- Volume 140, Issue 7, 1994
Volume 140, Issue 7, 1994
- Physiology And Growth
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Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora
More LessWhen grown in liquid cultures allowing the formation of nematode traps, the fungus Arthrobotrys oligospora produced two extracellular proteases hydrolysing the chromogenic substrate Azocoll. The protease activity was separated into two fractions (FI and FII) using anion-exchange chromatography. In bioassays, protease(s) present in FII immobilized the free-living nematode Panagrellus redivivus indicating that the enzyme(s) might be involved in the infection of nematodes. A protease designated PII was purified from FII to apparent homogeneity by hydrophobic interaction and size-exclusion chromatography, resulting in an approximately 15-fold increase in specific activity. The purified enzyme was glycosylated, had a molecular mass of approximately 35 kDa (gel filtration) and an isoelectric point of pH 4·6. PII immobilized P. redivivus in bioassays and hydrolysed proteins of the purified cuticle. The enzyme hydrolysed several protein substrates including casein, bovine serum albumin and gelatin, but not native collagen. Examination of substrate specificity with synthetic peptides showed that PII readily hydrolysed tripeptides with aromatic or basic amino acids including N-benzoyl-L-phenylalanyl-L-valyl-L-arginine-4-nitroanilide (Bz-Phe-Val-Arg-NA) and succinyl-glycyl-glycyl-L-phenylalanine-4-nitroanilide (Suc-Gly-Gly-Phe-NA). Mono-peptides were hydrolysed at considerably slower rates. PII had an optimum activity between pH 7 and 9 and was susceptible to autodegradation. PII was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride (PMSF), chymostatin and antipain. The protease was N-terminally blocked, but the sequence of one internal peptide showed a high homology with a region containing the active site histidine residue of the subtilisin family of serine proteases.
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Iron uptake and molecular recognition in Pseudomonas putida: receptor mapping with ferrioxamine B, coprogen B and their biomimetic analogues
This study shows that Pseudomonas putida possesses active uptake systems for Fe3 -ferrioxamine B (FOB) and Fe3 -coprogen B (Cop B). These systems were characterized using natural and synthetic siderophores as structural probes. The synthetic analogues p178, p191, p239, p254 and p271 are a family of systematically modified linear retro-trishydroxamates that have shorter links between the ion binding groups relative to the natural compounds and possess chiral centres. They form a lower number of isomeric Fe3+ complexes relative to the natural compounds, and may be regarded as their specific conformers. Growth promotion and facilitated 55Fe3+ uptake using both natural and synthetic siderophores were studied. The results obtained, along with those from competition experiments between the natural and the synthetic analogues demonstrate that: (i) the FOB and Cop B uptake systems share common transport determinants; (ii) FOB and Cop B make use of separate receptors; (iii) the Cop B receptor is conformationally more demanding than the FOB receptor; and (iv) the FOB receptor has preference for the A-cis configuration although the natural siderophore is achiral. These results also demonstrate the usefulness of the synthetic analogues as structural probes. Some of these analogues simulate the natural counterparts as Fe3+ carriers, while others merely inhibit the action of the natural compounds by competing for the respective siderophore receptor.
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Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764
More LessCell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalysed the degradation of cyanide into products that included CO2. formic acid, formamide and ammonia. Cyanide-degrading activity (CDA) was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM (2600 mg CN I 1). At least two different CDAs could be distinguished by: (i) the determination of reaction product stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent K m for cyanide of 1·2 mM. The second activity generated formic acid (and NH3) plus formamide as reaction products, was oxygen independent, and had an apparent K m of 12 mM for cyanide. The first enzymic activity was identified as cyanide oxygenase as previously described [Harris, R. E. & Knowles, C. J. (1983) FEMS Microbiol Lett 20, 337-341] whereas the second activity is believed to consist of two enzymes, a cyanide nitrilase (dihydratase) and hydratase (EC4·2.1·66). In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (EC 1·2.1·2) thereby providing a means of recycling NADH utilized by cyanide oxygenase. A mutant strain having lost the ability to grow on cyanide as a nitrogen source was isolated and shown to be defective in cyanide oxygenase, but not the cyanide nitrilase/hydratase enzymes. This finding together with results showing that the substrate affinity of cyanide oxygenase was tenfold greater than for the nitrilase/hydratase enzymes, indicates that it is this enzyme that is most important in cyanide assimilation. A cyanate-defective mutant was also isolated and shown to be unaffected in cyanide assimilation, indicating that the metabolism of these two compounds is physiologically distinct.
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3-Sulphocatechol 2,3-dioxygenase and other dioxygenases (EC 1·13.11·2 and EC 1·14.12.-) in the degradative pathways of 2-aminobenzenesulphonic, benzenesulphonic and 4-toluenesulphonic acids in Alcaligenes sp. strain 0-1
More LessAlcaligenes sp. strain 0-1 utilizes three sulphonated aromatic compounds as sole sources of carbon and energy for growth in minimal salts medium - benzenesulphonate (BS), 4-toluenesulphonate (TS) and 2-aminobenzenesulphonate (2AS). The degradative pathway(s) in 2AS-grown cells are initiated with membrane transport, NADH-dependent dioxygenation and meta ring cleavage. The specific activity of the NADH-dependent dioxygenation(s) varied with the growth phase and was maximal near the end of exponential growth for each growth substrate. Cells were harvested at this point from BS-, TS- and 2AS-salts medium. Cells grown with each sulphonated substrate could oxygenate all three compounds, but only 2AS-grown cells consumed 2 mol O2 per mol 2AS or BS or TS. BS- and TS-grown cells consumed 2 mol O2 per mol BS or TS but failed to oxygenate the product of oxygenation of 2AS, 3-sulphocatechol (3SC). These observations were repeated with cell extracts and we concluded that there were two sets of desulphonative pathways in the organism, one for 2AS and one for BS and TS. We confirmed this hypothesis by separating the degradative enzymes from 2AS-, BS- or TS-grown cells. A 2AS dioxygenase system and a 3SC-2,3-dioxygenase (3SC230) were detected in 2AS-grown cells only. In both BS- and TS-grown cells a dioxygenase system for BS and TS was observed as well as a principal catechol 2,3-dioxygenase (C230-III), neither of which was present in 2AS-grown cells. The 3SC230 was purified to near homogeneity, found to be monomeric (M r 42000), and to catalyse 2,3-dioxygenation to a product that decayed spontaneously to sulphite and 2-hydroxymuconate. The 2AS dioxygenase system could cause not only deamination of 2AS but also desulphonation of BS and TS. The BS dioxygenase could desulphonate BS and apparently either desulphonate or deaminate 2AS. Strain 0-1 thus seems to contain two putative, independently regulated operons involving oxygenation and spontaneous desulphonation(s). One operon encodes at least the 2AS dioxygenase system and 3SC230 whereas the other encodes at least the BS/TS dioxygenase system and C230-III.
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Is the Kluyver effect in yeasts caused by product inhibition?
Candida utilis CBS 621 exhibits the Kluyver effect for maltose, i.e. this yeast can respire maltose and is able to ferment glucose, but is unable to ferment maltose. When glucose was pulsed to a maltose-grown, oxygen-limited chemostat culture of C. utilis, ethanol formation from glucose started almost instantaneously, indicating that the enzymes needed for alcoholic fermentation are expressed in maltose-grown cells. However, the addition of glucose inhibited maltose metabolism. To eliminate a possible catabolite inhibition and/or repression of enzyme activities involved in maltose metabolism, the effect of simultaneously feeding glucose and maltose to an oxygen-limited, maltose-grown chemostat culture was studied. In this case, the glucose concentration in the culture remained below 0·1 mM, which makes glucose catabolite repression unlikely. Nevertheless, maltose metabolism appeared to cease when the culture was switched to the mixed feed. Based on the outcome of the mixed-substrate studies, it was postulated that the Kluyver effect may be caused by feedback inhibition of maltose utilization by ethanol, the product of fermentative maltose metabolism. If ethanol suppresses the utilization of non-fermentable disaccharides, this would provide a phenomenological explanation for the occurrence of the Kluyver effect: accumulation would then not occur and the rate of maltose metabolism would be tuned to the culture's respiratory capacity. This hypothesis was tested by studying growth of C. utilis CBS 621 and Debaryomyces castellii CBS 2923 in aerobic batch cultures on mixtures of sugars and ethanol. With both yeasts diauxic growth was indeed observed on mixtures of ethanol and a disaccharide that gives rise to the Kluyver effect, with ethanol being the preferred substrate. In contrast, sugars which could be fermented were either utilized simultaneously with ethanol or preferred over this substrate.
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Adaptive acid tolerance response (ATR) in Aeromonas hydrophila
More LessAeromonas hydrophila, a gastrointestinal pathogen of humans, was shown to exhibit a significant adaptive acid tolerance response (ATR) capable of protecting cells from severe acid at a pH of 3·5. The ATR was induced by exposure to a relatively mild pH level of 5·0 for 20 min. Adaptation required protein synthesis since treatment with chloramphenicol during adaptation to pH 5·0 prevented the development of acid tolerance. The adaptation to acid environment was found to be a non-transient phenomenon. Also, iron was not required for acid adaptation in A. hydrophila. Two-dimensional protein analyses revealed an increased production of 28 proteins and decreased synthesis of 10 following pH shifts from 7·2 to 5·0. The mild pH treatment must act as a signal to A. hydrophila to adapt and survive in acid environments by producing ‘protective’ proteins. The adaptation and survival of this pathogen in low pH may provide valuable information about its ability to withstand acid environments in nature and in the human gastrointestinal tract.
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Altered amino acid metabolism in Irp mutants of Escherichia coli K12 and their derivatives
More LessAn Escherichia coli Irp mutant, lacking the leucine-responsive regulatory protein and the global response it controls, is deregulated in the expression of many genes, but is nevertheless able to grow in glucose-minimal medium at 37 °C. In the presence of isoleucine and valine, the growth rate of the Irp mutant at 37 °C is significantly increased by exogenous L-serine or L-leucine (or both), suggesting that synthesis of these amino acids is limiting. In the absence of isoleucine and valine, however, growth is severely inhibited by both L-serine and L-leucine. A shift to 42 °C or to anaerobiosis makes the Irp mutant auxotrophic for L-serine. Three double mutants carrying Irp and another known mutation, acquire new auxotrophies: Irp relA, lacking the stringent response to amino acid limitation, requires leucine; Irp ssd with numerous metabolic perturbations and antibiotic resistances, requires serine and leucine; and Irp pnt, lacking pyridine nucleotide transhydrogenase, requires glutamate or aspartate (or the corresponding amides). The Irp mutant, although able to achieve balanced growth in some conditions, is clearly on the edge of a metabolic precipice, unable to tolerate many physiological and genetic perturbations which are inocuous to wild-type E. coli.
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GDP-mannose dehydrogenase is the key regulatory enzyme in alginate biosynthesis in Pseudomonas aeruginosa: evidence from metabolite studies
More LessThe Pseudomonas aeruginosa enzyme GDP-mannose dehydrogenase (GMD) is encoded by the algD gene, and previous genetic studies have indicated that it is a key regulatory and committal step in the biosynthesis of the polysaccharide alginate. In the present study the algD gene has been cloned into the broad-host-range expression vector pMMB66EH and GMD overexpressed in mucoid and genetically-related non-mucoid strains of P. aeruginosa. The metabolic approach of P. J. Tatnell, N. J. Russell & P. Gacesa (1993), J Gen Microbiol 139, 119-127, has been used to investigate the subsequent effect of GMD overexpression on the intracellular concentrations of the key metabolites GDP-mannose and GDP-mannuronate, which have been related to GMD activity and total alginate production. The overexpression of algD in mucoid and non-mucoid strains resulted in elevated GMD activities compared to wild-type strains; there was a concomitant reduction in GDP-mannose concentrations and greatly increased GDP-mannuronate concentrations. However, significantly, alginate biosynthesis was detected only in mucoid strains and GMD overexpression resulted in only a marginal increase in exopolysaccharide production. The GDP-mannuronate concentrations in mucoid strains which overexpressed GMD were always significantly greater than those of GDP-mannose, indicating that GMD was no longer the major kinetic control point in the biosynthesis of alginate by these genetically-manipulated strains. The small but significant increase in alginate production by such strains together with the increased GDP-mannuronate concentrations is interpreted as meaning that a later enzyme of the alginate pathway has become the major kinetic control point and now determines the extent of alginate production. This study has provided direct metabolic evidence that GMD is the key regulatory enzyme in alginate biosynthesis in P. aeruginosa.
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Unusual in vivo turnover of transfer RNA in Vibrio cholerae
More LessTwo lines of evidence suggest that, unlike in other organisms, the transfer RNAs of Vibrio cholerae undergo rapid turnover in vivo. Firstly, the tRNA content of V. cholerae cells treated with rifampicin (an inhibitor of initiation of RNA synthesis) decreased rapidly and continuously. Secondly, the newly synthesized tRNAs were rapidly degraded even under normal conditions of growth; the average half life of tRNA was 11·8 min. The degradation is mediated by an enzyme(s), present in V. cholerae cytoplasm, that apparently degrades tRNA completely. Rapid turnover is balanced by an enhanced rate of tRNA biogenesis, which was calculated to be 2·5 times higher than that in Escherichia coli.
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K+ transport in Vibrio alginolyticus: isolation of a mutant defective in an inducible K+ transport system
More LessWhen grown in a synthetic medium containing more than 3 mM K+, the marine bacterium Vibrio alginolyticus exhibited a K+ transport system with apparent K m and maximum velocity (V max) of 3·0 mM and 1·5 μmol min-1 (mg cell protein)-1, respectively. The growth rate of this organism in synthetic medium containing less than 0·2 mM K+ was dependent on K+ concentration and was half-saturated at about 50 μM K+. The cells grown at low concentrations of K+ induced another K+ transport system with K m and V max values of 0·3 mM and 0·6 μmol min-1 (mg cell protein)-1 respectively. The high-affinity system appeared when cells were grown at concentrations less than 2·0 mM K+ and was fully induced at 0·1 mM K+ and below. A mutant strain (FS181) unable to grow at 0·1 mM K+ was isolated and found to be defective in the inducible K+ transport system.
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- Plant-Microbe Interactions
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Germination triggers of Metarhizium anisopliae conidia are related to host species
More LessThe role of selectable strain variations in the development of pathogen strategies was examined using lines of Metarhizium anisopliae isolated from homopteran (isolate 549) or coleopteran (isolate 808) hosts. Conidia of strain 549 germinated in either alanine, glucose, cyclic AMP or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The non-metabolizable glucose analogues, 3-O-methylglucose and 6-deoxyglucose, did not allow germination by themselves but stimulated germination when added to IBMX. By contrast, 2-deoxyglucose (dGlc) blocked germination on glucose or IBMX and inhibited hyphal growth on other carbon sources including alanine and glycerol. Conidia of strain 808 germinated rapidly in alanine but responded slowly to glucose or IBMX in the medium and were resistant to the growth inhibitory effects of dGIc. Radioactive dGIc was taken up by conidia of strains 549 and 808 at similar rates and was recovered mainly as 2-deoxyglucose 6-phosphate. Competition experiments utilizing both strains demonstrated that glucose, dGIc and 3-O-methylglucose were transported by the same system. Fructose was much less able than glucose to inhibit uptake of dGIc indicating that fructose is taken up by a different transport system than that for glucose. It is unlikely, therefore, that the resistance of strain 808 to dGIc is explained by reduced sugar transport compared with strain 549 but that strains 549 and 808 differ in the regulation of carbon metabolism with some systems in strain 808 showing resistance to the catabolite-repressing effects of glucose. Apparently, catabolite repression is subdivided into different segments as glucose inhibited the derepression of a number of catabolite repressible enzymes in strain 808, including the pathogenicity determinant protease Pr1. The same effect was produced by dGIc but not by 3-O-methylglucose, indicating that the trigger for catabolite repression occurs at the level of transport-associated glucose phosphorylation. A comparative study of 26 isolates indicated that most lines from coleopteran hosts were dGIc resistant and germinated poorly on glucose. Conversely, isolates germinating well on glucose (mostly from hemipteran and lepidopteran hosts) were dGIc susceptible.
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- Systematics
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Lautropia mirabilis gen. nov., sp. nov., a Gram-negative motile coccus with unusual morphology isolated from the human mouth
An organism that seems to be identical to ⊘rskov's 'Sarcina mirabilis' [⊘rskov J. (1930) Acta Pathol Microbiol Scand Suppl III, 519-541] has been rediscovered in specimens from the upper respiratory tract of humans. Six strains were studied, and the results, which conformed to ⊘rskov's description of S. mirabilis, were as follows. Rough to smooth colonies grow on many plated media and show extremely polymorphic cell morphology with round cells wit diameters from 1 to > 10 μm. The smallest cells were often motile with circular movements. Strains were Gram-negative, facultatively anaerobic, oxidase and urease positive, and weakly catalase positive. Nitrate and nitrite were reduced and glucose, fructose, sucrose and mannitol were fermented. Polysaccharide was produced on sucrose agar. Electron microscopy showed coccoid cells with a bundle of three to nine flagella, a Gram-negative cell-wall morphology, and aggregates of irregular cells held together by a common surface layer. The mean mol% (G + C) of the organisms was 65·0. 16S-ribosomal RNA sequencing revealed that the organism belongs to the beta subgroup of Proteobacteria, separate from all other described genera, but most closely related to Burkholderia. The name Lautropia mirabilis is proposed for this organism.
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