- 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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The pyruvate dehydrogenase complex of the chemolithoautotrophic bacterium Thiobacillus ferrooxidans has an unusual E2-E3 subunit fusion
More LessSummary: The genes encoding pyruvate dehydrogenase (PDH) of Thiobacillus ferrooxidans were previously located by cloning and sequence analysis of the region upstream of the genes encoding the citrate synthase and -glutamylcysteine synthetase genes. The pdh genes of T. ferrooxidans were able to complement an Escherichia coli aroP-lpd mutant for growth on minimal medium lacking acetate, indicating that the T. ferrooxidans PDH complex was functional in E. coli. The predicted amino acid sequence of the T. ferrooxidans PDH complex contained three ORFs. The first ORF encoded a 36.7 kDa homologue of the PDH complex E1α subunit, the second ORF a 37.4 kDa E1α subunit and the third ORF an unusual 102 kDa fusion of the E2 and E3 subunits. In spite of T. ferrooxidans being a Gram-negative bacterium, its PDH complex had more features in common with Gram-positive bacteria and eukaryotes.
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The cydR gene product, required for regulation of cytochrome bd expression in the obligate aerobe Azotobacter vinelandii, is an Fnr-like protein
More LessSummary: The cytochrome bd complex in the obligately aerobic diazotroph Azotobacter vinelandii is an oxidase, which, in vivo, has a low affinity for oxygen and is required for respiratory protection of nitrogenase. Mutations caused by insertion of Tn5-B20 upstream of the structural genes (cydAB) for cytochrome bd result in over-expression of this oxidase and, for unexplained reasons, inability of the organism to grow microaerobically. Cloning and sequencing of this upstream region revealed a gene, cydR. The deduced amino acid sequence of CydR indicates that it is a new member of the Fnr class of regulators and that it represses cydAB expression. Refined mapping data for three insertions in cydR are presented. The cloned cydR gene complemented anaerobic growth of Escherichia coli fnr mutants and strongly enhanced expression of a narG-lacZ fusion in an E. coli fnr mutant.
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Regulation of the TCA cycle and the general amino acid permease by overflow metabolism in Rhizobium leguminosarum
More LessSummary: Mutants of Rhizobium leguminosarum were selected that were altered in the uptake activity of the general amino acid permease (Aap). The main class of mutant maps to sucA and sucD, which are part of a gene cluster mdh-sucCDAB, which codes for malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD) and components of the 2-oxoglutarate dehydrogenase complex (sucAB). Mutation of either sucC or sucD prevents expression of 2-oxoglutarate dehydrogenase (sucAB). Conversely, mutation of sucA or sucB results in much higher levels of succinyl-CoA synthetase and malate dehydrogenase activity. These results suggest that the genes mdh-sucCDAB may constitute an operon. suc mutants, unlike the wild-type, excrete large quantities of glutamate and 2-oxoglutarate. Concomitant with mutation of sucA or sucD, the intracellular concentration of glutamate but not 2-oxoglutarate was highly elevated, suggesting that 2-oxoglutarate normally feeds into the glutamate pool. Elevation of the intracellular glutamate pool appeared to be coupled to glutamate excretion as part of an overflow pathway for regulation of the TCA cycle. Amino acid uptake via the Aap of R. leguminosarum was strongly inhibited in the suc mutants, even though the transcription level of the aap operon was the same as the wild-type. This is consistent with previous observations that the Aap, which influences glutamate excretion in R. leguminosarum, has uptake inhibited when excretion occurs. Another class of mutant impaired in uptake by the Aap is mutated in polyhydroxybutyrate synthase (phaC). Mutants of succinyl-CoA synthetase (sucD) or 2-oxoglutarate dehydrogenase (sucA) form ineffective nodules. However, mutants of aap, which are unable to grow on glutamate as a carbon source in laboratory culture, show wild-type levels of nitrogen fixation. This indicates that glutamate is not an important carbon and energy source in the bacteroid. Instead glutamate synthesis, like polyhydroxybutyrate synthesis, appears to be a sink for carbon and recluctant, formed when the 2-oxoglutarate dehydrogenase complex is blocked. This is in accord with previous observations that bacteroids synthesize high concentrations of glutamate. Overall the data show that the TCA cycle in R. leguminosarum is regulated by amino acid excretion and polyhydroxybutyrate biosynthesis which act as overflow pathways for excess carbon and reductant.
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A Pneumocystis carinii multi-gene family with homology to subtilisin-like serine proteases
More LessSummary: Copies of a multi-gene family, named PRT1 (protease 1, encoding a subtilisin-like serine protease were cloned from the opportunistic fungal pathogen Pneumocystis carinii. Comparison of the nucleotide sequence of a genomic clone and a cDNA clone of PRT1 from P. carinii f. sp. carinii revealed the presence of seven short introns. Several different domains were predicted from the deduced amino acid sequence: an N-terminal hydrophobic signal sequence, a pro-domain, a subtilisin-like catalytic domain, a P-domain (essential for proteolytic activity), a proline-rich domain, a serine/threonine-rich domain and a C-terminal hydrophobic domain. The catalytic domain showed high homology to other eukaryotic subtilisin-like serine proteases and possessed the three essential residues of the catalytic active site. Karyotypic analysis showed that PRT1 was a multi-gene family, copies of which were present on all but one of the P. carinii f. sp. carinii chromosomes. The different copies of the PRT1 genes showed nucleotide sequence heterogeneity, the highest level of divergence being in the proline-rich domain, which varied in both length and composition. Some copies of PRT1 were contiguous with genes encoding the P. carinii major surface glycoprotein.
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Cloning and disruption of the ornithine decarboxylase gene of Ustilago maydis: evidence for a role of polyamines in its dimorphic transition
More LessSummary: The gene encoding ornithine decarboxylase (ODC) from Ustilago maydis was cloned. A conserved PCR product amplified from U. maydis DNA was synthesized and used to screen a genomic library of the fungus. Alignment of its deduced protein sequence with those of other cloned ODCs showed a high degree of homology. Gene replacement was obtained by removal of a central part of the gene and insertion of the hygromycin resistance cassette. The null mutant thus obtained displayed no ODC activity and behaved as a polyamine auxotroph. This result is evidence that a single ODC gene exists in the fungus, and that U. maydis utilizes the ODC pathway as the only mechanism for polyamine biosynthesis. When grown in polyamine-containing media, the null mutant accumulated a polyamine pool which further sustained its normal rate of growth in polyamine-free media for approximately 12-16 h. When putrescine concentrations lower than 0.5 mM were employed, the mutant grew at a normal rate but was unable to engage in the dimorphic transition. Under conditions favourable for mycelial growth, the mutant grew with a yeast-like morphology in liquid media, and formed smooth colonies consisting of yeast cells on solid media. Reversion to normal dimorphic phenotype required high concentrations of putrescine or spermidine. These results are evidence that concentrations of polyamines higher than those necessary to sustain vegetative growth are required for the dimorphic transition in U. maydis.
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The Aspergillus fumigatus mepB gene encodes an 82 kDa intracellular metalloproteinase structurally related to mammalian thimet oligopeptidases
More LessSummary: Aspergillus fumigatus produces an 82 kDa intracellular metalloproteinase that hydrolyses the Pz-peptide, 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg, a typical substrate of members of the thimet oligopeptidase family which is ubiquitously distributed across animal species. The A. fumigatus mepB gene encoding this 82 kDa metalloproteinase was cloned and sequenced. Analysis of the deduced amino acid sequence of mepB showed that the MepB protein is a cytosolic zinc metalloproteinase of the thimet oligopeptidase family (M3) and as such is probably involved in the intracellular degradation of small peptides. An A. fumigatus mutant that lacks the MepB Pz-peptidolytic activity was constructed by gene disruption at the mepB locus. Analysis of this mutant did not reveal any detectable phenotype.
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Regulation of the inducible acetamidase gene of Mycobacterium smegmatis
More LessSummary: The inducible acetamidase of Mycobacterium smegmatis NCTC 8159 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. After induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.
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Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A
More LessSummary: The strain Enterococcus faecium T136 produces two bacteriocins, enterocin A, a member of the pediocin family of bacteriocins, and a new bacteriocin termed enterocin B. The N-terminal amino acid sequences of enterocins A and B were determined, and the gene encoding enterocin B was sequenced. The primary translation product was a 71 aa peptide containing a leader peptide of the double-glycine type which is cleaved off to give mature enterocin B of 53 aa. Enterocin B does not belong to the pediocin family of bacteriocins and shows strong homology to carnobacteriocin A. However, sequence similarities in their leader peptides and C-termini suggest that enterocin B and carnobacteriocin A are related to bacteriocins of the pediocin family. Enterocins A and B had only slightly different inhibitory spectra, and both were active against a wide range of Gram-positive bacteria, including listeriae, staphylococci and most lactic acid bacteria tested. Both had bactericidal activities, but survival at a frequency of 10−44-10−2 was observed when sensitive cultures were exposed to either bacteriocin. The number of survivors was drastically reduced when a mixture of the two bacteriocins was added to the cells.
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The Staphylococcus aureus allelic genetic loci for serotype 5 and 8 capsule expression contain the type-specific genes flanked by common genes
More LessSummary: The nucleotide sequences of two gene clusters, cap5 and cap8, involved in the synthesis of Staphylococcus aureus type 5 and type 8 capsular polysaccharides (CPs), respectively, were determined. Each gene cluster contained 16 ORFs, which were named cap5A through cap5P for type 5 CP and cap8A through cap8P for type 8 CP. The cap5 and cap8 loci were allelic and were mapped to the Smal-G fragment in the standard Smal map of Staph, aureus strain NCTC 8325. The predicted gene products of cap5A through cap5G and cap5L through cap5P are essentially identical to those of cap8A through cap8G and cap8L through cap8P, respectively, with very few amino acid substitutions. Four ORFs located in the central region of each locus are type-specific. A comparison of the predicted amino acid sequences of cap5 and cap8 with sequences found in the databases allowed tentative assignment of functions to 15 of the 16 ORFs. The majority of the capsule genes are likely to be involved in amino sugar synthesis; the remainder are likely to be involved in sugar transfer, capsule chain-length regulation, polymerization and transport.
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Generalized transduction in the potato blackleg pathogen Erwinia carotovora subsp. atroseptica by bacteriophage M1
Summary: Using enrichment methods, a new bacteriophage (M1) was isolated, which is capable of generalized transduction in Erwinia carotovora subsp. atroseptica (Eca) strain SCRI1043. M1 is probably a virulent phage and contains double-stranded DNA of approximately 43 kb. Transduction frequencies for a number of chromosomal markers and plasmid pHCP2 were established, and conditions for transduction optimized. UV irradiation of the lysates prior to transduction enhanced the transduction frequency. M1 infected over 25% of Eca strains tested and so may be useful both for the genetic analysis of a number of Eca isolates and for the transductional transfer of selectable markers between strains.
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- Pathogenicity And Medical Microbiology
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The MtrD protein of Neisseria gonorrhoeae is a member of the resistance/nodulation/division protein family constituting part of an efflux system
Summary: The mtr (multiple transferable resistance) system of Neisseria gonorrhoeae mediates resistance of gonococci to structurally diverse hydrophobic agents (HAs) through an energy-dependent efflux process. Recently, complete or partial ORFs that encode membrane proteins (MtrC, MtrD, MtrE) forming an efflux pump responsible for removal of HAs from gonococci were identified and appeared to constitute a single transcriptional unit. In this study, the complete nucleotide sequence of the mtrD gene was determined, permitting the characterization of the MtrD protein. The full-length MtrD protein has a predicted molecular mass of nearly 114 kDa, putatively containing a 56 amino acid signal peptide. MtrD displays significant amino acid sequence similarity to a family of cytoplasmic, membrane proteins, termed resistance/nodulation/division (RND) proteins, which function as energy-dependent transporters of antibacterial agents and secrete bacterial products to the extracellular fluid. The predicted topology of the MtrD transporter protein revealed 12 potential membrane-spanning domains, which were clustered within the central and C-terminal regions of the primary sequence. Loss of MtrD due to insertional inactivation of the mtrD gene rendered gonococci hypersusceptible to several structurally diverse HAs, including two fatty acids (capric acid and palmitic acid) and a bile salt (cholic acid), but not hydrophilic antibiotics such as ciprofloxacin and streptomycin. Since gonococci often infect mucosal sites rich in toxic fatty acids and bile salts, the expression of the mtr efflux system may promote growth of gonococci under hostile conditions encountered in vivo.
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Involvement of the gonococcal MtrE protein in the resistance of Neisseria gonorrhoeae to toxic hydrophobic agents
More LessSummary: Low-level resistance of Neisseria gonorrhoeae to toxic hydrophobic agents (HAs), including some antibiotics, is chromosomally mediated via the multiple transferable resistance (mtr) efflux system. The gene encoding the 48.3 kDa outer-membrane protein MtrE, which is associated with the mtr phenotype, was identified and is homologous to export-associated outer-membrane proteins, including the OprM (formerly OprK) lipoprotein of Pseudomonas aeruginosa. Insertional inactivation of the mtrE gene in N. gonorrhoeae strain FA19 resulted in the loss of the outer-membrane protein, with concomitant hypersusceptibility of the mutant strain to a range of HAs. The properties of this mutant confirmed the role of MtrE in multidrug resistance mediated by an active efflux mechanism. Secondary structure predictions for MtrE indicated a largely hydrophilic protein with a single α-helical transmembrane region. A transposon-like element, similar to that found downstream of the region containing the promoters for mtrR and mtrC in Neisseria meningitidis, was identified 63 bp downstream of the mtrE gene.
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Tn5-induced lipopolysaccharide mutations in Bordetella pertussis that affect outer membrane function
More LessSummary: An LPSB-specific mAb was used to screen for ten Tn5 insertion mutants ofBordetella pertussis which have LPS which is phenotypically distinct from either wild-type LPSAB or LPSB. Silver-stained SDS-PAGE gels showed nine different LPS phenotypes, six of which contain two clinically undocumented LPS bands, designated IntA and IntB based on their proximity to the LPSA and LPSB bands, respectively. Binding assays with LPSA- and LPSB-specific mAbs established changes in epitope exposure for the various mutant LPS, both in cell-free form and as presented on the surface of whole cells. The possible involvement of a number of genes, both structural and regulatory, was indicated in production of the altered phenotypes. PFGE and Southern blotting showed that the Tn5 inserts of seven mutants mapped to a region of the B. pertussischromosome shown previously to encode the bpl gene products of LPS biosynthesis. Mutants MLT3, MLT5 and MLT8, however, mapped to distinctly different parts of the chromosome. In addition, mutants MLT2 and MLT3 contributed to an accelerated frequency in the appearance of avirulent phase organisms despite their Tn5 inserts being over 1000 bp from the bvglASR locus. The alterations in LPS structure in the mutants changed their reactivity to strain-specific mAbs and their sensitivity to hydrophobic and hydrophilic antibiotics.
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The effects of adherence to silicone surfaces on antibiotic susceptibility in Staphylococcus aureus
More LessSummary: Sensitivity of Staphylococcus aureus to the antibiotics tetracycline, benzylpenicillin and vancomycin was found to decrease by 2-10-fold when cells were grown adherent to silicone catheter surfaces. Sensitivity to rifampicin and fusidic acid was not significantly altered in adherent cells. Susceptibility further decreased with increased adherence time prior to antibiotic challenge. The resistance observed was not genotypic, or due to the presence of a specialized subpopulation of bacteria, as it disappeared when the bacteria were removed from the catheter, subcultured and retested. Also, adherent bacteria were found to grow more slowly than bacteria growing planktonically. It is concluded that the decrease in antibiotic susceptibility of adherent bacteria is a function of the physiological status of the individual cells rather than a function of biofilm formation or slime production. The decrease in growth rate of the adherent bacteria is a result of the adherence process rather than a result of nutrient depletion. The decrease in growth rate is implicated, but is not the sole factor, in the decreased antibiotic susceptibility of adherent bacteria.
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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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- Physiology And Growth
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Role of the colicin A lysis protein in the expression of the colicin A operon
More LessSummary: The involvement of the cal gene, which encodes the colicin A lysis protein, in the expression of the colicin A operon is demonstrated. Colicin A synthesis by Escherichia coli was studied at various temperatures in cells containing either the wild-type colicin A operon or the colicin A operon with the cal gene deleted. The amount of colicin A produced was lower in cells containing the colicin A operon devoid of the cal gene than in wild-type cells. In cells treated with the antibiotic globomycin, the synthesis of colicin A was blocked in null cal mutants at all temperatures. It was blocked only at low temperature in cells containing the wild-type colicin A operon, but not in cells subjected to heat shock or azide treatment. The cal gene product may be an activator of colicin A expression and of its own expression. An unidentified product, possibly a heat-shock protein, may also be involved and could complement the cal gene product in some situations.
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The starvation-stress response of Vibrio (Listoneila) anguillarum
More LessSummary: The starvation-stress response of Vibrio (Listonella) anguillarum was investigated and characterized with regard to changes in cell morphology and the ability of V. anguillarum to survive starvation, heat shock, exposure to H2O2 and exposure to ethanol. The ability of V. anguillarum to survive exposal to the latter three stresses after initiation of starvation was also examined. Results of these experiments indicated that when starved for carbon, nitrogeand phosphorus, the c.f.u. of V. anguillarum declined by about one order of magnitude over the first 5-7 d of starvation; starvation for an additional 3-4 weeks resulted in a gradual decline in c.f.u. by another order of magnitude. Examination of starved cells by electron microscopy revealed that while most cells formed spherical ultramicrocells during starvation, some of the cells elongated to form short spirals. While cross-protection against other stresses such as oxidative stress (exposure to H2O2) and exposure to ethanol developed only a small degree of resistance to heat shock developed. Moreover, in all cases these resistances disappeared during prolonged starvation (usually > 5 d). Additionally, the rate of protein synthesis per c.f.u., measured by [35S]methionine incorporation, declined during the initial 6 h of starvation and increased to over 70% of the rate measured in exponentially growing cells by 5 d of starvation. It was concluded that the starvation-stress response of V. anguillarum differs significantly from those starvation responses reported for other bacteria, including responses displayed by other Vibrio species.
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Novel thermophilic bacteria producing nitrile-degrading enzymes
More LessSummary: The first known report of the isolation of thermophilic bacteria which produce nitrile-degrading enzymes is presented. One of the strains isolated was studied in detail. Strain Dac521, classified as Bacillus pallidus, was capable of growth on acetonitrile, benzonitrile, propionitrile, acetamide, benzamide and propionamide as the sole carbon and nitrogen source in minimal nutrient media. The strain produced separate aliphatic-nitrile (e.g. acetonitrile)- and aromatic-nitrile (e.g. benzonitrile)-degrading activities. Acetonitrile-degrading activity was produced constitutively and enzyme production was not enhanced by the addition of substrate. Under conditions where benzonitrile was the sole carbon and nitrogen source in minimal nutrient media, acetonitrile-degrading enzyme activity was completely inhibited and benzonitrile-degrading activity was induced. Growth on substrates as sole carbon and nitrogen sources, together with the substrate specificity of cell-free extracts, suggested that acetonitrile and benzonitrile degradation may have occurred via nitrile hydratase and nitrilase pathways, respectively. Both the acetonitrile- and benzonitrile-degrading enzyme systems were significantly more thermostable in whole-cell preparations and cell-free extracts compared to their mesophilic counterparts.
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Evidence for metabolism of o-xylene by simultaneous ring and methyl group oxidation in a new soil isolate
More LessSummary: An o-xylene-utilizing Rhodococcus, strain B3, was isolated from enrichments with o-xylene. The pathway for o-xylene degradation was investigated by simultaneous adaptation experiments, studies of product formation by a mutant and fortuitous oxidation studies using trimethylbenzene isomers as substrates. Two pathways were found to operate simultaneously and both were inducible. The first pathway involved the oxidation of a methyl group to form 2-methylbenzyl alcohol, followed by oxidation via the corresponding acid to 3-methylcatechol. The second pathway involved oxidation of the aromatic ring to form a dimethylcatechol. The bulk of the evidence suggests that the initial reaction was catalysed by a monooxygenase rather than a dioxygenase, and that 2,3-dimethylphenol was produced as an intermediate.
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Effect of growth rate, nutrient limitation and succinate on expression of TOL pathway enzymes in response to m-xylene in chemostat cultures of Pseudomonas putida (pWW0)
More LessSummary: Previous studies have shown that expression of the toluene and m- and p-xylene degradation pathway in Pseudomonas putida (pWW0) is subject to catabolite repression by succinate. We report here that the expression level of the upper part of this so-called TOL pathway in cells grown in chemostat culture is strongly influenced by nutrient limitation when m-xylene is the sole carbon and energy source. The benzylalcohol dehydrogenase (BADH) levels in cells that are growth-limited by anabolic processes [sulphate (S)-, phosphate (P)- or nitrogen (N)-limiting conditions] were 3-12% of those in cells growing under oxygen limitation (when catabolism limits growth). BADH levels under S-, P- and N-limitation were further decreased (three- to fivefold) when succinate was supplied in addition to m-xylene. Levels of the meta-cleavage pathway enzyme catechol 2,3-dioxygenase were less affected by the growth conditions but the general pattern was similar. Dilution rate also influenced the expression of the TOL pathway: BADH levels gradually decreased with increasing dilution rates, from 1250 mU (mg protein)−1 at D = 0.05 h−1 under m-xylene limitation to 290 mU (mg protein)−1 at D = 0.58 h−1 (non-limited growth). BADH levels were shown to be proportional to the specific affinity whole cells for m-xylene. It may, therefore, be expected that natural degradation rates are adversely affected by anabolic nutrient limitations, especially at relatively low concentrations of the xenobiotic compound.
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Physiology of poly-3-hydroxybutyrate (PHB) production by Alcaligenes eutrophus growing in continuous culture
More LessSummary: Alcaligenes eutrophus was grown in continuous culture (34 °C, pH 6.8) under various conditions with respect to dilution rate, nutrient limitation and carbon substrate. Poly-3-hydroxybutyrate (PHB) content, the rate of PHB production (q PHB) and the rate of carbon substrate utilization (q s) during growth on glucose were maximum at low dilution rate under ammonia limitation (ammonia limitation > potassium/oxygen limitation > glucose limitation). PHB content decreased in a linear manner as a function of dilution rate, from approximately 80% at D 0-025 h−1 during ammonia-limited growth to approximately 5% during growth at the maximum specific growth rate (μmax) in batch culture. PHB content, q PHB and qs varied with the nature of the carbon substrate during ammonia-limited growth at fixed dilution rate, and were maximum during growth on lactate [lactate>pyruvate>glucose/gluconate>fructose; highest q PHB 0.38 g PHB (g non-PHB biomass)−1 h−1]. qPHB was related in an approximately linear manner to the q s in excess of that required solely for the production of non-PHB biomass. This surplus q s was higher during growth on lactate than on glucose because q s was approximately equal to the maximum rate of carbon substrate utilization (q smax) during growth on lactate, but much lower than q smax during growth on glucose. The relationship between q PHB and surplus q s was confirmed by the effect of adding formate (as an additional source of NADH and/or ATP) and the uncoupling agent carbonyl cyanide-m-chlorophenylhydrazone (CCCP) to ammonia-limited cultures. It is concluded that A. eutrophus is unable to regulate the rate at which it takes up excess carbon substrate to match that required solely for growth, particularly during growth on lactate at low dilution rate, and thus produces PHB as a means of avoiding the potentially deleterious effects of generating high concentrations of intracellular metabolites. Possible ways of further increasing PHB production are discussed.
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Anaerobic pathways of glycerol dissimilation by Enterobacter agglomerans CNCM 1210: limitations and regulations
Summary: Continuous cultures of Enterobacter agglomerans CNCM 1210 were performed under regulated pH conditions (pH 7.0) with glycerol or glucose (20 gl−1) as carbon source. Cultures grown on glucose produced mainly acetate, ethanol and formate. In contrast, 1,3-propanediol (PPD) was the main product with glycerol. The carbon flow distribution at branching metabolic points was investigated. Higher PPD yields with increased dilution rate were correlated with an important increase in the relative ratio of glycerol dehydratase to glycerol dehydrogenase. Determination of intracellular triose-phosphate and fructose 1,6-biphosphate concentrations demonstrated that glyceraldehyde-3-phosphate dehydrogenase is the limiting step in glycerol dissimilation. At the pyruvate branching point, pyruvate dehydrogenase (PDH) activity was systematically detected. The pyruvate flow shifted to PDH is suspected to represent up to 22% of the acetyl-CoA formed. In addition, this enzyme pattern combined with the enhanced in vivo lactate dehydrogenase activity at high growth rates, was correlated with a decrease in the pyruvate formate-lyase activity. A regulation of this latter enzyme by the accumulation of triose-phosphate is suspected.
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Mitogenic factor secreted by Streptococcus pyogenes is a heat-stable nuclease requiring His122 for activity
More LessSummary: The gene encoding a mitogenic factor, termed MF, was cloned from Streptococcus pyogenes and the recombinant MF was overexpressed in Escherichia coli. Both the natural and recombinant MF had heat-resistant nuclease activity. The nuclease activity of MF was characterized using the recombinant protein. MF showed endonuclease activity, digesting ssDNA, dsDNA and tRNA. The optimal pH for the DNase activity of MF was 9.5. The DNase activity was enhanced approximately tenfold by the simultaneous presence of two divalent cations, Mg2+ and Ca2+, compared to either alone and was inhibited by EDTA or NaCI. The heat stability of MF was biphasic; the DNase activity was heat-stable from 0 to 50 °C and over 80 °C but very unstable at around 60 °C. DNA digested by MF possessed 5′-phosphorylated and 3′-hydroxylated termini, identical to those obtained by digestion of DNA by pancreatic deoxyribonuclease I. A mutant clone revealed that His122 was a residue essential to the nuclease activity.
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Protein kinase Sck1 is involved in trehalase activation by glucose and nitrogen source in the fission yeast Schizosaccharomyces pombe
More LessSummary: Trehalase activity is markedly enhanced upon addition of glucose and a nitrogen source to cells of the fission yeast Schizosaccharomyces pombe. This increase corresponds to a post-translational activation of the enzyme, which is controlled by cAMP-dependent and cAMP-independent pathways. Recent work has shown that overexpression of SCK1 in Schiz. pombe is able to suppress mutations that result in reduced Pka1 (cAMP-dependent protein kinase A activity, suggesting that Sck1 (suppressor of loss of cAMP-dependent protein kinase) might be a functional analogue of Pka1 in the fission yeast. Here, an analysis of the possible role of Sck1 in the activation of trehalase triggered by glucose and a nitrogen source is reported in cells that were deficient in either Pka1, Sck1 or both protein kinases. The results showed that, except in repressed cells, Sck1 probably mediates a cAMP-independent activation of trehalase following the signal(s) triggered by glucose and the nitrogen source. The absence of functional Sck1 in derepressed cells renders trehalase insensitive to activation by glucose and the nitrogen source even in the presence of Pka1, indicating that the Sck1-dependent, cAMP-independent pathway is the main signalling pathway controlling trehalase activation under derepression conditions. It is proposed that, during the activation of trehalase induced by glucose or a nitrogen source, the cAMP-Pka1 activation pathway previously characterized is to some extent parallel to this newly described one which includes Sck1 as ohosphorylating enzyme. Neither of these two pathways, however, plays a key role in the heat-induced increase in trehalase activity.
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- Genome Analysis
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Molecular characterization of the restriction endonuclease gene (scrFIR) associated with the ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503
More LessSummary: The nucleotide sequence of the chromosomally encoded type II ScrFI restriction/modification system from Lactococcus lactis subsp. cremoris UC503 was completed. The ScrFI restriction endonuclease (ENase) has previously been shown to specifically recognize 5’ CCNGG 3’ sites, cleaving after the second cytosine and the degenerate central base. The ENase gene (scrFIR; 862 bp) was located between, and co-directionally transcribed with, two formerly characterized 5-methylcytosine methyltransferase genes, which encode proteins that independently confer protection against ScrFI digestion. scrFIR codes for a protein of 272 amino acids with a predicted molecular mass of 31470 Da, which agrees favourably with a previously estimated molecular mass of 34 kDa for this enzyme. The deduced sequence of this protein did not show any significant homology with known protein sequences, including the isoschizomeric SsoII ENase from ShigeIIa sonnei. The ENase gene was cloned and expressed in Escherichia coli and Lactococcus; however, no in vivo restriction of phage was observed, suggesting that expression of the ENase gene may be repressed, or that the appropriate expression signals may be absent in the cloned constructs. The ability of ScrFI to cleave non-canonically modified 5’ CCNGG 3’ sequences suggested that some ScrFI sites may require complex modifications to fully impair digestion by this enzyme.
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- Corrigendum
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- Guidelines For Authors
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Volumes and issues
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Volume 170 (2024)
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Volume 169 (2023)
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Volume 168 (2022)
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Volume 167 (2021)
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Volume 166 (2020)
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