- Volume 143, Issue 7, 1997
Volume 143, Issue 7, 1997
- Sgm Special Lecture
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Challenging food microbiology from a molecular perspective
More LessSummary: Two key themes within food microbiology are bacterial detection and control. There is a raft of sub-headings under each of these themes, but in the last decade molecular approaches within each have made a significant contribution to the field. This is a personal review of the author’s past and present contributions and future ideas for challenging food microbiology from a molecular perspective.
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- Microbiology Comment
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- Biochemistry
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The role of sulfoacetaldehyde sulfo-lyase in the mineralization of isethionate by an environmental Acinetobacter isolate
More LessSummary: An environmental Acinetobacter isolate, strain ICD, utilized isethionate at concentrations up to at least 20 mM as carbon and energy source, with essentially quantitative sulfate accumulation. The initial step in isethionate metabolism is likely to be its oxidation to sulfoacetaldehyde since inducible sulfoacetaldehyde sulfo-lyase activity was demonstrated in isethionate-grown cells by in vitro assay and gel zymography; sulfoacetaldehyde itself did not induce the enzyme. Isethionate-grown cells of Acinetobacter sp. ICD, unlike those of most other C-S bond-cleaving strains described, also contained an inducible sulfite-oxidizing activity. The results provide further evidence that sulfoacetaldehyde sulfo-lyase plays a central role in the mineralization of biogenic sulfonates.
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Degradation of chlorophenoxyacetic acids by the lignin-degrading fungus Dichomitus squalens
More LessSummary: We have examined the degradation of 14C ring- and side-chain-labelled 2,4,5-trichlorophenoxyacetic acid by Dichomitus squalens and Phanerochaete chrysosporium. The effects of Mn2+ on the degradation of these radiolabeled substrates by D. squalens and the effect of nitrogen limitation on their degradation by D. chrysosporium suggested that in both fungi, side-chain cleavage was catalysed by a mechanism independent of the lignin degradation system, whereas the degradation of the aromatic ring was dependent on the lignin degradative system. Using unlabelled substrates, a pathway for the degradation of chlorophenoxyacetic acids was elucidated in D. squalens. Time courses for the degradation of unlabelled chlorophenoxyacetic acids by D. squalens demonstrated that the corresponding chlorophenol was the initial product formed. The chlorophenol intermediate was xylosylated to form the chlorophenolxyloside. In turn, the chlorophenolxyloside could be hydrolysed by an intracellular -xylosidase to regenerate the chlorophenol. The chlorophenol product of the xylosidase reaction was oxidatively dechlorinated to form 2-chloro-p-benzoquinone which could undergo subsequent further dechlorination and ring-opening reactions, as has been reported previously for P. chrysosporium.
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An outer-membrane porin inducible by short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus
More LessSummary: The fmdA and fmdB genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium Methylophilus methylotrophus were recloned with additional flanking DNA (pSW1). fmdC, encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream of fmdAB. The derived amino acid sequence of mature FmdC (Mr 39204) showed that it was rich in -sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (M r 40000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane of M. methylotrophus under conditions where these nitrogen sources are present at very low concentration.
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The properties and localization of Saprolegnia monoica chitin synthase differ from those of other fungi
More LessSummary: The presence of non-fibrillar α-chitin in celluiosic fungi (class Oomycetes) poses intriguing questions as to its role, subcellular localization and evolutionary significance. Previous studies reported on the similarity of chitin synthase from Saprolegnia monoica with that of other fungi. The present work describes important dissimilarities. There was no evidence that the chitin synthase of S. monoica was present in small low-density vesicles (chitosomes). Chitin synthase sedimented with membranous components of high specific gravity (sp. gr. 1.177) that could be partially but distinctly separated from membranes harbouring most of the 1,3–glucan synthase in the cell csp. gr. 1.158). In contrast to other fungi, the chitin synthase from S. monoica was greatly stimulated by digitonin: both membrane-bound and dissociated chitin synthase showed little activity in the absence of digitonin. As in other fungi, the chitin synthase from S. monoica was solubilized by digitonin and remained zymogenic after dissociation. However, unlike the enzyme from other fungi, the solubilized chitin synthase of S. monoica had a lower sedimentation coefficient, was not stimulated by phospholipids and was not inhibited by high concentrations of digitonin. Unlike the enzyme from Mucor rouxii. the solubilized chitin synthase from S. monoica did not bind to a cation exchanger. The enzyme was partially purified by a four-step scheme that included sucrose density-gradient centrifugation, a single passage through a strong anion exchanger and two consecutive passages through a weak anion exchanger. The final preparation contained five to seven polypeptide bands that cochromatographed with the chitin synthase activity, some of which may be part of a presumed chitin synthase macromolecular complex.
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A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins
More LessSummary: Porphyromonas gingivalis has been associated with the development of adult periodontitis and cysteine proteinases with trypsin-like specificity have been implicated as major virulence factors. We have extracted the major cell-associated trypsin-like proteolytic activity of P. gingivalis W50 using mild sonication. Anion-exchange and gel-filtration FPLC of the sonicate revealed that Arg- and Lys-specific proteinase activity was associated with a 300 kDa complex which could be dissociated into seven bands (48, 45, 44, 39, 27, 17 and 15 kDa) by SDS-PAGE with the 44 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on Arg-Sepharose the majority of the complex eluted from the affinity column as an undissociated complex. However, a small amount dissociated such that the Lys- and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa protein of the complex was purified by further anion-exchange FPLC in the presence of octyl–D-glucopyranoside and was shown to be an Arg-specific, thiol-activated, calcium-stabilized cysteine proteinase. The 48 kDa protein was also further purified in a similar fashion and shown to be a Lys-specific cysteine proteinase that was not inhibited by EDTA. The two 44 kDa and the 39, 27, 17 and 15 kDa proteins of the complex exhibit amino acid sequence homology and are proposed to be haemagglutinins/adhesins. The 45 kDa Arg-specific proteinase and one of the 44 kDa adhesins as well as the 15, 17 and 27 kDa adhesins are processed from the single polyprotein encoded by the gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. Similarly, the 48 kDa Lys-specific proteinase, the 39 and 15 kDa adhesins as well as the other 44 kDa adhesin of the 300 kDa complex are encoded by a single gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. The 39, 15 and 44 kDa adhesins of PrtK all exhibit high homology with the 44, 15, 17 and 27 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, designated PrtR-PrtK, is therefore composed of the two gene products, the mature PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteolytically processed (most likely autolytically) to release proteinase and adhesin domains that remain non-covalently associated. The fully processed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 and PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtRIS, PrtR17, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingivalis
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- Bioenergetics And Transport
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Energetics of methanogenic benzoate degradation by Syntrophus gentianae in syntrophic coculture
More LessSummary: Growing cocultures of Syntrophus gentianae with Methanospirillum hungatei degraded benzoate to CH4 and acetate. During growth, the change of free energy available for Syntrophus gentianae ranged between -50 and -55 kJ mol−1. At the end-point of benzoate degradation, a residual concentration of benzoate of 0.2 mM was found, correlating with a free energy change of -45 kJ mol−1 available to the fermenting bacterium. Benzoate thresholds were also observed in dense cell suspensions. They corresponded 1 a final energy situation in the range -31.8 to -45.8 kJ mol−1 for the fermentin bacterium. Addition of a H2-oxidizing sulfate reducer to the methanogenic coculture inhibited by bromoethanesulfonate (BES) resulted in benzoate degradation to below the limit of benzoate detection (10 μM). Accumulated acetate proved to be thermodynamically inhibitory; removal of acetate by Methanosaeta concilii in methanogenic or molybdate-inhibited sulfate-reducing cocultures led to degradation of residual benzoate with a final δG’ -45.8 kJ mol−1. In methanogenic cocultures, the residual Gibbs free energy (δG’) available for the fermenting bacterium at the end of benzoate degradation correlated with the concentration of acetate built up during the course of benzoate degradation; higher concentrations led to more positive values for δG’. Addition of different concentrations of propionate resulted in different values for δG when benzoate degradation had ceased; higher concentrations led to more positive values for δG’. Addition of acetate or propionate to benzoate-degrading cocultures also lowered the rate of benzoate degradation. The protonophore carbonylcyanide chlorophenylhydrazone (CCCP) facilitated further benzoate degradation in methanogenic BES-inhibited cocultures until a δG’ of -31 kJ mol−1 was reache We conclude that the minimum energy required for growth and energy conservation of the benzoate-fermenting bacterium S. gentianae is approximately -45 kJ (mol benzoate)−1, equivalent to two-thirds of an ATP unit. Both hydrogen and acetate inhibit benzoate degradation thermodynamically, and acetate also partly uncouples substrate degradation from energy conservation.
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Medium- and long-chain fatty acid uptake and utilization by Streptomyces coelicolor A3(2): first characterization of a Gram-positive bacterial system
More LessSummary: The first characterization of fatty acid uptake in a Gram-positive bacterium is reported. Streptomyces coelicolor A3(2) utilizes fatty acids of different chain length (C4-C18) as sole carbon and energy sources. In vivo β-oxidation studies and the assay of two enzymes of the β-oxidation cycle proved that fatty acid degradation is constitutive in this micro-organism. Uptake of the medium-chain fatty acid octanoate showed the characteristics of simple diffusion, whereas the uptake of palmitate, a long-chain fatty acid, occurred by both simple diffusion and active transport. After correcting for non-mediated transport, palmitate uptake measured over a wide range of concentrations followed Michaelis-Menten kinetics. The apparent K m for palmitate was 97.8 μM and the V max was 19.3 nmol min−1 (mg protein)−1. Competition experiments showed specificity of the mediated transport component for long-chain fatty acids (> C10). Metabolic inhibitors such as oligomycin, NaF and vanadate, and the ionophores gramicidin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited palmitate uptake to different degrees, consistent with the existence of an active transport mechanism. Uptake rates measured at different pH values indicated that both the ionized and the unionized forms of octanoate crossed the cytoplasmic membrane by simple diffusion. Palmitate in its ionized form appears to be transported by an active mechanism, whereas the unionized molecule diffuses through the membrane. When present in the medium, glucose stimulated the degradation of long-chain fatty acids by increasing the rate of uptake and the level of acyl-CoA synthetase.
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- Biotechnology
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Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp. and metal accumulation in vitro by liposomes containing entrapped enzyme
More LessSummary: A heavy-metal-accumulating Citrobacter sp. has been used for the treatment of metal-laden industrial wastes. Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate. A phosphatase-deficient mutant accumulated little UO2+ 2, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate). The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions. Enzyme was also associated with the outer membrane and found extracellularly. Accumulated uranyl phosphate was visible as cell-surface- and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO2PO4.4H2O. Nuclaation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation. Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of ‘IMMR-silent’ 112Cd2+. Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate. It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.
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- Development And Structure
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Monoclonal antibodies as probes for fungal wall structure during morphogenesis
More LessSummary: Three monoclonal antibodies (mAbs), S4D1, S3B3 and S1E5, were produced from hybridoma cell limes raised from mice immunized with hyphal walls of Neurospora crassa and one (Pax-1) from mice immunized with hyphal walls of Paxillus involutus. In immunofluorescence studies, the three N. crassa mAbs recognized epitopes with different patterns of distribution at the hyphal surface of N. crassa. S4D1 recognized an epitope which was present on the surface of both conidia and hyphae; S3B3 recognized an epitope seen only at the ends of conidia or in the septal region of hyphae and conidial chains; and S1E5 recognized an epitope present on the surface of hyphae, but not on mature conidia. mAb Pax-1 reacted with hyphal wall fragments of Pax. involutus and with N. crassa conidia in a similar way to S3B3. S4D1 reacted with an epitope found in 1,3-α-glucan preparations from hyphal walls of different fungi. The surface distribution of this epitope varied: it was found of the surface of both conidia and hyphae of N. crassa and Aspergillus nidulans, on the basidiospore surface only of Amanita muscaria, and on the hyphae but not the conidia of Penicillium chrysogenum. Immunogold studies revealed tha the epitope was present throughout the wall of conidia and hyphae of N. crassa. mAbs S3B3, S1E5 and Pax-1 also reacted with other fungi: for example Pax-1 cross-reacted with all fungi tested except for a member of the Zygomycota. Immunogold studies revealed that epitopes of these three mAbs were present within the inner layers of the walls of conidia and hyphae of N. crassa.
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- Environmental Microbiology
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Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor
More LessSummary: A novel metabolic pathway for anaerobic ammonium oxidation with nitrite as the electron acceptor has been elucidated using 15N-Iabelled nitrogen compounds. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor. The hydroxylamine itself is most likely derived from nitrite. Batch experiments in which ammonium was oxidized with hydroxylamine transiently accumulated hydrazine. The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating electron equivalents for the reduction of nitrite to hydroxylamine. During the conversion of ammonium, a small amount of nitrate was formed from some of the nitrite. The addition of NH2OH to an operating fluidized bed system caused a stoichiometric increase in the ammonium conversion rate (1 mmol I−1 h−1) and a decrease in the nitrate production rate (0.5 mmol I−1 h−1). Addition of hydrazine also caused a decrease in nitrate production. On the basis of these findings, it is postulated that the oxidation of nitrite to nitrate could provide the anaerobic ammonium-oxidizing bacteria with the reducing equivalents necessary for CO2 fixation.
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Reduction of ATPase activity accompanied by photodecomposition of ergosterol by near-UV irradiation in plasma membranes prepared from Saccharomyces cerevisiae
More LessSummary: When plasma membranes prepared from the yeast Saccharomyces cerevisiae were exposed to near-UV radiation, photodecomposition of ergosterol and reduction of ATPase activity occurred simultaneously. The V max for ATPase activity decreased markedly with increasing near-UV dosage while the K m value remained constant. When ATPase solubilized from the plasma membrane was exposed to near-UV, the activity remained constant irrespective of dosage, indicating that the ATPase molecule itself was not damaged by near-UV irradiation. The relationship between content of ergosterol and ATPase activity was examined using liposomes constructed with lipids extracted from the membrane. Maximum activity of ATPase was seen at 5% ergosterol in liposomes; this activity was 2.5 times greater than that in liposomes without ergosterol. Activity of ATPase bound to liposomes with 5% ergosterol was reduced after near-UV irradiation, while the activity remained unchanged in the case of the liposomes without ergosterol. Fluidity of the liposomes with 5% ergosterol also decreased with increasing near-UV dosage. Dosage-response curves for reduction of ATPase activity and for decrease in fluidity were similar to that for photodecomposition of ergosterol. These results suggested that the reduction of ATPase activity in the membrane by near-UV irradiation was not caused by photochemical degradation of the primary structure of the ATPase molecule, but was attributable to conformational change resulting from an alteration in the higher-order structure of the membrane due to photochemical decomposition of ergosterol.
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- Genetics And Molecular Biology
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The Clostridium perfringens enterotoxin gene is on a transposable element in type A human food poisoning strains
More LessSummary: The Clostridium perfringens enterotoxin gene (cpe) is rarely found in naturally isolated strains. In human food poisoning strains, cpe is found on the chromosome, and is located episomally in animal isolates. Observations that the gene was somewhat unstable and could be gained or lost suggested that the gene was on a mobile element. An IS200-like element, IS1469, is almost always upstream of cpe. A new insertion element was identified, IS1470, a member of the IS30 family, which is found both up- and downstream of cpe in the type A strain NCTC 8239. PCR results confirmed that this configuration was conserved in type A human food poisoning strains. The enterotoxin gene was on a 6.3 kb transposon which, in addition to the two flanking copies of IS1470, included IS1469 and two 1 kb stretches, one on each side of cpe, with no open reading frames. Results indicated that 14 bp was copied from the genome during insertion. Details of the configuration of DNA in this transposon are presented, and the possible connection of this transposon with the movement of the enterotoxin gene is discussed.
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A spectinomyciin resistance determinant from the spectinomycin producerStreptomyces flavopersicus
More LessSummary: The spectinomycin (Sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphoryiates Sp and actinamine, the aminocyclitol moiety of Sp.
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Conjugative transfer of tet(S) between strains of Enterococcus faecalis is associated with the exchange of large fragments of chromosomal DIMA
More LessSummary: The tetracycline resistance determinant tet(S) was first detected in antibiotic multiresistant Listeria monocytogenes BM4210 and subsequently in strains of Enterococcus faecalis. Transfer of tet(S) from clinical isolate E. faecalis BM4242 to E. faecalis strains JH2-2 and OG1RF was found to require the presence in the donor strain of the 55 kb conjugative plasmid pIP825. Comparison of restriction endonuclease generated maps of the donor, the two recipients, and of four transconjugants indicated that transfer of tet(S) (i) was from chromosome to chromosome, (ii) resulted in the acquisition of an approximately 40 kb element in the same chromosomal region and (iii) was associated with the exchange of large chromosomal fragments. Similar observations were made following conjugal transfer of tet(S) from four other E. faecalis clinical isolates.
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bctA: a novel pBF4 gene necessary for conjugal transfer in Bacteroides spp.
More LessSummary: pBF4 is a 41 kb conjugative R-plasmid that confers MLS (macrolide-lincosamide-streptogramin B) resistance in Bacteroides spp. To identify pBF4 genes governing conjugation, recombinational mutagenesis using a suicide vector carrying fragments of the pBF4 plasmid was employed. One of six independent insertion mutants of pBF4 isolated using this method was found to be conjugation-deficient. Nucleotide sequence analysis around the insertion site on this plasmid revealed a 2.8 kb ORF that encoded a putative 110 kDa protein. A corresponding protein was observed when a 12 kb DNA fragment containing this ORF was used to program an in vitro transcription-translation system. Both the ORF and the predicted protein were novel when compared to available database sequences. This gene was designated bctA (Bacteroides conjugal transfer). Polyclonal rabbit antibodies that recognized a sub-sequence polypeptide of BctA reacted with a 55 kDa protein in Western blot analysis using a total protein extract from Bacteroides fragilis containing pBF4. The protein was not present in a B. fragilis strain containing the conjugation-deficient insertion mutant of pBF4. The 55 kDa protein was associated with the membrane fraction of B. fragilis. Although the cellular and biochemical basis of bctA-promoted conjugation remains unknown, this work demonstrates the existence of a heretofore unrecognized gene in bacterial conjugation, and the mutagenesis system used provides the means to isolate and characterize other genes involved in conjugal transfer in Bacteroides spp.
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Divergence and conservation of the partitioning and global regulation functions in the central control region of the IncP plasmids RK2 and R751
More LessSummary: The central control region (Ctl) of IncP plasmids is associated with two phenotypes: the coordinate expression of replication and transfer genes; and the ability to increase the segregational stability of a low-copy-number test plasmid. This region of the IncP plasmid R751 shows significant sequence divergence from the IncPα plasmid RK2 sequence, and two genes, korF and korG, present in the IncPα region are missing in the IncP Ctl. In other respects the organization of the Ctl is basically the same. Although the two key global regulatory genes korA and korB are highly conserved, studies on their ability to repress transcription from a variety of IncPα and IncP plasmid promoters suggest differences in operator recognition by KorA and synergy with other repressors. The products of kfrA, upf54.8 and upf54.4 genes are conserved; KfrA shows least conservation and, while retaining the ability to act as a transcriptional repressor, appears to have completely different DNA-binding specificity. The genes required for the plasmid segregational stabilization (partitioning) phenotype - incC, korB and the KorB operator OB3 - are conserved and contribute to a more efficient plasmid stabilization than the IncPα equivalents. This may indicate that the Ctl plays an especially important role in partitioning of IncP plasmids, since they lack the second stability region (parlmrs) found in IncP plasmids.
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Alanyl-tRNA synthetase gene of the extreme acidophilic chernolithoautotrophic Thiobacillus ferrooxidans is highly homologous to alaS genes from all living kingdoms but cannot be transcribed from its promoter in Escherichia coli
More LessSummary: The alaS gene of Thiobacillus ferrooxidans has been cloned and sequenced and its expression in Escherichia coli and T. ferrooxidans analysed. The same genomic organization to that in E. coli (recA-recX-alaS) has been found in T. ferrooxidans. The recA and alaS genes cannot be transcribed from their own promoters in E. coli. In addition to the well-known homology at the protein level between AlaS proteins from various organisms, a strong homology was found between all the known alaS genes from bacteria, archaea and eucarya. Two regions, one of which corresponds to the catalytic core, are particularly well-conserved at the nucleotide sequence level, a possible indication of strong constraints during evolution on these parts of the genes.
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