- Volume 143, Issue 8, 1997
Volume 143, Issue 8, 1997
- Microbiology Comment
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- Biochemistry
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The form of folate affects the mechanisms of methotrexate resistance in Enterococcus faecium
More LessSummary: Several mechanisms have been described to explain the resistance of cells to methotrexate (MTX); however, the basis for the heterogeneity of mechanisms has been obscure. It was hypothesized that the type of MTX resistance in a single species can be influenced by the form of extracellular folate supplied during the development of resistance. Two strains of MTX-resistant Enterococcus faecium were developed by transferring the bacteria to media containing increasing concentrations of MTX in the presence of constant concentrations of either 5-formyl-5,6,7,8-tetrahydropteroylglutamic acid (5-HCO-H4PteGlu) or pteroylglutamic acid (PteGlu). These resistant strains were designated E. faecium/MTX/5-HCO-H4PteGlu and E. faecium/MTX/PteGlu, respectively. The mechanisms of MTX resistance included: (1) increased folic acid reductase (FAR) activity in both resistant strains but increased dihydrofolate reductase (DHFR) activity only in E. faecium/MTX/PteGlu; (2) decreased synthesis and intracellular retention of MTX containing two glutamyl residues; (3) decreased uptake of MTX accompanied by decreased uptake of folates; and (4) reduction of folate-binding capacity. Among these, the form of folate present in the media during the development of resistance affected DHFR and FAR activities and the transport of folates. These findings, together with data from other laboratories, suggest that it may be important to use a reduced form of folate, a more physiological form than oxidized PteGlu, in the media during the development of resistance for the study of the mechanisms of MTX resistance in cultured cells.
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A reassessment of the genetic determinants, the effect of growth conditions and the availability of an electron donor on the nitrosating activity of Escherichia coli K-12
More LessSummary: Anaerobic, but not aerobic, cultures of Escherichia coli K-12 catalysed the rapid nitrosation of the model substrate 2,3-diaminonaphthalene when incubated with nitrite. Formate and lactate were effective electron donors for the nitrosation reaction, which was inhibited by nitrate. Optimal growth conditions for the expression of nitrosation activity by various strains and mutants were determined. Highest activities were found with bacteria that had been grown anaerobically in a minimal medium rather than in Lennox broth, with glycerol and fumarate rather than glucose as the main carbon and energy source, and in the presence of a low concentration of nitrate. Bacteria harvested in the early exponential phase were more active than those harvested in later stages of growth. Well-characterized mutants defective in the synthesis of one or more anaerobically induced electron transfer chains were screened for nitrosation activity under these optimal growth conditions: only the respiratory nitrate reductase encoded by the narGHJI operon was implicated as a major contributor to nitrosation activity. Due to the limited sensitivity of the assays currently available, a minor contribution from the two alternative nitrate reductases or even other molybdoproteins could not be excluded. The role of formate in nitrosation was complex and was clearly not limited simply to that of an electron donor in the bacterial reduction of nitrite to nitric oxide: at least two further, chemical roles were inferred. This extensive study of more than 400 independent cultures of E. coli K-12 and its derivatives resolved some, but not all, of the apparently conflicting data in the literature concerning nitrosation catalysed by enteric bacteria.
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- Bioenergetics And Transport
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Computer-based analyses of the protein constituents of transport systems catalysing export of complex carbohydrates in bacteria
More LessSummary: Bacteria synthesize and secrete an array of complex carbohydrates including exopolysaccharides (EPSs), capsular polysaccharides (CPSs), lipopolysaccharides (LPSs), lipo-oligosaccharides (LOSs) and teichoic acids (TCAs). We have analysed the families of homologous proteins that appear to mediate excretion of complex carbohydrates into or across the bacterial cell envelope. Two principal families of cytoplasmic-membrane transport systems appear to drive polysaccharide export: polysaccharide-specific transport (PST) systems and ATP-binding cassette-2 (ABC-2) systems. We present evidence that the secretion of CPSs and EPSs, but not of LPSs, LOSs or TCAs via a PST or ABC-2 system requires the presence of a cytoplasmic-membrane-periplasmic auxiliary protein (MPA1 or MPA2, respectively) in both Gram-negative and Gram-positive bacteria as well as an outer-membrane auxiliary (OMA) protein in Gram-negative bacteria. While all OMA proteins are included within a single family, MPA1 and MPA2 family proteins are not demonstrably homologous to each other, even though they share common topological features. Moreover, MPA1 family proteins (which function with PST systems), but not MPA2 family proteins (which function with ABC-2 systems), possess cytoplasmic ATP-binding domains that may either exist as separate polypeptide chains (for those from Gram-positive bacteria) or constitute the C-terminal domain of the MPA1 polypeptide chain (for those from Gram-negative bacteria). The sizes, substrate specificities and regions of relative conservation and hydrophobicity are defined allowing functional and structural predictions as well as delineation of family-specific sequence motifs. Each family is characterized phylogenetically.
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- Biotechnology
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Cadmium-specific formation of metal sulfide ‘Q-particles’ by Klebsiella pneumoniae
Summary: Klebsiella pneumoniae overcomes cadmium toxicity through the ‘biotransformation’ of cadmium ions into photoactive, nanometre-sized CdS particles deposited on the cell surface. The kinetics of particle formation during batch culture growth was monitored by electron microscopy (EM), energy-dispersive X-ray analysis and electronic absorption spectroscopy (EAS). During the deceleration phase of bacterial growth, the presence of CdS particles on the outer cell wall of K. pneumoniae (≥ 5 nm in diameter) was detected by EM. The size of these electron-dense particles continued to increase throughout the stationary phase of growth, with some of the particles reaching a diameter >200 nm. The formation of the extracellular CdS particles contributed to around 3-4% of the total cell biomass. EAS undertaken on these extracellular ‘bio-CdS’ particles suggested that the large ‘superparticles’ observed by EM, e.g. 200 nm, were aggregates of smaller particles termed ‘Q-particles’, ~ 4 nm in diameter. Metal sulfide particles were not formed in batch cultures of K. pneumoniae grown in the presence of lead, zinc, mercury, copper or silver ions. Growth in the presence of lead ions resulted in the formation of extracellular electron-dense particles containing lead but not sulfide or phosphate. Intracellular phosphorus-containing electron-opaque particles were formed during growth in the presence of copper and mercury. Intracellular electron-dense particles were formed in the presence of zinc ions but these did not contain phosphorus. From these results it was thought that metal sulfide formation in K. pneumoniae showed some cadmium-specificity. When cadmium and zinc ions were both added to the growth medium, metal sulfide particles were formed that were predominantly composed of cadmium, e.g. 48.6% cadmium and 0.04% zinc. Similarly, when cadmium and lead ions were both present during growth only CdS particles formed. In both cases analysis of the cells by EAS confirmed the presence of CdS only. These observations suggest that the mechanism of CdS formation is unlikely to occur simply through a cadmium-induced release of hydrogen sulfide by the cells into the external environment. If hydrogen sulfide production was the mechanism of sulfide formation then metal sulfide particles containing lead and zinc ions in addition to cadmium ions should have been produced.
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Expression of the eicosapentaenoic acid synthesis gene cluster from Shewanella sp. in a transgenic marine cyanobacterium, Synechococcus sp.
More LessSummary: The eicosapentaenoic acid (EPA) synthesis gene cluster isolated from a marine bacterium, Shewanella putrefaciens strain SCRC-2738, was cloned and expressed in the marine cyanobacterium Synechococcus sp. A broad-host-range cosmid vector, pJRD215 (10.2 kb, Smr Kmr), was used to clone a 38 kb insert, pEPA, containing the EPA synthesis gene cluster, creating plasmid pJRDEPA (approx. 48 kb). This plasmid was transferred to the cyanobacterial host at a frequency of 2.2 x 10−7. Cyanobacterial transconjugants grown at 29 °C produced 0.12 mg EPA (g dry weight)−1, whereas those grown at 23 °C produced 0.56 mg EPA (g dry weight)−1. The yield was further improved to 0.64 mg (g dry weight)−1 by incubation for 1 d at 17 °C. This is believed to be the first successful cloning and expression of such a large heterologous gene cluster in a marine cyanobacterium.
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- Environmental Microbiology
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Tn5041: a chimeric mercury resistance transposon closely related to the toluene degradative transposon Tn4651
Summary: This paper reports the discovery and characterization of Tn5041, a novel-type transposon vehicle for dissemination of mercury resistance in natural bacterial populations. Tn5041 (14876 bp), identified in a Pseudomonas strain from a mercury mine, is a Tn3 family mercury resistance transposon far outside the Tn21 subgroup. As in other Tn3 family transposons, Tn5041 duplicates 5 bp of the target sequence following insertion. Tn5041 apparently acquired its mer operon as a single-ended relic of a transposon belonging to the classical mercury resistance transposons of the Tn21 subgroup. The putative transposase and the 47 bp terminal inverted repeats of Tn5041 are closely related to those of the toluene degradative transposon Tn4651 and fall into a distinct subgroup on the fringe of the Tn3 family. The amino acid sequence of the putative resolvase of Tn5041 resembles site-specific recombinases of the integrase family. Besides the mer operon and putative transposition genes, Tn5041 contains a 4 kb region that accommodates a number of apparently defective genes and mobile elements.
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Adhesion of Pseudomonas aeruginosa to silicone rubber in a parallel plate flow chambe in the absence and presence of nutrient broth
More LessSummary: The physico-chemical cell-surface properties of Pseudomonas aeruginosa Al and its adhesion to silicone rubber under flow were compared for cells suspended in phosphate-buffered saline (PBS) or PBS supplemented with 2% nutrient broth. Addition of 2% nutrient broth to cells suspended in PBS yields minimal growth and did not significantly change the mean zeta potential of the organisms, which was around -13 mV. However, a comparatively larger proportion of the organisms had more negative zeta potentials in the presen of nutrient broth. This change was concurrent with a slight decrease in cell-surface hydrophobicity, as measured by water contact angles, from 119° to around 112°. The initial deposition rate of P. aeruginosa AK1 to silicone rubber, as studied in a parallel plate flow chamber, increased from 344 cm−2 s−1 in the absence of nutrient broth to 505 cm−2 s−1 in its presence. No stationary level of adhesion was observed in the presence of nutrient broth, instead the number of adhering cells increased steadily at a rate of approximately 85 cm−2 s−1. Fluorescent staining of adhering cells demonstrated that for adhesion from buffer only 2% of the adhering cells were metabolically active, whereas in case of deposition from PBS supplemented with nutrient broth, 67% of the adhering cells were metabolically active. It is concluded that the deposition rates measured in the parallel plate flow chamber with 2% nutrient broth added to the PBS suspension represent an interplay of adhesion and surface-associated growth.
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- Genetics And Molecular Biology
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Association of newly discovered IS elements with the dichloromethane utilization genes of methylotrophic bacteria
More LessSummary: Dichloromethane (DCM) dehalogenases enable facultative methylotrophic bacteria to utilize DCM as sole carbon and energy source. DCM-degrading aerobic methylotrophic bacteria expressing a type A DCM dehalogenase were previously shown to share a conserved 4.2 kb BamHI DNA fragment containing the dehalogenase structural gene, dcmA, and dcmR, the gene encoding a putative regulatory protein. Sequence analysis of a 10 kb DNA fragment including this region led to the identification of three types of insertion sequences identified as IS 1354, IS1355 and IS1357, and also two ORFs, orf353 and orf192, of unknown function. Two identical copies of element IS 1354 flank the conserved 4.2 kb fragment as a direct repeat. The occurrence of these newly identified IS elements was shown to be limited to DCM-utilizing methylotrophs containing a type A DCM dehalogenase. The organization of the corresponding dcm regions in 12 DCM-utilizing strains was examined by hybridization analysis using IS-specific probes. Six different groups could be defined on the basis of the occurrence, position and copy number of IS sequences. All groups shared a conserved 5.6 kb core region with dcmA, dcmR, orf353 and orf192 as well as IS1357. One group of strains including Pseudomonas sp. DM1 contained two copies of this conserved core region. The high degree of sequence conservation observed within the genomic region responsible for DCM utilization and the occurrence of clusters of insertion sequences in the vicinity of the dcm genes suggest that a transposon is involved in the horizontal transfer of the DCM-utilization character among methylotrophic bacteria.
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Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases
Summary: Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using β-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity. bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungue, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J. T. (1995). Trends Biochem Sci 20, 178-179]. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CeIE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.
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The KIPHO5 gene encoding a repressible acid phosphatase in the yeast Kluyveromyces lactis: cloning, sequencing and transcriptional analysis of the gene, and purification and properties of the enzyme
More LessSummary: A secreted phosphate-repressible acid phosphatase from Kluyveromyces lactis has been purified and the N-terminal region and an internal peptide have been sequenced. Using synthetic oligodeoxyribonucleotides based on the sequenced regions, the genomic sequence, KIPHO5, encoding the protein has been isolated. The deduced protein, named KIPho5p, consists of 469 amino acids and has a molecular mass of 52 520 Da (in agreement with the data obtained after treatment of the protein with endoglycosidase H). The purified enzyme shows size heterogeneity, with an apparent molecular mass in the range 90-200 kDa due to the carbohydrate content (10 putative glycosylation sites were identified in the sequence). A 16 amino acid sequence at the N-terminus is similar to previously identified signal peptides in other fungal secretory proteins. The putative signal peptide is removed during secretion since it is absent in the mature secreted acid phosphatase. The gene can be induced 400-600-fold by phosphate starvation. Consensus signals corresponding to those described for Saccharomyces cerevisiae PHO4- and PHO2-binding sites are found in the 5′ region. Northern blot analysis of total cellular RNA indicates that the KIPHO5 gene codes for a 1.8 kb transcript and that its expression is regulated at the transcriptional level. Chromosomal hybridization indicated that the gene is located on chromosome II. The KIPHO5 gene of K. lactis is able to functionally complement a pho5 mutation of Sacch. cerevisiae. Southern blot experiments, using the KIPHO5 gene as probe, show that some K. lactis reference strains lack repressible acid phosphatase, revealing a different gene organization for this kind of multigene family of proteins as compared to Sacch. cerevisiae.
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Mycobacteriophage D29 contains an integration system similar to that of the temperate mycobacteriophage L5
More LessSummary: A mycobacteriophage D29 DNA fragment cloned in pRM64, a shuttle plasmid that transforms Mycobacterium smegmatis, was sequenced. The determined sequence was 2592 nucleotides long and had a mean G+C content of 63.7 mol%, similar to that of mycobacterial DNA. Four ORFs were identified: one with strong homology to dCMP deaminase genes; one homologous to mycobacteriophage L5 gene 36, whose function is unknown; one encoding a possible excisase; and one encoding an integrase. The intergenic region between the putative excisase gene and the integrase gene had a lower than average G+C content and showed the presence of the same attP core sequence as mycobacteriophage L5. Transformation experiments using subclones of pRM64 indicated that the integrase gene and all the intergenic region were essential for stable transformation. A subclone containing the integrase gene and the core attP sequence was able to transform but recombinants were highly unstable. Southern analysis of total DNA from cells transformed with pRM64 and its derivatives showed that all the plasmids were integrated at one specific site of the bacterial chromosome. A recombinant exhibiting a high level of resistance to the selective drug kanamycin had two plasmids integrated at different sites. These results demonstrated that the D29 sequences contained in pRM64 were integrative, indicating that the generally held view of D29 as a virulent phage must be reviewed.
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Efficient secretion of the model antigen M6-gp41E in Lactobacillus plantarum NCIMB 8826
Summary: Four Lactobacillus strains (Lb. plantarum NCIMB 8826, Lb. paracasei LbTGS1.4, Lb. casei ATCC 393 and Lb. fermentum KLD) were tested for their ability to produce and secrete heterologous proteins. These strains were first screened with an α-amylase reporter under the control of a set of expression or expression/secretion signals from various lactic acid bacteria. With most of the constructions tested, the level of extracellular production was highest in Lb. plantarum NCIMB 8826, and lowest in Lb. paracasei LbTGS1.4. These two strains were next assayed using a model antigen consisting of the N-terminal part of the M6 protein from Streptococcus pyogenes fused to the linear epitope ELDKWAS from human immunodeficiency virus gp41 protein. Secretion of this heterologous protein was inefficient in Lb. paracasei LbTGS1.4, which accumulated a large intracellular pool of the unprocessed precursor, whereas Lb. plantarum NCIMB 8826 was able to secrete the antigen to a level as high as 10 mg I−1.
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SpoOA represses transcription of the cry toxin genes in Bacillus thuringiensis
More LessSummary: The DNA regions upstream from the genes encoding polypeptides of Bacillus thuringiensis subsp. israelensis larvicidal crystals (cry4A, cry4B, cry11A) contain sequences with similarities to the spoOA box of Bacillus subtilis (or ‘OA’ box) and the promoter recognized by the σH-associated RNA polymerase of B. subtilis. Expression of cry-lacZ transcriptional fusions was analysed in various B. thuringiensis genetic backgrounds. The early transcription of the toxin genes was not sporulation-dependent, whereas the late-stage expression at t 4-6 was σE-dependent. Primer extension analysis confirmed that the cry4-and cry11-type toxin genes were weakly transcribed during the transition phase; expression analysis of a cry11A'-lacZ transcriptional fusion in B. subtilis sporulation mutants confirmed the involvement of the σH-RNA polymerase. Primer extension analysis showed that in B. thuringiensis subsp. israelensis, the cry4A and cry11A gene transcription observed at the end of the growth stage was turned off at the beginning of the sporulation phase. The DNA region located upstream from the cry11A gene promoter including the putative ‘OA’ box was deleted. This led to a derepression of the expression of the cry11A operon. These results suggest that the cry4A, cry4B and cry11A toxin genes of B. thuringiensis subsp. israelensis are transcribed during the transition phase by the RNA polymerase associated with the σH factor and are subject to SpoOA repression.
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Mutational analysis of the early forespore/mother-cell signalling pathway in Bacillus subtilis
More LessSummary: Intercellular communication is a crucial phenomenon during spore development in Bacillus subtilis. It couples the establishment of a compartment-specific genetic program to the transcriptional activity of a σ factor in the other compartment. It also keeps σ factor activation in register with the morphological process. This study used directed mutagenesis to analyse the pathway that couples σE activation in the mother-cell to activation of σF in the forespore following asymmetric septation. Targets for mutagenesis in SpollGA (the receptor) were chosen based on the predicted topology of the protein when associated with the cell membrane. The results showed that a residue near the N terminus (D6), predicted to be exposed outside the cell, is required for receptor activity, whereas the major extracellular loop (between membrane domains IV and V) is dispensable for function. In contrast, mutations in SpollR (the signal) that partially blocked protein release (but not membrane translocation) had no effect on signal transduction. These results do not rule out the possibility that uncharacterized molecules intervene in the signalling pathway that establishes the mother-cell-specific developmental program during the early stage of sporulation.
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Molecular characterization of the Serratia marcescens OmpF porin, and analysis of S. marcescens OmpF and OmpC osmoregulation
More LessSummary: Serratia marcescens is a nosocomial pathogen with a high incidence of β-lactam resistance. Reduced amounts of outer-membrane porins have been correlated with increased resistance to β-lactams but only one porin, OmpC, has been characterized at the molecular level. In this study we present the molecular characterization of a second porin, OmpF, and an analysis of the expression of S. marcescens porins in response to various environmental changes. Two porins were isolated from the outer membrane using urea-SDS-PAGE and the relative amounts were shown to be influenced by the osmolarity of the medium and the presence of salicylate. From a S. marcescens genomic DNA library an 8 kb EcoRI fragment was isolated that hybridized with an oligonucleotide encoding the published N-terminal amino acid sequence of the S. marcescens 41 kDa porin. A 41 kDa protein was detected in the outer membrane of Escherichia coli NM522 carrying the cloned S. marcescens DNA. The cloned gene was sequenced and shown to code for a protein that shared 60-70% identity with other known OmpF and OmpC sequences. The upstream DNA sequence of the S. marcescens gene was similar to the corresponding E. coli ompF sequence; however, a regulatory element important in repression of E. coli ompF at high osmolarity was absent. The cloned S. marcescens OmpF in E. coli increased in expression in conditions of high osmolarity. The potential involvement of micF in the observed osmoregulation of S. marcescens porins is discussed.
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Sequence, localization and characteristics of the replicator region of the symbiotic plasmid of Rhizobium etli
Summary: The replicator region of the symbiotic plasmid of Rhizobium etli CFN42 was cloned and sequenced. A plasmid derivative (pH3) harbouring a 5.6 kb HindIII fragment from the symbiotic plasmid was found to be capable of independent replication and eliminated the symbiotic plasmid when introduced into a R. etli CFNX101 strain (a recA derivative). The stability and the copy number of pH3 were the same as that of the symbiotic plasmid, indicating that the information required for stable replication and incompatibility resides in the 5.6 kb HindIII fragment. The sequence analysis of this fragment showed the presence of three ORFs similar in sequence and organization to repA, repB and repC described for the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b and for the R. leguminosarum cryptic plasmid pRL8JI. Hybridization studies showed that p42d-like replicator sequences are found in the symbiotic plasmids of other R. etli strains and in a ‘cryptic’ plasmid of R. tropici.
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Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis
More LessSummary: Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated XIn) which ranged in size from 2 kb to approximately 26 kb. Instability of DNA inserts in RP4::XIn hybrid plasmids was observed. The smallest insert present in a stable RP4::XIn6 hybrid plasmid, termed XIn6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA. Sequence analysis of XIn6 led to the discovery of an open reading frame with homology to the maturases of group II introns. The XIn6 insert also exhibited several features characteristic of a group II intron. These included the presence of the consensus sequence GUGYG at the 5′ end and RAY at the 3′ end of the intron. RNA secondary structure modelling of XIn6 also revealed the presence of perfectly conserved domains V and VI. Differences were detected in the XIn6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol. In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the XIn6 group II intron. This is the first reported group II intron isolated from Pseudomonas spp. and the first time that the mobility of a bacterial group II intron has been demonstrated.
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- Pathogenicity And Medical Microbiology
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Heat shock response and groEL sequence of Bartonella henselae and Bartonella quintana
More LessSummary: Transmission of Bartonella species from ectoparasites to the mammalian host involves adaptation to thermal and other forms of stress. In order to better understand this process, the heat shock response of Bartonella henselae and Bartonella quintana was studied. Cellular proteins synthesized after shift to higher temperatures were intrinsically labelled with [35S]methionine and analysed by gel electrophoresis and fluorography. The apparent molecular masses of three of the major heat shock proteins produced by the two Bartonella species were virtually identical, migrating at 70, 60 and 10 kDa. A fourth major heat shock protein was larger in B. quintana (20 kDa) than in B. henselae (17 kDa). The maximum heat shock response in B. quintana and B. henselae was observed at 39 °C and 42 °C, respectively. The groEL genes of both Bartonella species were amplified, sequenced and compared to other known groEL genes. The phylogenetic tree based on the groEL alignment places B. quintana and B. henselae in a monophyletic group with Bartonella bacilliformis. The deduced amino acid sequences of Bartonella GroEL homologues contain signature sequences that are uniquely shared by members of the Gram-negative α-purple subdivision of bacteria, which live within eukaryotic cells. Recombinant His6-GroEL fusion proteins were expressed in Escherichia coli to generate specific rabbit antisera. The GroEL antisera were used to confirm the identity of the 60 kDa Bartonella heat shock protein. These studies provide a foundation for evaluating the role of the heat shock response in the pathogenesis of Bartonella infection.
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- Physiology And Growth
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Measurement of cytoplasmic pH of the alkaliphile Bacillus lentus C-125 with a fluorescent pH probe
More LessSummary: A method was established to measure the cytoplasmic pH of the facultative alkaliphilic strain, Bacillus lentus C-125. The bacterium was loaded with a pH-sensitive fluorescent probe, 2′,7′-bis-(2-carboxyethyl)-5 (and -6)-carboxyfluorescein (BCECF), and cytoplasmic pH was determined from the intensity of fluorescence of the intracellular BCECF. The activity of the organism to maintain neutral cytoplasmic pH was assessed by measuring the cytoplasmic pH of the cells exposed to various pH conditions. The cytoplasmic pH maintenance activity of C-125 increased with increasing culture pH, indicating that the activity was regulated in response to the culture pH.
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Spatial interactions between subsurface bacterial colonies in a model system: a territory model describing the inhibition of Listeria monocytogenes by a nisin-producing lactic acid bacterium
More LessSummary: The effect of spatial separation on interactions between subsurface bacterial colonies was tested using a model system: the inhibition of Listeria monocytogenes by nisin-producing and nisin-non-producing Lactococcus lactis subsp. lactis. Separation distance was controlled by altering the number of inoculum organisms within the agar. Mean separation distance was calculated by determining the mean volume available to each cell at the start of the experiment. Inhibition was assessed by comparing the growth of L. monocytogenes in pure culture with its growth in the presence of Lac. lactis subsp. lactis. Increasing the distance between colonies resulted in an exponential decrease in inhibition. When L. monocytogenes and Lac. lactis subsp. lactis colonies were within 100 μm of each other, the increase in cell numbers per L. monocytogenes colony was only 0.6 c.f.u. (which indicated some cells had become non-viable). This was a log reduction of 3.5 compared to the pure culture control. A separation distance of 1000 μm resulted in a L. monocytogenes colony growth increment of 2.5 × 102 c.f.u. per colony, a log reduction of 3.0 compared to the control. Increasing the separation distance to 3000 μm resulted in a L. monocytogenes colony growth increment of 1.3 × 106 c.f.u. per colony, a log reduction of 0.9 compared to the control. The effects of nisin and acidity were investigated by using a nisin-non-producing strain of Lac. lactis subsp. lactis and by buffering the medium. Data were obtained for the effect of separation on inhibition, as well as competition between colonies of the same species. The inhibition was mathematically described in terms of a simplified ‘territory’ model of immobilized bacterial growth. There was a strong qualitative agreement between the mathematical model and the experimental data. It was concluded that the phenomenon of propinquity is of important consideration when modelling and predicting microbial growth within solid food systems.
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A cytoplasmic lectin produced by the fungus Arthrobotrys oligospora functions as a storage protein during saprophytic and parasitic growth
More LessSummary: It was recently shown that the nematode-infecting fungus Arthrobotrys oligospora contains a saline-soluble lectin (designated AOL) that is a member of a novel family of fungal lectins sharing similar primary sequences and binding specificities. During saprophytic growth in liquid cultures, levels of AOL and AOL mRNA were found to vary depending on the growth phase of the mycelium and the carbon/nitrogen (C/N) ratio of the medium. AOL was not detected in young mycelium. In older mycelium (stationary growth phase) grown in media with low C/N ratios (1 or 6), AOL comprised 5-20% of the total amount of saline-soluble proteins present in the mycelium. Neither the lectin nor its transcript was detected in mycelia grown in medium with higher C/N ratios (≥150). Under conditions of nitrogen starvation, AOL was preferentially degraded in relation to the total amount of saline-soluble proteins present in the mycelium. During the infection of nematodes, the level of AOL protein and AOL mRNA increased significantly once the nematodes had been penetrated and digested. Large amounts of AOL accumulated in the trophic hyphae growing inside the nematode as visualized by immunofluorescence microscopy. Later, AOL labelling was detected outside the digested nematodes, preferentially in strands of aggregated hyphae and in newly developed trap cells. Electron microscopy showed that AOL was localized to the cytoplasm and the nucleus of both vegetative mycelium and trap cells, and in the trophic hyphae growing inside the infected nematodes. These results indicate that AOL functions as a storage protein during both saprophytic and parasitic growth.
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The Sch9 protein kinase in the yeast Saccharomyces cerevisiae controls cAPK activity and is required for nitrogen activation of the fermentable-growth-medium-induced (FGM) pathway
Summary: In cells of the yeast Saccharomyces cerevisiae, trehalase activation, repression of CTT1 (catalase), SSA3 (Hsp70) and other STRE-controlled genes, feedback inhibition of cAMP synthesis and to some extent induction of ribosomal protein genes is controlled by the Ras-adenylate cyclase pathway and by the fermentable-growth-medium-induced pathway (FGM pathway). When derepressed cells are shifted from a non-fermentable carbon source to glucose, the Ras-adenylate cyclase pathway is transiently activated while the FGM pathway triggers a more lasting activation of the same targets when the cells become glucose-repressed. Activation of the FGM pathway is not mediated by cAMP but requires catalytic activity of cAMP-dependent protein kinase (cAPK; Tpk1, 2 or 3). This study shows that elimination of Sch9, a protein kinase with homology to the catalytic subunits of cAPK, affects all target systems in derepressed cells in a way consistent with higher activity of cAPK in vivo. In vitro measurements with trehalase and kemptide as substrates confirmed that elimination of Sch9 enhances cAPK activity about two- to threefold, in both the absence and presence of cAMP. In vivo it similarly affected the basal and final level but not the extent of the glucose-induced responses in derepressed cells. The reduction in growth rate caused by delation of SCH9 is unlikely to be responsible for the increase in cAPK activity since reduction of growth rate generally leads to lower cAPK activity in yeast. On the other hand, deletion of SCH9 abolished the responses of the protein kinase A targets in glucose-repressed cells. Re-addition of nitrogen to cells starved for nitrogen in the presence of glucose failed to trigger activation of trehalase, caused strongly reduced and aberrant repression of CTT1 and SSA3, and failed to induce the upshift in RPL25 expression. From these results three conclusions can be drawn: (1) Sch9 either directly or indirectly reduces the activity of protein kinase A; (2) Sch9 is not required for glucose-induced activation of the Rasadenylate cyclase pathway; and (3) Sch9 is required for nitrogen-induced activation of the FGM pathway. The latter indicates that Sch9 might be the target of the FGM pathway rather than cAPK itself.
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Suppression of Escherichia coli formate hydrogenlyase activity by trimethylamine N-oxide is due to drainage of the inducer formate
More LessSummary: The effect of the addition of trimethylamine N-oxide (TMAO) in the growth medium on Escherichia coli anaerobic fermentative and respiratory pathways was examined. Formate dehydrogenase H (FDH-H) activity was totally repressed by the addition of 40 mM TMAO, whereas the overall hydrogenase (HYD) activity was reduced by 25%. Accordingly, expression of lacZ operon fusions with the fdhF and hycB structural genes specifying FDH-H and HYD3 was reduced sevenfold and eightfold, respectively, leading to suppression of an active formate hydrogenlyase system. In contrast, global respiratory formate-dependent phenazine methosulphate reductase (FDH-PMS) activity, which consists of both the major anaerobic FDH-N enzyme and the aerobic FDH-Z isoenzyme, was increased approximately twofold. This was corroborated by a 2.5-fold stimulation of the sole fdoG-uidA transcriptional fusion which reflects the synthesis of the respiratory aerobic FDH-Z enzyme. In fdhD, fdhE or torA mutants lacking either FDH-PMS activity or TMAO reductase (TOR) activity, the formate hydrogenlyase pathway was no longer inhibited by TMAO. In addition, introduction of 30 mM formate in the growth medium was found to relieve the repressive effect of TMAO in the wild-type strain. When TMAO was added as terminal electron acceptor a significant enhancement of anaerobic growth was observed with the wild-type strain and the fdoG mutant. It was associated with the concomitant suppression of the formate hydrogenlyase enzymes. This was in contrast to the fdnG and torA mutants whose growth pattern and fermentative enzymes remained unaffected. Taken together, these results strongly suggest that formate-dependent reduction of TMAO via FDH-N and TOR reduces the amount of formate available for induction of the formate hydrogenlyase pathway.
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Salmonella typhi mutants defective in anaerobic respiration are impaired in their ability to replicate within epithelial cells
More LessSummary: By using MudJ (Kan, lac)-directed operon fusion technology, mutants of Salmonella typhi whose gene expression is induced under anaerobic growth conditions were isolated. Characterization of their phenotypes and regulatory properties revealed that two of the mutants were unable to use nitrate as a terminal electron acceptor in the absence of oxygen, suggesting that they were defective in nitrate reductase activity. Anaerobic induction of these fusions did not further increase in response to nitrate. Strains carrying an additional mutation in oxrA were constructed. They showed a lower level of β-galactosidase expression both aerobically and anaerobically; however, the ratios of anaerobic induction remained unaltered. These MudJ insertions mapped to the 17-19 min region of the chromosome. Based upon their phenotypes and mapping, one of the mutants probably possessed a modC (chlD):: MudJ insertion and the other a moaA (chlA):: MudJ insertion. A third mutant was unable to use either nitrate or fumarate as a terminal electron acceptor. All three mutants showed a reduced ability to enter into and proliferate within HEp-2 epithelial cells. The oxrA mutation enhanced entry and proliferation of both the wild-type cells and the three mutants. Taken together, these results suggest that anaerobic respiration plays a role in S. typhi invasiveness.
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The Klebsiella pneumoniae cytochrome bd’ terminal oxidase complex and its role in microaerobic nitrogen fixation
More LessSummary: Cytochrome bd’ has been implicated in having an important role in microaerobic nitrogen fixation in the enteric bacterium Klebsiella pneumoniae, where it is expressed under all conditions that permit diazotrophy. In this paper the sequence of the genes encoding this terminal oxidase (cydAB) of Klebsiella pneumoniae and the characterization of a cyd mutant are reported. The deduced amino acid sequences support the proposal that His 19, His 186 and Met 393 provide three of the four axial ligands to the Fe of the three haems in the oxidase complex. The nitrogen-fixing ability of the mutant was severely impaired in the presence of low concentrations of oxygen compared with the wild-type bacterium. Only the wild-type organism was capable of microaerobic nitrogenase activity supported by fermentation products. It is proposed that formate dehydrogenase-O may be involved in supplying electrons to a respiratory chain terminated by the bd-type oxidase, which would remove inhibitory oxygen and supply ATP for nitrogenase activity.
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Kinetic analysis of morphological differentiation and protease production in Streptomyces albidoflavus SMF301
More LessSummary: The effects of specific growth rate and specific nutrient uptake rate on morphological differentiation of Streptomyces albidoflavus SMF301 were determined in chemostat cultures. Production of three types of proteases: chymotrypsin-like protease (CTP), trypsin-like protease (TLP) and metalloprotease (MTP) were analysed in relation to mycelium growth and spore formation. Production of CTP was closely linked to mycelium growth, whereas spore formation, TLP synthesis and MTP synthesis were inversely related to growth. Evaluation of various kinetic parameters [specific production rates of spores (q spo), TLP (q TLP), MTP (q MTP) and CTP (q CTP)] showed that mycelium growth rate and CTP production were optimal at 0.1 h−1, but submerged spore formation, TLP production and MTP production were optimal at 0.025 h−1. Changes in specific nutrient uptake rates [glucose (q glu), ammonium ion (q amn) and phosphate (q pho)] affected sporulation and protease production; limitation of carbon, nitrogen and phosphate stimulated spore, TLP and MTP production.
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A Streptomyces griseus gene (sgaA) suppresses the growth disturbance caused by high osmolality and a high concentration of A-factor during early growth
More LessSummary: A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone), produced in a growth-dependent manner, switches on secondary metabolite formation and morphological differentiation in Streptomyces griseus, presumably by binding to the A-factor receptor protein (ArpA)-DNA complex and releasing the repression caused by ArpA. In the A-factor-deficient mutant strain S. griseus HH1 a large deletion includes afsA which is required for A-factor production. Growth and aerial mycelium formation of strain HH1 on media containing high concentrations of sucrose, sorbitol, mannitol, KCI or NaCI was disturbed by the presence of a large amount of A-factor supplied either exogenously or by a high-copy-number plasmid carrying afsA. This disturbance did not occur on media of normal osmolality and was observed only when A-factor was supplied during the very early stage of growth, about 8 h after inoculation. In addition, neither the wild-type strain nor S. griseus KM7 defective in ArpA exhibited the disturbance. These observations suggest that the presence of a large amount of A-factor during the very early stage of growth, probably during the A-factor-sensitive stage, triggered abrupt and disordered expression of some genes. The effect was apparently mediated through ArpA in the A-factor regulatory cascade and disturbed the physiology of strain HH1 under high osmolality. A gene that suppressed the disturbance was identified 5.5 kb upstream of the afsA locus in the wild-type strain. The gene, named sgaA, encoded a protein of 264 aa with a calculated molecular mass of 28 kDa.
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- Plant-Microbe Interactions
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Restoration of wild-type virulence to Tri5 disruption mutants of Gibberella zeae via gene reversion and mutant complementation
More LessSummary: Gibberella zeae is a pathogen of small grain crops and produces trichothecene mycotoxins in infected host tissue. The role of trichothecenes in the virulence of G. zeae was previously investigated using trichothecene-non-producing mutants that were generated via transformation-mediated disruption of a gene (Tri5) that encodes the first enzyme in the trichothecene biosynthetic pathway. The mutants were less virulent on some hosts than the wild-type strain from which they were derived. Here, we used two approaches to determine whether the reduced virulence of mutants was due specifically to Tri5 disruption or to non-target effects caused by the transformation process. First, we generated a revertant from a Tri5 disruption mutant by allowing the mutant to pass through the sexual phase of its life cycle. In approximately 2% of the resulting progeny the disrupted Tri5 had reverted to wild-type; however, only one of three revertant progeny also regained the ability to produce trichothecenes. In the second approach, we complemented the Tri5 mutation in a disruption mutant by transforming the mutant with a plasmid carrying a functional copy of Tri5. In all transformants examined, the ability to produce trichothecenes was restored. The restoration of trichothecene production in the revertant progeny and in the complemented mutant was accompanied by restoration of wild-type or near wild-type levels of virulence on wheat seedlings (cultivar Wheaton). The results indicate that the reduced virulence of the mutants was caused by disruption of Tri5 rather than non-target effects resulting from the transformation process. The results also provide further evidence that trichothecenes contribute to the virulence of plant-pathogenic fungi.
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A new nos gene downstream from nosDFY is essential for dissimilatory reduction of nitrous oxide by Rhizobium (Sinorhizobium) meliloti
More LessSummary: Rhizobium (Sinorhizobium) meliloti strains capable of dissimilatory nitrous oxide reduction (Nos+) carry a nosRZDFY gene cluster on a 10.1 kb EcoRI fragment of the nod megaplasmid near the fixGHIS genes. These nos genes are arranged in three complementation groups and the 10.1 kb EcoRI fragment is sufficient to confer Nos activity to R. meliloti strains lacking such activity. An overlapping HindIII fragment containing the nosRZDFY genes but missing a 0.6 kb Hin dIII-EcoRI downstream segment was found incapable of imparting Nos activity to strains unable to reduce nitrous oxide, suggesting the presence of other nos gene(s) in this region. Tn5 introduced near the HindIII site resulted in mutants with a Nos− phenotype. Complete sequence analysis of nosY showed that it was well-conserved with respect to that of Pseudomonas stutzeri. Two previously unreported genes downstream of nosY in R. meliloti were also revealed. Contiguous with nosY was a sequence showing 63% identity with the ORFL protein of P. stutzeri. It appeared to be in the same operon as nosDFY and was predicted to encode a membrane lipoprotein similar to the putative NosL of P. stutzeri. Unlike the latter protein, however, amino acid sequences typical of metal-binding sites and cysteine residues indicative of the active site of protein disulphide isomerase were absent in the predicted NosL of R. meliloti. The Tn5 mutations resulting in a Nos− phenotype were localized within a 966 nucleotide gene 31 nucleotides downstream of nosDFYL with the same orientation. The new gene, nosX, was determined to be in a separate complementation group. It encoded a periplasmic protein with homology in the C-terminal domain with RnfF of Rhodobacter capsulatus and with a hypothetical Escherichia coli protein, YOJK. It was concluded that there are seven genes constituting the nos cluster in R. meliloti. They are organized in four complementation groups and in the same orientation, spanning a distance of about 9 kb on the nod megaplasmid.
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- Systematics
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Diversity and differential distribution of IS231, IS232 and IS240 among Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides
More LessSummary: Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides are very closely related bacteria, generally considered as subspecies of B. cereus sensu lato. Different transposable elements have been isolated from B. thuringiensis, including IS231, IS232 and IS240 and their variants. The distribution of these three insertion sequences (IS) within the B. cereus group has been investigated in 90 strains of B. thuringiensis (representing 61 serovars), in 30 reference strains of B. cereus and in 33 strains of B. mycoides. Since these IS elements art delimited by well-conserved and specific inverted repeats, the use of primers corresponding to these ends allowed their amplification by PCR. The results showed that IS231 is the most abundant element in the three taxa, whereas IS232 is apparently exclusively associated with B. thuringiensis. Hybridization and Dral RFLP analysis of the PCR products confirmed and extended knowledge of the heterogeneity previously observed among iso-IS231 elements. Moreover, a similar diversity was observed among iso-IS240 elements. This contrasted with the relative homogeneity displayed by iso-IS232 elements. No specific association appeared to exist between any particular iso-element and a specific strain or serotype.
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Genetic relationships among Pasteurella trehalosi isolates based on multilocus enzyme electrophoresis
More LessSummary: Genetic diversity among 60 British Pasteurella trehalosi isolates representing the four recognized capsular serotypes, T3, T4, T10 and T15, and recovered predominantly from sheep suffering from systemic pasteurellosis, was estimated by analysing electrophoretically demonstrable allelic variation at structural genes encoding 19 enzymes. Thirteen of the loci were polymorphic and 20 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The population structure of P. trehalosi is clonal and its genetic diversity is limited compared with most other pathogenic bacteria. ETs represent clones, and isolates of the same ET were generally associated with the same combination of serotype, LPS type and outer-membrane protein (OMP) type. The genetic diversity of isolates within each of the capsular serotypes varied. Serotype T10 was represented by 18 isolates in two related ETs and exhibited little diversity. By contrast, serotype T15 was represented by 18 isolates in nine ETs and was almost as diverse as the species as a whole. Serotype T4 was represented by 18 isolates in five ETs and was less diverse than serotype T15. Although serotype T3 was more diverse than serotype T15 it was represented by only three isolates. With the exception of the T10 isolates and those recovered from healthy sheep, 35 disease isolates belonged to 16 ETs, each of which was represented by only one to four isolates. The fact that a high proportion of disease is caused by a relatively large number of clones suggests that P. trehalosi is essentially an opportunistic pathogen. In addition to having the same capsular structure, isolates belonging to the two T10 clones were characterized by possession of similar, if not identical, O-antigens (LPS types 2 and 4). The occurrence of 18 serotype T10 isolates in only two ETs suggests that the T10 capsule and type 2/4 O-antigen confer enhanced virulence on members of these two clones. Multilocus enzyme electrophoresis (MLEE) had greater resolving power than did capsule/LPS/OMP analysis, being able to distinguish 20 rather than 14 sub-divisions within P. trehalosi. The technique demonstrated genetic identity or non-identity among strains of the same or different serotypes from different geographic localities within the UK and was a useful epidemiological tool.
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- Genome Analysis
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Sequence and analysis of a 31 kb segment of the Bacillus subtilis chromosome in the area of the rrnH and rrnG operons
More LessSummary: A 31 141 bp continuous nucleotide sequence in the region from trnl to pNEXT52 in the Bacillus subtilis 168 genome was determined. In the region, there were 22 ORFs, two complete rRNA operons, and five tRNA genes. It was deduced that the function of one of the ORFs was similar to that of a sigma factor belonging to the ECF (extra-cytoplasmic functions) subfamily. The gene cluster feuA, B, C reported previously for other strains of B. subtilis was also found in strain 168 and located in this region.
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Analysis of the Bacillus subtilis genome: cloning and nucleotide sequence of a 62 kb region between 275° (rrnB) and 284° (pai)
Summary: In the framework of the international project aimed at the sequencing of the Bacillus subtilis genome, five DNA fragments in the region between rrnB (275° and pai (284°) were cloned by inverse and combinatorial long-range PCR and their nucleotide sequences were determined and analysed. Together these sequences constituted a contig of 62229 bp. On the basis of the position of NotI and SfiI restriction sites, the orientation and order of known genetic markers was determined to be pai (284°) - degQ comQ comP comAA comAB - pbpD - kapB kinB patB - mcpB tlpA mcpA tlpB - rrnB (275°). Fifty-four ORFs were detected. Thirteen of these coincided with known B. subtilis genes, and 41 new ORFs were found. Of the predicted new gene products, 12 showed no significant similarity to other known proteins, whereas ten showed strong similarity to proteins of other organisms with unknown function. Nineteen predicted proteins showed strong similarity to known proteins of other organisms, for instance a Na+/H+ antiporter system of Bacillus alcalophilus, a sugar transport system found in Mycoplasma genitalium, NADH-dependent butanol dehydrogenase of Clostridium acetobutylicum, glucose-6-phosphate isomerase A of B. subtilis, exo-1,4-α-glucosidase activity of Bacillus stearothermophilus and L-rhamnose isomerase of Escherichia coli.
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A 32 kb nucleotide sequence from the region of the lincomycin-resistance gene (22°-25°) of the Bacillus subtilis chromosome and identification of the site of the lin-2 mutation
More LessSummary: A 32 kb nucleotide sequence in the region of the lincomycin-resistance gene, located from 22° to 25° on the Bacillus subtilis chromosome, was determined. Among 32 putative ORFs identified, four [lipA for lipase, natA, natB and yzaE (renamed yccK)] have already been reported, although the functions of NatA, NatB and YccK remain to be characterized. Six putative products were found to exhibit significant similarity to known proteins in the databases, namely L-asparaginase precursor, protein aspartate phosphatase, x-glucosidase, two tellurite-resistance proteins and a hypothetical protein from B. subtilis. The region of the tellurite-resistance gene, consisting of seven ORFs, seems to correspond to an operon. The products of 14 ORFs exhibited considerable or limited similarity to known proteins. The sequenced region seems to be rich in membrane proteins, since at least 16 gene products appeared to contain membrane-spanning domains. The site of the lin-2 mutation (two nucleotide replacements) was mapped and identified by sequencing. This site is located between a putative promoter and the SD sequence of ImrA (yccB)[a putative repressor of the Imr operon, which consists of ImrA and ImrB (yccA)]. LmrB is a homologue of proteins involved in drug-export systems and seems likely to be the protein responsible for resistance to lincomycin.
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Genomic rearrangements during evolution of the obligate intracellular parasite Rickettsia prowazekii as inferred from an analysis of 52015 bp nucleotide sequence
More LessSummary: In this study a description is given of the sequence and analysis of 52 kb from the 1.1 Mb genome of Rickettsia prowazekii, a member of the α-Proteobacteria. An investigation was made of nucleotide frequencies and amino acid composition patterns of 41 coding sequences, distributed in 10 genomic contigs, of which 32 were found to have putative homologues in the public databases. Overall, the coding content of the individual contigs ranged from 59 to 97%, with a mean of 81%. The genes putatively identified included genes involved in the biosynthesis of nucleotides, macromolecules and cell wall structures as well as citric acid cycle component genes. In addition, a putative identification was made of a member of the regulatory response family of two-component signal transduction systems as well as a gene encoding haemolysin. For one gene, the homologue of metK, an internal stop codon was discovered within a region that is otherwise highly conserved. Comparisons with the genomic structures of Escherichia coli, Haemophilus influenzae and Bacillus subtilis have revealed several atypical gene organization patterns in the R. prowazekii genome. For example, R. prowazekii was found to have a unique arrangement of genes upstream of dnaA in a region that is highly conserved among other microbial genomes and thought to represent the origin of replication of a primordial replicon. The results presented in this paper support the hypothesis that the R. prowazekii genome is a highly derived genome and provide examples of gene order structures that are unique for the Rickettsia.
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Volumes and issues
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Volume 170 (2024)
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