- Volume 134, Issue 1, 1988

The ε-caprolactone hydrolases (EC 3.1.1-) induced by growth of Acinetobacter NCIB 9871 and Nocardia globerula CL1 with cyclohexanol were purified to homogeneity. Both enzymes constituted approximately 1% of the soluble protein of the bacteria. Each was formed from two electrophoretically indistinguishable subunits and each had a closely similar M r value (≈60000). Both enzymes had high turnover numbers typical of carboxyesterases, broad pH-activity spectra and very restricted substrate specificities. In contrast to other bacterial lactone hydrolases they catalysed irreversible lactone hydrolysis and were not inhibited by thiol-reactive compounds. Their sensitivity to Paraoxon (diethyl p-nitrophenylphosphate) suggested that they, in common with mammalian acetylcholinesterase and carboxyesterases, have a functional catalytic centre serine.

Chitinases were isolated from the grains of wheat, barley and maize, and compared with those obtained from Serratia marcescens, Streptomyces griseus and Pseudomonas stutzeri for antifungal activity and enzyme specificity. The six enzymes were tested for antifungal activity using an assay based upon inhibition of hyphal extension of the fungi Trichoderma reesei and Phycomyces blakesleeanus. Antifungal activity was observed with as little as 1 μg of each of the grain chitinases, whereas none of the bacterial chitinases had any effect on hyphal extension, even at 50 #x03BC;g chitinase per assay. This difference in antifungal activity correlated with the different mechanisms of action of the two classes of enzymes. In common with other plant chitinases, the grain chitinases functioned as endochitinases and contained lysozyme activity. In contrast, the bacterial enzymes were exochitinases and hydrolysed the chromogenic trisaccharide analogue p-nitrophenyl-β-D-N, N′-diacetylchitobiose, which proved to be an excellent substrate for assaying bacterial chitinases. These experiments strengthen the hypothesis that plant chitinases function to protect the host against fungal infections.

The cellular fatty acid composition of Deleya halophila, a moderately halophilic bacterium, grown at different temperatures and NaCl concentrations is reported. When the temperature was lowered the amounts of monounsaturated C16:1 and C18:1 fatty acids increased with a corresponding decrease in the amounts of saturated C16:0 and C18:0 fatty acids. Increasing the salt concentration in the medium resulted in an increase of cyclopropane fatty acids, with a concomitant decrease in the monounsaturated fatty acids, suggesting that cyclopropane synthetase is activated or induced by high levels of salt.

The major polar lipids from haloalkaliphilic archaebacteria of the genera Natronobacterium and Natronococcus have been analysed by spectroscopic methods, including 13C NMR, to establish unequivocal structural detail. Diether forms of phosphatidylglycerol (PG) and phosphatidyl-glycerophosphate (PGP) are the major polar lipids; PGP is the main component for all species. Natronobacterium spp. show a preponderance of the 2-O-sesterterpanyl-3-O-phytanyl glycerol diether form (C25, C20) of both PG and PGP, whereas Natronococcus occultus has a preponderance of the diphytanylglycerol diether form (C20, C20) for both. In all cases, PGP (C25, C20 and C20, C20 forms) eluted from silica columns in association with glycine betaine (trimethylglycine).

5 May 1987

Two novel antibacterial substances (designated mutalipocins) have been isolated from the culture supernatant of Streptococcus mutans strain 32K (serotype c). The mutalipocins were purified by extraction of the culture supernatant with light petroleum (b.p. range 30舑60 ŶC), followed by Lobar column chromatography on Lichroprep RP-8. HPLC indicated that both mutalipocin preparations (ML-I and ML-II) were homogeneous. The M r values of ML-I and ML-II were less than 1000. Both mutalipocins were unaffected by treatment over the pH range 3·0–10·0, or with phospholipase A or proteolytic enzymes, but were partially inactivated by treatment with lipase or phospholipase C. ML-II was resistant to heat treatment. TLC indicated that ML-I and ML-II contained unsaturated, aldehyde and/or ketone, and ester groups. The inhibition of S. mutans by ML-I and ML-II was due to bactericidal, rather than bacteriostatic, activities. The antibacterial spectra of ML-I and ML-II were narrower and more species-specific than those of bacteriocins produced by other Gram-positive bacteria.

Growth of Mycobacterium phlei under low oxygen tension resulted in specific activities two to twenty times lower for formate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase, lactate oxidase and NADH dehydrogenase than when cultures were grown under high aeration. An increase in fumarate reductase and succinate dehydrogenase occurred with M. phlei grown under low oxygen tension. Malate: vitamin K dehydrogenase and glucose-6-phosphate dehydrogenase activity were not significantly affected by the oxygen tension used to grow the bacteria, and neither culture contained a lactate dehydrogenase. With growth of M. phlei in conditions of low oxygen tension, cytochrome a was not detected, but cytochrome b was prominent in membranes and cytochrome c was present in the soluble fraction

The spoIVC locus of Bacillus subtilis was analysed. Fourteen spoIVC mutants isolated following nitrosoguanidine mutagenesis were used along with two previously characterized spoIVC mutants to construct a fine structure genetic map of the locus. The recombination index (RI) measured between extreme mutations was 0·26; no recombination could be detected between four of the mutations. Complementation analysis showed that all the mutations fall into two cistrons. The RI between extreme mutations in cistron A was about 0·17 and that between extreme mutations in cistron B was about 0·05. In respect of biochemical markers, the spoIVC mutations all produced similar phenotypes, irrespective of their location. However, in both cistrons oligosporogenous and asporogenous mutations mapped close together.

We previously reported the cloning of a 1·6 kb HindIII fragment (containing the junction of the repeating unit) from chromosomal DNA of Bacillus subtilis strain B7 in which tandem amplification of a 16 kb region occurred, and the induction of B7-type gene amplification by competence transformation with this cloned fragment. Based on this result, we designed, on the B. subtilis chromosome, a gene amplification of the 22 kb repeating unit containing the a-amylase structural gene (amyE), the tunicamycin-resistance gene (tmrB) and the shikimate kinase structural gene (aroI). We cloned only two short DNA fragments from both termini of the 22 kb region, constructed a junction structure of the designed repeating unit on pBR327 and transformed a B. subtilis wild-type strain by this constructed plasmid. As a result, we succeeded in obtaining tunicamycin-resistant (Tmr) transformants in which the designed gene amplification of 22 kb occurred on the chromosome. The Tmr transformants showed high productivity of α-amylase and shikimate kinase. The copy number of the repeating unit was estimated to be 10–20. This system may provide an effective means of amplifying long (> 20 kb) DNA regions on the chromosome.

The cyclomaltodextrin glucanotransferase (CGTase, EC 2.4.1.19) gene from the alkalophilic Bacillus sp. strain no. 38-2 was cloned in Escherichia coli using pBR322. A plasmid, pCS8, was isolated from a transformant producing CGTase and the cloned CGTase gene was found to be in a 5·3 kb DNA fragment. The nucleotide sequence of a 2·5 kb segment encoding the CGTase was determined. This segment showed an open reading frame which would encode a polypeptide of 712 amino acids. The pCS8 CGTase had the same enzymic properties as those of the extracellular CGTase produced by the alkalophilic Bacillus sp. strain no. 38-2. The nucleotide and amino acid sequences of this CGTase gene and gene product, respectively, have strong homology with those of the Bacillus macerans CGTase.

Genetic transformation at the solid/liquid interface was studied using Bacillus subtilis 1G20 (trpC2) with a flow-through system of columns filled with chemically pure sea sand. Studies were done at 23 °C. In one type of experiment, competent cultures were incubated with sand-adsorbed DNA, and in another, competent cultures were exposed to sand and then incubated with dissolved DNA for transformation. Of the applied cells, around 10% were retained in columns filled with DNA-loaded sand and around 1% in columns with pure sand. Reversible attachment of some of the cells to surfaces of sand grains could be demonstrated. The overall transformation frequencies obtained were 25- to 50-fold higher than in a standard liquid culture procedure. In this standard procedure, transformation was sensitive to DNAase I concentrations above 50 ng ml−1, whereas in sand columns it was resistant to DNAase I concentrations up to 1 g ml−1. Quantification of transformants eluting from columns indicated that sand-attached cells detach at some point after DNA binding or uptake.

An internal 0·9 kb segment of Rhizobium meliloti insertion sequence ISRm1 was used as a probe to determine the distribution of ISRm1 in strains of R. meliloti and other Gram-negative bacteria. The insertion sequence was detected in 80% (12/15) of R. meliloti strains from different parts of the world. Its copy number ranged from one to at least eleven. The ISRm1 copies detected showed variation in their internal restriction sites and their degree of homology to the probe. ISRm1 was found in a variety of genomic restriction fragments, and was detected in plasmids, including the nod and exo megaplasmids of R. meliloti. Other rhizobia found to contain ISRm1 were a strain of R. leguminosarum biovar phaseoli and two Rhizobium isolates capable of nodulating both Medicago sativa and Phaseolus vulgaris. It was also found in a diazotrophic soil bacterium isolated from the roots of wetland rice.

Cosmid gene libraries of the obligate methylotrophs Methylophilus viscogenes and Methylophilus methylotrophus AS1 were generated by ligating Sau3A partial digests of chromosomal DNA into the broad-host-range cosmid vector pLA2917. Genetic linkage groups were identified for each organism by complementation following mobilization of the recombinant cosmids to a wide range of Pseudomonas aeruginosa and Escherichia coli auxotrophs. The linkage data thus obtained confirmed and extended those previously established by prime plasmid complementation, and the two methylotrophs showed very similar arrangements for four of the gene clusters studied. However, comparison of DNA sequences which complemented the same auxotrophic markers failed to demonstrate restriction fragment identity or substantial DNA-DNA homology between the two methylotrophs.

The effect of purified human interferon-? on the responsiveness of human neutrophils was investigated. Pre-incubation of neutrophils with 100 U interferon ml-1 for 10 min at 37C resulted in a 2.5-fold increase in N-formylmethionyl-leucyl-phenylalanine-stimulated reactive oxygen metabolite generation (as assayed by luminol-dependent chemiluminescence). Pretreatment of neutrophils with interferon also potentiated their ability to kill Staphylococcus aureus, and thus it is proposed that this lymphokine may also enhance neutrophil function in vivo under certain pathological conditions.

The role of motility as a virulence factor in Pseudomonas aeruginosa burn wound sepsis was examined using mutants deficient in the Fla or Mot phenotype. Physiological profiles of parental strains and Fla- and Mot- mutants were similar with respect to antibiograms, O antigen types, growth rates, and proteolytic, exotoxin A and phospholipase activities, providing evidence for isogenicity. Lethality studies using a subcutaneous mouse burn model showed that three Fla- mutants and one Mot- mutant were much less virulent (102 to 105 times) than the parent wild-type. Topical challenges in the flame burn model showed that a Fla- mutant of strain M-2 was approximately tenfold less virulent. A reduction in virulence, although somewhat less than tenfold, was also observed in the scald burn model for M-2 Fla-, and Mot- strains. Tissue colonization experiments revealed a characteristic, rapidly systemic infection in burned mice challenged with wild-type organisms. Nonmotile mutants similarly proliferated in the burn wound, but the characteristic bacteraemia and systemic invasion were markedly absent. The infection remained localized in the skin wound and the mice survived. The pattern of infection by nonmotile mutants in the colonization studies was very similar to that obtained with Fla+ cells in burned animals passively treated with antiflagellar antibody. These results add substantial support to the concept of motility as a P. aeruginosa virulence factor in invasive infections.

The gene coding for coagulase (coa) was cloned from Staphylococcus aureus 8325-4 in a λ replacement vector in Escherichia coli. Coagulase (plasma-clotting) activity was measured in λ coa lysates and an immunoreactive protein of 60 kDa was detected by Western immunoblotting with anti-coagulase serum. This protein comigrated with the major immunoreactive protein in supernatants of S. aureus 8325-4. The coa gene was subcloned in pUC vectors. One recombinant expressed a 60 kDa immunoreactive protein and plasma-clotting activity. A putative β-galactosidase-coagulase fusion protein and truncated peptides were expressed by variants formed by subcloning. These results are consistent with previously published biochemical data that the prothrombin-binding domain of coagulase is located in the N-terminus of the protein. The cloned coa gene was transferred into S. aureus on a shuttle plasmid. Expression of coagulase was higher in a strain with a mutation in the agr locus, which controls the level of several exoproteins in S. aureus, suggesting that agr normally regulates coagulase expression negatively.

A tissue-destructive protease of Legionella pneumophila was assayed for in the lungs of experimentally infected guinea-pigs by ELISA. It was found in amounts equivalent to the known lethal dose of purified protease administered by the intranasal route. The identity of the protease was confirmed by immunoblot analysis. This is further evidence that Legionella pneumophila protease may play a major role in the pathogenesis of Legionnaires’ disease.