicillin N (IPN) epimerase, an enzyme involved in cephalosporin and cephamycin biosynthesis that converts IPN into penicillin N, was extracted from Nocardia lactamdurans and purified 88-fold. The enzyme was unstable but could be partially stabilized by addition of pyridoxal phosphate. The purified enzyme did not require ATP for activity in contrast to other amino acid racemases. The enzyme had an M r of 59000 as determined by gel filtration; IPN epimerase from Streptomyces clavuligerus had an M r of 63000. A protein band of M r 59000 was found to be enriched in SDS-PAGE of active fractions from N. lactamdurans. The optimal temperature of the epimerase was 25°C and the optimal pH 7·0. The apparent K m for IPN was 270 μM. Fe2+, Cu2+, Hg2+ and Zn2+ strongly inhibited enzyme activity. α-Aminoadipic acid, valine, glutamine, glycine, aspartic acid and glutathione do not affect enzyme activity, whereas ammonium sulphate was inhibitory. The epimerase activity was partially inhibited by several thiol-specific reagents.

Azotobacter vinelandii strain 206 degrades benzoate via the meta-cleavage pathway. In a genomic library derived from this organism a clone was obtained which carried and expressed the gene for the third enzyme in this pathway, catechol 2,3-dioxygenase (EC 1.13.11.2), on a 5·9 kb SalI restriction fragment. The structural gene was more precisely mapped on an internal 1·6 kb EcoRI fragment which, after insertion into expression vectors, directed the synthesis of a 33 kDa polypeptide. The gene showed very little or no homology with isofunctional genes derived from Pseudomonas. Comprehensive substrate specificity analysis showed significant differences between the specific activities obtained from the cloned gene product and extracts derived from Azotobacter itself.

It was established that Candida albicans grew rapidly in a simple medium containing yeast extract (0·2 %, w/v) plus glucose (2 %, w/v). These cultures were in or near to a state of nitrogen limitation and the concentration of secreted aspartate proteinase increased rapidly (within 3–4 h) on addition of BSA. Synthesis and secretion were apparently controlled both positively (induction by albumin or, more probably, the peptides produced from it) and negatively (repression by NH4Cl). A small intracellular pool of the enzyme was detected during production of the enzyme and this pool decreased with the cessation of synthesis and secretion. A stable mutant, IR24, was isolated which secreted less than 0·3% of the amount of the proteinase exported by the parent strain ATCC 10261. The LD50 values for mutant IR24 and the parent strain administered intravenously to mice were > 1·0 × 109 and 1·6 × 106 c.f.u. kg−1 respectively.

WHI2mRNA levels were followed through the growth cycle in WHI2 mutant and wild-type cells of Saccharomyces cerevisiae. Levels were high during the first (glucose) phase of growth, and were reduced sharply during the second (ethanol) phase of growth. Transcript levels of the glycolytic genes PDC1 and PYK1 were also measured; they each showed a pattern similar to that of WHI2, whereas transcript levels of the CDC7 gene remained constant throughout the cycle, showing that a decrease in transcription is not a general feature of genes. These results make it unlikely that the WHI2 product acts as an inhibitor of cell proliferation which is activated upon carbon starvation. No difference was observed between the pattern of expression of mutant and wild-type strains, showing that the mutant phenotype was not the result of a change in regulation at the transcriptional level.

The chitinolytic properties of a facultatively anaerobic bacterium isolated from the hindgut of plaice were compared with those of Clostridium sp. strain 9·1, a bacterium isolated from anoxic estuarine sediment. The chitinolytic enzyme systems of the gut isolate and strain 9·1 both released N,N′-diacetylchitobiose (NAG2) as the major hydrolysis end-product. During the hydrolysis of chitin, there was transient accumulation of a non-sedimentary chitin fraction which was not detectable by high-performance liquid chromatography. Growth on NAG2 repressed chitinase synthesis in the gut isolate but not in the Clostridium species. Thiol reagents were strongly inhibitory to the chitinase of the strict anaerobe but did not affect the hydrolytic enzymes of the gut isolate. When the two bacteria were cocultured with chitin as the sole carbon and energy source, Clostridium sp. strain 9·1 was always outcompeted. Experiments with batch and phauxostat cultures showed that the competitiveness of strain 9·1 could be improved dramatically by the inclusion in the cocultures of a non-chitinolytic bacterium capable of fermenting chitin oligomers. The cooperation between the oligomer-fermenting species and the Clostridium sp. is discussed in relation to the regulation of chitinolytic activity in the latter organism.

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage λ. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.