- Volume 139, Issue 9, 1993

SUMMARY: The products of condensing organic hydroxyl groups (ROH) with the mineral acids (hydrochloric, nitric, phosphoric and sulphuric) collectively constitute a major part of the output of the synthetic organic chemical industry, with a wide diversity of applications including surfactants, pesticides, herbicides, dyes, methylating agents, explosives and pharmaceuticals. Compounds containing similar or related structures also occur as natural products. Phosphate esters are, of course, exceptional for their ubiquity in the living world, ranging from the simple intermediary metabolites of glycolysis, through phospholipids, to the backbone of DNA. Sulphate esters too are abundant (Dodgson et al., 1982) and naturally occurring halogenated compounds are also being detected in increasing numbers (for examples, see Strunz, 1984; Engvild, 1986; Neidleman & Geigert, 1986; Harper & Hamilton, 1988). It is therefore no surprise that living organisms have evolved phosphatase (Boyer, 1971), sulphatase (Dodgson & Rose, 1975; Dodgson et al., 1982) and dehalogenase (Neilson, 1990; Hardman, 1991) enzyme systems for initiating biodegradation of such compounds by the removal of the mineral moiety. In marked contrast, we are unable to find any examples of naturally occurring nitrate esters, so that the introduction of such compounds into the environment during their industrial production and usage constitutes a true xenobiotic challenge to microorganisms. This raises intriguing questions about microbial capability for biotransformation/biodegradation of nitrate esters, not only from an academic viewpoint but also because of the wide industrial usage of these compounds and their likely impact on the environment.

SUMMARY: Eleven strains of Pseudomonas were isolated by selective enrichment on 4-nitrotoluene (4NT). They all utilized 4NT, 4-nitrobenzyl alcohol (4NBA) or 4-nitrobenzoate (4NBZate) as sole sources of carbon and nitrogen. One strain, TW3, was used for more detailed studies. 4NT-grown cells of TW3 take up O2 when incubated in the presence of 4NBA, 4-nitrobenzaldehyde (4NBZ) and 4NBZate. HPLC analysis of culture supernatants showed that 4NBZ and 4NBZate were formed when 4NT-grown cells were incubated with 4NBA, whereas only 4NBZate was found when they were incubated with 4NBZ. Two dehydrogenases were detected in extracts of 4NT-grown cells. 4NBA dehydrogenase could be assayed by a dye-linked assay whereas 4NBZ dehydrogenase activity was linked to NAD+ reduction. No nitrite was detected in supernatants of 4NBZate-grown cells incubated with 4NBZate but the nitrogen appeared as ammonium. The only aromatic ring-cleavage dioxygenase that was induced during growth on the nitroaromatics was protocatechuate 3,4-dioxygenase. It is proposed that the pathway for 4NT catabolism proceeds via 4NBA, 4NBZ and 4NBZate and ultimately to protocatechuate with release of the nitro group as ammonium.

SUMMARY: A Lactobacillus plantarum bacteriocin, plantaricin A, has been purified to homogeneity by ammonium sulphate precipitation, binding to cation exchanger and Octyl-Sepharose, and reverse-phase chromatography. The bacteriocin activity was associated with two peptides, termed a and , which were separated upon reverse-phase chromatography. Bacteriocin activity required the complementary action of both the a and peptides. From the N-terminal end, 21 and 22 amino acid residues of a and , respectively, were sequenced. Further attempts at sequencing revealed no additional amino acid residues, suggesting that either the C terminus had been reached or that modifications in the next amino acid residue blocked the sequencing reaction. Judging from their amino acid sequence, a and may be encoded by the same gene, since a appeared to be a truncated form of . Alanine, the first amino acid residue at the N-terminal end of was not present at this position in a. Otherwise the sequences of a and appeared to be identical. The calculated molecular masses of the sequenced part of a and were 2426 and 2497 Da, respectively. The molecular masses of a and as determined by mass spectroscopy were 2687 30 and 2758 30 Da, respectively, indicating that (i) the only difference between a and was the presence of the N-terminal alanine residue in , and that (ii) in addition to the sequenced residues, two to three unidentified amino acid residues are present at the C-terminal ends of the a and peptides. Both a and may form amphiphilic a-helices, suggesting that they are pore-forming peptides that create cell membrane channels through a barrel-stave' mechanism.

SUMMARY: Extracts of methanol-grown cells of Amycolatopsis methanolica and Mycobacterium gastri oxidized methanol and ethanol with concomitant reduction of N, N'-dimethyl-4-nitrosoaniline (NDMA). Anion-exchange chromatography revealed the presence of a single enzyme able to catalyse this activity in methanol- or ethanol-grown cells of M. gastri. A. methanolica, however, possessed two different enzymes, one of which was similar to the single enzyme found in M. gastri. The methanol: NDMA oxidoreductases (MNO) were purified to homogeneity from methanol-grown cells of A. methanolica and M. gastri. Both enzyme preparations showed similar relative molecular masses with subunits of M r 50000 and 49000, and native enzymes of M r 268000 and 255000 (gel-filtration data for A. methanolica and M. gastri, respectively). Both enzymes also displayed a similar substrate specificity. They were active with methanol and various other primary alcohols (yielding the corresponding aldehydes), polyols and formaldehyde. In addition, the MNO enzymes produced methylformate from methanol plus formaldehyde, and catalyzed formaldehyde dismutase and NADH-dependent formaldehyde reductase reactions. They did not possess NAD(P)+- or dye-linked alcohol dehydrogenase or oxidase activities.

SUMMARY: Streptomyces cyaneus produces extracellular xylanases in good yield when grown on ball-milled straw and xylan. Ball-milled straw induced nearly twice as much of these activities as did xylan, but phenolic substances released from the straw impeded purification of enzymes. Three extracellular xylanases, I, II and III, were identified in xylan-grown culture supernatants. Xylanases I and II were each purified to give single bands by SDS-PAGE. They had M r values of 37500 and 34000 respectively, while that of III was apparently 45000. pI values were 5.1 (I) and 5.3 (II) while pH and temperature optima, in 10 min assays, were 8.5, 72° (I) and 6.5, 65 °C (II).

SUMMARY: Thiosulphate dehydrogenase (EC 1.8.2.2; thiosulphate:acceptor oxidoreductase) was purified to apparent homogeneity from Thiobacillus acidophilus by a combination of ammonium sulphate precipitation, hydrophobic interaction chromatography, anion-exchange chromatography and gel filtration. The enzyme catalysed the oxidation of thiosulphate (S2O2- 3) to tetrathionate (S4O2- 6) with potassium ferricyanide as an artificial electron acceptor. The molecular mass of the native enzyme, as determined by gel filtration, was 102 ± 4.2 kDa. The enzyme contained two different subunits with a molecular mass of 24 ± 0.9 and 20 ± 1.0 kDa (SDS-PAGE), respectively. Both subunits contained c 553-type haem with absorption bands at 553, 524 and 416 nm. A 77 K spectrum of purified thiosulphate dehydrogenase revealed that the absorption at 553 nm is due to different haem groups. A cytochrome content of 5.3 mole c-type haem per mole of native enzyme was calculated. The pH optimum of the purified enzyme was 3. Apart from ferricyanide, Wurster's blue (the free radical of tetramethyl p-phenylenediamine) and horse heart cytochrome c could also serve as electron acceptors, though less effectively than ferricyanide. At pH 7.0, the K m for thiosulphate was 0.54 mM. The K m could not be determined at the pH optimum due to the chemical reactivity of thiosulphate at low pH values. Sulphite was a potent inhibitor of enzyme activity.

SUMMARY: Enriched preparations of chitin synthase were obtained from cell homogenates from Saprolegnia monoica. Chitin synthase was solubilized from a mixed membrane fraction by two successive digitonin treatments. Glycerol gradient centrifugation of the solubilized proteins separated the chitin synthase activity from the majority of proteins and from -1,3 and -1,4 glucan synthases. The properties of chitin synthase from this Oomycete fungus are similar to those reported for the enzymes of chitinous fungi. The solubilized enzyme catalysed the synthesis in vitro of spindle-like crystals of chitin. Biophysical analysis (by electron and X-ray diffractometry and infrared spectroscopy) demonstrated that the polymer synthesized in vitro was a-chitin. These results and those previously reported demonstrate unambiguously that chitin synthase and chitin are normal components of the cell wall of the cellulosic fungus S. monoica.

SUMMARY: The cell wall of Trichosporon cutaneum consists of 11% protein, 63% neutral carbohydrate, 9% glucosamine and 13% glucuronic acid. The sugars include glucose (32%), mannose (6%) and traces of xylose and galactose. The cell wall was fractionated with alkali to yield a mixture of alkali-soluble matrix components, and an alkali-insoluble glucan associated with chitin. The alkali-insoluble glucan contained a mixture of (1-3) and (1-6) glycosidic linkages. It was only partly susceptible to digestion by the β(1-3) glucanase, Zymolyase. The alkali-soluble fraction contained glucan, mannan and acidic polymers. The glucan was (1-3)-linked with no (1-6) linkages and only trace amounts of (1-3-6)-linked glucose. It was resistant to digestion by Zymolyase. Extensive hydrolysis of this fraction with trifluoroacetic acid released a high-molecular-mass glucuronan which had 1H- and 13C-NMR profiles matching those of the β(1-4) glucuronan, mucoric acid. Xylomannan was purified from isolated cell walls and from whole cells. It contained glucose, mannose, xylose, and D-glucuronic acid. It was very similar in composition and structure to the capsular polysaccharides of Cryptococcus neoformans, and to an extracellular polysaccharide produced by another yeast described as T. cutaneum. Electron microscopy showed that the cell wall of T. cutaneum has a lamellar structure characteristic of a basidiomycetous yeast rather than the electron-dense ‘fuzzy coat’ seen in Candida albicans.

SUMMARY: Glutathione reductase was purified to homogeneity from the unicellular alga Chlamydomonas reinhardtii. The enzyme was a monomer with a molecular mass of 54-56 kDa as judged by gel filtration and SDS-PAGE. The activity was maximal at pH 8.2 and 49 °C. The enzyme was specific for NADPH, but not for NADH. The reverse reaction with NAD(P)+ and GSH (glutathione, reduced form) was not observed in the pH range 4.8-8.2. K m values for NADPH and GSSG (glutathione, oxidized form) were 10.6 μ;m and 54.1 μ;M, respectively. Thiol inhibitors and metal ions such as Hg2+ and Cu2+ markedly inhibited the enzyme activity. Activity was lost when the apoenzyme was prepared by dialysis, but was restored to 40% of the original activity by the addition of 50 μ;M-FAD. The enzyme reaction proceeded via a branching mechanism. Upon immunoprecipitation, glutathione reductase activity of C. reinhardtii was inhibited 50% and 90% by antibodies generated against spinach and Euglena glutathione reductases, respectively. Both antibodies cross-reacted with C. reinhardtii glutathione reductase in an immunoblot analysis.

SUMMARY: Accumulation of Cs+ by Chlorella salina was 28-fold greater in cells incubated in the presence than in the absence of 0.5 M-NaCl. An approximate 70% removal of external Cs+ resulted after 15 h incubation of cells with 50 μ;M-CsCl and 0.5 M-NaCl. LiCl also had a stimulatory effect on Cs+ uptake, although mannitol did not. Cs+ influx increased with increasing external NaCl concentration and was maximal between 25-500 mM-NaCl at approximately 4 nmol Cs+ h−1 (106 cells)−1. Little effect on Cs+ uptake resulted from the presence of Mg2+ or Ca2+ or from varying the external pH, and Cs+ was relatively non-toxic towards C. salina. At increasing cell densities (from 4 × 105 to 1 × 107 cells ml+1), decreasing amounts of Cs+ were accumulated per cell although the rate of Cs+ removal from the external medium was still greatest at the higher cell densities examined. Freely suspended C. salina and cell-loaded alginate microbeads accumulated similar levels of Cs+, however, 46% of total Cs+ uptake was attributable to the calcium-alginate matrix in the latter case. When Cs+-loaded cells were subjected to hypoosmotic shock, loss of cellular Cs+ occurred allowing easy Cs+ recovery. This loss exceeded 90% of cellular Cs+ when cells were washed with solutions containing ≤ 50 mM-NaCl between consecutive Cs+ uptake periods; these cells subsequently lost their ability to accumulate large amounts of Cs+. Maximal Cs+ uptake (approximately 85.1% removal after three 15 h incubations) occurred when cells were washed with a solution containing 500 mM-NaCl and 200 mM-KCl between incubations. The relevance of these results to the possible use of C. salina in a salt-dependent biological Cs-removal process is discussed.

SUMMARY: The ultrastructure of Candida albicans cells induced to secrete extracellular proteinase (EPR) has been studied. Electron microscopy employing alkaline bismuth staining, a method which stains polysaccharides, clearly revealed Golgi-like bodies and secretory vesicles in C. albicans cells. After EPR induction, there was no apparent increase in the number of these structures. Instead, many flocculent granules appeared at the periphery of induced cells. The granules were similar to secretory vesicles in size, but were more irregular in shape. Similar granules were observed in non-induced cells, though less frequently than in induced cells. Brefeldin A, a specific inhibitor of membrane transport in the secretory pathway, caused the accumulation of EPR and Golgi-like bodies in EPR-induced cells, but did not affect the accumulation of the granules. These results suggest that the granules are unrelated to EPR secretion. Electron microscope immunocytochemistry with affinity-purified anti-EPR antibodies showed that the granules in EPR-induced cells were recognized by the antibodies. This recognition was completely inhibited by the presence of glycogen, suggesting that antibodies cross-react with glycogen-like polysaccharides in the granules. Although the location of EPR within the cells remains unclear, the results suggest that EPR might be secreted via the constitutive secretory pathway, and that EPR is glycosylated to give a structure with some similarity to glycogen.

SUMMARY: Lake Geneva is a large, holomictic, eutrophic lake with a maximum depth of about 300 m. The sediments in the central basin have a pillow-like appearance. The soft elevations containing the major portion of the recently sedimented detritus are separated by trenches of 5 to 15 cm depth in which the top sediment layers seem to be missing. Bottom-dwelling fishes (Lota lota) prefer the trenches as their habitat and might partly be responsible for the turbation of the trench sediment layers. Thus, within distances of 10 to 30 cm two sediment types can clearly be distinguished. They differ with respect to morphology and chemical stratification. Concentration depth profiles of nitrate, manganese(II), iron(II), sulphate, and methane dissolved in the interstitial water reveal the location within the sediment of microbially catalysed redox processes. The redox transition zone (RTZ) from aerobic to anaerobic is located only a few millimetres below the sediment surface in the pillow sediments, which contain the bulk of the organic detritus. The RTZ is at a depth of approximately 6 cm in the trench sediments, which are poor in oxidizable organic matter. The same thermodynamic sequence of microbially catalysed redox reactions can be observed in both sediment types. As a consequence, microbial activities as well as diffusion fluxes of dissolved substances in and out of the different sediment regions vary greatly. This leads to small-scale horizontal differences in the sediment's abilities to supply nutrients to the bottom water, which is probably a major controlling factor for sediment-borne eutrophication of this lake.

SUMMARY: A polyclonal antiserum was produced by immunization with nitrite reductase (NiR) purified from Pseudomonas stutzeri (ATCC 14405) and tested for specificity among known denitrifying strains. The antiserum was nearly strain-specific, identifying NiR only in some, but not all, other P. stutzeri strains. Denitrifying isolates from water column and sediment environments were also screened; several isolates from an intertidal microbial mat reacted with the NiR antiserum. Activity assays for NiR in polyacrylamide gels demonstrated that strains with apparently very similar NiR proteins did not react with the antiserum. These results imply that the NiR protein is more variable even among closely related strains than previously suspected. A DNA probe for a 721 bp region of the NiR structural gene was obtained by PCR amplification of P. stutzeri (ATCC 14405) DNA and used to screen denitrifying strains and isolates. The probe hybridized with a greater variety of strains than did the antiserum, implying that the DNA probe may be a more broadly useful and functional probe in environmental samples, whilst the NiR antiserum is nearly strain- or, at most, species-specific. Limits for detection of the enzyme and gene in seawater were estimated and NiR DNA was detected in DNA extracted from natural seawater. The hybridization data imply that in the order of 1-10 in 1000 cells in natural seawater possess homology with the NiR gene probe.

SUMMARY: Mixtures of 2-aminobenzenesulphonate (trivial name orthanilic acid, OA) and 3-methylbenzoic acid (3-MB), which are degraded by enzymes of plasmid-encoded pathways, can exert inhibition of growth and respiration in Alcaligenes sp. O-1 and Pseudomonas putida mt-2 depending on the ratio of their concentrations. The pronounced inhibition of Alcaligenes sp. O-1 growing on OA by the addition of equimolar amounts of 3-MB is characterized by a rapid inactivation of the OA-converting desulphonation activity. The exconjugant Alcaligenes sp. O/T was selected for simultaneous breakdown of OA and 3-MB by assembling the catabolic pathways from the plasmids pSAH (OA) and pWW0 (3-MB) of the above strains. The transpositional insertion of the TOL catabolic genes (Tn4651) from pWW0 into the recombinant plasmid of the exconjugant O/T was detected by Southern blot hybridization using the TOL plasmid as a probe. The exconjugant showed a rapid inactivation of OA desulphonation activity similar to the parent strain. However, following induction of the TOL catabolic genes and mineralization of 3-MB, the exconjugant O/T recovered and displayed high desulphonation activity, thus allowing sequential breakdown of both substrates. Our results clearly extend the expression range of the TOL catabolic genes, but not the replication ability of the plasmid, to the genus Alcaligenes.

SUMMARY: Study of Mycobacterium leprae, the causative agent of leprosy, has been advanced by the isolation of genes encoding mycobacterial proteins including dnaK encoding the M. leprae 70 kDa heat shock protein. The sequence downstream from dnaK revealed a second open reading frame coding for a protein of 389 amino acids with a calculated molecular mass of 41.2 kDa. Sequence analysis demonstrated significant DNA homology with the dnaJ gene of other organisms. High amino acid sequence identity was obtained between the DnaJ protein of M. leprae and M. tuberculosis (89 %) with significant divergence between the two occurring only at the C-terminal end. The expressed recombinant DnaJ protein had a molecular mass of 42 kDa.

SUMMARY: β-Glucanase synthesis in Bacillus subtilis was repressed by glucose and other substrates of glycolysis. Experiments with different pts mutants showed that the phosphoenolpyruvate:sugar phosphotransferase system is not involved in carbon catabolite repression of β-glucanase synthesis. Carbon catabolite repression of β-glucanase synthesis was completely abolished in a ccpA mutant. An operator structure similar to those upstream of amyE and the xyl operon was found and was shown by site-directed mutagenesis to be the target for carbon catabolite repression of β-glucanase synthesis. The presence of this operator on a multi-copy plasmid resulted in a reduced repression of both β-glucanase and α-amylase synthesis. It seems likely that the gene encoding these enzymes are part of one regulon with respect to catabolite repression.

SUMMARY: As described previously, in Rhodococcus sp. (formerly Nocardia opaca) strains MR11 and MR22, the ability to grow as an aerobic hydrogen bacterium (the Aut character) is located on giant conjugative linear plasmids - on pHG201 (270 kb) in strain MR11 and on pHG205 (280 kb) in strain MR22. In an autotrophic transconjugant originating from MR22 a smaller plasmid, pHG207 (225 kb), was detected and shown to be a recombination product of the wild-type plasmids pHG204 and pHG205. A donor carrying pHG207 as the sole plasmid transferred the Aut marker at a 1000-fold frequency compared to the wild-type plasmid pHG205. Analysis of the plasmid ends revealed that plasmid pHG207 carries proteins at both ends; the proteins are linked to the 5' ends of the strands. The cloned end fragments of about 2 kb were sequenced and found to contain highly homologous sequences within the terminal 583 bp (left end part) and 560 bp (right end part). Several potential reading frames were detected, but database searching gave no indication about possible functions.

SUMMARY: The streptococcal M protein family, a number of cell surface molecules that interact with the human immune system, can be divided into two major classes, A and C, characterized by different types of repeats in the central part of the molecule. Class A and class C molecules are known to have a variable N-terminal region and a more conserved C-terminal region, but little is known about the mechanisms that give rise to this structural variation. In this report, we show that two variants of protein Arp, an IgA receptor in class C of the M protein family, have virtually identical signal sequences and C-terminal halves, but unrelated N-terminal sequences. Comparison of the sequences of the two genes and their flanking regions also demonstrates the presence of well-defined variable and conserved regions. Our results strongly suggest that the N-terminal sequence variation between the two variants of protein Arp was generated through an intergenic recombination event, rather than through intragenic recombination or accumulation of mutations.

SUMMARY: Metarhizium anisopliae isolates from several insect hosts and from various sugar cane growing areas of Queensland, Australia, were examined for genetic diversity using random amplified polymorphic DNA (RAPD) markers. Thirty isolates of M. anisopliae var. anisopliae and one isolate of M. anisopliae var. majus were examined. Ten randomly chosen 10mer or 11mer primers were used and RAPD banding patterns were compared. Thirty distinct genotypes could be distinguished amongst the 31 isolates tested on the basis of RAPD patterns. Six of the isolates classified as M. anisopliae var. anisopliae exhibited closer similarity to the M. anisopliae var. majus isolate than to other anisopliae strains tested. Isolates exhibiting similar (> 80 % similarity) RAPD profiles tended to be isolated from the same geographic area and evidence for the persistence of particular fungal genotypes in specific geographical localities was obtained. Pathogenicity assays suggested that, in some instances, RAPD groupings may also indicate insect host range. The mean similarity amongst isolates measured by band sharing in all pairwise comparisons was 41% and the most distinct pair of isolates shared only 9% of their RAPD bands. We conclude that the isolates tested belonging to the species M. anisopliae, as assessed on morphological grounds, represent a very diverse genetic group. The results also suggest that RAPD markers may be useful for the tracking of specific biocontrol strains in the field.

SUMMARY: The Sc7 and Sc14 genes are specifically expressed in the dikaryon of the basidiomycete fungus Schizophyllum commune during fruiting. These genes are closely linked (within 6 kb) and highly similar in gene structure and nucleotide sequence (70% identical nucleotides in their coding regions). The encoded proteins (204 and 214 amino acids, respectively) have 87% similarity in amino acids (56% of the amino acids are identical). They contain putative signal sequences for secretion, are rich in aromatic amino acids which are generally located at similar positions, and they are generally hydrophilic. Inspection of databanks showed similarities with pathogenesis-related proteins (PR1) from plants, testis-specific proteins from mammals and venom allergen proteins from insects. An antibody raised against a Sc7 fusion protein showed the presence of the Sc7 protein in the culture medium and in the fruit bodies where it is apparently loosely associated with hyphal walls.