- Volume 141, Issue 9, 1995
Volume 141, Issue 9, 1995
- Sgm Special Lecture
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- Microbiology Comment
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- Biochemistry
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a-D-Glucuronidases from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum
More Lessa-D-Glucuronidases were purified from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum. This enzyme activity was found to be intracellular in each organism, with T. saccharolyticum producing much greater total activity. The specific activities of the purified enzymes (10 U mg-1 T. saccharolyticum; 1.7 U mg-1 C. stercorarium) differed by a factor of approximately 5. For the determination of enzyme activities, 4-O-methyl-a-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by a-D-glucuronidase action was quantified by a colorimetric procedure. 4-O-Methyl-a-D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of 4-O-methyl-a-D-glucuronoxylan with xylanases of C. stercorarium. Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected. Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-nitrophenyl-a-D-glucuronoside. Both a-D-glucuronidases have a dimeric structure, with monomeric molecular masses of 72 and 76 kDa for C. stercorarium and of 71 kDa for T. saccharolyticum. The pl was estimated to be 4.3 for each enzyme. While both enzymes exhibited a similar pH optimum (pH 5.5-6.5) they differed in their thermostabilities. At 60 C, half-lives of 14 and 2.5 h, respectively, were determined for the a-D-glucuronidases of C. stercorarium and T. saccharolyticum. This description of a-D-glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi.
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L-Arginine transport and metabolism in Giardia intestinalis support its position as a transition between the prokaryotic and eukaryotic kingdoms
More LessArginine is metabolized by the arginine dihydrolase pathway in Giardia intestinalis trophozoites and is an important metabolic fuel for this parasite. Radiolabelled arginine was used to characterize the transport of arginine into Giardia intestinalis trophozoites. The transporter had a high affinity for arginine (K m 15 üM) and a high activity [V max 76 nmol min-1 (mg protein)-1 at 25 °C]. Substrate specificity studies indicated an absolute requirement for the α-amino and carboxyl groups, but a tolerance for some substitutions in the guanidino group. The use of non-metabolized arginine analogues in combination with HPLC amino acid analysis of intra- and extracellular pools demonstrated that the arginine transporter is an arginine-ornithine antiport. Investigations of the first step of arginine metabolism, involving arginine deiminase, revealed a relatively high affinity for arginine (K m 0.16 mM) and a large maximal velocity [V max 550 nmol min-1 (mg protein)-1 at 37 °C]. Substrate specificity studies showed that the arginine deiminase had a characteristically different substrate recognition profile to that of the arginine transporter. Overall, the combination of the transporter and the deiminase result in very low intracellular arginine concentrations and their properties are consistent with the rapid transport of arginine for metabolism via the arginine dihydrolase pathway.
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Bacterial [Cu,Zn]-superoxide dismutase: phylogenetically distinct from the eukaryotic enzyme, and not so rare after all!
More LessCopper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens - members of the Haemophilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.
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St-PepA, a Streptococcus thermophilus aminopeptidase with high specificity for acidic residues
More LessThe proteolytic system of lactic acid bacteria has been extensively studied over the past 10 years and peptidases from lactococci are now well known. The situation is, however, different for Streptococcus thermophilus from which only a few peptidases have been purified and characterized. The present work was conducted to characterize an aminopeptidase of S. thermophilus CNRZ 302, called St-PepA, with high specificity for acidic amino acids. St-PepA was purified by a three-step procedure from a spheroblast extract of S. thermophilus CNRZ 302. Its molecular mass was estimated to be 360 kDa by gel filtration and 45 kDa by SDS-PAGE, indicating that it had an octameric structure. Its activity against aspartate-p-nitroanilide was maximal at pH 8.5 and 62 C and highly enhanced by Zn2+, Co2+ and Mg2+. St-PepA was inhibited by metal-chelating reagents and, to a lesser extent, by agents modifying sulfhydryl groups. It showed an activity towards p-nitroanilide derivatives, di-and tripeptides, and larger peptides such as fragment 43-58 of as1-casein. It had a high substrate specificity towards N-terminal acidic amino acid residues but it could also release serine and malic acid, the a-hydroxy acid homologue of aspartic acid. Kinetic studies revealed that the affinity of St-PepA was more than 18-fold higher for aspartic acid-p-nitroanilide (K m = 0.42 mM) than for glutamic acid-p-nitroanilide (K m = 7.65 mM) with a similar V max for both substrates [about 40 mol min-1 (mg enzyme)-1]. St-PepA is heat stable, with a maximal loss of activity of 15% after incubation for 120 min at 50 C and 60C. It is likely to be involved in the nitrogen metabolism of S. thermophilus and in the development of the organoleptic characteristics of cheese and yoghurt.
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Biosynthesis of glycoproteins in Candida albicans: activity of dolichol phosphate mannose synthase and protein mannosylation in a mixed membrane fraction
More LessA mixed membrane fraction (MMF) was isolated from yeast cells of Candida albicans with the ability to synthesize dolichol phosphate mannose (Dol-P-Man) from GDP-Man and dolichol phosphate (Dol-P) and transfer the sugar to proteins. Temperature of incubation (20-37 °C) did not affect the synthesis of Dol-P-Man but protein mannosylation occurred better at physiological temperatures (28 °C and 37 °C). Most of the sugar (87-93 %) in the mannoproteins was O-linked as judged by its release by β-elimination. Mannose was identified as the sole product after this treatment. Following incubation of MMF with the sugar donor, parallel levels of Dol-P-Man and mannosylated proteins were detected up to 30 min. Thereafter, Dol-P-Man levels reached a steady value whereas mannoproteins rapidly accumulated. Lipid-linked oligosaccharides were also detected in incubation mixtures, though in much lower amounts than those of Dol-P-Man or mannoproteins. Dol-P-Man synthase activity increased proportionally in response to increasing concentrations of either of the two enzyme substrates. A K m value of 0·36 μM for GDP-Man was calculated. MMF failed to use exogenous Dol-P-Man for protein glycosylation. Specific inhibition of Dol-P-Man synthesis with amphomycin was concomitant with a parallel decrease in protein mannosylation, indicating that most of the sugar is transferred to protein via the carrier lipid. Results are discussed in terms of the role of Dol-P-Man in protein glycosylation in C. albicans.
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Substrate specificity of nine NAD+-dependent alcohol dehydrogenases in Aspergillus nidulans
More LessIn Aspergillus nidulans three alcohol dehydrogenases (ADHs) have been described. ADHI is induced by ethanol and is the physiological enzyme of ethanol utilization, ADHII has not been attributed a function but is repressed by ethanol. The ALCR regulatory protein acts positively to induce ADHI, and negatively in its control of ADHII. ADHIII is specifically induced by anaerobic stress. We have characterized the substrate specificity of these three enzymes by looking at their staining profile on polyacrylamide gels with a range of alcohols. In addition to these enzymes we have observed six other NAD+-dependent ADHs, two of which, propan-2-ol dehydrogenase and pentan-2-ol dehydrogenase, share similar control with ADHII. The inducibility of these enzymes with some alcohols has also been investigated. The profile of ADHs with NADP+ as an electron acceptor is also reported.
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- Environmental Microbiology
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Selective recovery of Streptosporangium fragile from soil by indirect immunomagnetic capture
More LessA polyclonal antibody raised to Streptosporangium fragile spores reacted strongly and specifically with the immunizing strain and to a number of related species of Streptosporangium, as determined by dot immunoblotting. An indirect immunomagnetic capture method was developed for the recovery of the target organism from sterile and non-sterile soil, using sheep anti-rabbit M-280 Dynabeads. The effects of different soil blocking agents, antibody labelling concentrations and spore/Dynabead capture times on the recovery of S. fragile spores were investigated. Pre-blocking of antibody binding sites within the soil, with either 2% partially hydrolysed gelatin or 10% skimmed milk, was essential prior to immunomagnetic capture. Increasing the capture time from 15 to 60 min did not affect spore recovery; however, a 10-fold decline in the magnetic bead concentration did result in a significantly lower recovery of spores from soil. S. fragile was selectively enriched (1:190-fold) when present as a mixed population with Arthrobacter oxydans in sterile soil. The indirect immunomagnetic capture method was used to selectively recover S. fragile spores seeded into non-sterile soil, although some background binding of non-target bacteria was noted. The target was successfully recovered from a sterile soil microcosm after 14 d incubation and the capture rate was increased by the inclusion of an initial soil dispersion and biomass concentration procedure, using the ion-exchange resin Chelex 100.
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- Genetics And Molecular Biology
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Distribution of the smpA gene from Serpulina hyodysenteriae among intestinal spirochaetes
More LessForty intestinal spirochaete strains were investigated for nucleotide sequences related to the smpA locus from Serpulina hyodysenteriae by Southern hybridization of chromosomal DNA using the smpA locus from S. hyodysenteriae strain P18A as a probe and by PCR using primers internal to the smpA gene. The intensity of the hybridization signal at high stringency and positive PCR results suggested that 12 S. hyodysenteriae strains possessed a similar nucleotide sequence. PCR was negative for another 12 S. hyodysenteriae strains and the hybridization signal obtained from 11 of these was weak and one was negative. All S. hyodysenteriae strains hybridized under low stringency conditions. These results indicated that there is variation among the smpA loci of S. hyodysenteriae strains. Among seven strains of S. innocens, and the proposed species ‘S. intermedius’ and ‘S. murdochil’, hybridization was weak and no PCR products were obtained, suggesting that these species have sequences related to, but divergent from, the smpA sequences of strains of S. hyodysenteriae. Both gene probe hybridization and PCR analysis of nine strains of the proposed new genus ‘Anguillina’, including isolates from pigs and humans, gave negative results.
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A murC gene in Porphyromonas gingivalis 381
The gene encoding a 51 kDa polypeptide of Porphyromonas gingivalis 381 was isolated by immunoblotting using an antiserum raised against P. gingivalis alkaline phosphatase. DNA sequence analysis of a 2.5 kb DNA fragment containing a gene encoding the 51 kDa protein revealed one complete and two incomplete ORFs. Database searches using the FASTA program revealed significant homology between the P. gingivalis 51 kDa protein and the MurC protein of Escherichia coli, which functions in peptidoglycan synthesis. The cloned 51 kDa protein encoded a functional product that complemented an E. coli murC mutant. Moreover, the ORF just upstream of murC coded for a protein that was 31% homologous with the E. coli MurG protein. The ORF just downstream of murC coded for a protein that was 17% homologous with the Streptococcus pneumoniae penicillin-binding protein 2B (PBP2B), which functions in peptidoglycan synthesis and is responsible for antibiotic resistance. These results suggest that P. gingivalis contains a homologue of the E. coli peptidoglycan synthesis gene murC and indicate the possibility of a cluster of genes responsible for cell division and cell growth, as in the E. coli mra region.
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Listeria monocytogenes exists in at least three evolutionary lines: evidence from flagellin, invasive associated protein and listeriolysin O genes
More LessRegions of the genes encoding flagellin (flaA), the invasive associated protein (iap), listeriolysin O (hly) and 23S rRNA were sequenced for a range of Listeria monocytogenes isolates of different origin and serotypes. Several nucleotide sequence variations were found in the flaA, iap and hly genes. No differences were found for the rRNA genes, but our approach does not exclude the existence of differences between single copies of these genes. Based on the sequence differences, the L. monocytogenes strains can be divided into three distinct sequence types. Further, the presence of only a small number of sequence differences within each group indicates a strong degree of conservation within the groups. There was a complete correspondence among the groups of strains formed according to the analysis of the flaA, iap and hly genes, and the grouping correlates with serotype, pulsed field gel electrophoretic and multilocus enzyme electrophoretic data. Analysis of the region encoding the threonine-asparagine repeat units in the iap gene revealed some striking features. Sequence type 1 strains were found to have 16-17 repeats, sequence type 2 strains had 16-20 repeats whereas the two sequence type 3 strains analysed had only 11 repeats. Furthermore, within a 19 bp segment there was a 37% difference between the sequences of type 1 and 2 strains and that segment was absent in type 3 strains. Within the threonine-asparagine repeat region the nucleotide differences gave rise to four amino acid changes; however, all were changes among the three amino acids present in the repeat structure indicating a strong selective pressure on the composition of this region.
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Expression of foreign genes and selection of promoter sequences in Acholeplasma laidlawii
More LessThe stable maintenance and expression of foreign genes in mollicutes (mycoplasmas) have been difficult to achieve due to the lack of suitable vectors. In this paper we show for the first time that a replicating vector can been used to express foreign genes other than antibiotic resistance genes in Acholeplasma laidlawii. Plasmids derived from the lactococcal vector pNZ18 could introduce and maintain four different genes for many generations in A. laidlawii. One of these, encoding the dominant membrane lipoprotein spiralin from the mollicute Spiroplasma citri, was expressed; however, expression was weak, the signal peptide of spiralin was not cleaved and the protein was not covalently modified by fatty acids. This resulted in a hydrophilic character of spiralin and its cytoplasmic localization in A. laidlawii. To increase the expression of foreign genes, random A. laidlawii DNA fragments were cloned into a pNZ18-related plasmid and expression signals were selected using the Bacillus licheniformis a-amylase gene as a probe. Selection was done in Escherichia coli as well as directly in A. laidlawii. Active recombinants from E. coli were also able to express a-amylase activities and an enzyme of native size in A. laidlawii. The highest activity was obtained from a recombinant selected directly in A. laidlawii. This is the first example of a promoter sequence selected in a mollicute. Analysis of the putative promoters in seven clones revealed similar -10 and -35 regions, and similar spacer distances in A. laidlawii, Acholeplasma oculi, Lactococcus and E. coli. Vectors related to pNZ18 should be useful for the genetic analysis of specific A. laidlawii proteins and functions.
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Delineation of the virulence-related locus (vrl) of Dichelobacter nodosus
Dichelobacter nodosus is the primary pathogen implicated in ovine footrot. In this paper we have delineated a 27 kb locus, termed the virulence-related locus (vrl), that was essentially specific for virulent D. nodosus isolates. The precise ends of this locus were mapped and the sequences of the junction regions from the virulent strain A198 were compared to corresponding sequences from the benign isolate C305. The left end of the vrl locus was located in a sequence similar to that of the small stable 10Sa RNA molecule of Escherichia coli, next to a phage-attachment-site-like sequence, which indicated that the vrl locus might have arisen by the integration of a phage. However, no attachment-like sequence could be found at the right end of the vrl locus. In the chromosome of the benign strain the sequences bordering vrl were not contiguous but were separated by about 3 kb. It was concluded that the divergence of the benign and virulent strains at this locus was a multi-step process. Several potential ORFs were identified at the junction regions but only one ORF, encoding a 126 kDa protein, was expressed in a T7 expression system in E. coli.
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The Cowdria ruminantium groE operon
More LessA Cowdria ruminantium genomic DNA library was constructed in the expression vector γZAPII, and an immunoreactive clone, designated γCr9.4, was isolated by screening with serum from a C. ruminantium -infected goat. Sequencing of the insert from this clone revealed two open reading frames, encoding peptides of 10462 and 58697 kDa respectively. Database searching indicated that the two genes were homologues of groES and groEL, genes encoding a group of heat shock proteins involved in protein processing, export and assembly. Western blotting experiments showed that the recombinant GroEL protein was recognized by sera raised against four isolates of C. ruminantium which originate from South Africa, West Africa and the Caribbean, but not by antisera to the closely related Ehrlichia species (E. ovina, E. [Cytoecetes] ondiri, E. bovis, E. phagocytophila) of African and European ruminants which can be expected to occur in similar geographical areas to C. ruminantium. This suggests that this protein may be useful in development of serodiagnostic tests for C. ruminantium infection which are not subject to cross-reactions with antibodies to Ehrlichia species. The cloning and expression of the GroE operon will also facilitate further study of the roles of the GroE proteins in the immune response to C. ruminantium.
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Characterization of the Mycobacterium tuberculosis erp gene encoding a potential cell surface protein with repetitive structures
Using the phoA gene fusion methodology adapted to mycobacteria, several Mycobacterium tuberculosis DNA fragments encoding exported proteins were recently identified. In this paper, the molecular cloning, genomic positioning, nucleotide sequence determination and transcriptional start site mapping of a new M. tuberculosis gene, identified by this methodology, are reported. This gene was called erp (for exported repetitive protein) and has a sequence similar to that of the Mycobacterium leprae 28 kDa antigen irg gene M. tuberculosis erp gene contains a putative iron box close to the mapped transcriptional start site. The predicted Erp protein displays a typical N-terminal signal sequence, a hydrophobic domain at the C-terminus and harbours repeated amino acid motifs. These structural features are reminiscent of cell-wall-associated surface proteins from Gram-positive bacteria. We found that these repeats are conserved among M. tuberculosis isolates, and are absent from the published M. leprae irg gene sequence. In addition to being present in M. leprae, erp sequences were found in other members of the M. tuberculosis complex, but not in other mycobacteria tested. These results suggest that erp might encode a cell surface component shared by major pathogenic mycobacteria.
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Species-specific identification of Mycobacterium bovis by PCR
More LessThe Random Amplified Polymorphic DNA (RAPD) technique was used in the identification of a species-specific fragment of Mycobacterium bovis. A fragment of approximately 500 bp was amplified from the genome of 15 different M. bovis strains, including M. bovis BCG Pasteur, but was shown to be absent in 26 different mycobacteria and 20 different clinical isolates of Mycobacterium tuberculosis. When the fragment was used as a probe in a Southern blot analysis, several radioactive bands common to M. tuberculosis and M. bovis were observed. However, this fragment hybridized specifically to a 2900 bp EcoRI fragment in the M. bovis genome, but failed to hybridize in either M. tuberculosis or M. avium chromosomal DNA. Based on a partial nucleotide sequence of the 500 bp fragment, two oligonucleotide primers were designed and a PCR assay was developed. Using purified mycobacterial DNA samples, only M. bovis and M. bovis BCG rendered a unique amplification band. This PCR assay is able to detect down to 10 fg purified M. bovis DNA, which corresponds roughly to two bacilli. The assay is also useful for identifying the bacilli directly from uncultured biological samples, such as milk.
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Nucleotide sequences of streptomycete 16S ribosomal DNA: towards a specific identification system for streptomycetes using PCR
More LessTo facilitate the differential identification of the genus Streptomyces, the 16S rRNA genes of 17 actinomycetes were sequenced and screened for the existence of streptomycete-specific signatures. The 16S rDNA of the Streptomyces strains and Amycolatopsis orientalis subsp. lurida exhibited 95-100% similarity, while that of the 16S rDNA of Actinoplanes utahensis showed only 88% similarity to the streptomycete 16S rDNAs. Potential genus-specific sequences were found in regions located around nucleotide positions 120, 800 and 1100. Several sets of primers derived from these characteristic regions were investigated as to their specificity in PCR-mediated amplifications. Most sets allowed selective amplification of the streptomycete rDNA sequences studied. RFLPs in the 16S rDNA permitted all strains to be distinguished.
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Distribution of the ardA family of antirestriction genes on conjugative plasmids
More LessThe ardA gene of I1 plasmid Collb-P9 was previously shown to alleviate DNA restriction by type I enzymes and to promote conjugative transmission of the unmodified plasmid to a restricting host. To clarify the ecological role of ardA, its distribution was determined on plasmids from 23 incompatibility groups using hybridization to the coding sequence as an assay. Hybridizing sequences, shown by nucleotide sequencing to have at least 60% identity with ardA, were detected on plasmids belonging to the I complex (IncB, I1 and K), the F complex (IncFV) and the IncN group. The ardA homologues were found to specify an antirestriction phenotype which was enhanced by genetic derepression of the plasmid transfer system. ardA loci map in plasmid leading regions but show no consistent association with a particular type of origin-of-transfer or a leading region gene of the ssb (single-stranded DNA-binding protein), psiB (plasmid SOS inhibition) and hok (host killing) families. It may be significant that ardA + plasmids are authentic enterobacterial plasmids and that type I restriction systems are associated historically with members of the Enterobacteriaceae.
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IS1500, an IS3-like element from Leptospira interrogans
More LessCopies of an insertion-sequence (IS)-like element were isolated from two closely related serovars of Leptospira interrogans sensu stricto. Nucleotide sequence analysis of the 1236 bp element showed a characteristic IS structure with terminal imperfect inverted repeats (IRs) flanking a 1159 bp central region. This element was designated IS 1500. Four open reading frames (orfA-orfD) were found in the central ‘unique’ region of IS 1500. Similarities were detected between ORFA and ORFB and the putative transposases from members of the IS3 family of transposable elements. IS 1500 or IS 1500-like sequences were also detected in all other pathogenic leptospiral serovars, but not in the saprophytic species L. biflexa. Differences in IS1500 copy numbers in members of the same species suggest that this element can transpose. Physical mapping of IS 1500 insertions in L. interrogans serovars icterohaemorrhagiae and pomona showed insertions were only on the large chromosomal replicon. The location of some IS1500 insertions coincides with regions of the genome that have undergone large rearrangements.
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Molecular genetic analysis of a thioredoxin gene from Thiobacillus ferrooxidans
More LessThe Thiobacillus ferrooxidans thioredoxin gene, trxA, was isolated by its ability to complement an Escherichia coli gshA trxA mutant which was otherwise unable to grow on minimal medium lacking glutathione. The T. ferrooxidans thioredoxin also enabled the in vivo reduction by E. coli of methionine sulfoxide to methionine, as well as the in vitro reduction of insulin. When present in E. coli, the T. ferrooxidans thioredoxin supported the replication of phage T7, but not the growth of phage M13. The T. ferrooxidans trxA gene was sequenced and the thioredoxin was found to be most like that of E. coli (71% identity) and Chromatium vinosum (70% identity). As in the case of E. coli, the gene was located immediately upstream of the gene for the rho transcriptional terminator. DNA:RNA blot hybridization and primer-extension analysis of the trxA gene in T. ferrooxidans and the cloned gene in E, coli indicated that it was transcribed as an independent unit and that the major transcriptional start sites were the same in both organisms.
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A group I intron in the terminase gene of Lactobacillus delbrueckii subsp. lactis phage LL-H
More LessAn 837 nt long group IA intron was discovered in the Lactobacillus delbrueckii subsp. lactis virulent phage LL-H genome. The LL-H intron conforms well to the secondary structure that is common to all group I introns. The only exception is that the extreme 3' nucleotide of the intron is an A residue instead of the usual G; despite this the intron is efficiently spliced in vivo. This LL-H intron contains an ORF, ORF168, which shows homology with endonucleases encoded by ORFs contained in Bacillus subtilis phage introns. At present, the LL-H intron is the only one found in the phages of lactic acid bacteria and the first one to be found in a phage belonging to the most abundant taxonomic group, group B or Siphoviridae. The LL-H intron interrupts gene terL, the product of which (50.5 kDa, TerL) is significantly homologous to the large subunit of B. subtilis phage SPP1 terminase. The product of the upstream gene, terS of LL-H (15.9 kDa, TerS), shows homology to small subunits of B. subtilis phage terminases.
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The bacteriophage 434 operator/repressor system in yeast
More LessThe ability of the bacteriophage 434 operator/repressor system to function in a eukaryotic cell has been explored. An idealized 434 operator was placed at various positions in the PGK promoter of Saccharomyces cerevisiae: within the upstream activator sequence, close to the TATA box, and downstream of the transcription-initiation site. Expression of the 434 cl gene from a 2 μm-based plasmid resulted in significant repression of gene expression from constructs containing the altered promoters linked to a β-galactosidase reporter gene. Attempts to use a variant of the 434 repressor that has the binding specificity of the P22 repressor (434P22) were unsuccessful, due to a severely inhibitory effect of this gene-product on the growth of the yeast cells.
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IMP2, a gene involved in the expression of glucose-repressible genes in Saccharomyces cerevisiae
More LessTwo mutants carrying different deletions of the IMP2 coding sequence of Saccharomyces cerevisiae, Ã1, which encodes a protein lacking the last 26 C-terminal amino acids, and Ã2, which completely lacks the coding region, were analysed for derepression of glucose-repressible maltose, galactose, raffinose and ethanol utilization pathways in response to glucose limitation. The role of the IMP2 gene product in the regulation of carbon catabolite repressible enzymes maltase, invertase, alcohol dehydrogenase, NAD-dependent glutamate dehydrogenase (NAD-GDH) and L-lactate:ferricytochrome-c oxidoreductase (L-LCR) was also analysed. The IMP2 gene product is required for the rapid glucose derepression of all above-mentioned carbon source utilization pathways and of all the enzymes except for L-LCR. NAD-GDH is regulated by IMP2 in the opposite way and, in fact, this enzyme was released at higher levels in both imp2 mutants than in the wild-type strain. Therefore, the product of IMP2 appears to be involved in positive and negative regulation. Both deletions result in growth and catalytic defects; in some cases partial modification of the gene product yielded more dramatic effects than its complete absence. Moreover, evidence is provided that the IMP2 gene product regulates galactose- and maltose-inducible genes at the transcriptional level and is a positive regulator of maltase, maltose permease and galactose permease gene expression.
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Functional analysis of the Bacillus subtilis purT gene encoding formate-dependent glycinamide ribonucleotide transformylase
More LessThe purT gene from Bacillus subtilis encoding the formate-dependent glycinamide ribonucleotide transformylase T was cloned by functional complementation of an Escherichia coli purN purT double mutant. The nucleotide sequence revealed an open reading frame of 384 amino acids. The purT amino acid sequence showed similarity to the enzyme phosphoribosylaminoimidazole carboxylase encoded by the purK gene but not to the N 10-formyltetrahydrofolate-dependent glycinamide ribonucleotide transformylase N enzyme encoded by the purN gene. The glycinamide ribonucleotide transformylase T level was repressed in cells grown in rich medium compared to minimal-medium-grown cells. However, when the culture entered the stationary-growth phase the enzyme level increased in rich medium and decreased in minimal medium. By comparing the deduced amino acid sequence of the B. subtilis purT gene product with translated nucleotide sequences in various databanks, evidence for the existence of putative purT genes in the Gram-negative bacteria Pasteurella haemolytica and Pseudomonas aeruginosa was obtained.
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The 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahM and nahO genes of the naphthalene catabolic plasmid pWW60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences
More LessWe report the complete nucleotide sequence and over-expression of the nahOM genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pWW60-22 from Pseudomonas sp. NCIMB9816. Additional partial sequence analysis of adjacent DNA shows the gene order within the operon to be nahNLOMK, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway of TOL plasmid pWWO and the phenol/methylphenol pathway of pVI150. The deduced amino acid sequences of NahO and NahM were significantly homologous to the equivalent enzymes encoded by other Pseudomonas meta pathways, although both were the most divergent in each comparison. The nahOM genes were inserted downstream of the T7 promoter in the expression vector pET3a and similar constructs were also made of the isofunctional regions from pVI150 (dmpFG) and TOL plasmid pDK1 (xylQK). High expression of all three gene pairs was detected by enzyme assays and by SDS-PAGE.
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Characterization of a nitrogen-fixation (nif) gene cluster from Anabaena azollae 1a shows that closely related cyanobacteria have highly variable but structured intergenic regions
More LessThe exact identity of cyanobacteria that have been cultured from symbiotic associations with the water fern Azolla spp., whether they are required in the symbiotic process, and their relationship to the symbiotic species, is a matter of some debate. We have characterized a 6 kb region containing the nifB operon and the nifH gene from cyanobacterium Anabaena azollae 1a, a putative symbiont of Azolla caroliniana. Five complete open reading frames have been sequenced. All are very highly conserved when compared with the corresponding regions of Anabaena sp. PCC 7120, with 93% to 97% identity at the nucleotide level and 93% to almost 100% at the amino-acid level. The intergenic regions, however, are not highly conserved (53-89% identity) when compared to the corresponding regions of Anabaena 7120: the A. azollae genome contains both more copies and more types of short tandemly repeated repetitive sequences than Anabaena 7120. The startpoints of transcription for both the nifB and nifH operons were mapped and found to be the same as those in Anabaena 7120. It was not possible to discern an improved consensus nif promoter sequence, but it was possible to define the likely extent of the promoter to within 40 bases upstream of the transcription start-point.
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Fructose phosphotransferase system of Xanthomonas campestris pv. campestris: characterization of the fruB gene
More LessIn the plant pathogen Xanthomonas campestris pv. campestris, fructose is transported by a specific phosphotransferase system (PTS). This PTS involves a multiphosphoryl transfer protein (MTP) encoded by the fruB gene, which was cloned and sequenced. fruB is part of a transcriptional unit together with the fruK gene, coding for 1-phosphofructokinase, which is located upstream from the fruA gene, coding for the fructose-specific permease (EIIB'BCFru). The amino acid sequence of the X. campestris MTP deduced from the fruB sequence shared 46% identical residues with an MTP identified in Rhodobacter capsulatus. The X. campestris MTP (837 amino acid residues) consists of three moieties: a fructose-specific enzyme-IIA-like N-terminal moiety (residues 1-148), followed by an HPr-like moiety (161-251) and an enzyme-l-like C-terminal moiety (274-837). The three domains are separated by two flexible hinge regions rich in proline and alanine residues. The construction of a fruB mutant confirmed the role of the MTP in fructose transport and phosphorylation in X. campestris.
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- Physiology And Growth
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Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms
More LessStreptococcus bovis had a diauxic pattern of glucose and lactose utilization, and both of these sugars were transported by the sugar phosphotransferase system (PTS). Lactose catabolism was inducible, and S. bovis used the tagatose pathway to ferment lactose. Since a mutant that was deficient in glucose PTS activity transported lactose as fast as the wild-type, it appeared that S. bovis has separate enzyme lls for glucose and lactose. The nonmetabolizable glucose analogue 2-deoxyglucose (2-DG) was a noncompetitive inhibitor of methyl β-D-thiogalactopyranoside (TMG) transport, and cells that were provided with either glucose or 2-DG were unable to transport TMG or lactose. Because the glucose-PTS-deficient mutant could ferment glucose, but could not exclude TMG, it appeared that enzyme IIGlC rather than glucose catabolism per se was the critical feature of inducer exclusion. Cells that had accumulated TMG as TMG 6-phosphate expelled free TMG when glucose was added, but 2-DG was unable to cause TMG expulsion. The glucose-PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion and TMG expulsion. Membrane vesicles that were electroporated with phosphoenolpyruvate (PEP) and HPr retained TMG for more than 3 min, but vesicles that were electroporated with PEP plus HPr and fructose 1,6-diphosphate (FDP) (or glycerate 2-phosphate) lost their ability to retain TMG. Because FDP was able to trigger the ATP-dependent phosphorylation of HPr, it appeared that inducer expulsion was mediated by an FDP-activated protein kinase. This conclusion was further supported by the observation that mutant forms of HPr differed in their ability to faciliate inducer expulsion. S46DHPr, a mutant HPr with aspartate substituted for serine at position 46, promoted TMG expulsion from membrane vesicles in the absence of FDP better than wild-type HPr or S46AHPr, a mutant form with alanine substituted for serine at position 46. Based on these results, it appeared that glucose catabolism was needed for inducer expulsion, but not inducer exclusion.
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An extreme creA mutation in Aspergillus nidulans has severe effects on D-glucose utilization
More LessAspergillus nidulans wild-type and the extreme carbon catabolite derepressed mutant creAd-30 were characterized with respect to enzyme activities, metabolite concentrations and polyol pools all related to glycolysis, after growth on D-glucose. In the creAd-30 strain the enzymes hexokinase and fructose-6-phosphate reductase showed a two- and threefold increase in activity, respectively, whereas phosphofructokinase and pyruvate kinase activity decreased two- and threefold, respectively, in comparison with the wild-type strain. The most notable changes in metabolite concentrations were that fructose 2,6-bisphosphate and fructose 1,6-bisphosphate showed a 2.5-fold increase, whereas both pyruvate and citrate decreased in the creAd-30. Striking differences were found for the polyol concentrations measured for the two strains tested. Intracellular glycerol and arabitol concentrations were 10-fold higher and erythritol fivefold higher in creAd-30, whereas intracellular trehalose and mannitol were both decreased. The total internal polyol concentration appears to be constant at ~ 700 mol (g dry wt)-1. All polyols were also detected in high amounts in the culture filtrate of the creAd-30 mutant strain but no extracellular trehalose was found. The overall production of polyols in this strain was therefore much higher than in the wild-type. The high level of polyols produced and the changes in metabolite concentrations in the creAd-30 strain suggest that the differences in enzyme activities result in an altered flow through glycolysis leading to a more rapid formation of polyols which are subsequently secreted.
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Novel Neurospora crassa mutants with altered synthesis of polyunsaturated fatty acids
Five new mutants of Neurospora crassa that require supplementation with unsaturated fatty acids have been isolated. The mutants, designated pfa, are impaired in the synthesis of the polyunsaturated fatty acids α-linoleic or α-linolenic acid, but are able to synthesize oleic acid. The pfa mutants are thus distinct from previously described ufa mutants, which are unable to synthesize oleic acid. The five pfa mutants map to distinct loci, and have characteristic patterns of incorporation of [14C]acetate and [14C]oleate into their fatty acids.
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Physarum polycephalum haemagglutinins: effect of nutrition on synthesis, and their possible role in nature
More LessThe activity of haemagglutinins in plasmodia of Physarum polycephalum was measured under different culture conditions. The activity was markedly increased when the plasmodia were incubated in a non-nutrient salt medium. During starvation, significant amounts of haemagglutinins were found in the slime layer on the surface of the plasmodia. An increase in activity was not observed in the presence of actinomycin D or cycloheximide. Under starvation conditions, plasmodia are known to differentiate into either sclerotia (spherules) or fruiting bodies. Acceleration of haemagglutinin synthesis, however, was not always observed during spherulation and fruiting-body formation. Attempts to detect endogenous glycoconjugates that bind to the haemagglutinins were unsuccessful but we found that the haemagglutinins could bind to acidic polysaccharides produced by Escherichia coli K12. The bacterial glycoconjugates were purified and partially characterized. They contained N-acetylhexosamine residues which appeared to be important for binding with the haemagglutinins. It is possible that the haemagglutinins play a physiological role in the interaction with these organisms.
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An arginine/ornithine exchange system in Spiroplasma melliferum
More LessSpiroplasma melliferum cells utilize arginine via the arginine dihydrolase pathway. L-Arginine uptake by intact cells was a saturable process both as a function of time and arginine concentration (Km = 40 μM). Uptake was not affected by pH in the range pH 5.0-8.0, or by L-citrulline, D-arginine, L-histidine or L-canavanine at concentrations tenfold higher than that of L-arginine. In contrast, L-arginine uptake was markedly inhibited by L-ornithine and partially inhibited by L-lysine. Uptake was neither affected by protonophores nor by cation ionophores, but was inhibited by protease treatment or by the sulfhydryl reagents p-chloromercuribenzoate or N-ethylmaleimide. Sealed membrane vesicles prepared by fusing isolated S. melliferum membranes with asolectin-cholesterol vesicles catalysed a rapid exchange (t1/2 = 1 min) between arginine and ornithine. Exchange did not require ATP and could be demonstrated in both directions, i.e. with either arginine or ornithine trapped within vesicles. These observations suggest that the driving forces for arginine uptake by whole cells are the concentration gradients of arginine and ornithine formed by arginine metabolism.
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Protein burden in Zymomonas mobilis: negative flux and growth control due to overproduction of glycolytic enzymes
More LessIncreasing the expression of various glycolytic operons in Zymomonas mobilis caused a significant decrease rather than increase in the glycolytic flux and growth rate. Because the relative decrease depended on the amount of overexpressed protein, and was independent of which enzyme was overexpressed, we attributed it to a protein burden effect. More specifically, we examined if the decrease in glycolytic flux could be explained by a decreased concentration of other glycolytic enzymes (for which glucokinase was used as a marker enzyme). Using the summation theorem of metabolic control theory we predicted the extent of this protein burden effect. The predictions were in good agreement with the experimental observations. This suggests that the negative flux control is caused either by a simple competition of the overexpressed gene with the expression of all other genes or by simple dilution. Furthermore, we determined the implications of protein burden for the determination of the extent to which an enzyme limits a flux. We conclude that a protein burden can cause a significant underestimation of the flux control coefficient, especially if the enzyme under investigation is a highly expressed enzyme.
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Glucose metabolism in ‘Sphingomonas elodea’: pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant
More Less‘Sphingomonas (formerly Pseudomonas) elodea’ produces the industrially important polysaccharide gellan when grown in media containing glucose. Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium. Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways. Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation. Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways. Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production. The zwf gene was cloned from ‘S. elodea’ and partially sequenced, and a null zwf mutant was constructed. This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain. Potential explanations for this finding are provided. Clones carrying the pgi gene were isolated fortuitously.
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- Plant-Microbe Interactions
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Induction of heat shock response in Vibrio cholerae
More LessGeneral properties of the heat shock response in Vibrio cholerae were examined. Enhanced or de novo synthesis of 24 proteins was observed upon heat shock from 30 °C to 42°C in cells labelled with [35S]methionine. A similar response could also be induced by a rise in temperature from 30 °C to 37 °C. Of these heat shock proteins, two were determined to be homologues of GroEL and DnaK, based upon their immunological cross-reactivity with antibodies raised against the Escherichia coli proteins. Three proteins, of molecular sizes 38, 44 and 48 kDa, which were undetectable in the 30 °C grown culture, appeared de novo after the heat shock. As in other prokaryotic systems thermal induction of many of the proteins was transient, but both DnaK and GroEL remained induced for at least 28 min after heat shock. DNA hybridization studies revealed that genes analogous not only to dnaK and groEL but also to dnaJ of E. coli exist in V. cholerae. Heat shock induced thermotolerance in V. cholerae but made the cells more sensitive to UV radiation. Unlike in E. coli, however, heat shock had no effect on the progeny virus yield in V. cholerae.
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Restriction site polymorphism of the genes encoding the major 25 kDa and 36 kDa outer-membrane proteins of Brucella
More LessSeventy-seven Brucella reference and field strains from different geographic origins and hosts representing the six recognized species and their different biovars were analysed for diversity of their genes encoding the major 25 and 36 kDa outer-membrane proteins (OMPs) by PCR-RFLP. The 25 kDa OMP is encoded by a single gene (omp25) whereas two closely related genes (omp2a and omp2b) encode and potentially express the 36 kDa OMP. Analysis of PCR products of the omp25 gene digested with nine restriction enzymes revealed two species-specific markers, i.e. the absence of the EcoRV site in all Brucella melitensis strains and an ~ 50 bp deletion at the 3' terminal end of the gene in all Brucella ovis strains. Analysis of PCR products of the omp2a and omp2b genes digested with 13 restriction enzymes indicated a greater diversity than the omp25 gene among the six Brucella species and within the Brucella abortus, Brucella suis, B. melitensis and B. ovis species. Greater polymorphism was also detected for the omp2b than for the omp2a gene, especially in B. ovis which seemed to carry two similar (but not identical) copies of omp2a instead of one copy each of omp2a and omp2b for the other Brucella species as was previously suggested by Ficht et al. (1990; Mol Microbiol 4, 1135-1142). Results of PCR-RFLP indicated that distinction can be made between Brucella species and some of their biovars, except between B. canis and B. suis bv. 3 and 4, on the basis of the size and diversity of their major OMP genes, and that it could be of importance for diagnostic, epidemiological and evolutionary study purposes.
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Elaboration of flavonoid-induced proteins by the nitrogen-fixing soybean symbiont Rhizobium fredii is regulated by both nodD1 and nodD2, and is dependent on the cultivar-specificity locus, nolXWBTUV
More LessGenistein and other flavonoids from host legumes are known to stimulate cells of the nitrogen-fixing soybean symbiont Rhizobium fredii to synthesize Nod factors, which function as signals during nodule initiation. Flavonoids also trigger R. fredii to secrete a set of signal-responsive (SR) proteins into the environment. By insertion mutagenesis, we showed that secretion of SR proteins by this organism has an absolute dependence on the regulatory gene nodD1. We isolated and sequenced nodD1 and nodD2 of R. fredii USDA257 and constructed strains containing additional, plasmid-borne copies of these genes. Extra copies of nodD1 had no effect on secretion of SR proteins, but extra copies of nodD2 rendered the process constitutive. Extracts from seeds of the soybean cultivars McCall and Peking can substitute for purified flavonoids as inducers of SR proteins. The nolXWBTUV locus is known to control cultivar-specific nodulation of McCall soybean in a negative, flavonoid-dependent manner. Inactivation of any of these genes prevented SR proteins from accumulating in culture fluids. Protein secretion in response to host signals was a characteristic of nine out of ten R. fredii strains tested. Immunological probes failed to detect SR3 or SR5 in mature soybean or cowpea nodules. Although the functions of these proteins remain unknown, their potential role in symbiosis is strengthened by the discovery that their accumulation depends on nodD1, nodD2 and nolXWBTUV.
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- Genome Analysis
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Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B. subtilis Marburg strain
A DNA fragment containing the aroA(G) gene of Bacillus subtilis 168, encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase-chorismate mutase, was cloned and sequenced. The N-terminus of the protein encoded by aroA(G) showed homology with chorismate mutase encoded by aroH of B. subtilis and with the chorismate mutase parts of proteins encoded by the pheA and tyrA genes of Escherichia coli. The C-terminus of the aroA(G) product has sequence simililarity with 3-deoxy-D-manno-octulosonate 8-phosphate synthase of E. coli. It was shown that the proteins encoded by the aroA(G) gene of B. subtilis 168 and the aroA gene of B. subtilis ATCC 6051 Marburg strain are identical, so the observed differences in DAHP synthase activity from these two strains must result from other changes.
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- Corrigendum
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