- Volume 135, Issue 3, 1989

In in vitro tests, two strains of Trichoderma harzianum failed to parasitize colonies of Fusarium oxysporum f. sp. vasinfectum and F. oxysporum f. sp. melonis. However, these strains were strongly mycoparasitic on Rhizoctonia solani and Pythium aphanidermatum. When grown in liquid cultures containing laminarin, chitin or fungal cell walls as sole carbon sources, both strains of T. harzianum released, 1-3-β-glucanase and chitinase into the medium. Higher levels of these enzymes were induced in strain T-203 than in T-35 by hyphal cell walls of F. oxysporum. When the lytic enzymes produced by T-35 were incubated with hyphal cell walls of the test fungi, more glucose and N-acetyl-d-glucosamine was released from cell walls of R. solani and Sclerotium rolfsii than from those of F. oxysporum. Treatment of F. oxysporum cell walls with 2 m-NaOH, protease or trypsin prior to their incubation with the lytic enzymes of T. harzianum significantly increased the release of glucose and N-acetyl-d-glucosamine. The effect of these treatments on R. solani and S. rolfsii cell walls was much lower. These results suggest that proteins in the cell walls of F. oxysporum may make these walls more resistant than those of R. solani or S. rolfsii to degradation by extracellular enzymes of T. harzianum.

Cell-free extracts of eighteen fermentative and nonfermentative Mollicutes were examined for enzyme activities associated with the hexose monophosphate shunt (HMS) and Embden---Meyerhof---Parnas (EMP) pathway. All Acholeplasma spp. had glucose-6-phosphate (G6P) dehydrogenase (EC 1.1.1.49), 6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44) and hexokinase (EC 2.7.1.1) activity. Of these three enzyme activities, hexokinase was also detected in Mycoplasma sp. Let. 1 but in no other fermentative or nonfermentative Mycoplasma spp. The Acholeplasma and fermentative Mycoplasma spp. possessed all other HMS and EMP activities examined. All Acholeplasma spp. possessed a pyrophosphate (PPi)-dependent phosphofructokinase (PFK) (EC 2.7.1.90) while fermentative Mycoplasma spp. possessed an ATP-dependent PFK (EC 2.7.1.11). Transaldolase (EC 2.2.1.2) activity was detected in some, but not all Acholeplasma and fermentative Mycoplasma spp. 2-Deoxyribose-5-phosphate aldolase (EC 4.1.2.4) activity was present in all mollicute extracts tested except for Mycoplasma gallisepticum and Mycoplasma sp. Let. 1. The two nonfermentative Mycoplasma spp. lacked all enzyme activities of the HMS pathway except for ribulose-5-phosphate epimerase activity, and of the EMP pathway only phosphoglucose isomerase and the enzymes converting glyceraldehyde 3-phosphate (G3P) to phosphoenolpyruvate (PEP) were detected. We believe that the three major observations of this study are: (1) all Mycoplasma spp. lack G6P and 6PG dehydrogenase activities, suggesting a reduction in their NADPH pool, which may relate to the lipid growth requirement of this genus; (2) the fermentative Mycoplasma spp. have an ATP-dependent PFK activity, while the fermentative Acholeplasma spp. have a PPi-dependent PFK activity; and (3) the nonfermentative Mycoplasma spp. lack ATP and PPi-dependent PFK and fructose-1,6-bisphosphate aldolase activities but, like the fermentative Mollicutes, can convert three-carbon compounds, G3P to PEP through the three-carbon arm of the EMP pathway.

A recently described extremely thermophilic, sulphate-reducing archaebacterium, Archaeoglobus fulgidus, was investigated for the presence of respiratory lipoquinones. Initial screening using thin-layer chromatography and UV spectroscopy indicated that naphthoquinones were present. A more detailed study, using a combination of HPLC, mass spectroscopy and NMR techniques, indicated that the major respiratory lipoquinone present in this organism is a menaquinone with a fully saturated heptaprenyl side chain (MK-7H14).

Glycerol: NADP+ 2-oxidoreductase (EC 1.1.1.156) was isolated from Schizosaccharomyces pombe, purified and characterized. It had an M r of 57000, and SDS-PAGE revealed two polypeptides, of M r 25000 and 30000. Its coenzyme requirement was satisfied exclusively by NADP. The pH optimum for glycerol oxidation was 9·5, for dihydroxyacetone reduction 6·0. Rates of oxidation with some structurally related diols were three- to six-fold lower than for glycerol, while glyceraldehyde and other carbonyl compounds showed negligible rates of reduction. Neither monovalent nor divalent cations activated the enzyme. Apparent K m and V max values were determined. The enzyme is similar to glycerol dehydrogenases isolated from Mucor javanicus and from Dunaliella parva but differs considerably from the glycerol: NAD+ 2-oxidoreductase of S. pombe.

Effects of Na+ on respiratory NADH oxidase were studied in membranes prepared from Gram-negative marine bacteria belonging to the genera Alcaligenes, Alteromonas, Flavobacterium and Vibrio. NADH oxidases of 9 out of the 10 strains examined were found to require Na+ for maximum activity. Without exception, the Na+-dependent site was located at the level of the NADH: quinone oxidoreductase region of the respiratory chain and was highly sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). NADH dehydrogenases in the Na+-dependent segment were sensitive to Ag+ and able to oxidize reduced nicotinamide hypoxanthine dinucleotide as a substrate. Since the NADH dehydrogenase was neither dependent on Na+ nor sensitive to HQNO, it was suggested that reduction of quinone by the NADH: quinone oxidoreductase involves the formation of an intermediate, presumably semiquinone, and that electron transfer from the intermediate requires Na+ and is sensitive to HQNO. These results showed a striking similarity to those obtained with the Na+-dependent NADH: quinone oxidoreductase, the Na+ pump, of Vibrio alginolyticus, indicating that this enzyme is widely distributed among Gram-negative marine bacteria and shares common properties.

Gel-stabilized model sediment ecosystems were prepared using agar and colloidal silica as gelling agents and were employed in studies of the effects and degradation of xenobiotics in marine and freshwater sediments. All models produced physicochemical and microbiological profiles characteristic of sediments. The fate of 3-nitrophenol was studied in a freshwater system and the compound was found to be readily distributed through the gel column and to be rapidly degraded. The release of the nitro-group during metabolism resulted in the accumulation of nitrate in the aerobic portion of the gel column. The fate of a 1:1 hexadecane/naphthalene mixture was investigated using a seawater model system. The metabolism of these compounds resulted in oxygen depletion in the gel column and in a decrease in the population size of aerobic heterotrophic bacteria. Conversely, the populations of anaerobic heterotrophic bacteria and sulphate-reducing bacteria were significantly increased. The data are discussed with particular respect to the practical uses of gel-stabilized model ecosystems in research into the microbiology of sediments.

The virulence region of the Salmonella dublin 50 MDa plasmid shared homology with 678 of 1021 salmonellae tested in colony hybridization experiments. The majority of S. dublin, S. typhimurium and S. enteritidis isolates tested hybridized with the region whereas, with the exception of S. hessarek, S. pullorum and S. gallinarum, other serotypes did not. Homologous virulence regions were plasmid encoded. In S. typhimurium a common 60 MDa plasmid was present in all phage types tested but not in DT4, DT37 and DT170. Smaller plasmids showing partial homology were found in DT12, DT18, DT193 and DT204C. In S. enteritidis a distinct plasmid profile for each of eight phage types was observed. Hybridizing plasmids were found in DT3, DT4, DT8, DT9 and DT11 whereas DT7, which was plasmid free, and DT10 and DT14, which harboured plasmids, did not hybridize. The extent of homology shared between S. dublin, S. typhimurium and S. enteritidis virulence plasmids was about 10 MDa and appeared conserved. Virulence plasmids from S. typhimurium and S. enteritidis did not show homology with a region of the S. dublin 50 MDa plasmid which was not associated with virulence functions whereas plasmids of about 24 MDa and 38 MDa in some S. typhimurium phage types did. The association of conserved virulence regions upon differing plasmids within salmonellae is discussed with reference to possible mechanisms of distribution and evolution of virulence genes.

Plasmid DNAs from 25 Aeromonas salmonicida strains (14 ‘typical’ and 11 so-called ‘atypical’ strains) were analysed using two plasmid isolation techniques in conjunction with restriction enzyme digestion and agarose gel electrophoresis. ‘Atypical’ strains carried two to four plasmid species, which were different from the plasmids carried by ‘typical’ strains, and corresponded to isolate source and biotype, suggesting that plasmid content may be a useful epidemiological marker for ‘atypical’ A. salmonicida. The ‘typical’ group was extremely homogeneous in plasmid content. In addition to a single large (70–145 kb) structurally related plasmid species, all strains carried a highly conserved group of three low-M r plasmids (pAsa1, 5·0 kb; pAsa2, 5·2 kb; pAsa3, 5·4 kb). The pAsa plasmids had different restriction maps, and directed the synthesis of four polypeptides when assayed in vitro using a transcription/translation system and [3 5S]methionine radiolabelling. A partially conserved 6·0 kb plasmid directed synthesis of an additional polypeptide. Total cellular plasmid DNA from virulent ‘typical’ strain A. salmonicida A449 was also cloned in Escherichia coli, using the vector pBR322. When analysed using a minicell expression system, 17 plasmid-encoded proteins, ranging in size from 12 to 90 kDa, were identified, including two putative exported proteins, and a chloramphenicol acetyltrans-ferase which was encoded by the 145 kb conserved, non-conjugative, plasmid.

Twenty-six wild-type Streptomyces strains tested for resistance to arsenate, arsenite and antimony(III) could be divided into four groups: those resistant only to arsenite (3) or to arsenate (2) and those resistant (8) or sensitive (13) to both heavy metals. All strains were sensitive to antimony. The structural genes for the ars operon of Escherichia coli were subcloned into various Streptomyces plasmid vectors. The expression of the whole ars operon in streptomycetes may be strain-specific and occurred only from low-copy-number plasmids. The arsC gene product could be expressed from high-copy plasmids and conferred arsenate resistance to both E. coli and Streptomyces species. The ars operon expressed in S. lividans and the arsC gene expressed in S. noursei did not render the synthesis of undecylprodigiosin and nourseothricin, respectively, phosphate-resistant. In addition in wild-type strains of Streptomyces phosphate sensitivity of antibiotic biosynthesis did not show strong correlation with resistance of growth to arsenicals.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 °C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20–30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200–500 stable transformants per μg of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.

Five crn loci have been identified in Fusarium moniliforme. Mutants at these loci were chlorate resistant and able to utilize nitrate. The crnl locus was closely linked to, and may be allelic with, nit3, a regulatory gene controlling induction of the nitrate reductase pathway. The crn2 locus was linked to, but distinct from, the nit5 gene coding for a molybdenum-containing cofactor. The crn3 and crn4 loci were unlinked to any known gene affecting nitrate reduction. The crn5 locus was clearly linked to the nit1 gene coding for the nitrate reductase enzyme. Mutants of crn1 and crn5 had low but detectable levels of nitrate reductase activity; these mutations may be leaky alleles of nitrate reduction genes. crn2 mutants had normal nitrate reductase activity, and may be altered in nitrate uptake.

Twenty-six strains belonging to the five main species of the genus Listeria were examined for production of thiol-dependent exotoxins. All strains of L. monocytogenes cultured in charcoal-treated broth secreted a haemolytic factor at a level ranging from 200 to 800 haemolytic units (HU) ml-1, except for the strain EGD (1500 HU ml−1) and the type strain CIP 82110T (10 HU ml−1). The haemolytic activity reached a maximum level by 8–10 h and then rapidly declined as soon as bacterial exponential growth ceased. The titres of haemolytic activity were markedly reduced when bacteria were grown in charcoal-untreated broth. The haemolytic factor produced by L. monocytogenes strains was characterized as listeriolysin O (M r about 60000), a member of the group of thiol-dependent exotoxins. Strains of Listeria ivanovii also produced high levels of thiol-dependent exotoxin (about 2500 HU ml−1), in both charcoal-treated and untreated broth. Small amounts of haemolytic factor (about 9–30 HU ml−1) were also produced by Listeria seeligeri in charcoal-treated broth. The haemolysin produced by L. seeligeri was identified for the first time as a thiol-dependent exotoxin of M r about 60000, antigenically related to listeriolysin O. As expected, we failed to detect thiol-dependent exotoxin in the two nonhaemolytic species, Listeria innocua and Listeria welshimeri.

Larvae of Galleria mellonella (Lepidoptera) displayed a strong cellular reaction to the walled-stage hyphal bodies of the entomopathogenic fungus Entomophaga aulicae (Entomophthorales). In contrast, no reaction was observed against the wall-less protoplasts. This phenomenon was not due to an inhibition of the cellular response by secretion of a toxin by protoplasts. Furthermore, no molecular mimicry was observed between host and protoplast antigens. Host macromolecules which could protect them from being recognized as foreign did not attach to the protoplasts. Ultrastructural and chemical studies of hyphal body and protoplast surfaces of E. aulicae demonstrated the presence of 1,3-β-glucan and chitin on the hyphal wall, whereas these components were absent on the protoplast surface. Since purified 1,3-β-glucan and chitin isolated from E. aulicae and from another entomophthoralean species, Conidiobolus obscurus, induced their haemocytic encapsulation in vitro, it is suggested that these wall components are responsible for the recognition of hyphal body elements, and that their absence accounts for the ability of protoplasts to escape encapsulation.

A genomic library of Streptococcus sanguis, strain G9B, was constructed and expressed in Escherichia coli using a λgt11 expression vector. The amplified library was probed with polyclonal anti-G9B IgG and 13 antigen-positive clones were isolated. A lysate of one clone, designated PP39, absorbed the adhesion-inhibitory activity of anti-G9B IgG. This clone contained an insert of approximately 2000 bp and expressed unique 200 and 53 kDa proteins that reacted with monospecific anti-adhesin antibody. The 200 kDa protein also reacted with anti-β-galactosidase IgG, indicating that it is a fusion protein of which 84 kDa represents the streptococcal adhesin. The 84 and 53 kDa proteins are similar in size to the major polypeptides in a streptococcal antigen complex which is associated with the adhesion of G9B to salivacoated hydroxyapatite. The 53 kDa fragment may result from post-translational cleavage of the recombinant polypeptide.