- Volume 137, Issue 9, 1991

Comamonas testosteroni T-2 utilizes p-toluate (TC, 4-toluenecarboxylate) as sole source of carbon and energy for growth. Cells grown in TC-salts medium oxygenated terephthalate (PcB, 4-carboxybenzoate) and contained protocatechuate 4,5-dioxygenase but no detectable (methyl)catechol dioxygenase. The intermediates 4-carboxybenzyl alcohol (COL), 4-carboxybenzaldehyde (CYD) and PcB were detected during the metabolism of TC. A TC methyl-monooxygenase system, a COL dehydrogenase and a CYD dehydrogenase were detected, analogous to the known degradative pathway and enzymes for 4-toluenesulphonate (TS) to 4-sulphobenzoate (PSB) (Locher et al., Journal of General Microbiology 135, 1969-1978, 1989). Genetic evidence indicated that the steps from TS to PSB and from TC to PcB were catalysed by the same enzymes. This hypothesis was substantiated by purifying or separating the appropriate enzymes from cells grown in TS-salts and TC-salts media. The behaviour of pairs of enzymes was effectively identical in all chromatographic and catalytic properties that were compared. The data support the existence of a novel pathway for the degradation of TC, with the same initial pathway enzymes being used to metabolize TS.

Summary: Phanerochaete chrysosporium produced several intracellular NADH:quinone oxidoreductases under agitated, nitrogen-limited cultivation conditions. One of the quinone reductases was purified and shown to have a molecular mass of 69 kDa by SDS-PAGE, while the molecular mass determined by gel filtration was 47 kDa. This reductase was separated by IEF into four protein bands, each with quinone reductase activity. The isoelectric points of the proteins were 5·7, 5·9, 6·0 and 6·3. The proteins reduced several quinones to the corresponding hydroquinones, but none of them was specific to any one of the quinones tested. Mycelial extracts of P. chrysosporium contained several more quinone reductases, with isoelectric points of 4·4, 4·7, 5·0, 5·3, 5·5 and 6·6. Quinone reductase activity could be induced by adding vanillic acid or 2-methoxy-1,4-benzoquinone to the growth medium in nitrogen-limited cultures and in carbon-limited cultures.

Summary: Extracellular lipase was purified from a Tween 80-limited continuous culture of Pseudomonas aeruginosa EF2 by ultrafiltration of the culture supernatant followed by anion-exchange and gel-filtration FPLC. The lipase was composed of a single subunit (M r29000, pI 4·9), which was capable of a variable degree of aggregation, and which exhibited both lipase activity, measured with the insoluble substrate olive oil (predominantly triolein), and esterase activity, measured with the soluble substrates p-nitrophenyl acetate and Tween 80. Lipase activity was approximately eight times higher than either type of esterase activity (k cat, approximately 3000 s−1for the hydrolysis of olive oil). The enzyme showed a marked regiospecificity for the 1,3-oleyl residues of radiolabelled triolein, was relatively stable at moderate temperatures (exhibiting a biphasic loss of activity with an initial t 1/2 of 17·5 min at 60 °C) and was very stable to freezing and thawing. Lipase activity was only weakly inhibited by the serine-active reagent 3,4-dichloroisocoumarin, and was not inhibited by the chelating agent EDTA (1 mm). The N-terminal amino acid sequence of the Ps. aeruginosa EF2 lipase showed a marked similarity to those of several other bacterial lipases.

Summary: Twenty-nine strains of 14 species of rumen bacteria were screened for their ability to hydrolyse Ala2. Ala5, Gly Arg-4-methoxy-2-naphthylamide (GlyArg-MNA) and Leu-MNA. Several species, notably Megasphaera elsdenii, were active against Ala2, and a smaller number, including Bacteroides ruminicola, Butyrivibrio fibrisolvens, Ruminococcus flavefaciens, Lachnospira multipara and Ruminobacter amylophilus, broke down Ala5. Streptococcus bovis had an exceptionally high leucine arylamidase activity. However, only Ba. ruminicola hydrolysed GlyArg-MNA. Further investigation revealed that only Ba. ruminicola and Bu. fibrisolvens hydrolysed Ala5 to Ala3 and Ala2, with little Ala4 being produced, in a manner similar to rumen fluid. The activity of Ba. ruminicola against synthetic peptidase substrates, including GlyArg-MNA, LysAla-MNA, ArgArg-MNA, GlyPro-MNA, LeuVal-MNA, and Ala3-p-nitroanilide, was similar to that of rumen fluid, whereas the activity of Bu. fibrisolvens was quite different. Since the main mechanism by which peptides are broken down in the rumen is similar to dipeptidyl aminopeptidase type I, for which GlyArg-MNA is a diagnostic substrate, it was concluded that Ba. ruminicola was the most important single species in peptide breakdown in the rumen.

In S. coelicolor there are mutants (named bld) which fail to form aerial mycelium and which are also pleiotropically blocked for synthesis of several antibiotics, including the blue compound actinorhodin. Using a high copy-number vector, a DNA fragment was cloned which restored actinorhodin production, but not aerial mycelium formation, to bldA, D, F, G and H mutants. Subcloning and Southern blotting revealed that the fragment was from the region (actII) that regulates actinorhodin biosynthesis. The ability to elicit actinorhodin production and to complement an actII mutant was localized to a 1·3 kb fragment. These results indicate that the structural genes for actinorhodin biosynthetic enzymes do not contain targets for the bldA, D, F, G and H gene products, and suggest that the corresponding bld genes control actinorhodin synthesis principally via a single gene in the actII regulatory region.

In order to study the genetic control of chitinolytic activity in Streptomyces, chitinase genes were cloned from S. lividans TK64 into the multicopy plasmid pIJ702 and their expression monitored in their natural host by measuring increases in chitinase productivity. Four independent clones were obtained, and the plasmids named pEMJ1, pEMJ5, pEMJ7 and pEMJ8. Restriction endonuclease digestion showed that although two of the plasmids (pEMJ7 and pEMJ8) shared a common DNA fragment, there were no substantial similarities between the inserts of plasmids pEMJ1, pEMJ5 and pEMJ7. This was confirmed by DNA-DNA hybridization studies. Four chitinases (A, B, C, and D) were identified, with molecular masses of 36, 46, 65, and 41 kDa, respectively. Production of chitinases A and B was specified by the plasmids pEMJ1 and pEMJ5, respectively. Genes for the other two chitinases (C and D) were carried by plasmid pEMJ7. Although significant differences were observed between chitinases A, B, and C in terms of optimum pH for activity and mode of digestion of substrates, chitinases C and D were very similar in these respects. Cloned genes were also expressed in S. coelicolor M130 and in Escherichia coli.

The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half that produced in wild-type E. coli cells. The production of E. coli SPase I in B. subtilis was increased approximately fivefold by cloning the lep gene into a high-copy-number plasmid. The expression of E. coli SPase I in B. subtilis did not appear to increase the rate of processing of two hybrid secretory precursor proteins. Two observations may explain the failure of E. coli SPase I to stimulate processing of exported proteins in B. subtilis. First, the E. coli SPase I was apparently not exposed on the outside of the B. subtilis cytoplasmic membrane, indicating its incorrect insertion into the membrane. Second, in vitro processing studies, using cell-free extracts of B. subtilis producing E. coli SPase I, suggested that the enzyme was not active. A further outcome of this study was that conditions favouring processing of precursors by SPase I in cell-free extracts of E. coli did not favour processing by the corresponding enzyme in B. subtilis cell-free extracts. This suggests that significant differences exist between the two enzymes. The observation that antibodies directed against E. coli SPase I did not cross-react with B. subtilis membrane proteins supports this idea.

We have developed a homologous transformation system for the wheat-pathogenic fungus Septoria nodorum based on a benomyl-(MBC-) resistant allele of the β-tubulin gene. The β-tubulin gene was isolated by heterologous hybridization from a cosmid library prepared from an MBC-resistant mutant. Cosmids carrying the gene conferred MBC resistance when introduced into a sensitive strain, demonstrating that resistance to MBC fungicides in S. nodorum may be determined by the β-tubulin gene. This MBC resistant allele of the β-tubulin gene (tubAR) was subcioned into pUC18 and used as a dominant selectable marker for transformation of wild-type sensitive strains. Transformants arose at frequencies of approximately 5 per μg of DNA, were integrative in nature and were mitotically stable. Some transformants showed a marked reduction in vigour, both in the presence and absence of MBC; this is thought to arise from overproduction of β-tubulin. The S. nodorum tubAR gene also conferred MBC resistance on the related species Leptosphaeria maculans, a pathogen of Brassica, following its introduction by cotransformation. Probing digested S. nodorum DNA with tubAR at low stringency revealed only a single β-tubulin gene. We anticipate that tubAR will prove a useful tool for the investigation of the pathogenicity of S. nodorum and other fungi.

pCG100, a 3 kb cryptic plasmid of Corynebacterium glutamicum ATCC 13058, probably identical with pSR1 from C. glutamicum ATCC 19223, was characterized. The minimum region for autonomous replication was shown to be contained on a 1·9 kb BglII-NcoI fragment; a 380 bp HindIII-SphI fragment can replicate in the presence of the parental plasmid, which presumably provides a trans-acting replication factor. Derivatives of pCG100 are able to replicate in several Corynebacterium, Brevibacterium and Arthrobacter strains. pCG100 is compatible with pBL1, a cryptic plasmid of Brevibacterium lactofermentum. Shuttle plasmid vectors, containing the kanamycin-resistance gene from Tn903 or from Streptococcus faecalis as selectable markers and the AmpR, TetRor lacZα genes for insertional inactivation, were constructed using the minimum replication fragment of pCG100.

Some wild isolates of Neurospora show microcycle conidiation in liquid culture under continuous agitation. Macroconidia from agar-grown mycelial cultures germinated in liquid and the germlings spontaneously produced conidia with no intervening mycelial phase. Three types of microcycle conidiation were seen among progeny of N. crassa Vickraman A × N. crassa a wild-type: (1) multinucleate blastoconidia produced by apical budding and septation, (2) multinucleate arthroconidia produced by holothallic septation and disarticulation of cells, and (3) uninucleate microconidia produced directly from conidiogenous cells of the germlings. Two genes were identified which control specific patterns of microcycle conidiogenesis. A single gene mcb in linkage group VR near al-3 (3·2% recombination) controls blastoconidiation. This gene is epistatic to gene mcm located in linkage group IIL, very near ro-7 (1·4%). mcm controls both microconidiation and arthroconidiation depending on temperature. Strains of genotype mcm produce microconidia almost exclusively at 18–22 °C, but arthroconidia with few or no microconidia at 30 °C. Because they result in rapid and synchronized conidiation in liquid culture, the two genes should be useful for studies of developmental gene regulation. mcm makes it possible to obtain large quantities of pure microconidia rapidly for experimentation.

We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs, 1D10 and 2A6, were shown to be highly protective. It was also demonstrated, by means of a competitive enzyme immunoassay, that these mAbs recognized different epitopes in the preparation. Examination of a dose-response curve for mAbs 1D10 and 2A6 revealed that optimal levels of protection were achieved using 1 mg of either 1D10 or 2A6, or 0·5 mg 1D10 and 0·5 mg 2A6 given together. Immunological reactivity of these mAbs with a preparation of M-like protein showed that the antigens they recognized were comparable in size to some of the antigens recognized by convalescent horse serum antibodies. The role of immunoglobulin isotype in conferring protection is discussed.

The intra-generic inhibition of bacterial growth observed previously in vivo and in vitro with strains of Salmonella, Citrobacter and E. coli was studied in vitro using S. typhimurium strain F98. There was complete inhibition of multiplication of S. typhimurium when it was added to stationary-phase broth cultures of different Salmonella serotypes, but only partial inhibition when added to broth cultures of E. coli. The degree of inhibition between different mutants of F98 was affected by the numbers of bacteria of the inhibiting strain, but this was not the only factor, since exponential-phase bacterial cells were less inhibitory than stationary-phase cells. The inhibitory effect was produced at temperatures between 20 °C and 40 °C. The complete inhibition of growth observed between F98 mutants was abolished by ampicillin, rifampicin and streptomycin, but not by nalidixic acid. Inhibition was also prevented by separating the two cultures by a dialysis membrane. A TnphoA insertion mutant of F98 was produced which did not show inhibition in vitro but was still inhibitory in vivo. It is suggested that this complete inhibition of bacterial multiplication between organisms of the same genus, which is greater than that produced between organisms from different genera, is mediated by a cell surface protein.

Summary: Fluconazole, ketoconazole and tioconazole were shown to act synergistically in vitro with the antibiotic nikkomycin X/Z on the pathogenic fungus Candida albicans. The phenomenon was demonstrated using a checkerboard technique and growth inhibition experiments. The azole antifungal agents, even at concentrations not affecting growth, decreased the incorporation of the 14C-label from [14C]glucose into chitin of the candidal cell wall. After 3 h incubation with tioconazole, 1 μg ml−1, the incorporation of the radiolabelled glucose into chitin of intact cells and regenerating spheroplasts of C. albicans was inhibited by 43% and 30%, respectively. Moreover, the relative chitin content was approximately 45% lower than that of control cells. The chitin content increased after prolonged incubation with azoles, thus confirming the known phenomenon of azole-induced uncoordinated chitin synthesis and deposition. On the other hand, azole derivatives had very little effect on the rate of nikkomycin transport into C. albicans cells. A sequential blockade mechanism of synergism is proposed.

Summary: The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0·5%, at 28 °C, pH 6·0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.

Non-homogeneous cell suspensions in a continuous culture may result in a difference between the biomass concentrations in the culture vessel and in the effluent. This will have important consequences for values calculated for stoichiometric and kinetic coefficients. For the bacterium Acinetobacter calcoaceticus it was shown that the cells tend to be buoyant, and because of this, if the vessel overflow device is not designed properly, cells will be selectively removed from the reactor. Two different effluent removal devices were compared, only one of which functioned well. A mathematical model is proposed to explain the observations.