bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype‐specific antibody responses
Authors: McCarvil J. , McKenna A. J. , Grief C. , Hoy C. S. , Sesardic D. , Maiden M. C. J. , Feavers I. M. 
Published: October 2006

SummaryThe class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole‐cell enzyme‐linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole‐cell dot‐blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype‐specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype‐specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.
DNA rearrangements and phenotypic switching in prokaryotes
Authors: Dybvig K. 
Published: October 2006

SummaryMicroorganisms have numerous strategies for coping with environmental changes. In many systems, a single cell has the capacity to generate a seemingly infinite array of phenotypic variants in just a few generations of growth. The resulting heterogeneous population is well equipped for sudden environmental change; even if only a few cells in the population possess a phenotype needed for survival, these cells have the capacity to regenerate a similarly diverse population. Phenotypic switching in these systems usually results from high‐frequency DNA rearrangements which are the subject of this review.
Topoisomerase mutations affect the relative abundance of many Escherichia coli proteins
Authors: Steck Todd R. , Franco Robert J. , Wang Jian‐Ying , Drlica Karl 
Published: October 2006

SummaryThe relative abundance of 88 proteins was measured in extracts from three strains of Escherichia coli K‐12 that are isogenic except for the topA and gyrB genes. Mutations in these genes slightly raise or lower, respectively, steady‐state DNA supercoiling levels but have little effect on growth rate. Altered protein abundances were observed in the mutant strains relative to wild type. Many proteins exhibited minimum abundance at wild‐type supercoiling levels, and other proteins exhibited maximal abundance at relaxed levels. A smaller number showed maximal abundance at elevated levels of supercoiling. These data suggest that small, non‐lethal changes in DNA supercoiling can have widespread effects on patterns of gene expression.
A novel strategy for the isolation of luxl homologues: evidence for the widespread distribution of a LuxR:Luxl superfamily in enteric bacteria
Authors: Swift S. , Winson M. K. , Chan P. F. , Bainton N. J. , Birdsall M. , Reeves P. J. , Rees C. E. D. , Chhabra S. R. , Hill P. J. , Throup J. P. , Bycroft B. W. , Salmond G. P. C. , Williams P. , Stewart G. S. A. B. 
Published: October 2006

SummaryThe pheromone N‐(3‐oxohexanoyl)‐L‐homoserine lactone (OHHL) regulates expression of bioluminescence in the marine bacterium Vibrio fischeri, the production of carbapenem antibiotic in Erwinia carotovora and exoenzymes in both E. carotovora and Pseudomonas aeruginosa. A characteristic feature of this regulatory mechanism in V. fischeri is that it is cell density‐dependent, reflecting the need to accumulate sufficient pheromone to trigger the induction of gene expression. Using a lux plasmid‐based bioluminescent sensor for OHHL, pheromone production by E. carotovora, Enterobacter agglomerans, Hafnia alvei, Rahnella aquatilis and Serratia marcescens has been demonstrated and shown also to be cell density‐dependent. Production of OHHL implies the presence in these bacteria of a gene equivalent to luxl. Chromosomal banks from all five enteric bacteria have yielded clones capable of eliciting OHHL production when expressed in Escherichia coli. The luxl homologue from both E. carotovora (carl) and E. agglomerans (eagl) were characterized at the DNA sequence level and the deduced protein sequences have only 25% identity with the V. fischeri Luxl. Despite this, carl, eagl and luxl are shown to be biologically equivalent. An insertion mutant of eagl demonstrates that this gene is essential for OHHL production in E. agglomerans.
The rpsL gene and streptomycin resistance in single and multiple drug‐resistant strains of Mycobacterium tuberculosis
Authors: Nair Jaygopal , Rouse David A. , Bai Gill‐Han , Morris Sheldon L. 
Published: October 2006

SummaryThe recent emergence of indolent and rapidly fatal drug‐resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal SR protein (rpsL gene) from streptomycin‐resistant strains and their streptomycin‐sensitive parental strains. We have demonstrated that five singly SmRM. tuberculosis strains and an SmR isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer SmR in Escherichia coli. In contrast, two other multiple drug‐resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug‐sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.
Cloning of Bordetella bronchiseptica urease genes and analysis of colonization by a urease‐negative mutant strain in a guinea‐pig model
Authors: Monack D. M. , Falkow S. 
Published: October 2006

SummaryThe genes encoding urease were cloned from Bordetella bronchiseptica and the 5.2 kb of DNA essential for expression analysed in a T7 RNA polymerase transcription‐translation system. At least four poly‐peptides with predicted molecular weights of 69 000, 26 000, 12 200 and 11 000 were found. Partial DNA sequence of the gene encoding the 69 000 Da polypeptide revealed high amino acid identity to the α‐subunit of Proteus mirabilis urease, UreC and jack bean urease. A stable, unmarked deletion was constructed in this gene to create a urease‐negative mutant of B. bronchiseptica. To assess colonization in a guinea‐pig model, the urease‐negative strain was inoculated with the urease‐positive parental strain in a mixed infection. The urease‐negative strain out competed the urease‐positive strain in the trachea, lungs and caecum. We demonstrate that urease is not essential for B. bronchiseptica colonization of the guinea‐pig respiratory and digestive tracts.
Correlation of gene transcription with the time of initiation of chromosome replication in Escherichia coli
Authors: Theisen Patrick W. , Grimwade Julia E. , Leonard Alan C. , Bogan Joseph A. , Helmstetter Charles E. 
Published: October 2006

SummaryTranscriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dnaA prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation.
A proline‐rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae
Authors: Donnelly Sean F. H. , Pocklington Michael J. , Pallotta Dominick , Orr Elisha 
Published: October 2006

SummaryA gene (VRP1) encoding a novel proline‐rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline‐rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild‐type VRP1 allele with a mutant allele results in strains that grow slower than wild‐type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verprolin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.
Restoration of attachment, virulence and nodulation of Agrobacterium tumefaciens chvB mutants by rhicadhesin
Authors: Swart Saskia , Smit Gerrit , Lugtenberg Ben J.J. , Kijne Jan W. 
Published: October 2006

SummaryIn contrast to wild‐type Agrobacterium tumefaciens strains, β‐1,2‐glucan‐deficient chvB mutants were found to be unable to attach to pea root hair tips. The mutants appeared to produce rhicadhesin, the protein that mediates the first step in attachment of Rhizobiaceae cells to plant root hairs, but the protein was inactive. Both attachment to root hairs and virulence of the ChvB mutants could be restored by treatment of the plants with active rhicadhesin, whereas treatment of plants with β‐1,2‐glucan had no effect on attachment or virulence. Moreover, nodulation ability of a chvB mutant carrying a Sym plasmid could be restored by pretreatment of the host plant with rhicadhesin. Apparently the attachment‐minus and avirulence phenotype of chvB mutants is caused by lack of active rhicadhesin, rather than directly being caused by a deficiency in β‐1,2‐glucan synthesis. The results strongly suggest that rhicadhesin is essential for attachment and virulence of A. tumefaciens cells. They also indicate that the mechanisms of binding of Agrobacterium and Rhizobium bacteria to plant target cells are similar, despite differences between these target cells.
Suppression of temperature‐sensitive assembly mutants of heat‐labile enterotoxin B subunits
Authors: Sandkvist Maria , Bagdasarian Michael 
Published: October 2006

SummaryDeletions or substitutions of amino acids at the carboxyl‐terminus of the heat‐labile enterotoxin B sub‐unit (EtxB) affect its assembly into pentamers in a temperature‐dependent manner. At 42°C, the mutations prevent the B subunits from achieving their final pentameric structure resulting in membrane association of the monomers. However, mutant B subunits produced at 30°C assemble, in the periplasm, into pentamers that remain stable when transferred to 42°C, indicating that the mutant pentamers are stable under conditions where their formation is inhibited. The mutant pentamers are, similarly to wild‐type pentamers, SDS‐resistant and stable, in vitro, at temperatures up to 65°C. This suggests that although the C‐terminal amino acids are part of the subunit interface, they appear not to contribute significantly to the stability of the final pentameric complex, but are instead essential for the formation or stabilization of an assembly intermediate in the pentamerization process. Single second site mutations suppress the assembly defect of mutant EtxB191.5, which carries substitutions at its C‐terminus. The Thr→IIe replacement at position 75 in the α2‐helix probably restores the van der Waals contact between residues 75 and 101, which had been greatly reduced by the Met→Leu substitution at position 101 in the β6‐strand of EtxB191.5. Interaction between the α2‐helix and β6‐strand which contains the c‐terminus probably stabilizes a conformation essential for assembly and is therefore required for the formation of pentamers.
pH‐dependent permeabilization of the plasma membrane of mammalian cells by anthrax protective antigen
Authors: Milne Jill C. , Collier R. John 
Published: October 2006

SummaryProtective antigen (PA) of anthrax toxin forms ion‐conductive channels in planar lipid bilayers and liposomes under acidic pH conditions. We show here that PA has a similar permeabilizing action on the plasma membranes of CHO‐K1 and three other mammalian cell lines (J774A.1, RAW264.7 and Vero). Changes in membrane permeability were evaluated by measuring the efflux of the K+ analogue, 86Rb+, from prelabelled cells, and the influx of 22Na+. The permeabilizing activity of PA was limited to a proteolytically activated form (PAN) and was dependent on acidic pH for membrane insertion (optimal at pH 5.0), but not for sustained ion flux. The flux was reduced in the presence of several known channel blockers: tetrabutyl‐, tetrapentyl‐, and tetrahexylammonium bromides. PAN facilitated the membrane translocation of anthrax edema factor under the same conditions that induced changes in membrane permeability to ions. These results indicate that PAN permeabilizes cellular membranes under conditions that are believed to prevail in the endosomal compartment of toxin‐sensitive cells; and they provide a basis for more detailed studies of the relationship between channel formation and translocation of toxin effector moieties in vivo.
The XbaI‐BlnI‐CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2
Authors: Liu Shu‐Lin , Hessel Andrew , Sanderson Kenneth E. 
Published: October 2006

SummaryWe have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed‐field gel electrophoresis. The chromosome of 4600kb was analysed by XbaI (16 fragments), I‐CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb. Cleavage sites of I‐CeuI, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K‐12, and the XbaI and BinI sites in glt‐tRNA are also conserved, but other sites are less conserved. Transposon Tn10, located at 60 different positions in the chromosome of S. typhimurium, was transduced by bacteriophage P22 into S. enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10. Gene order in S. enteritidis is identical to S. typhimurium LT2 and similar to E. coli K‐12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2. Endpoints are in the NDZs, or non‐divisible zones, in which inversion endpoints were not detected in experiments in E. coli K‐12 and S. typhimurium LT2. This inversion resembles the inversion between S. typhimurium and E. coli, but is longer at both ends.
The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin‐like PulG protein into a multiprotein complex
Authors: Pugsley Anthony P. , Possot Odile 
Published: October 2006

SummaryIt has been proposed that the four type IV pilin‐like proteins that are required for extracellular protein secretion by the general secretory pathway (GSP) might assemble into a trans‐periplasm complex resembling a type IV pilus. To test this idea, we examined the subcellular distribution and oligomeric state of PulG, one of the type IV pilin‐like proteins required for pullulanase secretion in Klebsiella oxytoca. Fractionation of Escherichia coli cells carrying a single copy of each pul gene showed that PulG protein was located in two distinct envelope fractions corresponding to the outer and cytoplasmic membranes. The protein was partially released by treating the membranes with Triton X‐100 + EDTA or at high pH, but not by Triton X‐100 atone or by 8M urea, 6M guanidine hydrochloride or 1 M NaCl. Like type IV pilins, non‐sedimentable PuiG that had been released from the membranes at high pH could be sedimented by centrifugation when the pH was lowered. Treatment of whole cells, sphaeroplasts or isolated membranes with a cleavable cross‐linking agent produced mainly PulG homodimers. Previous studies showed that both PulO, which cleaves and N‐methylates the PulG precursor, and PulE, a putative ATP‐binding protein, share extensive sequence identity with proteins known to be required for type IV pilus processing and assembly. However, mutations which disrupted either pulE or pulO, or indeed the complete absence of all other components of the pullulanase secretion apparatus, had little or no effect on any of the properties of PulG protein described above. We conclude that there is no evidence that PulG protein assembles into a stable multiprotein complex or that processing of the PulG precursor causes a detectable change in its subcellular distribution.
Chromosomal domains of supercoiling in Salmonella typhimurium
Authors: Pavitt† Graham D. , Higgins Christopher F. 
Published: October 2006

SummaryThe chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains. It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled. We have addressed this question genetically, using a supercoiling‐sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome. We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling. These findings have implications for the organization of the bacterial genome.
Light‐induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region
Authors: McGowan Simon J. , Gorham Hazel C. , Hodgson David A. 
Published: October 2006

SummaryThe carR region encodes a light‐inducible promoter, a negative regulator of the promoter and a trans‐acting activator that controls the light‐inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans‐acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence‐specific DNA‐binding motifs. The sequence of the light‐inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response (‘heat‐shock’) promoters.
Specific transcriptional requirements for positive regulation of the anaerobically inducible pfl operon by ArcA and FNR
Authors: Sawers Gary 
Published: October 2006

SummaryExpression of the pfl operon of Escherichia coli Is induced 12‐ to 15‐fold by anaerobiosis and transcription is mediated by seven co‐ordinately regulated promoters. The 5′ non‐translated regulatory region of the operon is approximately 450 bp in length and contains two of the seven promoters, termed promoter 6 and promoter 7. Site‐directed mutagenesis was used to aid the identification of DNA sequences important in directing transcription from the two promoters and to examine the effects such mutations had on the regulation of anaerobic pfl operon expression. Introduction of chromosomal mutations either in the FNR‐binding site or ‐10 region of promoter 6 blocked transcription from this promoter, as determined by primer extension. Similarly, mutation of the ‐10 region or the putative FNR half‐site located at ‐50 relative to the transcription start site of promoter 7 severely reduced transcription from that promoter. Prevention of transcription from promoter 6 by the ‐10 box mutation had no influence on promoter 7 transcription. Surprisingly, however, alteration of the FNR‐binding site at promoter 6 did reduce transcription from promoter 7. Thus, a cis mutation located 280bp downstream on the DNA had a profound effect on promoter 7 transcription. This effect would be commensurate with this mutation disrupting an important interaction between proteins bound at promoter 7 with those bound at promoter 6. Primer extension demonstrated that the promoter 7 mutations had no apparent influence on promoter 6 transcription. By using pfl–lacZ gene fusions it could be shown that the FNR‐binding site and ‐10 region mutations at promoter 6 abolished FNR‐dependent anaerobic regulation of pfl operon expression. The equivalent mutations at promoter 7 caused a 25% reduction in anaerobic expression. The residual anaerobic expression in such constructs was FNR‐, but no longer ArcA‐dependent. A construct in which the ‐10 region of both promoters 6 and 7 was mutated showed no anaerobic induction of pfl operon expression. This indicates that transcription from both promoters is required for maximal anaerobic regulation by ArcA and FNR.
Identification of a gene required for the oxygen‐regulated formation of the photosynthetic apparatus of Rhodobacter capsulatus
Authors: Pollich Michael , Jock Susanne , Klug Gabriele 
Published: October 2006

SummaryThe pigment‐binding proteins of Rhodobacter capsulatus are encoded by the polycistronic puf and puc operons. Both operons show higher expression under low oxygen tension than under high oxygen tension in the wild‐type strain. The Tn5 mutant strain AH2 shows only low levels of puf and puc mRNA under high and low oxygen tension, indicating that it lacks a gene product required for stimulation of puf and puc gene expression under low oxygen tension. The formation of wild‐type levels of photosynthetic complexes and normal oxygen regulation could be restored by the expression in trans of a 1.7 kb fragment of the R. capsulatus wild‐type chromosome or by addition of 10μg I‐1 vitamin B12 to the growth medium. An open reading frame of 798 nucleotides containing the Tn5 insertion was identified on the 1.7kb fragment. This open reading frame shows no homology to known genes and has a remarkably high GC content of 76%.