- Volume 140, Issue 1, 1994

Summary: An NADP-dependent constitutive alcohol dehydrogenase that can oxidize hexan-1-ol was detected in several Gram-positive and Gram-negative eubacteria and in two yeasts. The enzyme was purified to homogeneity from Acinetobacter calcoaceticus NCIB 8250 and from Saccharomyces cerevisiae D273–10B. The bacterial enzyme appears to be a tetramer of subunit M r 40 300 and the yeast enzyme appears to be a monomer of subunit M r 43 500. The N-terminal amino acid sequence of the bacterial enzyme has 34% identity with part of the sequence of a fermentative alcohol dehydrogenase from Escherichia coli. The pl value of the bacterial enzyme was 5.7 and the pH optimum was 10.2. Both the bacterial and yeast enzymes were shown to transfer the pro-R hydrogen to/from NADP(H). The substrate specificities of the two enzymes were similar to each other, both oxidizing primary alcohols and some diols, but not secondary alcohols. The maximum velocities of both enzymes were with pentan-1-ol as substrate and there was very low activity with ethanol; the maximum specificity constants were found with primary alcohols containing six to eight carbon atoms. Neither enzyme was significantly inhibited by metal-binding agents but some thiol-blocking compounds inhibited them. It appears that these two alcohol dehydrogenases, one prokaryotic and one eukaryotic, are structurally, kinetically and functionally different from members of the major known groups of alcohol dehydrogenases.

Summary: The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent that one of these, a chymoelastase PR1, is a determinant of pathogenicity. We have shown previously that the wide-domain regulatory circuits of carbon and nitrogen derepression regulate PR1 production. In the present work we have established in addition that PR1 is specifically induced by insect cuticle, but not by other soluble or insoluble proteinaceous substrates. The feeding of elastin or collagen to derepressed established mycelium (starved for carbon and nitrogen) did not enhance PR1 production significantly and the soluble proteins BSA and gelatin rapidly and completely repressed PR1. The carbohydrate polymers cellulose and xylan gave derepressed basal levels of PR1. However, addition of locust cuticle enhanced PR1 production to a level approximately 10-fold that of derepressed mycelium. In order to establish if the enhancing effect of insect cuticle on PR1 production was due to specific induction or merely a reflection of enhanced growth on this insoluble dual carbon and nitrogen source, ergosterol was used as a measure of fungal growth. Expressing enzyme activity per mg dry weight showed that PR1 production in cuticle cultures increased approximately five- and ninefold after 12 and 24 h growth compared with elastin-grown cultures. Thus, the substantial increase in PR1 production on cuticle was shown not to be a function of fungal growth and this confirms that PR1 is induced by a component of insect cuticle; we believe this is the first report of induction by a specific substrate for any microbial protease.

Summary: The obligately methylotrophic bacterium Methylophilus methylotrophus readily uses formamide as a source of nitrogen for growth. Physiological investigations using batch, fed-batch and continuous cultures indicated that the organism contains a discrete formamidase (formamide amidohydrolase; EC 3.5.1.49), in addition to the previously characterized aliphatic amidase (acylamide amidohydrolase; EC 3.5.1.4) which is specific to short-chain aliphatic amides such as acetamide, propionamide and acrylamide. Formamidase synthesis was induced by formamide and acetamide, and repressed by ammonia. The enzyme was purified using anion-exchange and gel-filtration FPLC and shown to exhibit a narrow substrate specificity (high activity with formamide, little activity with short-chain aliphatic amides, no activity with urea). SDS-PAGE and gel-filtration FPLC showed that the enzyme comprises a single type of subunit and probably exists as a homodimer. A 3·2 kbp Pstl restriction fragment of chromosomal DNA from M. methylotrophus was cloned in pUC19 and expressed in Escherichia coli JM109. The purified gene product exhibited essentially identical properties to those of the M. methylotrophus formamidase in terms of its chromatographic behaviour, native M r (123 000), subunit M r (51 000), K cat (58 cf. 64 s−1), K m (1·6 cf. 2·1 mM), substrate specificity and N-terminal amino acid sequence (MKTIV-).

Summary: The ability of flow cytometry to detect and enumerate viable bacteria during survival in a lakewater microcosm was assessed using Staphylococcus aureus as a model organism. Counts of colony-forming units (c.f.u.) on nutrient agar were not significantly different from those obtained by flow cytometric detection of rhodamine 123 stained bacteria and there was no evidence for a viable but nonculturable state using these methods. However c.f.u. were significantly lower when estimated using mannitol salts agar compared with nutrient agar. S. aureus was also enumerated immunofluorescently after staining with FITC-lgG. There was no significant difference between the population estimated immunofluorescently and by acridine orange direct counting, and unlike estimations of viability, only slight reductions in total cell numbers were observed. Changes in the protein and nucleic acid content of S. aureus during survival were also measured by flow cytometry to investigate any potential heterogeneity arising within the starved population. Flow cytometric determinations were found to correlate significantly with their respective chemical determinations. These results demonstrate the ability of flow cytometry to detect viable bacteria during starvation and to study changes in macromolecular content. They also illustrate the importance of using appropriate methods for the detection of viable bacteria in environmental samples.

Summary: Extant Escherichia coli K12 strains are phenotypically rough, their lipopolysaccharide having a complete core structure, but no O antigen. We used DNA hybridization and DNA sequencing to show that the rough phenotype of this strain is due to the presence of one of two independent mutations in the rfb gene cluster. The rfb-50 mutation, consisting of an IS5 insertion at the downstream end of rfb, is present in strain EMG2, which is representative of most K12 derivatives. The rfb-51 mutation is a deletion at the upstream end of rfb, and was found in strain WG1. A gene cloned from strain WG1 could complement the rfb-50 mutation in strain EMG2, and the complemented strain produced O antigen which was typed as O16 with cross reaction to O17.

Summary: Subcloning, transposon insertion, and deletion analysis revealed that the Escherichia coli 09 rfb region is about 12 kb in size. The region encodes at least seven polypeptides of 89, 74, 55, 50, 44, 41 and 39.5 kDa. Southern hybridization analysis of rfb regions of E. coli 08 and 09, and Klebsiella 03 and 05 serotypes (all of these O polysaccharides are mannose homopolymers and the structures of the repeating unit of E. coli 09 and Klebsiella 03 are identical) showed that a central region specific for E. coli 09 and Klebsiella 03 is flanked by two regions common to all four. Complementation experiments using strains with known defects and specific tests for the enzymic activity showed that the 50 and 55 kDa polypeptides, encoded by the common region, are phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), respectively. Nucleotide sequencing of the region revealed the presence of two genes, rfbK and rfbM, analogous to the corresponding genes of Salmonella typhimurium. In E. coli 09, rfbK and rfbM encode proteins of 460 amino acids (50809 Da) and 471 amino acids (52 789 Da). The amino acid sequence of GMP was conserved in RfbMs of E. coli 07 and Salmonella groups B, C1 and C2, CpsB of S. typhimurium, AlgA of Pseudomonas aeruginosa, and XanB of Xanthomonas campestris. The phylogenetic trees of PMM and GMP were different in topology and in the evolutionary distances from ancestors.

Summary: Penicillin-binding protein (PBP) 6 is anchored to the periplasmic face of the Escherichia coli inner membrane. Analysis of the C-terminal 20 amino acids of PBP 6 implies the presence of a C-terminal amphiphilic α-helical anchor comparable to that of PBP 5. A C-terminal deletion of PBP 6 was constructed; it resulted in the release of the protein from the inner membrane into the periplasm, thus confirming that this region is essential for anchoring. Treatment of E. coli K12 membrane vesicles with various reagents was used to probe the membrane-binding characteristics of both PBP 5 and PBP 6. The results indicate that, although the strength of membrane anchoring of PBP 6 is weaker than that of PBP 5, both modes of anchoring involve a large hydrophobic element and have similar membrane-binding characteristics. This is in agreement with the hypothesis that both proteins exhibit the same novel method of anchoring.

Summary: A number of non-replicative, integrational plasmids have been introduced into the chromosome of the NCIMB 8052 strain of Clostridium acetobutylicum. Integration occurred via homologous recombination, apparently by a Campbell-like mechanism. For plasmids containing internal fragments of gutD or spoOA, insertion was mutagenic leading, respectively, to an inability to use sorbitol as sole source of carbon for energy and growth or an inability to form endospores. One insertion was used to determine the map location of a randomly cloned DNA fragment and another underwent amplification in the bacterial chromosome. These results suggest that integrational plasmids will be very useful tools for genetic analysis in Clostridium acetobutylicum. To our knowledge, this is the first demonstration of gene transfer involving homologous recombination with the chromosome of any species of Clostridium.

Summary: Enterotoxin production in Clostridium perfringens is both strain dependent and sporulation associated. Underlying these phenotypic observations must lie a genetic and molecular explanation and the principal keys will be held within the DNA sequence both upstream and downstream of the structural gene cpe. In accordance with the above we have sequenced 4·1 kbp of DNA upstream of cpe in the type strain NCTC 8239. A region of DNA extending up to 1·5 kb 5′ to cpe is conserved in all enterotoxin-positive strains. This region contains a putative ORF with substantial homology to an ORF in the Salmonella typhimurium IS200 insertion element and, in addition, contains multiple perfect consensus DNA-binding sequences for the Bacillus subtilis transition state regulator Hpr. The detailed structural elements revealed by the sequence analysis are presented and used to develop a new perspective on the molecular basis of enterotoxin production in this important food-poisoning bacterium.

Summary: We have examined the heat shock response of Rhizobium leguminosarum. After normal growth at 28 °C, a 10 min heat shock at 37 °C induced the synthesis of proteins with approximate M r values of 90000, 70000, 60000, 58000, 19000, 17000 and 13000. A monoclonal antibody raised against the E. coli Cpn60 cross-reacted with proteins of M r 60000 and 58000 in R. leguminosarum, suggesting that both were Cpn60 homologues. Hybridization of an E. coli cpn60 probe to total DNA from Rhizobium leguminosarum also showed evidence for at least two cpn60 homologues. One of these was cloned and completely sequenced, and showed close homology to cpn60 sequences from other prokaryotes. The expression of this gene in E. coli failed to complement a cpn60 mutation, either for growth at high temperature or for growth of bacteriophage δ. Hybridization of total R. leguminosarum DNA with a probe from this gene revealed the presence of a third putative cpn60 gene. Two further hybridizing clones were analysed and found to consist of two additional cpn60 sequences plus upstream regions containing putative cpn10 genes.