- Volume 142, Issue 9, 1996
Volume 142, Issue 9, 1996
- Review Article
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- Antigens And Immunity
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A newly identified immunodominant membrane protein (pMB67) involved in Mycoplasma bovis surface antigenic variation
More LessMycoplasma bovis is a bovine pathogen able to cause systemic disease. It possesses a series of prominent, structurally related yet clearly distinguishable membrane lipoproteins on the cell surface. These variable surface proteins (Vsps) undergo highly dynamic and spontaneous changes in size and expression and are key immunogenic components. They may play a critical role as mediators of adherence to host cells and in escaping immune destruction. In this report, we define a novel, Vsp-unrelated membrane protein also associated with M. bovis surface antigenic variation. This protein has an apparent molecular mass of 67000 Da in the type strain PG45 and was designated pMB67. Immunological and biochemical characterization of pMB67 demonstrated that it: (i) contains a specific epitope, (ii) is not modified by lipid but does contain cysteine, (iii) does not contain a Vsp-like repetitive periodic protein structure, (iv) is a predominant antigen recognized during M. bovis infections, (v) undergoes a high rate of phase variation in vitro and (vi) is size-variable. These results showed that M. bovis employs two types of specialized membrane proteins for surface diversification. The pMB67 protein may be useful in diagnostic assays and as a vaccine component.
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- Biochemistry
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Degradative pathways for p-toluenecarboxylate and p-toluenesulfonate and their multicomponent oxygenases in Comamonas testosteroni strains PSB-4 and T-2
Three multicomponent oxygenases involved in the degradation of p-toluenesulfonate and p-toluenecarboxylate and the regulation of their synthesis have been examined in three strains (T-2, PSB-4 and TER-1) of Comamonas testosteroni. Strain T-2 utilizes p-toluenesulfonate as a source of carbon and energy for growth via p-sulfobenzoate and protocatechuate, and p-toluenecarboxylate via terephthalate and protocatechuate, and has the unusual property of requiring the reductase (TsaB) of the toluenesulfonate methyl monooxygenase system (TsaMB) in an incompletely expressed sulfobenzoate dioxygenase system (PsbAC) [ Schläfli Oppenberg, H. R., Chen, G., Leisinger, T. & Cook, A. M. (1995) . Microbiology 141, 1891-1899]. The independently isolated C. testosteroni PSB-4 utilized only sulfobenzoate and terephthalate via protocatechuate. Mutant TER-1, derived from strain T-2, utilized only terephthalate via protocatechuate. We detected no enzymes of the pathway from toluenesulfonate to sulfobenzoate in strains PSB-4 and TER-1, and confirmed by PCR and Southern blot analysis that the genes (tsaMB) encoding toluenesulfonate monooxygenase were absent. We concluded that, in strain PSB-4, the regulatory unit encoding the genes for the conversion of toluenesulfonate to sulfobenzoate was missing, and that generation of mutant TER-1 involved deletion of this regulatory unit and of the regulatory unit encoding desulfonation of sulfobenzoate. The degradation of sulfobenzoate in strain PSB-4 was catalysed by a fully inducible sulfobenzoate dioxygenase system (PsbACPSB-4), which, after purification of the oxygenase component (PsbAPSB-4), turned out to be indistinguishable from the corresponding component from strain T-2 (PsbAT-2). Reductase PsbCPSB-4, which we could separate but not purify, was active with oxygenase PsbAPSB-4 and PsbAT-2. Oxygenase PsbAPSB-4 was shown by electron paramagnetic resonance spectroscopy to contain a Rieske [2Fe-2S] centre. The enzyme system oxygenating terephthalate was examined and the oxygenase component purified and characterized. The oxygenase component in strains T-2 (and mutant TER-1) and PSB-4 were indistinguishable. The reductase component, which we separated but failed to purify, was active with the oxygenase from all strains. Gains and losses of blocks of genes in evolution is discussed.
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Defect in export and synthesis of the periplasmic galactose receptor MgIB in dnaK mutants of Escherichia coli, and decreased stability of the mgIB mRN
More LessThe high-affinity galactose permease, which comprises the periplasmic galactose receptor MgIB, the membrane translocator MgIC and the membrane-associated ATPase MgIA, displayed a reduced activity in a dnaK temperature-sensitive mutant of Escherichia coli. This reduced transport activity correlated with a reduction in the quantity of MgIB. At 42 °C, an accumulation of pre-MgIB in the dnaK temperature-sensitive mutant reflected a defect in MgIB export. In addition, an accumulation of pre-MgIB in secB, secA and secY mutants suggested that SecB and the Sec translocase are also involved in export of the periplasmic galactose receptor. At 30 °C, there was no accumulation of pre-MgIB in the dnaK mutant, but there was still a decreased amount of MgIB in the periplasm. The reduction in MgIB expression was not the result of a decrease in its stability, nor was it the result of a general defect in translation or transcription, since the MgIA protein (which is expressed from the same operon as MgIB) was synthesized in normal amounts. Two mRNAs are implicated in the expression of the mgl genes, a polycistronic mgIBAC mRNA, and a more stable and more abundant mgIB mRNA, produced by 3′-5′ degradation of the mgIBAC mRNA (R. W. Hogg, C. Voelker & I. von Carlowitz, 1991, Mol Gen Genet 229, 453–459). The mgIB mRNA is protected against exonucleases by a REP (Repetitive Extragenic Palindrome) sequence located at its 3′ extremity, which is responsible for the higher expression of MgIB compared to MgIA and MgIC. The decreased MgIB expression in the dnaK mutant at 30 °C in the present work correlated with a reduced stability of the mgIB mRNA, which may have resulted from a defective stabilization by the REP sequence, or from a defect in translation of the mgIB gene.
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- Bioenergetics And Transport
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Cytochrome c550 expression in Paracoccus denitrificans strongly depends on growth condition: identification of promoter region for cycA by transcription start analysis
More LessThe periplasmic cytochrome c 550 content of Paracoccus denitrificans has been shown by immunological detection to be strongly dependent on the mode of growth. Cells grown under anaerobic, denitrifying conditions or methylotrophically in the presence of oxygen contained substantially more cytochrome c 550 than cells grown aerobically on multicarbon substrates. A similar pattern was observed when expression of the cycA gene (encoding cytochrome c 550), was monitored using an Escherichia coli alkaline phosphatase gene (phoA) fusion as a reporter of cycA promoter activity. The increase in cycA expression observed during growth on C1 substrates was substantially diminished if succinate was also present. These results reveal that expression of cycA is subject to multiple regulatory controls and suggest that cytochrome c 550 has a general role in electron transfer to periplasmic reductases required for anaerobic denitrifying growth and from dehydrogenases required for aerobic growth on C1 compounds. Two major transcriptional initiation start points for the cycA gene have been identified.
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- Biotechnology
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Lactococcin 972: a homodimeric lactococcal bacteriocin whose primary target is not the plasma membrane
More LessLactococcus lactis subsp. lactis IPLA 972 was shown to produce a bacteriocin which had a bactericidal effect on sensitive lactococci. Production of lactococcin 972 reached a maximum during the late-exponential phase of growth. The bacteriocinogenic activity was heat-sensitive, active in the pH range 4·0–9·0 and showed low susceptibility to proteases. Purification of the bacteriocin rendered a single polypeptide of 7·5 kDa (monomer) as shown by SDS-PAGE. Gels overlaid with a lawn of sensitive bacteria showed inhibitory activity at a point corresponding to 15 kDa. Changes in the electrophoretic conditions allowed the detection of a band at a position corresponding to that expected for a hypothetical dimer. Sequencing of the NH2-terminal end of lactococcin 972 revealed the sequence NH2-EGTWQHGYGV, which is not related to any other bacteriocin sequence present in the databases. Finally, lactococcin 972 did not induce the efflux of compounds previously incorporated into the cytoplasm of sensitive cultures nor did it inhibit macromolecular synthesis, suggesting that, in contrast to other bacteriocins, its primary target is not the plasma membrane.
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- Environmental Microbiology
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Environmental gasoline-utilizing isolates and clinical isolates of Pseudomonas aeruginosa are taxonomically indistinguishable by chemotaxonomic and molecular techniques
More LessA total of 42 Pseudomonas aeruginosa strains was isolated previously from clinical sources (27 strains) and from a gasoline-contaminated aquifer (15 strains). Selected strains were subjected to taxonomic tests involving chemical and molecular biological techniques, including membrane fatty acid analysis, phage-sensitivity, growth temperature range, presence of plasmids, and PCR-amplification and sequencing of a species-specific 16S–23S rDNA internal transcribed spacer region. The clinical and environmental isolates formed a coherent taxonomic group with few distinguishing characteristics. Of the phenotypes observed, a consistent difference was the ability of the aquifer strains to utilize gasoline supplied in the gas phase as sole carbon source and, conversely, the inability of the clinical strains to do so. Fourteen of the 15 environmental strains possessed similar-sized cryptic plasmids. The clinical isolates either lacked detectable plasmids or contained plasmids of a different size. The observation that the clinical and environmental isolates of P. aeruginosa were taxonomically indistinguishable is discussed in terms of its relevance to environmental-regulatory guidelines because P. aeruginosa, a known opportunistic pathogen, is a prime candidate for use in bioremediation processes involving deliberate release of this organism to the environment.
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The phylogenetic position and ultrastructure of the uncultured bacterium Achromatium oxaliferum
More LessAchromatium oxaliferum is a large, morphologically conspicuous, sediment-dwelling bacterium. Nothing is known concerning its phylogeny and it has eluded all attempts at laboratory cultivation. The limited physiological description of A. oxaliferum has been based on morphological features of the bacterium such as the presence of intracellular sulphur inclusions. A. oxaliferum cells were purified from a wetland region close to Rydal Water (Cumbria, UK). Scanning and transmission electron microscopy revealed that a number of morphologically distinct A. oxaliferum cell-types, based on cell surface features and the size and abundance of calcite and sulphur inclusions within the cells, were present in a single sample of purified cells. PCR was used to amplify almost full-length 16S rRNA gene sequences from DNA extracted from A. oxaliferum cells directly purified from sediments. The PCR products were cloned and partial sequences (approx. 400 bp) were determined for seven of the clones. Three different sequence clusters were recovered from the clone libraries. A near full-length (1489 bp) 16S rRNA gene sequence was determined for a representative clone of the most dominant sequence-type (52 % of the sequences). Comparative sequence analysis showed A. oxaliferum to form a deep branching lineage within the γ-subdivision of the Proteobacteria. A. oxaliferum was related most closely to the Chromatium assemblage that includes sulphur-oxidizing symbiotic bacteria, purple sulphur bacteria, and sulpur- and iron-oxidizing thiobacilli. Phylogenetic inferences made using distance, parsimony and maximum likelihood methods all placed A. oxaliferum with this group of bacteria. Bootstrap support for a relationship with any particular lineage within the assemblage was weak. The seven clone sequences recovered from the A. oxaliferum cells however formed a monophyletic group well supported by bootstrap analysis (85–100% support depending on the analysis done). It was concluded that A. oxaliferum was related to organisms of the Chromatium assemblage but constituted a novel lineage within this group of bacteria. A. oxaliferum cells were confirmed as the source of the 16S rRNA sequence obtained, by the use of a fluorescently-labelled 165 rRNA-targeted oligonucleotide specific for the A. oxaliferum rRNA sequence.
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Influence of ionic strength and substratum hydrophobicity on the co-adhesion of oral microbial pairs
More LessCo-adhesion between oral microbial pairs (i.e. adhesion of a planktonic micro-organism to a sessile organism adhering to a substratum surface) has been described as a highly specific interaction, mediated by stereochemical groups on the interacting microbial cell surfaces, and also as a non-specific, critical colloid-chemical interaction. In a colloid-chemical approach, microbial co-adhesion is considered as an interplay between, amongst others, hydrophobic and electrostatic interactions. The aim of this paper was to determine the influence of ionic strength on the co-adhesion of Streptococcus oralis 34 to either Actinomyces naeslundii T14V-J1 or its mutant strain 5951 adhering to glass in a parallel-plate flow chamber. To this end, the ionic strength of the suspension was varied by the addition of KCl. Another aim was to investigate whether substratum hydrophobicity affected the co-adhesion between the organisms by allowing the sessile organisms (in this case the actinomyces) to adhere either to hydrophilic or to hydrophobic, dimethyldichlorosilane (DDS)-coated glass. The kinetics of co-adhesion of S. oralis 34 to the actinomyces decreased with increasing ionic strength, expressed as the ratio, χ, between the local and non-local initial deposition rates of the streptococci in the vicinity of, or far away from, the adhering actinomyces, respectively. In a stationary end-point of co-adhesion, ionic strength appeared not to be a determinant factor for the co-adhesion of S. oralis 34 with A. naeslundii 5951, either when the actinomyces were adhering to hydrophilic glass or to hydrophobic, DDS-coated glass. However, for S. oralis 34 co-adhering in a stationary end-point with A. naeslundii T14V-J1 in the high-ionic-strength (250 mM KCl) suspension, co-adhesion was far less on hydrophobic, DDS-coated glass than on hydrophilic glass. It is possible that the hydrophobic fibrils on A. naeslundii T14V-J1 bearing the lectin responsible for co-adhesion were immobilized in the latter case by adsorption to the hydrophobic substratum, making them less available for interaction with the streptococci.
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- Genetics And Molecular Biology
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Gene transfer and transposition mutagenesis in Streptomyces roseosporus: mapping of insertions that influence daptomycin or pigment production
More LessStreptomyces roseosporus, the producer of the cyclic lipopeptide antibiotic daptomycin, was shown to be a suitable host for molecular genetic manipulation. S. roseosporus does not appear to express significant restrictic barriers based upon bacteriophage plaque formation studies. Plasmid DNA can be introduced into S. roseosporus by bacteriophage-FP43-mediated transduction and by conjugation from Escherichia coli. The streptomycete transposons Tn5096 and Tn5099, derived from IS493, transpose in S. roseosporus, and Tn5099-induced transposition mutants altered in the production of daptomycin, red pigment or black pigment were identified, and mapped to Dral and Asnl fragments. Three auxotrophic mutations (argB1, ade-1 and metB1) were identified among 100 individual Tn5096 insertions. Alignment and physical mapping of several Tn 5099 insertions in Dral-E and Asnl-B fragments was facilitated by the presence of Dral and Asnl cleavage sites in Tn5099.
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The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus
More LessPreviously we have shown that the type strain of Lactobacillus acidophilus possesses two S-protein-encoding genes, one of which is silent, on a chromosomal segment of 6 kb. The S-protein-encoding gene in the expression site can be exchanged for the silent S-protein-encoding gene by inversion of this slp segment. In this study the presence of S-protein and corresponding S-protein-encoding genes of strains belonging to species that are closely related to L. acidophilus was determined. All strains investigated were identified by numerical comparison of highly standardized one-dimensional SDS-PAGE whole-cellular-protein patterns. Western blot and Southern blot methods were used to identify the presence of, and homology between, S-proteins and S-protein-encoding genes. From these analyses we conclude that strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum possess an S-layer and contain two slp genes. Strains of L. helveticus possess an S-layer but have only one intact slp gene. Strains of L. gasseri, L. johnsonii and L. delbrueckii subsp. bulgaricus have neither an S-layer nor S-protein-encoding genes hybridizing with probes derived from the L. acidophilus slpA or slpB region. The presence of a highly conserved 5′ region in the slp genes of strains of L. acidophilus, L. crispatus, L. amylovorus and L. gallinarum suggests that S-layer variation is a common feature for strains of these species.
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Molecular analysis of the regulation of nisin immunity
More LessThe genetic determinants controlling immunity to nisin are coordinately regulated, along with biosynthesis genes, in response to an environmental signal, nisin or a nisin analogue. The nisR gene product, the putative response regulator of nisin biosynthesis, was found to be a vital component of this induction mechanism. This protein forms part of a two-component regulatory system which controls the expression of genes involved in nisin immunity and biosynthesis. Analysis of the structural requirements of the external signal, using nisin fragments and engineered nisin variants, indicated that the 12 amino-terminal residues of the molecule are a minimum requirement for induction, with an intact ring A being an essential component. Changes throughout the molecule also affected its induction capacity. The production of certain variant nisins by engineered lactococcal strains is reduced in paralle with the strains’ immunity to nisin. This can be attributed to inefficient induction by the variant molecule. Treating growing cultures with nisin restored full immunity and maximized the yields of nisin variants by the producer strains.
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Curing of F-like plasmid TP181 by plumbagin is due to interference with both replication and maintenance functions
More LessF-like plasmid TP181 is particularly susceptible to curing by the naphthoquinone derivative plumbagin, which may attack DNA gyrase. TP181 should provide particular insight into the basis of plasmid elimination, which has application in a number of contexts. Curing was found to be optimal at pH 7·2. An EcoRl fragment containing the RF1A replicon of TP181 was joined to a KmR determinant (giving miniTP181). MiniTP181 had the same increased susceptibility to curing by plumbagin when compared to miniF as TP181 had relative to F. Plumbagin interfered with replication of miniTP181, depressing its copy number and increasing the rate of segregation. Plumbagin also blocked the lethal effect of the ccd locus after rifampicin treatment, which mimics production of plasmid-free segregants, so that more of these plasmid-free cells would survive. Restriction mapping and DNA sequence analysis indicated that the ccd locus of TP181 is almost identical to that of F but that TP181 lacks the repC gene present in F which is needed to activate replication from oriV. Thus the sensitivity of TP181 may be due to its dependence on both a replicon which is hypersensitive to perturbation of supercoiling by plumbagin and a host-killing system which is blocked by plumbagin.
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4-Methylphthalate catabolism in Burkholderia (Pseudomonas) cepacia Pc701: a gene encoding a phthalate-specific permease forms part of a novel gene cluster
More LessWe have determined the entire nucleotide sequence of a 4·4 kbp fragment of pMOP, a plasmid involved in 4-methylphthalate catabolism in Burkholderia cepacia (formerly Pseudomonas cepacia) Pc701. Two complete ORFs were identified and termed mopA and mopB. mopB encodes a 4-methylphthalate permease which is a member of a superfamily of symport proteins found in both prokaryotes and eukaryotes. Functionality was assigned to MopB by detailed analysis of the predicted amino acid sequence, resulting in the identification of 12 hydrophobic membrane-spanning domains and motifs associated with this class of protein. An assay was developed to demonstrate MopB function in substrate uptake. Of 4-methylphthalate, 4-hydroxyisophthalate, benzoate, p-toluate and phthalate, only uptake of 4-methylphthalate and phthalate was demonstrated, suggesting that two carboxyl groups in the ortho position are essential for substrate recognition. The predicted protein MopA showed significant levels of homology to reductase proteins implicated in aromatic and aliphatic catabolism, and contained motifs recognized as binding the ADP and flavin moieties of FAD/NAD. Northern hybridization experiments determined that mopA and mopB are cotranscribed, but expression was only seen in cells grown on 4-methylphthalate and not in cells grown on closely related structural analogues, including phthalate. mopA and mopB may be situated at the 3′-terminus of a cistron about 10 kbp in size. The isolation and characterization of a 4-methylphthalate permease gene may lead to the identification of other permeases involved in bacterial biodegradation processes and possibly the construction of strains with enhanced degradative abilities.
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Stringent and growth-rate-dependent control of the gua operon of Escherichia coli K-12
More LessThe promoter of the gua operon has been located by transcript mapping using primer extension with reverse transcriptase. The surrounding nucleotide sequence has features characteristic of promoters under stringent and growth-rate-dependent regulation, namely a GC-rich discriminator next to the − 10 hexamer, an upstream AT-rich sequence (the UP element) and potential FIS-binding sites. Transcriptional activity of the gua promoter was examined using transcriptional fusions to lacZ placed at a single chromosomal location. Expression from gua was reduced under stringent conditions in vivo, and varied with growth rate. Growth-rate control was independent of guanine-mediated repression. A fusion in which the GC-rich discriminator was mutated by insertion of an AT-rich oligonucleotide was used to demonstrate the importance of this region in control. Both stringent and growth-rate-dependent controls were abolished by the mutation. Other potential regulatory signals in the vicinity of the gua promoter are a pur operator (binding site for the PurR repressor), a gua operator, a DnaA-binding site and a CRP/FNR-binding sequence. The gua promoter lies back-to-back with the promoter for xseA (exonuclease VII), the two promoters being separated by only 20 bp.
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The Mycobacterium tuberculosis purine biosynthetic pathway: isolation and characterization of the purC and purL genes
Genes from the Mycobacterium tuberculosis purine biosynthetic pathway were identified using purine auxotrophic mutants of Mycobacterium smegmatis obtained by Tn 611 transposon mutagenesis. Two approaches were followed in parallel. The first consisted of the complementation of the M. smegmatis purine auxotrophs using a M. tuberculosis H37Rv shuttle cosmid library. In the second approach, specific probes corresponding to the regions adjacent to the insertion sites of Tn 611 in the M. smegmatis genome were used to screen a M. tuberculosis plasmid library by colony hybridization for inserts carrying homologous DNA fragments. Nucleotide sequence analysis of two M. tuberculosis genes isolated by these methods revealed high similarities with purC and purL genes from other bacterial and fungal sources. Transcriptional start sites were mapped for both genes, which revealed similar −10 boxes but with a higher GC content than the Escherichia coli σ70 consensus.
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Molecular analysis of a new insertion sequence from Actinobacillus (Haemophilus) actinomycetemcomitans FDC Y4
More LessWe have found a new insertion sequence (IS), designated IS Aa1, downstream of the S10 operon in Actinobacillus (Haemophilus) actinomycetemcomitans FDC Y4. IS Aa1, the first IS element characterized in this organism, is 705 bp long and lacks terminal inverted repeats. This element displayed significant homology with IS200. Hybridization patterns of genomic DNA of seven A. actinomycetemcomitans strains with an internal ISAa1 probe varied depending on the serotypes, suggesting that IS Aa1 might be a useful tool for epidemiological studies.
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Mycobacterium xenopi IS1395, a novel insertion sequence expanding the IS256 family
More LessAn insertion sequence (IS) of Mycobacterium xenopi has been isolated and sequenced. This 1323 bp element, designated IS1395, is present in up to 18 copies in the M. xenopi genome and may be harboured in an M. xenopi extra-chromosomal element. It encodes a putative transposase of 415 amino acids which displays sequence homology to the Staphylococcus aureus IS256 family. Members of this class of elements have been described in the genus Mycobacterium - for example IS1081 is present in the M. tuberculosis complex, IS1245-IS1311 in M. avium, and IS6120 in M. smegmatis; these elements exhibit an 89%, 45% and 16% amino acid identity with IS1395, respectively. Investigation of the host range of IS1395 by Southern blot analysis revealed additional IS1395-related repeated sequences in M. gordonae and M. celatum. Moreover, IS1395 represents a useful epidemiological tool for M. xenopi strain typing as it provides a diversity of restriction fragment length polymorphism patterns.
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Isolation of Borrelia burgdorferi genes encoding homologues of DNA-binding protein HU and ribosomal protein S20
More LessLinear DNA with covalently closed ends is the predominant form of DNA in the spirochaete Borrelia burgdorferi. All bacteria examined to date have small DNA-binding proteins related to the Escherichia coli IHF and HU proteins that appear to play roles in DNA compaction and replication, but such proteins had not been isolated from bacteria with linear genomes. We found a single gene in B. burgdorferi (named hbb) whose product (named Hbb) complements the defects for λ DNA packaging found in E. coli strains mutant in the genes for IHF and HU. The sequence of the predicted B. burgdorferi protein is similar to those of HU and IHF-like proteins in other bacteria. The gene appears to be in an operon with the order rpsT-hbb-orfH, where the rpsT gene is a homologue of the E. coli gene encoding ribosomal protein S20 and the orfH gene encodes a protein of unknown function. This operon is located upstream of the previously identified B. burgdorferi rho homologue.
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Identification of an EF-Tu protein that is periplasm-associated and processed in Neisseria gonorrhoeae
More LessA 44 kDa protein is a dominant component of periplasmic extracts of Neisseria gonorrhoeae. Peptide sequence generated from a cyanogen-bromide-cleaved fragment of this protein indicated sequence homology with elongation factor-Tu (EF-Tu). Polyclonal antiserum was made against the 44 kDa protein purified from periplasm extracts of N. gonorrhoeae. The preabsorbed antiserum was immunoblotted against whole-cell lysates on two-dimensional gels. A 44 kDa protein and a smaller 37 kDa protein were recognized by this antiserum. A N. gonorrhoeae λ phage DNA library was screened and a clone expressing a 44 kDa protein was identified. The DNA insert in this clone contained several genes homologous to genes contained in the str operon of Escherichia coli. One ORF product with a calculated molecular mass of 43 kDa was highly homologous to the EF-TuA of E. coli. A synthetic peptide antiserum specific for a portion of the C terminus of EF-Tu confirmed that the 37 kDa protein in whole-cell lysates of N. gonorrhoeae was a processed form of EF-Tu. Deletion of the tufA gene homologue in N. gonorrhoeae was attempted but was unsuccessful.
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Isolation of the mRNA-capping enzyme and ferric-reductase-related genes from Candida albicans
The mRNA-capping enzyme (mRNA 5′-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1∆ null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1∆ null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5′-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1∆ null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.
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Manipulation of the tuf gene provides clues to the localization of sequence element(s) involved in the thermal stability of Thermotoga maritima elongation factor Tu
More LessTruncated versions of the tuf gene for elongation factor Tu (EF-Tu; 400 aa) from the hyperthermophilic bacterium Thermotoga maritima have been produced by progressive 3′→5′ trimming. The truncated genes have been expressed in Escherichia coli and the thermal stability of the gene products has been assayed by monitoring their GDP-binding capacity after preheating the cell-free extracts at various temperatures (65–95 °C). One of the truncated proteins, corresponding to the nucleotide-binding domain (G domain aa 1–200) appears to be only slightly less stable than the full-length EF-Tu. Replacement of the first 90 N-terminal residues of both the full-length Thermotoga EF-Tu and the isolated G domain with the corresponding sequence of the mesophilic bacterium E. coli, drastically destabilizes both the complete and the truncated protein, indicating that sequence element(s) that are crucial for the attainment of a thermally stable conformation of the Thermotoga EF-Tu lie well within the initial portion of the G domain between residues 1 and 90. The relevant residues defy identification, however, as no amino acid preferences, or exclusive sequence element(s), appear to distinguish the N-terminal region of the thermophilic proteins from those of mesophilic counterparts. It is suggested that the thermal stability of Thermotoga EF-Tu is critically dependent upon unique tertiary structural interactions involving certain N-terminal residues of the molecule.
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Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes
Recombinant Escherichia coli clones displaying thermostable β-glucanase activity were isolated from two different gene libraries of the hyperthermophilic bacterium Thermotoga maritima MSB8 (DSM 3109), and the nucleotide sequence of a 1,4-β-glucanase gene designated celA was determined. Amino-terminal sequencing of cellulase I previously detected in T. maritima cells indicated that the celA gene encodes this β-glucanase, which is now designated CelA. CelA, which has a calculated molecular mass of 29732 Da, was purified from a recombinant E. coli strain to apparent homogeneity as judged by SDS-PAGE with a 44% yield. The enzyme was most active against soluble substrates such as mixed-linkage β-glucan and CM-cellulose. CelA displayed remarkable thermostability, which was enhanced in the presence of high concentrations of salt. Downstream of the celA gene we found a second open reading frame, celB, whose nucleotide sequence was 58% identical to celA. Experimental proof that celB also encodes a β-glucanase was obtained by separation from celA and expression in E. coli under the control of an efficient host promoter. According to the deduced amino acid sequences, CelB, in contrast to CelA, contains a signal peptide at the amino terminus. CelB and CelA had similar substrate specificities and temperature optima, but differed in their pH optima. Also, the addition of salt had a less stabilizing effect on CelB than on CelA. Nine 30 bp direct repeats, each itself representing a sequence with imperfect dyad symmetry, were detected upstream of the celA-celB cellulase gene cluster.
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The gene for γ-glutamylcysteine synthetase from Thiobacillus ferrooxidans has low homology to its Escherichia coli equivalent and is linked to the gene for citrate synthase
More LessThe gene for γ-glutamylcysteine synthetase (gshA) from Thiobacillus ferrooxidans was isolated from a family of cosmids by its ability to complement an Escherichia coli gshA trxA double mutant which was unable to grow on minimal medium lacking glutathione. The predicted sequence of the γ-glutamylcysteine synthetase was found to have only 18% amino acid sequence identity to the equivalent enzyme from E. coli. In spite of this low sequence homology, concentrations of GSH in a cell extract prepared from the E. coli gshA trxA mutant containing the cloned gene were almost as high as in a cell extract prepared from a wild-type E. coli strain. The gshA gene was found to be physically and transcriptionally linked to the T. ferrooxidans gene for citrate synthase (gltA). The T. ferrooxidans and E. coli citrate synthases shared 37% amino acid sequence identity and the cloned T. ferrooxidans citrate synthase gene was able to complement an E. coli gltA mutant.
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Unusual ribulose-1,5-bisphosphate carboxylase/oxygenase genes from a marine manganese-oxidizing bacterium
More LessThe Gram-negative bacterium strain SI85-9A1 is a novel marine α-proteobacterium that oxidizes manganese(II) to manganese(IV). Initial DNA hybridization screening showed that SI85-9A1 possesses a gene similar to cbbL, the gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO; EC 4.1.1.39). However, no RubisCO enzyme activity was found in cultures of SI85-9A1. Genes coding for both large (cbbL) and small (cbbS) subunits of a RubisCO enzyme were identified, isolated and sequenced. When these genes were introduced into an Escherichia coli host strain, ribulose-1,5-bisphosphate-dependent CO2 fixation occurred under control of a lac promoter, indicating that the protein is functional in E. coli. Although their function is unknown, this is the first direct evidence for the presence of RubisCO genes in a manganese-oxidizing bacterium. Phylogenetic analysis of the RubisCO genes of strain SI85-9A1 showed that they are divergent, but are more related to those from non-chlorophyte algal chloroplasts than are those from other bacteria. The fact that the RubisCO sequence of strain SI85-9A1 is not closely related to any other published RubisCO sequence suggests that the protein may be valuable for studies of the function and evolution of the RubisCO enzyme as well as its activity in the environment.
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An upstream activating sequence containing curved DNA involved in activation of the Clostridium perfringens plc promoter
The plc gene, which encodes phospholipase C (α-toxin) of Clostridium perfringens, possesses three poly(A) tracts forming an intrinsically curved DNA region immediately upstream of the promoter. The in vivo transcriptional activity of the plasmid-borne plc gene was stimulated by this curved-DNA-containing sequence, depending on its proper linear and rotational orientation. The in vitro transcriptional activity of the plc gene was also stimulated by the upstream sequence. In addition, the stimulatory effect of the sequence and the degree of DNA bending were greater at lower temperature, as was demonstrated by both in vitro and in vivo transcription assays, and a gel-mobility assay, respectively. A similar temperature effect was also observed with the chromosomal plc gene. These observations suggest that the upstream DNA curvature per se stimulates the initiation of transcription of the plc gene, possibly through direct contact with RNA polymerase.
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Aspartate transport by the Dct system in Rhizobium leguminosarum negatively affects nitrogen-regulated operons
More LessAmino acid uptake by the general amino acid permease (Aap) of Rhizobium leguminosarum strain 3841 was severely reduced by the presence of aspartate in the growth medium when glucose was the carbon source. The reduction in transport by the Aap appeared to be caused by inhibition of uptake and not by transcriptional repression. However, as measured with lacZ fusions, the Ntr-regulated gene glnll was repressed by aspartate. The negative regulatory effect on both the Aap and glnll was prevented by mutation of any component of the dicarboxylate transport (Dct) system or by the inclusion of a C4-dicarboxylate in the growth medium, including the non-metabolizable analogue 2-methylsuccinate. As measured by total uptake and with a dctA-lacZ fusion, aspartate was an efficient inducer of the Dct system, but slightly less so than succinate alone or succinate and aspartate together. Thus, aspartate does not cause overexpression of DctA leading to improper regulation of other operons. Transport measurements revealed that the Dct system has an apparent K m for succinate of 5 μM and an apparent K m for aspartate inhibition of succinate uptake of 5 mM. These data imply that the Dct-mediated accumulation of aspartate causes an unregulated build-up of aspartate or a metabolic product of it in the cell. This accumulation of aspartate is prevented either by mutation of the dct system or by the presence of a higher affinity substrate that will reduce access of aspartate to the carrier protein. Elevation or disruption of the intracellular aspartate pool is predicted to disrupt N-regulated operons and nitrogen fixation.
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Expression of the exoY gene, required for exopolysaccharide synthesis in Agrobacterium, is activated by the regulatory ros gene
More LessSome mutants of Agrobacterium radiobacter, defective in exopolysaccharide synthesis, were phenotypically complemented by two different regions of cloned chromosomal DNA. One of these had been shown to contain a gene termed ros, a novel class of transcriptional regulator. The other contains a gene termed exoY which encodes a glycosyltransferase that is involved in one of the early steps in exopolysaccharide synthesis. Mutations in ros reduced the expression of exoY and a model to account for the complementation of certain exo alleles by both ros and exoY is presented. Tn phoA insertions into exoY which expressed alkaline phosphatase activity were isolated and mapped, confirming the membrane location of the exoY gene product. Some of these mutations were dominant, causing merodiploids to be non-mucoid. exoY is linked to two genes, one encoding an ω-aminotransferase and the other encoding an aldehyde dehydrogenase.
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IdiA, a 34 kDa protein in the cyanobacteria Synechococcus sp. strains PCC 6301 and PCC 7942, is required for growth under iron and manganese limitations
More LessIn the cyanobacteria Synechococcus PCC 6301 and PCC 7942 a protein with an apparent molecular mass of about 34 kDa (called IdiA for iron-deficiency-induced protein A) accumulates under iron and manganese limitation. IdiA from Synechococcus PCC 6301 was partially sequenced, showing that the N-terminal amino acid is an alanine. Moreover, the gene encoding this protein in Synechococcus PCC 6301 has been identified and completely sequenced. The idiA gene codes for a protein starting with valine and consisting of 330 amino acid residues. Thus, IdiA is apparently synthesized as a precursor protein of 36·17 kDa and cleaved to its mature form of 35·01 kDa between two alanine residues at positions 9 and 10. IdiA is a highly basic protein having an isoelectric point of 10·55 (mature protein). Comparison of the amino acid sequence of IdiA with protein sequences in the database revealed that IdiA has similarities to two basic bacterial iron-binding proteins, SfuA from Serratia marcescens and Fbp from Neisseria gonorrhoeae. Insertional inactivation of the idiA gene in Synechococcus PCC 7942 resulted in a mutant which was unable to grow under iron- or manganese-limiting conditions. Manganese limitation of the mutant strain led to a drastic reduction of photosystem II activity (O2 evolution) within less than 48 h, while wild-type cells required a prolonged cultivation in Mn-deficient medium before an effect on photosystem II was observed. Thus, IdiA is a protein involved in the process of providing photosystem II with manganese.
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4-Dihydromethyltrisporate dehydrogenase from Mucor mucedo, an enzyme of the sexual hormone pathway: purification, and cloning of the corresponding gene
More LessWe have purified the NADP-dependent 4-dihydromethyltrisporate dehydrogenase from the zygomycete Mucor mucedo. The enzyme is involved in the biosynthesis of trisporic acid, the sexual hormone of zygomycetes, which induces the first steps of zygophore development. Protein was obtained from the (−) mating type of M. mucedo after induction with trisporic acid, and purified by gel filtration and affinity chromatography steps. On SDS-PAGE a band with an apparent molecular mass of 33 kDa was ascribed to the enzyme. After transferring onto PVDF membranes the protein was digested with endoprotease Lys-C, and several peptides were sequenced. Oligonucleotides derived from protein sequence data were used for PCR amplification of genomic M. mucedo DNA. The PCR fragment was used as probe for isolation of the corresponding cDNA and complete genomic DNA clones. Comparison of protein and DNA sequence data showed that the cloned fragment corresponded to the purified protein. Search for similarity with protein sequences of the Swiss-Prot database revealed a relationship to enzymes belonging to the aldo/keto reductase superfamily. Southern-blot analysis of genomic DNA with the labelled cloned fragment detected a single-copy gene in both mating types of M. mucedo. PCR with genomic DNA from other zygomycetes gave rise to several fragments. Hybridization analysis with the cloned M. mucedo fragment showed that a fragment of similar length cross-hybridized in Blakeslea trispora (Choanephoraceae) as well as in Parasitella parasitica and Absidia glauca (Mucoraceae). The promoter region of the gene contains DNA elements with similarity to a cAMP-regulated gene of Dictyostelium discoideum.
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MIG1 overexpression causes flocculation in Saccharomyces cerevisiae
More LessMIG1, encoding a C2H2 zinc-finger repressor protein involved in carbon catabolite repression, was found to play a role in non-sexual flocculation of Saccharomyces cerevisiae. Disruption of MIG1 in a flocculent mutant strain of NCYC 227, resulted in a non-flocculent phenotype. Expression of MIG1 on a 2 μ pRS426 vector in a non-flocculent strain, YM 4134, caused flocculation; MIG1 on a high-copy-number LEU2-d plasmid caused intense flocculation in the same strain. Mutations in the SSN6 and TUP1 genes confer a flocculent phenotype in non-flocculent strains of S. cerevisiae, and it has been shown that Mig1 can tether the Ssn6p-Tup1p complex to the regulatory regions of glucose-repressible genes. Mutations in tup1 in a MIG1 background caused flocculation while double mutants of TUP1 and MIG1 did not flocculate. Based on these results, a model for the role of MIG1 in flocculation gene regulation is proposed.
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- Pathogenicity And Medical Microbiology
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Immune responses to linear epitopes on the PorB protein of Neisseria meningitidis in patients with systemic meningococcal disease
Neisserial porins, the major protein constituents of the outer membrane capable of inducing antibody responses in humans, are considered to be meningococcal vaccine candidates, so it is important to map the relevant B-cell epitopes. For B-cell epitope analyses of the serotype 15 PorB protein in Neisseria meningitidis, paired sera from selected patients with systemic meningococcal disease (SMD) were screened with synthetic 12mer peptides spanning the PorB protein sequence, and/or its variable region 1 (VR1). A ‘SMD-related’ linear B-cell epitope was found within the VR1 region consisting of 14 residues (17svFHQNGQVTEvtt30). A 23mer soluble peptide (D63b2) that covered the VR1 region, including the complete 17svFHQNGQVTEvtt30 sequence, was recognized, whereas no detectable binding was observed to a 16mer peptide (D63a1) containing most of the essential sequence (19FHQNGQVTEvtt30). A low frequency of lgG responses specific for the PorB linear epitopes was found in convalescent-phase sera from 132 SMD patients studied, as judged from both immunoblotting studies (24/132; 18·2%) and reactivity with peptide D63b2 (18/132; 13·6%). Peptide D63B2 significantly inhibited lgG binding to the denatured PorB protein on immunoblots, suggesting that this B-cell epitope was one of the main linear epitopes on the PorB protein recognized by sera from some SMD patients.
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Characterization of epitopes involved in the neutralization of Pasteurella haemolytica serotype A1 leukotoxin
More LessDefined segments of the leukotoxin A gene (lktA) from an A1 serotype of Pasteurella haemolytica were cloned into a plasmid vector and expressed as LacZα fusion proteins. These fusion proteins were electrophoresed in SDS-PAGE gels and their immunoblotting reactivities with several monoclonal antibodies characterized. The epitope recognized by a strongly neutralizing monoclonal antibody was localized to a 32 amino acid region near the C terminus of the leukotoxin A (LktA) molecule. The epitope recognized by a non-neutralizing antibody was localized to a 33 amino acid region immediately adjacent. Smaller recombinant peptides containing these epitopes were not antigenic, but a polypeptide encompassing 229 amino acids at the C terminus evoked neutralizing antibodies when used to immunize specific-pathogen-free lambs. The distributions of linear epitopes recognized by this antiserum and by antisera raised to full-length recombinant LktA and to native LktA produced by P. haemolytica serotype A1 were determined by their reactivities with a set of overlapping 10 amino acid synthetic peptides. This revealed a complex distribution of linear epitopes at the C-terminal end of LktA. Toxin-neutralizing antibodies in convalescent sheep serum were shown to be directed against conformational epitopes by selective absorption of antibodies directed against linear epitopes.
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Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis
The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CA14 (∆ura3::imm434/∆ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.
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An extracellular enzyme with hyaluronidase and chondroitinase activities from some oral anaerobic spirochaetes
More LessTreponema denticola, Treponema vincentii and Treponema socranskii produce an enzyme that hydrolyses hyaluronic acid (HA) and chondroitin sulphate (CS). The secreted enzyme is specifically inhibited by gold sodium thiomalate and anti-bee-venom antibodies. The use of saturated substrate (HA or CS) transblots allowed the visualization of active enzyme directly from culture supernatants and is a useful tool in clarification of complex polysaccharide-degrading enzyme specificities. The affinity-purified extracellular enzyme of T. denticola contains a single molecular species with a molecular mass of 59 kDa. Since it hydrolyses both HA and CS, it can more appropriately be termed a hyaluronoglucosaminidase (HGase). The HGase has been localized at the cell surface by electron microscopy and may play an active role in the degradation of connective tissue ground substance in the initiation and progression of periodontal disease.
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- Physiology And Growth
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Adenosylcobalamin-dependent methylmalonyl-CoA mutase isozymes in the photosynthetic protozoon Euglena gracilis Z
More LessThe photosynthetic protozoon Euglena gracilis Z contains adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM) involved in propionate metabolism. The specific activity of the Euglena mutase was about 6·5-fold greater in propionate-adapted Euglena cells than in photoautotrophic cells (control). Although the control cells contained only one mutase (apparent M r 72000), the propionate-adapted cells contained two mutases with M r values of 72000 and 17000; both enzymes were located in the mitochondria. These results provide evidence that propionate-adapted Euglena contains two MCM isozymes. The induced mutase (M r 17000) permits photoassimilation of propionate.
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Cell cycle studies on the mode of action of yeast K28 killer toxin
More LessThe virally encoded K28 killer toxin of Saccharomyces cerevisiae kills sensitive cells by a receptor-mediated process. DNA synthesis is rapidly inhibited, cell viability is lost more slowly and cells eventually arrest, apparently in the S phase of the cell cycle with a medium-sized bud, a single nucleus in the mother cell and a pre-replicated (1n) DNA content. Cytoplasmic microtubules appear normal, and no spindle is detectable. Arrest of a sensitive haploid yeast strain by α-factor at START gave complete protection for at least 4 h against a toxin concentration that killed non-arrested cells at the rate of one log each 2.5 h. Cells released from α-factor arrest were killed by toxin at a similar rate; arrest occurred with medium-sized buds within the same cell cycle. Cells arrested by hydroxyurea, with unreplicated DNA, or by the spindle poison methylbenzimidazol-2yl-carbamate, with unseparated chromosomes, both arrest at the checkpoint at the G2/M boundary; these arrested cells were not protected against toxin, losing about one log of viability every 4 h. Following release from the cell cycle block, a majority of these toxin-exposed cells progressed through the cell cycle and arrested in the following S-phase, again with medium-sized buds. Killing by K28 toxin apparently requires entry into the nuclear division and bud cycles, but can result from inhibition of either early or late events in these cycles. Morphogenesis in moribund cells is uniformly blocked in early S-phase with an immature bud. Toxin action causes either independent blockage of both DNA synthesis and the budding cycle, or inhibits some unknown step required for both events.
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- Plant-Microbe Interactions
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Regulatory systems modulating the transcription of the pectinase genes of Erwinia chrysanthemi are conserved in Escherichia coli
More LessTo depolymerize plant pectin, the phytopathogenic enterobacterium Erwinia chrysanthemi produces five isoenzymes of pectate lyases encoded by the five genes pelA, pelB, pelC, pelD and pelE. In Er. chrysanthemi, all genes involved in pectin degradation are specifically controlled by the KdgR repressor and are induced in the presence of a pectin catabolic product, 2-keto-3-deoxygluconate (KDG). Transcription of the pectinase genes is dependent on many environmental conditions. Transcriptional fusions present on low-copy-number plasmids were used to study the regulation of the pel genes in a heterologous host, Escherichia coli. Some physiological regulations that take place in Er. chrysanthemi are conserved in E. coli. The five pel fusions in E. coli are affected by growth phase, catabolite repression and anaerobic growth conditions and are induced in the presence of galacturonate, a sugar whose catabolism leads to the formation of KDG, the inducer of pel transcription in Er. chrysanthemi. Expression of pelE increased with the osmolarity of the culture medium. In contrast, the regulation of pel expression by temperature or nitrogen starvation, observed in Er. chrysanthemi, was not conserved in E. coli, suggesting that the mechanisms responsible for these regulations are specific to Er. chrysanthemi. Analysis of different E. coli mutants allowed some regulators affecting the transcription of the pel genes to be identified. In E. coli, the growth-phase regulation of the pel genes is not dependent on the RpoS sigma factor and the fnr gene is not involved in the increase of pel expression in oxygen-limited conditions. The gene hns, involved in the regulation of numerous genes, appears to affect pel expression but the effects of E. coli hns mutations are not related to osmoregulation. In contrast, this analysis clearly demonstrates the interchangeability of two regulatory systems of E. coli and Er. chrysanthemi: the global control exerted by the catabolite activator protein CAP and the specific regulation mediated by the KdgR repressor.
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- Genome Analysis
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Organization of the Haemophilus ducreyi 35000 chromosome
More LessA physical and rudimentary genetic map of the Haemophilus ducreyi strain 35000 genome was constructed. Pulsed-field gel electrophoresis was used to separate restriction fragments of H. ducreyi DNA digested with SfiI, I-Ceul, or Sfil plus I-Ceul. The sizes of the fragments were determined, and the circular chromosome was estimated to be 1757 kbp. The six I-Ceul fragments and four Sfil fragments were ordered into macrorestriction maps using Southern blot hybridization with random H. ducreyi clones as probes. It was shown that both H. ducreyi and the distantly related Haemophilus influenzae have six rrn operons marked by the locations of the I-Ceul sites. However, the two species displayed distinct I-CeuI restriction patterns. A second H. ducreyi strain, CIP542, displayed an identical I-CeuI pattern to that of H. ducreyi 35000, but SfiI digests of the two strains were distinct. The orientation of the six rrn operons was determined and thirteen identified H. ducreyi genes positioned on the map of strain 35000.
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