- Volume 140, Issue 7, 1994
Volume 140, Issue 7, 1994
- Review Article
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- Environmental Microbiology
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Gene transfer in the aquatic environment: persistence and mobilization of the catabolic recombinant plasmid pD10 in the epilithon
More LessThis study investigated mobilization and persistence of the recombinant, catabolic plasmid pD10 in laboratory microcosms by natural mobilizing plasmids, recently isolated from epilithic bacteria by exogenous isolation in Pseudomonas putida, as opposed to their isolation in the strains in which they occur in situ. Initial experiments in simple, beaker microcosms were used to optimize conditions for selection of the donor, recipient and transconjugant populations. Studies in a recirculating stream microcosm showed that donor, P. putida KT2440(pD10, pQKH6), and recipient, P. putida UWC6, populations, although declining with time, were able to survive for three weeks and that colonization and survival was predominantly in the epilithon of the microcosm rather than in the liquid phase. Transconjugants UWC6(pD10, pQKH6) were also isolated from epilithon, showing that plasmid pD10 had been mobilized by plasmid pQKH6. The donor strain was able to survive at 100-fold greater numbers than the recipient, but transconjugants were not isolated after day 15. Separate inoculation of the donor and recipient strains into the microcosm showed that they were able to colonize other stones, where transconjugants then arose as a direct result of plasmid transfer within the epilithon. A catechol 2,3-dioxygenase gene, tdnC, was used to facilitate identification of donors and transconjugants. Preliminary laboratory matings showed that pD10 transferred to indigenous epilithic strains identified as Pseudomonas fluorescens, P. chlororaphis and P. aureofaciens. These results suggest that in the absence of selection, mobilization of introduced recombinant genes encoded by pD10 occurs at easily detectable frequencies, even in an oligotrophic environment.
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Carbon transformations by attached bacterial populations in granitic groundwater from deep crystalline bed-rock of the Stripa research mine
More LessThis paper presents and compares the assimilation rates of CO2 and lactate, and the lactate respiration rates, of attached bacterial populations growing in slowly flowing groundwater (1-3 mm s-1) from deep crystalline bed-rock of the Stripa research mine, Sweden. The bacteria studied grew in anoxic, high-pH (9-10) and low-redox artesian groundwater flowing up through tubing from two levels of a borehole designated V2, 812-820 m and 970-1240 m below ground. Bacteria were allowed to attach to and grow on sterile glass microscope slides in laminar-flow reactors connected to the flowing groundwater. Total numbers of bacteria were counted by acridine orange direct counts. The bacteria grew slowly, with doubling times of 34 d at 10 °C for the 812-820 m population, 23 d for the 970-1240 m population at 10 °C and 16 d for this population at 20 °C. Numbers of attached bacteria reached between 106 and 107 bacteria cm-2. The populations at the two levels of the borehole were different in physiology as well as in phylogeny and reflected the heterogeneity between the sampling levels. The earlier proposed presence of sulphate-reducing bacteria could not be confirmed. This is discussed in relation to results from 16S rRNA gene sequencing studies. The CO2 assimilation rates (as mol CO2 cm-2 h-1, using liquid scintillation techniques) increased with depth and temperature. The quotients calculated for inorganic/organic carbon utilization were between 0·07 and 0·25, indicating that autotrophy could not support the levels of growth observed and that heterotrophy was the dominant carbon transformation process for growth of the studied populations. The Stripa bacteria could further be seen not only to assimilate but also to catabolize lactate and release CO2, which added to the indications of a heterotrophic dominance in the Stripa environment.
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Characterization of attached bacterial populations in deep granitic groundwater from the Stripa research mine by 16S rRNA gene sequencing and scanning electron microscopy
More LessThis paper presents the molecular characterization of attached bacterial populations growing in slowly flowing artesian groundwater from deep crystalline bed-rock of the Stripa mine, south central Sweden. Bacteria grew on glass slides in laminar flow reactors connected to the anoxic groundwater flowing up through tubing from two levels of a borehole, 812-820 m and 970-1240 m. The glass slides were collected, the bacterial DNA was extracted and the 16S rRNA genes were amplified by PCR using primers matching universally conserved positions 519-536 and 1392-1405. The resulting PCR fragments were subsequently cloned and sequenced. The sequences were compared with each other and with 16S rRNA gene sequences in the EMBL database. Three major groups of bacteria were found. Signature bases placed the clones in the appropriate systematic groups. All belonged to the proteobacterial groups beta and gamma. One group was found only at the 812-820 m level, where it constituted 63% of the sequenced clones, whereas the second group existed almost exclusively at the 970-1240 m level, where it constituted 83% of the sequenced clones. The third group was equally distributed between the levels. A few other bacteria were also found. None of the 16S rRNA genes from the dominant bacteria showed more than 88% similarity to any of the others, and none of them resembled anything in the database by more than 96%. Temperature did not seem to have any effect on species composition at the deeper level. SEM images showed rods appearing in microcolonies. The conditions at the levels differ in pH, temperature, redox and flow rate, and in content of sulphate, iron and sulphide. The presence of one dominant species in the laminar-flow reactors at each level indicates that the environments might have offered restrictive physical or physiological conditions difficult to adapt to.
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- Genetics And Molecular Biology
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An increase in switching frequency correlates with an increase in recombination of the ribosomal chromosomes of Candida albicans strain 3153A
H. Ramsey, B. Morrow and D. R. SollWhen cells of Candida albicans strain 3153A are treated with a low dose of ultraviolet irradiation, they switch at high frequency (10-2) between a number of switch phenotypes discriminated by colony morphology. Clones from switching lineages exhibit continuous reorganization of their ribosomal chromosomes (R chromosomes), while clones from lineages maintaining the original smooth phenotype exhibit no reorganization. R chromosome reorganization results in decreases as well as increases in size of the chromosomes, but changes are not reciprocal between R chromosome homologues.
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A Candida albicans cyclic nucleotide phosphodiesterase: cloning and expression in Saccharomyces cerevisiae and biochemical characterization of the recombinant enzyme
More LessWe have cloned a Candida albicans gene, which encodes a cyclic nucleotide phosphodiesterase (PDEase), by complementation in a Saccharomyces cerevisiae PDEase-deficient mutant. The deduced amino acid sequence is similar to that of the low-affinity PDEase of S. cerevisiae (PDE1) and the cyclic nucleotide PDEase (PD) of Dictyostelium discoideum. Biochemical analysis of recombinant protein produced in S. cerevisiae indicated that the enzyme behaves as a PDE1 homologue: it hydrolyses both cAMP (K m = 0·49 mM) and cGMP (K m = 0·25 mM), does not require divalent cations for maximal activity and is only moderately inhibited by millimolar concentrations of standard PDEase inhibitors. Based on these data, we designate the C. albicans we have cloned, PDE1. Low-stringency genomic Southern blots showed cross-hybridization between C. albicans PDE1 and DNA from Candida stellatoidea, but not with DNA from S. cerevisiae or several closely related Candida species.
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Cloning and sequencing of the genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and the implications for the domain structure of the enzyme
More LessThe genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated pntAA, pntAB and pntB, whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from Escherichia coli (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from R. rubrum, equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the α polypeptide of E. coli transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the α polypeptide of the E. coli transhydrogenase. PntB corresponds to the E. coli β polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the E. coli and bovine mitochondrial enzymes. The two tryptic fragments from the native R. rubrum soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.
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Characterization of the chromosomes of Bacillus subtilis merodiploid strains by quantitative DNA-DNA hybridization
More LessThe position of junctions and the extent of the duplicated chromosomal regions in Bacillus subtilis merodiploid strains were studied by quantitative DNA-DNA hybridization. We describe a method which allows (i) the identification of genes present in two copies per chromosome and (ii) the measurement of the amount of additional DNA in chromosomes with relatively large duplicated regions (about 10% or more). Analysis of previously described B. subtilis merodiploid strains GSY1127, GSY1800 and GSY1835 revealed that the duplicated segments represent 29 ± 2%, 7 ± 2% and 13 ± 2% of the chromosome, respectively. Small discrepancies between these and previous genetic linkage data are discussed. Support for a role of prophage SPβ in the formation of merodiploid GSY1835 is provided. In conclusion, the described method confirmed the genetic maps of the merodiploids previously obtained by transduction and transformation crosses and showed that a duplication of a segment is not accompanied by large deletions of other chromosomal regions, providing direct evidence that a cell can accommodate genomes of substantially increased size.
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A rapid and simple method for Bacillus subtilis transformation on solid media
More LessCells of Bacillus subtilis strains 168 and W23 deprived of an amino acid or a base on a given solid medium were found to develop competence. We describe a rapid and simple method of genetic transformation of this organism consisting in spreading a sample containing 1 μg DNA and 107 exponentially growing cells of an auxotrophic mutant onto plates devoid of the required amino acid or base. After overnight incubation, about 100-200 prototrophic transformants per plate were obtained, i.e. a frequency of about 10-5, as compared to 10-4 routinely obtained by the method of transformation in liquid medium with frozen competent cells. Plasmids and other chromosomal or plasmid-borne markers, which cannot be directly selected for, were transferred by congression. The dependence of the transformation efficiency on cell density, medium richness, incubation time and the nature of transforming DNA was investigated. We conclude that the development of competence accompanies amino acid or base starvation of cells under appropriate physiological conditions.
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- Pathogenicity And Medical Microbiology
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A new extracellular protein of Pseudomonas aeruginosa PA103 regulated by regA
More LessThe expression of exotoxin A (ExoA) from Pseudomonas aeruginosa is influenced by iron and is under the control of the regulatory gene regA. To test whether regA plays a role in the expression of other iron-regulated proteins a RegA- mutant was constructed by insertional mutagenesis. The polypeptide pattern of this mutant (PA103R) was compared with the parental strain (PA103) and a trans-complemented strain PA103R(pREX18) after growth of the strains in conditions containing low or high concentrations of iron. An iron-regulated 42 kDa protein (RRP) was identified and purified from the culture supernatant of PA103 and PA103R(pREX18) which was missing in PA103R. Database analysis of the N-terminal sequence of this regA-regulated protein (RRP) revealed no similarity to other proteins. Preliminary investigations into the function of RRP revealed that it has no proteolytic or cytotoxic activity. Using two-dimensional electrophoretic analysis of whole cells, a technique which allowed separation of over 600 polypeptides, we were unable to identify any other iron-regulated protein whose expression was regulated by regA.
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Nucleotide sequences of the spacer-1, spacer-2 and trailer regions of the rrn operons and secondary structures of precursor 23S rRNAs and precursor 5S rRNAs of slow-growing mycobacteria
More LessThe single ribosomal RNA (rrn) operons of slow-growing mycobacteria comprise the genes for 16S, 23S and 5S rRNA, in that order. PCR methodology was used to amplify parts of the rrn operons, namely the spacer-1 region separating the 16S rRNA and 23S rRNA genes and the spacer-2 region separating the 23S rRNA and 5S rRNA genes of Mycobacterium avium, Mycobacterium intracellulare, ‘Mycobacterium lufu’ and Mycobacterium simiae. The amplified DNA was sequenced. The spacer-2 region, the 5S rRNA gene, the trailer region and the downstream region of the rrn operon of Mycobacterium tuberculosis were cloned and sequenced. These data, together with those obtained previously for Mycobacterium leprae, were used to identify putative antitermination signals and RNase III processing sites within the spacer-1 region. Notable features include two adjacent potential Box B elements and a Box A element. The latter is located within a sequence of 46 nucleotides which is very highly conserved among the slow-growers which were examined. The conserved sequence has the capacity to interact through base-pairing with part of the spacer-2 region. Secondary structures for mycobacterial precursor 23S rRNA and for precursor 5S rRNA were devised, based on sequence homologies and homologous nucleotide substitutions. All the slow-growers, including M. leprae, conform to the same scheme of secondary structure. A putative motif for the intrinsic termination of transcription was identified approximately 33 bp downstream from the 3'-end of the 5S rRNA gene. The spacer-1 and spacer-2 sequences may prove a useful supplement to 16S rRNA sequences in establishing phylogenetic relationships between very closely related species.
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- Physiology And Growth
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Structural organization of the components of the cell wall from Candida albicans
The organization of the components of the cell wall from Candida albicans was studied by means of sequential treatment with hot SDS, anhydrous ethylenediamine (EDA) and lytic enzymes, followed by chemical and microscopic analyses of the different separated fractions. The EDA-insoluble fraction retained the original morphology of the wall, which was destroyed by β-glucanase, but not by chitinase treatments. Staining with fluorescent lectins revealed distinct distributions of mannoproteins, glucans and chitin in the wall. Amino acid analysis of SDS-extracted walls, and the EDA-soluble and -resistant fractions gave similar results, with seven amino acids making up about 70% of the total protein weight. Treatment of the EDA-insoluble fraction with Zymolyase or chitinase released fragments of variable size whose susceptibility to these and other hydrolases suggests that they are made of glucan, chitin and mannan oligomers associated with proteins. Treatment of the Zymolyase-insoluble residue with chitinase released a series of low-molecular-mass oligomers made of neutral sugars, GIcNAc and amino acids, mainly lysine. It is suggested that they represent fragments of the core making up the scaffold of the cell wall of the fungus.
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Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation
More LessTwo forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 °C in medium containing N-acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. N-Acetylglucosaminidase A (32% total carbohydrate, M r 85000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, M r 132000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form, 350000; B form, 600000; A and B forms after endoglycosidase H (endo H) treatment, 180 000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of M r centred at ~ 100 000 (A) and ~ 150 000 (B) but were reduced to a single 58 000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of M r ~ 3000 that were endo H resistant. Therefore the difference in the size of N-acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. N-Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both p-nitrophenylGIcNAc (pNPGIcNAc) and pNPGalNAc at pH 4·0. The kinetic parameters k cat (s-1) and K m (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGIcNAc, 740, 0·77; pNPGalNAc, 910, 1·26; N,N'-diacetylchitobiose 620, 0·20; and N,N',N-triacetylchitotriose, 170, 0·044. The enzyme showed substrate inhibition with all substrates above 0·5 mM except for pNPGalNAc.
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Effect on lipopolysaccharide structure of aeration during growth of a plum isolate of Pseudomonas syringae pv. morsprunorum
More LessThe composition of lipopolysaccharide (LPS) extracted with aqueous phenol from a virulent English plum isolate of Pseudomonas syringae pv. morsprunorum varied according to the partial pressure of oxygen (pO2) in the culture medium at the time of harvest. When pO2 was low, the organism grew slowly and produced smooth LPS bearing rhamnan sidechains. As pO2 was raised, the rate of growth increased and smooth LPS was replaced by a rough species deficient in rhamnose, which co-extracted with a d-glucan. Organization of rhamnose and glucose into separate polymers was shown by the selective susceptibility of the rhamnose-containing polymer to hydrolysis by rhamnanase of the phage A7. By methylation analysis, GC-MS, and 1H- and 13C-NMR spectroscopy, the glucan was shown to consist of α(1 → 4)-linked residues with α(1 → 4,6)-branch points and non-reducing terminal residues in the approximate ratio 4:1:1, resembling glycogen in composition. A glucan which co-extracted with LPS using phenol/water from an avirulent plum isolate that was resistant to lysis by phages A1 and A7 was shown by methylation analysis to have a similar structure. Whether the effect on LPS composition was due directly to pO2, or was dependent on the rate of growth, has not been established. It is suggested that, because epiphytic growth would entail exposure to high pO2, English plum isolates growing on the surfaces of host plants might be unable to produce smooth LPS. Since cell surface composition affects virulence in plant-pathogenic pseudomonads, this effect could account for the observed failure of the English plum isolates to enter the host via leaf scars.
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The role of cGMP in photosensory and thermosensory transduction in Dictyostelium discoideum
More LessWeak and strong light/heat stimuli induced changes in cyclic guanosine monophosphate (cGMP) levels in vegetative and aggregation competent amoebae and in slug cells of the Dictyostelium discoideum strain X22. Mutant strains derived from X22 with mutations in the phototaxis loci phoA-phoK fell into four phenotypic classes with respect to cGMP responses to weak and strong light/heat stimuli. These results suggest an intermediary role for cGMP in photosensory and thermosensory processing in slugs and amoebae. The streamer F mutant NP368 which has previously been shown to exhibit a prolonged cGMP response to cAMP, showed a wild-type cGMP response to light and heat. All phototaxis mutant strains with altered cGMP responses to light and heat were unaltered in their cGMP response to cAMP. These results suggest that cAMP and light/heat regulate cGMP via independent pathways. The thermotaxis mutant HPF228 showed altered cGMP responses to heat but not to light stimuli. This suggests that the mutation in HPF228 affects thermosensory transduction before convergence with the phototaxis pathway and the subsequent cGMP response.
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Is mRNA sequestration involved in the regulation of progesterone 14α-hydroxylase cytochrome P-450 expression in Mucor hiemalis?
More LessDuring a 6 h incubation at 26 °C, Mucor hiemalis hydroxylated progesterone predominantly at the 14α site. Further incubation resulted in the production of several 14α-dihydroxyprogesterones and a novel microbial transformation product, 8(9), 14(15)-didehydroprogesterone (pregna-4,8[9], 14[15]-triene-3,20-dione). Azole inhibition indicates that the hydroxylases are cytochromes P-450. Mycelia transformed in the presence of the translation inhibitor cycloheximide failed to hydroxylate progesterone, whereas identical treatment with the transcription inhibitor actinomycin D, or heat-shock for 1 h at 37 °C prior to progesterone transformation, stimulated hydroxylation. 14α-Hydroxylation was stimulated if mycelia were pre-incubated with progesterone, transcription or translation inhibitors. These data are consistent with a model in which cytochrome P-450 steroid hydroxylase mRNA is stored in cells in a dormant form by a labile sequestering protein.
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Metabolism of [14C]glutamate and [14C]glutamine by the ectomycorrhizal fungus Paxillus involutus
More LessTo examine pathways of glutamate and glutamine metabolism in the ectomycorrhizal fungus Paxillus involutus, tracer kinetic experiments were performed using l-[U-14C]glutamate and L-[U-14C]glutamine and the enzyme inhibitors methionine sulfoximine (MSX), azaserine (AZA) and aminooxyacetate (AOA). When [14C]glutamate was supplied to fungal cultures, 25% of the radioactivity of the amino acid fraction was incorporated into glutamine after 5 min feeding, but MSX inhibited incorporation of 14C into glutamine by 85%, suggesting the rapid operation of glutamine synthetase. Conversely, when P. involutus was fed with [14C]glutamine, 46% of the label was found in glutamate within 30 min of feeding and AZA inhibited glutamate formation by 90%. Taken together, these data indicate that glutamate synthase (GOGAT) is the major enzyme of glutamine degradation. In addition, the strong inhibition of glutamine utilization by AOA indicates that glutamine catabolism in P. involutus might involve a transamination process as an alternative pathway to GOGAT for glutamine degradation. The high 14CO2 evolution shows that glutamate and glutamine are further actively consumed as respiratory substrates, being channelled through the tricarboxylic acid (TCA) cycle and oxidized as CO2. It appears that synthesis of amino acid precursors during TCA cycle operation is an essential step for aspartate and alanine synthesis through aminotransferase activities in P. involutus.
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The effect of phosphonate on the acid-soluble phosphorus components in the genus Phytophthora
More LessThe high concentrations of short-chain soluble polyphosphates, previously reported in Phytophthora palmivora, have been found in six other species of Phytophthora, and appear to be characteristic of this genus. The distribution of phosphorus among the various acid-soluble pools was similar in all species except P. infestans, which accumulated less polyphosphate than other species when grown in high phosphate medium, and contained several unique and as yet unidentified organic phosphorus compounds. When grown in the presence of potassium phosphonate sufficient to reduce growth by 50%, all species showed an increased accumulation of phosphorus in both pyrophosphate and polyphosphate, without parallel increases in sugar phosphate or nucleotide phosphorus pools. The internal concentration of phosphonate required to produce 50% inhibition varied with species, ranging from 4·6 to 52 μmol (g dry wt)-1. Assimilation of orthophosphate was only slightly reduced in the presence of phosphonate, resulting in an increased concentration of phosphorus per unit mass. Metalaxyl, which also inhibits growth of Phytophthora spp. did not cause accumulation of either pyro- or polyphosphate. Phosphonate treatment also resulted in the formation of a compound identified as isohypophosphate, a metabolite of phosphonate not previously reported in Phytophthora. Taken together, these observations suggest that the primary site of phosphonate inhibition in Phytophthora spp. lies in the metabolism of pyrophosphate.
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A cytosolic trehalase from the thermophilic fungus Humicola grisea var. thermoidea
More LessA cytosolic trehalase was purified from the thermophilic fungus Humicola grisea var. thermoidea. The apparent molecular mass of the purified native enzyme was estimated to be 360 kDa by gel filtration chromatography. A single polypeptide band of approximate molecular mass 120 kDa was detected by SDS-PAGE, suggesting that the native enzyme was composed of three identical polypeptides. The carbohydrate content of the enzyme was estimated to be 12%. The pl of the enzyme, estimated by electrofocusing, was about 4·0. The purified trehalase was specific for trehalose and its activity was stimulated by manganese, calcium and cobalt and inhibited by aluminium chloride, copper sulfate, ADP and ATP. Sugars such as cellobiose, lactose, sucrose, fructose and maltose also showed some inhibitory effect which was abolished in the presence of calcium. The purified cytosolic trehalase exhibited an apparent K m of 0·86 mM, a pH optimum of 5·5 and an optimum temperature of 60 °C. Treatment with calcium induced disaggregation of the enzyme into three components. Apparently, calcium-induced disaggregation was not a prerequisite for calcium-mediated activation of the enzyme. The disaggregated trehalase was more labile to temperature than the native enzyme. The properties of the two H. grisea trehalases: the cytosolic enzyme and the conidial one, were compared.
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Isolation and characterization of two xylitol dehydrogenases from Aspergillus niger
More LessAn NADPH-dependent L-xylulose reductase [xylitol:NADP 4-oxidoreductase; EC 1·1.1·10 (LXDH)] from Aspergillus niger was purified and characterized. It is an octamer with a monomeric molecular mass of 32 kDa, showing high specificity for L-xylulose, xylitol and NADP(H). The K m values for L-xylulose and xylitol are relatively high (16·5 and 925 mM, respectively). An NAD+-dependent xylitol dehydrogenase [xylitol:NAD+ 2-oxidoreductase; EC 1·1.1·9 (DXDH)] was partially purified from the same fungus. It has a monomeric molecular mass of 40 kDa and shows a high specificity for D-xylulose, xylitol and NAD(H). The K m values for D-xylulose and xylitol are relatively low (approximately 4 and 50 mM, respectively). The reactivity towards xylitol, the product or substrate these enzymes have in common, confirms their role in the L-arabinose catabolic pathway.
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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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