- Volume 137, Issue 9, 1991
Volume 137, Issue 9, 1991
- Biochemistry
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Defective mycolic acid biosynthesis in a mutant of Mycobacterium smegmatis
More LessSummary: A mutant of Mycobacterium smegmatis defective in mycolic acid biosynthesis was isolated following chemical mutagenesis. Fatty acids were extracted from the mutant and subjected to structural analysis by thin-layer chromatography and high-performance liquid chromatography (HPLC) of both methyl and p-bromophenacyl ester derivatives. Thin-layer chromatography did not show the presence of any fatty acid of R F comparable to that of standard methyl mycolate. The HPLC profile revealed a broad peak in the standard mycolic acid ester region. No characteristic peaks of mycolic acid esters comparable to the wild-type could be resolved. Mass spectral analysis of the HPLC-purified peak demonstrated the presence of shorter-chain fatty acids in the mutant. These data support the idea that the mutant accumulates precursors of mycolic acids and is incapable of carrying out the final conversion to mycolic acids of 60–90 carbon atoms.
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Degradation of p-toluic acid (p-toluenecarboxylic acid) and p-toluenesulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes in Comamonas testosteroni T-2
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.
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Purification and partial characterization of an intracellular NADH:quinone oxidoreductase from Phanerochaete chrysosporium
More LessSummary: 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.
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Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2
More LessSummary: 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.
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A survey of peptidase activity in rumen bacteria
More LessSummary: 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.
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- Biotechnology
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Lysogeny in Leuconostoc oenos
More LessThirty strains of Leuconostoc oenos were exposed to mitomycin C to induce lysogenic bacteriophages. Lysis curves typical for lysogenic strains were obtained with 19 strains. Indicator strains were found for 17 of these phages. Five were characterized by electron microscopy, lytic spectrum, molecular masses of the proteins, sequencing of five N-terminal amino acids of the two major proteins and DNA analysis (restriction patterns, cross hybridization). The results revealed a very close relationship between the phages. Hybridization experiments between the DNAs of the temperate phages and the appropriate lysogenic strains revealed phage-related sequences in the DNA of the lysogenic strain.
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- Genetics And Molecular Biology
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Rapid methods in bacterial DNA fingerprinting
More LessThe characterization and comparison of isolates of bacterial species by comparing restriction enzyme digests of their chromosomal DNA (fingerprints) is highly discriminatory for different strains and allows similarities between them to be readily determined. However, the utility of the technique is dependent on the selection of appropriate restriction enzyme(s) and on the method of determining the similarities between the fingerprints generated. We report here a system which circumvents these two problems. The restriction enzyme is selected from amongst those which have a suitable frequency of restriction for given enzyme-genome combinations. The frequencies of restriction enzyme recognition sites are calculated from the frequencies of di- and trinucleotides in sequenced genes from the species of interest using Markov chain analysis. Fingerprints are compared by dividing them up into sections with DNA size standards, scoring the number of bands in a few of these sections, and comparing these scores (numerical profiles) to establish similarities. In this way a single electrophoretic gel yields easily analysable data which can be compared with data from other gels. The time from the acquisition of bacterial isolates to their final characterization is much reduced in comparison to existing methods.
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Additional copies of the actII regulatory gene induce actinorhodin production in pleiotropic bld mutants of Streptomyces coelicolor A3(2)
More LessIn 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.
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Molecular cloning and characterization of chitinase genes from Streptomyces lividans 66
More LessIn 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.
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Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis
More LessThe 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.
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isolation of a benomyl-resistant allele of the β-tubulin gene from Septoria nodorum and its use as a dominant selectable marker
More LessWe 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.
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Structural organization of the Corynebacterium glutamicum plasmid pCG100
More LesspCG100, 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.
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Microcycle conidiation and its genetic basis in Neurospora crassa
More LessSome 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.
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- Pathogenicity And Medical Microbiology
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Identification of three gene regions associated with virulence in Dichelobacter nodosus, the causative agent of ovine footrot
Dichelobacter nodosus (formerly Bacteroides nodosus) is a Gram-negative strict anaerobe and is the primary pathogen involved in ovine footrot. A comparative hybridization strategy was used to isolate recombinant clones which hybridized to DNA from a virulent strain of D. nodosus but not with a benign isolate. Three virulence-associated gene regions were identified and one of these regions was shown to be present in multiple copies in the D. nodosus genome. Hybridization studies on 101 clinical isolates of D. nodosus showed that these strains could be divided into three hybridization categories which could be correlated with the virulence of the isolates. The recombinant clones have considerable potential for the development of a gene-probe-based method for the differential diagnosis of ovine footrot.
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Protection against Streptococcus equi infection by monoclonal antibodies against an M-like protein
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.
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- Physiology And Growth
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Resistance of Streptomyces cinnamonensis to butyrate and isobutyrate: production and properties of a new anti-isobutyrate (AIB) factor
More LessSummary: Butyrate and isobutyrate (after isomerization to n-butyrate) are specific precursors for the biosynthesis of monensin A in Streptomyces cinnamonensis. High concentrations of both butyrate and isobutyrate (greater than 20 and 10 mm, respectively) were toxic to S. cinnamonensis plated on solid medium. Spontaneous mutants resistant to these substances were isolated. These new strains produced monensins at even higher concentrations of butyrate or isobutyrate, with an increased yield of monensin A. S. cinnamonensis produced an anti-isobutyrate (AIB) factor, which was originally found to be excreted by some isobutyrate-resistant strains growing on solid medium containing isobutyrate. On plates, the AIB factor efficiently counteracted toxic concentrations not only of isobutyrate, but also of acetate, propionate, butyrate, 2-methylbutyrate, valerate and isovalerate against S. cinnamonensis as well as other Streptomyces species. Although the AIB factor enabled normal growth, sporulation and monensin production on plates, it did not have positive effects on submerged cultures of S. cinnamonensis with isobutyrate. The partial purification of the AIB factor was achieved. The role of the AIB factor during spore germination on solid medium containing isobutyrate or its homologues is discussed.
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In vitro characterization of intra-generic inhibition of growth in Salmonella typhimurium
More LessThe 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.
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Synergistic action of nikkomycin X/Z with azole antifungals on Candida albicans
More LessSummary: 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.
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Regulation of chitinase synthesis in Trichoderma harzianum
More LessSummary: 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.
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The bioreactor overflow device: an undesired selective separator in continuous cultures?
More LessNon-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.
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Topography of methanogenic subpopulations in a microbial consortium adapting to thermophilic conditions
More LessSummary: The microanatomy of a granular, methanogenic, microbial consortium (granule) was investigated by applying histochemical chemical techniques and light microscopy. The granules were obtained from a full-scale, mesophilic, upflow anaerobic-sludge blanket (UASB) bioreactor and were inoculated into laboratory-scale counterparts with a defined Substrate in which they were maintained for several weeks before shifting to thermophilic conditions. Samples for analysis were collected from the original inoculum, and from the laboratory-scale bioreactors before and after the temperature shift. Four months after this shift, the basic architectural plan of the granules was found to comprise a core or medulla, and a peripheral zone or cortex bounded by denser layers, and a spongy matrix formed by microbes and intercellular material. Also, five methanogenic subpopulations known to occur in these 4-month thermophilic granules were mapped to elucidate their spatial arrangement using immunofluorescence with antibody probes of predefined specificity spectra on histologic thin sections. Each subpopulation showed a distinctive distribution pattern; e.g. elongated surface and inner colonies for the methanogen antigenically related to Methanobacterium thermoautotrophicum ΔH, packets and cellular cords for the methanosarcina related to Methanosarcina thermophila TM1, bundles for Methanothrix rods, and dense or sparse lawns for the methanogens antigenically related to Methanobrevibacter arboriphilus AZ and Methanobrevibacter smithii ALI, respectively. These features were absent from, or much less developed in, the original inoculum granules, and before the temperature shift or even 1 week thereafter. All granules showed the same basic architecture involving a cortex and a medulla, but the inner texture of the mesophilic granules was considerably more compact than that of the 4-month thermophilic granules. Thus, the topography of methanogenic subpopulations and some structural details of the 4-month thermophilic granules Seem to be the result of adaptation to enrivonmental factors such as temperature, the same way as the array of methanogenic species and their numbers were found to be in previous work.
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Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2
More LessSummary: Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6·5, 35·5 †C, at a dilution rate of 0·04 h−1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)−1and a specific rate of lipase production (q Lipase) of 1·56 LU (mg cells)−1h−1(1 LU equalled 1 μmol fatty acid released min−1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.
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The use of acetate as an additional co-substrate improves methylotrophi growth of the acetogenic anaerobe Eubacterium limosum when CO2 fixation is rate-limiting
More LessSummary: Growth of the acetogenic anaerobe Eubacterium limosum on methanol/CO2 mixtures is limited by the rate at which CO2 can be assimilated. This limitation can be offset by the consumption of acetate as an additional co-substrate. Growth on methanol/CO2/acetate mixtures improves growth rates but stimulates production of an unidentified polymer leading to cell aggregation and wall growth under chemostat conditions. Production of butyrate as major fermentation end-product leads to growth inhibition visualized by an increased maintenance requirement as demonstrated by Y ATP estimations.
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The involvement of glutamate dehydrogenase and glutamine synthetase/glutamate synthase in ammonia assimilation by the basidiomycete fungus Stropharia semiglobata
More LessSummary: When grown on ammonia as sole nitrogen source, the basidiomycete fungus Stropharia semiglobata assimilated nitrogen via glutamate dehydrogenase (GDH). The two isoenzymes of GDH were both repressed by increasing concentrations of ammonia, but NADP-GDH (EC 1.4.1.4) was far more active than NAD-GDH (EC 1.4.1.2). Glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.7.1) were also active in S. semiglobata and these enzyme activities were independent of the ammonia concentration in the suspension. From enzyme activity and 1 5N-labelling studies of amino acids, it is concluded that both GDH and GS/GOGAT contribute to ammonia assimilation in this fungus.
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- Plant-Microbe Interactions
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Molecular genetics of Pseudomonas syringae pathovar pisi: plasmid involvement in cultivar-specific incompatibility
More LessSummary: A mutant (PF24) of the race 1 strain, 299A, of Pseudomonas syringae pv. pisi has been characterized in terms of its interactions with pea (Pisum sativum) cultivars. The mutant showed a changed reaction (avirulence to virulence) with a group of pea cultivars, including cvs. Belinda and Puget, previously thought to contain resistance genes R1 and R3. Avirulence towards cv. Puget was restored by transfer of any one of five cosmid clones from a race 3 (strain 870A) gene library to a rifampicin-resistant derivative of PF24. These observations were in agreement with a revised race-specific resistance genotype for Belinda and similar cultivars comprising a single resistance gene, R3. An incompatible interaction was observed between strain PF24 and cvs. Vinco (postulated to harbour race-specific resistance genes R1, R2, R3 and R5) and Hurst’s Greenshaft (R4 and possibly R1), indicating that the mutant retains at least one avirulence gene (A1 or A1 and A4). Mutant PF24 showed loss of a cryptic plasmid (pA V212) compared with its progenitor, strain 299A. A subclone (pAV233) of one of the race 3 restoration clones showed strong hybridization with similar-sized digestion fragments in race 3 plasmid DNA, confirming the A3 gene to be plasmid-borne. Strong cross-hybridization was also observed with a single 3·27 kb EcoRI fragment of plasmid DNA present in strain 299A but absent from strain PF24. This is consistent with the corresponding A3 determinant being located on pAV212 in the race 1 strain 299A. The novel avirulence gene corresponding to A3 in strain 870A is provisionally designated avrPpi3. A spontaneous race-change variant (strain 1759, which expressed no avirulence phenotype toward the pea differential cultivars) was derived from the race 3 strain 870A. This race 6 strain and a wild isolate of a race 6 strain both lacked plasmid DNA sequences corresponding to the insert in pA V233.
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Two different modes of attachment of Azospirillum brasilense Sp7 to wheat roots
More LessSummary: Two in vitro assays are described that can distinguish between two modes of attachment of Azospirillum brasilense Sp7 to wheat roots, and quantify the number of attached bacteria. The first assay measures A. brasilense adsorption (Ads), which reaches a maximal level within 2 h of incubation. Adsorption forces are weak, since adsorbed bacteria can be quantitatively removed by vortexing the root in water. In addition, adsorption is strongly reduced by pretreatment of A. brasilense cells with pronase E. The second assay measures A. brasilense anchoring (Anc), which begins only after 8 h of incubation and reaches a maximal level after 16 h. That adsorption and anchoring are different phenomena is further demonstrated by the properties of two classes ofA. brasilense attachment mutants. The first class of mutants, deficient in the production of a particular surface polysaccharide, has completely lost anchoring capability, but maintains wild-type adsorption capacity (Ads+Anc−). Mutants of the second class are defective in adsorption, but not in anchoring (Ads−Anc+). On the basis of these data, a two-step attachment mechanism of A. brasilense to wheat roots is proposed. The first step consists of a rapid and weak adsorption and depends on bacterial surface protein; the second step is, at least in vitro, independent of the first and consists of firm anchoring of adsorbed and free bacteria by means of bacterial extracellular polysaccharide.
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- Systematics
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An obligately anaerobic, coiled bacterium from Ace Lake, Antarctica
More LessSummary: A coiled or ‘C-shaped’, Gram-negative, non-motile bacterium was isolated from the anaerobic waters of Ace Lake, an Antarctic lake of salinity similar to seawater. The strain was obligately anaerobic and lacked oxidase, catalase and respiratory lipoquinones. It fermented glucose and peptones, and formed H2, CO2, and butyric, acetic and formic acids as major end-products. Pyruvate was metabolized to H2, CO2, and acetic and formic acids. The organism had an optimum temperature for growth of 15–16 °C, and no growth occurred at 22 °C. In synthetic medium, the generation time at 1·7 °C (in situ lake temperature) was 53 h. The optimum NaCl concentration for growth was 0·3 m, with poor growth at 0·1 m (5·8%, w/v). The mol% G + C of the DNA was 25·9 ± 0·4% (T m). The bacterium could not be accommodated within current bacterial species based on phenotypic criteria. Although coiled bacteria have been previously observed in Antarctic anaerobic environments, they had not been cultivated, and their metabolic capability and possible ecological role were unknown.
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Classification of acidophilic, neutrotolerant and neutrophilic streptomycetes by nucleotide sequencing of 5S ribosomal RNA
Summary: Complete 5S ribosomal RNA sequences were obtained for four acidophilic actinomycetes, seven neutrophilic streptomycetes and a strain of Streptoverticillium baldaccii. All of the organisms contained RNAs belonging to the 120 nucleotide type. An evolutionary tree was generated after combining the test data with results from similar studies on representative Gram-positive bacteria. The acidophilic, neutrotolerant and neutrophilic actinomycetes were recovered in a distinct cluster that was equated with the genus Streptomyces. The sequence data support the view that the genera Chainia, Elytrosporangium, Kitasatoa and Microellobosporia should be considered as synonyms of the genus Streptomyces. The recovery of the Streptoverticillium baldaccii strain on the fringe of the Streptomyces cluster is also consistent with current trends in the taxonomy of these organisms. Further work is needed to determine the taxonomic status of the two streptomycete subgroups that comprised the streptomycete cluster.
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- Corrigendum
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