- Volume 144, Issue 7, 1998
Volume 144, Issue 7, 1998
- Review Article
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- Biochemistry
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An aminopeptidase nutritionally important to Fusobacterium nucleatum
More LessThe properties of an aminopeptidase (AP) from Fusobacterium nucleatum were studied in view of the fact that this organism, along with other Gram-negative anaerobes involved in periodontal diseases, survives in the subgingival environment by obtaining energy via the fermentation of a small number of peptide-derived amino acids. The AP was found to be cell-associated and was isolated from disrupted chemostat-grown cells. It was purified by (NH4)2SO4 fractionation, two column chromatographic steps and IEF. The enzyme was found to have a molecular mass of 54 kDa, a pl of 5.1, a pH optimum between 7.5 and 8.0 and, using Leu-Ala as substrate, it gave Km and Vmax values of 0.66 mM and 0.12 μmol min-1 mg-1, respectively. No complete homology was found between the N-terminal sequence of the first 20 amino acids (MDXKXYVDLKERFLRYVKFN.) and any other published sequence, but residues 8--20 gave a 62% match with residues 9--21 of an AP from Haemophilus influenzae. The enzyme was inactivated by chelating agents, bestatin, p-hydroxymercuribenzoate and some heavy metals. Cobalt ions restored EDTA-inactivated activity but did not reverse inhibition by 1,10-phenanthroline. In addition, bestatin and 1, 10-phenanthroline had an inhibitory effect on the batch growth of F. nucleatum in a complex medium in which peptidase activities would be nutritionally essential. No such inhibition was observed in a chemically defined medium in which growth was not dependent upon peptidase activities. The peptidase described in this paper therefore appears to be a cobalt-activated metallo-AP which, together with other peptidases, is considered to be important in the survival of F. nucleatum in the subgingival environment of the mouth.
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SepA, the 110 kDa protein secreted by Shigella flexneri: two-domain structure and proteolytic activity
Shigellosis is characterized by a strong inflammatory response which is induced by bacteria invading the colonic mucosa. Characterization of a sepA mutant indicated that SepA, the major protein secreted by Shigella flexneri growing in laboratory media, might be involved in invasion and destruction of the host intestinal epithelium. The sequence of the first 500 residues of mature SepA (110 kDa) is homologous to that of the N-terminal region of lgA1 proteases. To investigate the potential proteolytic activity of SepA, the activity of the purified protein on a wide range of synthetic peptides was tested. SepA hydrolysed several of these substrates and the activity was inhibited by PMSF. Several peptides which were hydrolysed by SepA have been described as specific substrates for cathepsin G, a serine protease produced by polymorphonuclear leukocytes that was proposed to play a role in inflammation. However, unlike cathepsin G, SepA degraded neither fibronectin nor angiotensin I and had no effect on aggregation of human platelets. In addition, analysis of SepA hydrolysis by proteinase K suggested that the protein is composed of two domains of about 450 residues separated by a hinge region of 100 residues. The 47 kDa N-terminal domain was stable and endowed with proteolytic activity.
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- Biotechnology
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High exogenous concentrations of phenoxyacetic acid are crucial for a high penicillin V productivity in Penicillium chrysogenum
More LessA high-penicillin-yielding strain of Penicillium chrysogenum was grown in continuous culture on a chemically defined medium with glucose as the growth-limiting component. The cultivations were operated at a constant dilution rate of 0.05 h-1 and the feed concentration of the penicillin V sidechain precursor phenoxyacetic acid was varied between 0 and 6.5 g l-1. Subsequent formation of penicillin V and by-products related to the penicillin biosynthetic pathway was monitored at steady state. It was established that the concentration of phenoxyacetic acid in the growth medium had to be kept high to obtain a high productivity of penicillin V. The specific production rate of penicillin V as a function of the phenoxyacetic acid concentration followed Michaelis--Menten-type kinetics, from which an overall apparent Km value of 42 mM for the incorporation of intracellular phenoxyacetic acid into penicillin V could be obtained. High phenoxyacetic acid concentrations tended to lower the formation of the by-products 6-aminopenicillanic acid and 8-hydroxypenillic acid. Furthermore the undesirable loss of the pathway intermediate isopenicillin N into the extracellular medium was lowered, whereas the opposite effect was observed for the pathway intermediate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine and the by-product 6-oxo-piperidine-2-carboxylic acid, the δ-lactam form of α-aminoadipic acid.
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- Development And Structure
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Structural differences between two types of basidiomycete septal pore caps
The septal pore cap (SPC) of Trichosporon sporotrichoides CBS 8245 is vesicular-tubular, connected with flat-tubular endoplasmic reticulum (ER), and stains densely with zinc/iodine/osmium tetroxide, as does the ER. The SPC of Schizophyllum commune CBS 340.81 is more complex, about 600 nm in diameter, with perforations of 80--120 nm diameter, and stains less densely with zinc/iodine/osmium tetroxide than the ER. In high-pressure frozen and freeze-substituted hyphae of T. sporotrichoides the ER is present parallel to the dolipore septa, and electron-dense material occurs opposite the septal pore channel; the SPC rarely showed smooth vesicular-tubular membranes, suggesting that this is an ephemeral function of the SPC. The SPC of S. commune has a smooth outer and inner membrane, which enclose a matrix with a palisade-like substructure. A thin layer of electron-dense material covers the inner surface of the SPC of S. commune, from which beaded filamentous structures connect the SPC and the pore-occluding material. These filamentous structures may maintain the intracellular position of the SPC and possibly play a role in plugging the septal pore channel. The septal pore swellings of T. sporotrichoides contain more 1,6--glucan than the septum, and intracellular glucans are also present near the septal pore channel. This cytosolic 1,6--glucan in T. sporotrichoides may serve as a matrix to keep the tubular membranous structures of the SPC together. In contrast, 1,6--glucan is not observed in the SPC and in the pore-occluding material of S. commune, and hyphal septa of this species show less labelling of 1,6--glucan than the septal swelling. The evolutionary transition from simple to more complex types of SPCs may have resulted in a requirement for different components to maintain the morphological integrity and cell biological function.
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The aberrant positioning of nuclei and the microtubular cytoskeleton in Saccharomyces cerevisiae due to improper actin function
More LessAn excentric position of the nuclei, random orientation of mitoses, and multinuclear budding cells were identified in part of a population of temperature-sensitive (ts) Saccharomyces cerevisiae actin mutants at the permissive temperature of 23 ° by fluorescence and electron microscopy. The phenotype resembled that of mutants in -tubulin, dynein, JNM1, NUM1, ACT3, ACT5, myosins, profilin, tropomyosin 1, SLA2 and other genes. The question was addressed whether the cause was (i) defects in cell polarity in some ts actin mutants, manifested by lack of asymmetry of actin cortical patches, or (ii) lack of cytoplasmic or astral microtubules. The results indicated that in the cells with the nuclear defects, actin cortical patches showed the normal asymmetric distribution typical of undisturbed polarity. Cytoplasmic, astral and spindle microtubules were also preserved. The principal difference found between the wild-type and actin mutant cells was in actin cables, which in the actin mutants were developed insufficiently. It is suggested that actin cables serve as a ‘suspensory apparatus’ and/or ‘intracellular corridor’, predetermining: the location of the nucleus in the central position in interphase; the axis of nuclear movement to the bud neck before mitosis; the direction of the elongating nucleus during mitosis; and the motion of each nucleus from an excentric to a central position during cytokinesis, in cooperation with the above-mentioned and other gene products.
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A transposition-induced mutant of Nostoc ellipsosporum implicates an arginine-biosynthetic gene in the formation of cyanophycin granules and of functional heterocysts and akinetes
More LessIn strain NE1 of Tn5-1058-mutagenized Nostoc ellipsosporum, the transposon was found within a gene whose translation product is similar in amino acid sequence to the arginine-biosynthetic protein N-acetylglutamate semialdehyde dehydrogenase encoded by argC of Bacillus subtilis. The argC reported from Anabaena sp. strain PCC 7120 hybridized to a sequence different from the one interrupted by the transposon in NE1. The newly identified gene from N. ellipsosporum was denoted argL. The argL mutation renders certain processes in strain NE1 conditionally dependent on provision of L-arginine. Heterocysts and apparent akinetes that formed in the absence of added L-arginine failed to fix dinitrogen or to germinate, respectively, and lacked granules of cyanophycin, composed of copolymers of arginine and aspartic acid. However, apparent akinetes that differentiated upon growth of the mutant in the presence of L-arginine plus nitrate formed cyanophycin granules and could regenerate a new culture.
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- Environmental Microbiology
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Aggregation in Azospirillum brasilense: effects of chemical and physical factors and involvement of extracellular components
More LessA medium for consistent induction of aggregation of Azospirillum brasilense cells was developed and used to study the effects of chemical and physical factors as well as extracellular components involved in this phenomenon. Growth of A. brasilense strain Cd in a high C:N medium using fructose and ammonium chloride as C and N sources, respectively, resulted in flocculation visible to the naked eye after 24 h. No cell aggregates were formed after 72 h growth in low C:N medium. Aggregating cells, but not cells grown under low C:N, accumulated high amounts of poly--hydroxybutyrate and the cell envelope contained a well-defined electron-dense layer outside the outer membrane. Suspending the aggregates in 0.2 or 0.5 M urea was the only treatment effective for disrupting aggregates. The concentration of exopolysaccharide produced by four different strains of A. brasilense, differing in their capacity to aggregate, strongly correlated with the extent of aggregation. Electrophoretic protein profiles from different fractions of aggregating and non-aggregating cells were compared. Differences were observed in the pattern of low-molecular-mass proteins and in the polar flagellin that has previously been proposed to be involved in adhesion processes. However, a mutant lacking both lateral and polar flagella showed the strongest aggregation. The involvement of polysaccharides and/or proteins in aggregation of A. brasilense is discussed.
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- Genetics And Molecular Biology
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Regulation of the spvR gene of the Salmonella typhimurium virulence plasmid during exponential-phase growth in intracellular salts medium and at stationary phase in L broth
More LessThe authors previously showed that the SpvR-regulated spvABCD operon of the Salmonella typhimurium virulence plasmid is highly induced during exponential-phase growth by salmonellae intracellularly in mammalian cells and in a medium designed to mimic the intracellular environment of mammalian cells, intracellular salts medium (ISM), as well as at stationary phase in L broth (LB). The most relevant signal(s) for spv gene expression in vivo is not known. To elucidate the means by which salmonellae regulate the spv genes in response to the environment during the disease process, expression of the spvR gene, encoding the positive regulatory protein SpvR, was examined under these same growth conditions by using RNAse-protection analysis, spvR was expressed at a low, basal level during exponential growth in LB but was induced during exponential growth in ISM and during stationary phase in LB, the same conditions that increased expression of the spvABCD operon. Basal expression of spvR during exponential growth in LB was independent of both SpvR and the alternative sigma factor RpoS, whereas maximal induction of spvR was dependent on both SpvR and RpoS. In an RpoS-background, spvR message was decreased in stationary phase, whereas spvR exhibited residual RpoS-independent induction during exponential growth in ISM. Deletion of spvA from the virulence plasmid of S. typhimurium increased expression of spvR during stationary phase in LB, but not during exponential growth in ISM. These results suggest that expression of spvR is controlled by different regulatory factors, depending on the growth conditions encountered by the salmonellae.
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The role of the Corynebacterium glutamicum rel gene in (p)ppGpp metabolism
To investigate the metabolism of (p)ppGpp in amino-acid-producing coryneform bacteria, a PCR-based strategy using degenerate consensus oligonucleotides was applied to isolate the rel gene of Corynebacterium glutamicum ATCC 13032. The gene consists of 2283 nucleotides and encodes a protein of 760 amino acids with a molecular mass of 84.4 kDa. The amino acid sequence revealed extensive similarities to the related proteins RelA and SpoT of Escherichia coli, which are known to be involved in (p)ppGpp biosynthesis and degradation. The C. glutamicum rel gene is located downstream of the apt gene encoding an adenine phosphoribosyltransferase, and an ORF with similarities to dciAE, which represents part of a dipeptide transport system in E. coli. A C. glutamicum mutant strain carrying a defined deletion in the rel gene was constructed. This mutant failed to accumulate (p)ppGpp in response to amino acid starvation. When overexpressed in E. coli, the C. glutamicum rel gene was able to reverse growth defects caused by an overexpressed relA gene. It is proposed that the C. glutamicum rel gene encodes a bifunctional enzyme with (p)ppGpp synthetase and (p)ppGpp-degrading activities.
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A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum
More LessA simple system has been developed for generating Corynebacterium glutamicum strains containing stable replicative plasmids integrated into the chromosome via homologous recombination. The system is based upon extremely strong incompatibility between two plasmids, which cannot be comaintained even under antibiotic selective pressure. Integration of the resident plasmid that contained the trpD gene of C. glutamicum was achieved by introduction of a second plasmid and subsequent selection for the maintenance of both plasmids. Plasmid integrates positive for both plasmid markers were obtained at a frequency about 10-3 of the normal transformation frequency with selection for the maintenance of only the second plasmid. Southern analysis revealed that the integration had occurred through a single-crossover homologous recombination between the trpD regions of the host genome and the plasmid. On the basis of the Campbell-type integration, chromosome walking was attempted by using Escherichia coli replication origins that were also present in the integrated plasmid. The chromosomal DNA was digested, ligated, and used to transform E. coli, which enabled recovery of the expected adjacent genomic DNA regions. The plasmid integrate was stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated sequences carrying a replicon active in the host were maintained as a stable chromosomal insert in C. glutamicum.
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Evidence of past recombination events among the genes encoding the Erp antigens of Borrelia burgdorferi
More LessA single Borrelia burgdorferi bacterium may contain six or more different 32 kb circular plasmids (cp32s). Although these plasmids are homologous throughout much of their sequences, two loci have been identified at which they can vary significantly. The cp32 plasmids and their relatives each contain two adjacent genes, orfC and orf3, that vary in sequence between plasmids found within clones of individual bacteria. The orfC gene product is homologous to proteins involved in partitioning of bacterial plasmids, and the differences at this locus between plasmids may account for their compatibility. The orfC-orf3 loci are located approximately 5 kb from another variable locus called erp. The orfC-orf3 loci were used as physically linked markers to assess genetic rearrangements in the erp loci; this revealed examples of recombination involving both individual genes and entire erp loci. Recombination of the genes encoding the Erp antigens might contribute to the evasion of the mammalian immune response and could play roles in the establishment and persistence of B. burgdorferi infections in mammalian hosts.
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Sirohaem sulfite reductase and other proteins encoded by genes at the dsr locus of Chromatium vinosum are involved in the oxidation of intracellular sulfur
More LessThe sequence of the dsr gene region of the phototrophic sulfur bacterium Chromatium vinosum D (DSMZ 180T) was determined to clarify the in vivo role of ‘reverse’ sirohaem sulfite reductase. The dsrAB genes encoding dissimilatory sulfite reductase are part of a gene cluster, dsrABEFHCMK, that encodes four small, soluble proteins (DsrE, DsrF, DsrH and DsrC), a transmembrane protein (DsrM) with similarity to haem-b-binding polypeptides and a soluble protein (DsrK) resembling [4Fe---4S]-cluster-containing heterodisulfide reductase from methanogenic archaea. Northern hybridizations showed that expression of the dsr genes is increased by the presence of reduced sulfur compounds. The dsr genes are not only transcribed from a putative promoter upstream of dsrA but primary transcripts originating from (a) transcription start site(s) downstream of dsrB are also formed. Polar insertion mutations immediately upstream of dsrA, and in dsrB, dsrH and dsrM, led to an inability of the cells to oxidize intracellularly stored sulfur. The capability of the mutants to oxidize sulfide, thiosulfate and sulfite under photolithoautotrophic conditions was unaltered. Photoorganoheterotrophic growth was also unaffected. ‘Reverse’ sulfite reductase and DsrEFHCMK are, therefore, not essential for oxidation of sulfide or thiosulfate, but are obligatory for sulfur oxidation. These results, together with the finding that the sulfur globules of C. vinosum are located in the extracytoplasmic space whilst the dsr gene products appear to be either cytoplasmic or membrane-bound led to the proposal of new models for the pathway of sulfur oxidation in this phototrophic sulfur bacterium.
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Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger
More LessAcetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.
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A novel filamentous phage, fs-2, of Vibrio cholerae 0139
More LessA novel filamentous bacteriophage, fs-2, was isolated from Vibrio cholerae O139 strain MDO14. The fs-2 phage was a long filamentous particle 1200 nm long and 7 nm wide. The purified phage formed a turbid plaque when spotted on a lawn of the host organisms. The plaque-formation activity was stable following heating to 70 � but was inhibited by treatment with chloroform. fs-2 had a single-stranded DNA genome and was converted to a double-stranded replicative form in the host cell. Almost all V. cholerae O139 and O1 El Tor biotype strains tested were sensitive to the phage, but most O1 classical strains and non-O1 non-O139 strains were resistant. The fs-2 genome comprised 8651 nucleotides containing nine open reading frames, five of which had predicted protein products partially homologous to the reported protein products of other filamentous phages. Although the extent of the homology was not particularly high, the genetic organization of other filamentous phages appears to be preserved in fs-2. The phage was not integrated into the chromosome of its host, but a 715 nucleotide fragment located in the large intergenic region of fs-2 was highly homologous to a part of region RS2 (repetitive sequence 2) of the V. cholerae CTX sequence which is speculated to be required for integration of the phage into the V. cholerae chromosome at a specific site.
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Degradation pathway of CopA, the antisense RNA that controls replication of plasmid R1
More LessRNA decay in bacteria is carried out by a number of enzymes that participate in the coordinated degradation of their substrates. Endo- and exonucleolytic cleavages as well as polyadenylation are generally involved in determining the half-life of RNAs. Small, untranslated antisense RNAs are suitable model systems to study decay. A study of the pathway of degradation of CopA, the copy number regulator RNA of plasmid R1, is reported here. Strains carrying mutations in the genes encoding RNase E, polynucleotide phosphorylase (PNPase), RNase II and poly(A) polymerase I (PcnB/PAP I) -- alone or in combination -- were used to investigate degradation patterns and relative half-lives of CopA. The results obtained suggest that RNase E initiates CopA decay. Both PNPase and RNase II can degrade the major 3-cleavage product generated by RNase E. This exonucleolytic degradation is aided by PcnB, which may imply a requirement for A-tailing. RNase II can partially protect CopA's 3′-end from PNPase-dependent degradation. Other RNases are probably involved in decay, since in rnblpnp double mutants, decay still occurs, albeit at a reduced rate. Experiments using purified RNase E identified cleavage sites in CopA in the vicinity of, but not identical to, those mapped in vivo, suggesting that the cleavage site specificity of this RNase is modulated by additional proteins in the cell. A model of CopA decay is presented and discussed.
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Probing the receptor recognition site of the FimH adhesin by fimbriae-displayed FimH---FocH hybrids
More LessType 1 fimbriae are surface organelles of Escherichia coli which mediate D-mannose-sensitive binding to different host surfaces. This binding is conferred by the minor fimbrial component FimH. The binding domain of the FimH adhesin has been studied by constructing hybrids of FimH and a homologous protein, FocH, originating from F1C fimbriae. F1C fimbriae do not bind to D-mannosides or confer agglutination of any known types of erythrocytes or yeast. It was previously shown that the FocH protein can be readily substituted by the FimH adhesin, resulting in hybrid fimbriae with the same binding characteristics as type 1 fimbriae. The receptor binding of fimbriae-presented chimeric FimH--FocH hybrids was studied. FimH--FocH fusions encompassing 72% of the N-terminus of FimH fused to the complementary sector of FocH conferred agglutination of erythrocytes and yeast cells at a comparable level to FimH. Surprisingly, it was also found that similar fusions containing between 56 and 66% FimH still conferred binding to yeast cells, D-mannose--BSA and D-mannose--beads but did not give rise to agglutination. The receptor binding capacity of fusions containing 50% or less of the FimH N-terminal region was virtually abolished. The results point to the presence of a D-mannose-receptor-binding core domain in FimH, the affinity of which is modulated by other sectors of the protein to enable binding to extended mannose-containing targets.
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Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae
More LessMycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.
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Rhizobium leguminosarum contains a group of genes that appear to code for methyl-accepting chemotaxis proteins
More LessMethyl-accepting chemotaxis proteins (MCPs) play important roles in the chemotactic response of many bacteria. Oligonucleotide primers designed to amplify the conserved signalling domain of MCPs by PCR were used to identify potential MCP-encoding genes in Rhizobium leguminosarum. Using a PCR-derived probe created from these primers a genomic library of R. leguminosarum VF39SM was screened; at least five putative MCP-encoding genes (termed mcpB to mcpF) were identified and isolated from the library. One of these putative genes (mcpC) is located on one of the indigenous plasmids of VF39SM. Fifteen different cosmids showing homology to an mcpD probe were also isolated from a genomic library. The complete DNA sequences of mcpB, mcpC and mcpD were obtained. All three genes code for proteins with characteristics typical of MCPs. However, the protein encoded by mcpB has a relatively large periplasmic domain compared to that in other MCPs. Partial DNA sequences of mcpE and mcpF had strong similarity to sequences from the methylation domains of known MCPs. Mutants defective in mcpB, mcpC, mcpD or mcpE were created using insertional mutagenesis strategies. Mutation of mcpB resulted in impairment of chemotaxis to a wide range of carbon sources on swarm plates; phenotypes for the other three mutants have yet to be elucidated. The mcpB, mcpC and mcpD mutants were tested for loss of nodulation competitiveness. When co-inoculated with the wild-type, the mcpB and mcpC mutants formed fewer nodules than the wild-type, whereas the mcpD mutant was just as competitive as the wild-type. The results overall suggest that R. leguminosarum possesses mcp-like genes, and that at least some of these play a role in early steps in the plant-microbe interaction.
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A swarming-defective mutant of Proteus mirabilis lacking a putative cation-transporting membrane P-type ATPase
More LessThe motile TnphoA mutant IC24 of Proteus mirabilis U6450 generates an aberrant swarming colony, and was shown to be impaired in swarm cell differentiation, i.e. cell elongation and hyperflagellation, causing delayed and slower population migration across a solid growth medium. Levels of transcript from the flagellin filament gene fliC, the flagellar master operon flhDC, and the leucine-responsive regulatory protein gene Irp, a regulator of swarming differentiation, were reduced in IC24 mutant swarm cells. The transposon had inserted into a gene encoding a putative P-type ATPase closely related to those transporting cations across bacterial membranes. This ppa gene (Proteus P-type ATPase) was maximally expressed in differentiated swarm cells. The data suggest an effect of ion homeostasis on swarm cell differentiation, possibly mediated via the Irp--flhDC pathway.
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Transposon mutagenesis with IS6100 in the avermectin-producer Streptomyces avermitilis
More LessThe insertion sequence IS6100 was shown to undergo intermolecular transposition from a temperature-sensitive delivery plasmid to the genome of the avermectin-producer Streptomyces avermitilis, creating cointegrates. Evidence from both Southern hybridization and the range of auxotrophic mutations present in a transposon library was consistent with random transposition. It was not possible to increase transposase expression by readthrough transcription from a copy of the tipA promoter located adjacent to the insertion sequence. This was in part due to the absence of a homologue of the Streptomyces lividans transcriptional activator TipAL in S. avermitilis. However, recombinant S. avermitilis strains carrying the S. lividans tip operon were also deficient for induction of the promoter. The frequency of reversion of different auxotrophic mutations by precise excision, involving recombination across 8 bp direct repeats, was shown to vary by at least five orders of magnitude. This dependence of recombination frequency on chromosomal location may contribute to the stability of repetitive modular type I polyketide biosynthetic genes.
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Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator
More LessThe nucleotide sequence of a 2·4 kb BamHI--Sall fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.
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- Genomics
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Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes
More LessThe authors previously identified large plasmids in Mycobacterium xenopi, M. branderi and M. celatum which appeared to have a linear topology. This study has confirmed the presence of such linear plasmids in mycobacteria, including M. avium, and demonstrated that the ends of these replicons are covalently bound with protein(s), suggesting an invertron-like structure. The termini of one 25 kb plasmid, designated pCLP, from M. celatum were cloned and the first 500 bp of each terminus were sequenced. The termini of this plasmid show the characteristic features of invertrons with terminal inverted repeats of 45 bp (with imperfect matches) and several palindromic sequences. Moreover, similarity existed in the structure and terminal nucleotide sequence of pCLP and the termini of linear replicons of Streptomyces and Rhodococcus species, indicating a conservation of these linear extrachromosomal elements within the Actinomycetales.
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- Pathogenicity And Medical Microbiology
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Magnesium transport in Salmonella typhimurium: regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells
More LessSalmonella typhimurium contains two inducible Mg2+ transport systems, MgtA and MgtB, the latter encoded by a two-gene operon, mgtCB. Mg2+ deprivation of S. typhimurium increases transcription of both mgtA and mgtCB over a thousandfold and a similar increase occurs upon S. typhimurium invasion of epithelial cells. These increases are mediated by the phoPQ two-component signal transduction system, an essential system for S. typhimurium virulence. It was therefore hypothesized that expression of MgtA and MgtCB is increased upon invasion of eukaryotic cells because of a lack of intravacuolar Mg2+. However, when S. typhimurium was grown at pH 5.2, the capacity of the constitutive CorA transporter in mediating Mg2+ was greater than that at pH 7.4. Furthermore, induction of mgtA and mgtCB transcription was greater in the presence of a wild-type corA allele than in its absence. This implies that intravacuolar S. typhimurium could obtain sufficient Mg2+ via the CorA system. The effect of acid pH on mgtA and mgtCB transcription was also measured. Compared to induction at pH 7.4, exposure to pH 5.2 almost completely abolished induction of mgtA at low Mg2+ concentrations but diminished induction of mgtCB only twofold. Adaptation of cells to acid pH by overnight growth resulted in normal levels of induction of mgtA and mgtCB at low Mg2+ concentrations. These results imply an additional level of regulation for mgtA that is not present for mgtCB. Conversely, repression of mgtA and mgtCB expression by increased extracellular Mg2+ was relatively insensitive to acid. Transcription of both loci was strongly induced upon invasion of the Hep-2 or CMT-93 epithelial-like or J774 macrophage-like cell lines. However, the presence or absence of functional alleles of either or both mgtA or mgtCB had no effect on invasion efficiency or short-term survival of S. typhimurium within the eukaryotic cells. It was concluded that the strong Mg2+-dependent induction of mgtA and mgtCB upon invasion of eukaryotic cells is not required because S. typhimurium lacks sufficient Mg2+ during eukaryotic cell invasion and initial intravacuolar growth.
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Actin enhances the haemolytic activity of Escherichia coli
Act in is a major cytoskeletal protein of mammalian muscle and non-muscle cells. Exposure of cells to soluble factors that damage cell membranes results in the release of actin into the extracellular spaces. The α-haemolysin (HlyA) of Escherichia coli is the prototype RTX (repeat in toxin) toxin and is thought to be important in virulence because of its ability to lyse cells by formation of pores in the cell membrane. These studies were conducted to determine if actin influences growth and haemolytic activity of E. coli. Growth of E. coli in the presence of actin resulted in culture supernatant haemolytic activity that was 2.4-, 2.7- and 3.3-fold greater than that of E. coli grown in medium containing BSA, non-supplemented medium, or medium containing heat-denatured actin, respectively. The enhanced haemolytic activity occurred only when actin was present during the growth phase and there was no effect when actin was added to culture supernatants containing haemolysin. The increased haemolytic activity by actin was concentration-dependent, detectable in early-exponential-phase growth, and associated with increased concentrations of secreted HlyA by Western blotting. Actin induced a 2.9-fold increase in alkaline phosphatase activity in E. coli CC118 with a TnphoA insertion in the hlyB determinant of the recombinant haemolysin piasmid pWAM04. These results indicate that extracellular actin enhances haemolysin production by E. coli and may have implications in the pathogenesis of E. coli infections.
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- Physiology And Growth
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Changes in fatty acid composition of Neurospora crassa accompany sexual development and ascospore germination
More LessFatty acid composition was determined during several stages of sexual development in Neurospora crassa. Triacylglycerol was the predominant acyl lipid in cultures undergoing sexual development. The absolute amounts of triacylglycerol in fertilized cultures varied over time, in contrast to control (unfertilized or mock-fertilized) cultures, in which the amount of triacylglycerol decreased linearly with age. In cultures competent to undergo sexual development, -linoleate was the predominant fatty acid, ranging from 53 to 65% of the total fatty acid mass. -Linolenate was 3% or less of the total fatty acid, in marked contrast to the much higher levels (10--35%) typically reported for vegetative cultures. In fertilized cultures, a slightly higher mass ratio of oleate was also observed. This difference was due to the developing asci: in developing asci and mature ascospores, oleate replaced α-linoleate as the predominant fatty acid (45 to 50% of the total). In germinating ascospores, the fatty acid composition approached that of vegetative cultures 6 h after inducing germination by heat activation. These results show that the fatty acid composition of sexual tissues of Neurospora differs substantially from the composition of asexual tissues, and that extensive changes in fatty acid composition correlate with several events in the sexual stage of development.
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Antioxidant vitamins C and E affect the superoxide-mediated induction of the soxRS regulon of Escherichia coli
More LessThe mechanism of activation of Escherichia coli redox sensory protein SoxR still unclear: a [2Fe--25] cluster contained in a SoxR dimer is potentially redo sensitive, but the nature of the signal is unknown. Antioxidant vitamins C (ascorbate) and E (α-tocopherol) were used to explore the mechanism of activation of the SoxR protein in vivo. Treating E. coli cells with ascorbate o α-tocopherol increased their tolerance to paraquat (PQ, a redox-cycling compound), even in the absence of the soxRS locus, suggesting a radical-quenching activity. When using a soxS’:: lacZ fusion, whose expression is governed by activated SoxR, ascorbate and α-tocopherol also prevented the expression of α-galactosidase after PQ treatment. A secondary activity was observed in cells carrying soxR101, a mutation resulting in the constitutive expression of the sox regulon, where the overexpression of soxS’::lacZ was also reduced by ascorbate or α-tocopherol treatment. Additionally, different mechanisms of action were revealed as α-tocopherol was capable of preventing both PQ and menadione (MD) lethality, whilst ascorbate prevent PQ lethality but increased MD-mediated cell death. It is proposed that α-tocopherol, positioned in membranes, can prevent superoxide-dependent membrane damage; however, water-soluble ascorbate is unable to do so an can even increase the concentration of oxygen radicals reacting with release membrane-associated Fe(ll).
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Phenotypic variation of lipid composition in Burkholderia cepacia: a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid
More LessBurkholderia cepacia produces an unusual range of polar lipids, which includes two forms each of phosphatidylethanolamine (PE) and ornithine amide lipid (OL), differing in the presence or absence of 2-hydroxy fatty acids. By using chemostat cultures in chemically defined media, variations in the lipid content and the proportions of individual lipids have been studied as a function of (a) growth temperature, (b) growth rate and (c) growth-limiting nutrient (carbon, magnesium, phosphorus or oxygen). Total cellular lipid in carbon-limited cultures was lowest at high growth temperatures and low growth rates. Increases in growth temperature over the range 25--40 ° led to increases in the proportions of molecular species of PE and OL containing 2-hydroxy acids, without changing the PE: OL ratio. Growth temperature did not alter the balance between neutral and acidic lipids, but the contribution of phosphatidylglycerol to the latter increased with rising growth temperature and growth rate. Pigmentation of cells and the presence of flagella were also temperature-dependent. Change in growth rate also affected the PE: OL ratio and the extent to which monoenoic acids were replaced by their cyclopropane derivatives. Whereas similar lipid profiles were found for carbon-, magnesium-and oxygen-limited cultures, ornithine amides were the only polar lipids detected in phosphorus-limited cells.
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The catecholate siderophores of Azotobacter vinelandii: their affinity for iron and role in oxygen stress management
More LessIn iron-limited medium, Azotobacter vinelandii strain UW produces three catecholate siderophores: the tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate aminochelin. Each siderophore was found to bind Fe3+ preferentially to Fe2+, in a ligand:Fe ratio of 1:1, 3:2 and 3:1, respectively. Protochelin had the highest affinity for Fe3+, with a calculated proton-independent solubility coefficient of 10439, comparable to ferrioxamine B. Iron-limited wild-type strain UW grown under N2-fixing or nitrogen-sufficient conditions hyper-produced catecholate siderophores in response to oxidative stress caused by high aeration. In addition, superoxide dismutase activity was greatly diminished in iron-limited cells, whereas catalase activity was maintained. The ferredoxin I (Fdl)-negative A. vinelandii strain LM100 also hyper-produced catecholates, especially protochelin, under oxidative stress conditions, but had decreased activities of both superoxide dismutase and catalase, and was about 10 times more sensitive to paraquat than strain UW. Protochelin and azotochelin held Fe3+ firmly enough to prevent its reduction by.O- 2 and did not promote the generation of hydroxyl radical by the Fenton reaction. Ferric-aminochelin was unable to resist reduction by O- 2 and was a Fenton catalyst. These data suggest that under iron-limited conditions, A. vinelandii suffers oxidative stress caused by.O- 2. The catecholate siderophores azotochelin, and especially protochelin, are hyper-produced to offer chemical protection from oxidative damage catalysed by.O- 2 and Fe3+. The results are also consistent with Fdl being required for oxidative stress management in A. vinelandii.
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Starvation recovery of Staphylococcus aureus 8325-4
More LessNutrient limitation of Staphylococcus aureus induces a starvation-survival state which enables it to survive until sufficient nutrients become available to support growth. The response of starved S. aureus cells to nutritional upshift was analysed to characterize the recovery mechanism which results in the resumption of rapid growth. S. aureus 8325-4 starved for 7 d in a chemically defined medium limited for glucose was able to resume growth upon the addition of complex medium (brain heart infusion broth) or a mixture of amino acids and glucose. The addition of either glucose or amino acids alone did not lead to recovery of cells. Prior to the first cell division event, a lag period of about 120--150 min was observed, the duration of which was independent of the length of starvation survival. During this lag period, RNA synthesis increased immediately upon the addition of nutrients whilst protein synthesis was delayed by approximately 5 min. Cells rapidly enlarged within 30 min of recovery, and initiation of chromosome replication could be detected after 90 min. Changes in the profile of proteins expressed during the recovery period revealed that several starvation-specific proteins were down-regulated within 30 min, whilst other proteins were common to both starvation and recovery. Two proteins were identified which were only transiently expressed during the first 60 min of recovery. Protein synthesis could be detected during recovery even if the cells had been treated with the RNA synthesis inhibitor rifampicin for 30 min prior to the addition of recovery nutrients, demonstrating that several proteins are translated from long-lived mRNA transcripts present in starved cells.
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Phenol hydroxylase cloned from Ralstonia eutropha strain E2 exhibits novel kinetic properties
More LessRalstonia eutropha strain E2 (previously Alcaligenes sp.) is a phenol-degrading bacterium expressing phenol-oxygenating activity with a low Ks (the apparent half-saturation constant in Haldane's equation) and an extremely high KSI (the apparent inhibition constant). To identify the molecular basis for these novel cellular kinetic properties, a 9.5 kb DNA fragment that allowed Pseudomonas aeruginosa PAO1c (Phl- Cat+) to grow on phenol as the sole carbon source was cloned from strain E2 into plasmid pRO1614. PAO1c harbouring this plasmid (designated pROE217) transformed phenol to catechol, indicating that this fragment contains gene(s) for phenol hydroxylase. The cloned genes consist of eight complete ORFs, designated poxRABCDEFG. The products are homologous to those of dmpRKLMNOPQ of Pseudomonas sp. CF600, sharing 30--65% identity: this suggests that the phenol hydroxylase is a multicomponent enzyme. The kinetic constants for phenol-oxygenating activity of PA01c(pROE217) were determined, and these were compared with those of strain E2. The kinetic constants of PAO1c derivatives expressing different phenol hydroxylases were also determined. A comparison of these kinetic data suggests that phenol hydroxylase, the first enzyme in the phenol-degradative pathway, determines Ks and KSI values for the cellular phenol-oxygenating activity. It is thus suggested that the phenol hydroxylase cloned from strain E2 exhibits the novel kinetic properties that were observed with intact cells of strain E2.
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Oxygen protection of nitrogen fixation in free-living Azorhizobium caulinodans: the role of cytochrome aa3
The growth properties of Azorhizobium caulinodans wild-type and a cytochrome aa3 mutant strain, both growing with N2 as N source at fixed dissolved partial oxygen pressures in the range 0.5--4.0 kPa, were studied by making use of continuous cultures (chemostats and pH-auxostats) and transient cultures. In succinate-limited chemostats, the wild-type exhibited a higher growth yield than the aa3 mutant at every dissolved oxygen tension tested, indicating activity of cytochrome aa3 in this entire oxygen regime. The growth yield of both the wild-type and the aa3 mutant declined when the dissolved oxygen tension was raised. In contrast, for growth on ammonia at the same dilution rate, the wild-type showed an increase in growth yield with increasing dissolved oxygen tension, whereas the growth yield of the aa3 mutant remained constant. The transient changes in growth properties observed in chemostat cultures after pulsing with succinate pointed to a negative effect of oxygen on the maximum specific growth rate. This was studied further in steady-state pH-auxostat cultures. The specific growth rate of both strains decreased with increasing dissolved oxygen tension. The less steep decline in growth rate of the wild-type compared to the aa3 mutant confirmed that cytochrome aa3 is active in the wild-type. Again, the growth yield of both strains decreased with the dissolved oxygen tension, but in contrast to the results obtained with chemostats, no difference in growth yield was observed between wild-type and mutant at any oxygen tension. In either type of continuous culture a decrease in the overall P/O ratio with increasing dissolved oxygen tension is improbable for the wild-type, and even more so for the aa3 mutant. Therefore, the adverse effects of oxygen on the growth of A. caulinodans are not readily explained by respiratory protection; alternatively, it is proposed that the catalytic oxidation of nitrogen-fixation-specific redox enzymes by oxygen (auto-protection) enables the bacterium to deal with intracellular oxygen at the expense of reducing equivalents and free energy. To compensate for the loss of free energy, respiration increases and an active cytochrome aa3 contributes to this by keeping the P/O ratio high.
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