bluuebunny/crossref_metadata_2025_split huggingface dataset:
A recombinant C‐terminal toxin fragment provides evidence that membrane insertion is important for Clostridium perfringens enterotoxin cytotoxicity
Authors: Hanna P. C. , McClane B. A. 
Published: October 2006

Summary Clostridium perfringens enterotoxin (CPE) is believed to be involved in several important gastrointestinal illnesses. Recent studies have identified a number of distinct molecular events which occur after CPE treatment of eukaryotic cells or isolated membranes. Additional studies are underway to determine the temporal order and intrinsic importance of each CPE event for cytotoxicity. We now demonstrate that a truncated CPE fragment binds to membranes, but is unable to Insert into membranes or cause any other subsequent post‐insertion event. This is the first experimental evidence supporting the importance of membrane insertion for CPE cytotoxicity. Binding of the CPE fragment is also shown to be irreversible, strongly suggesting that the irreversible binding of wild‐type CPE is not due solely to insertion of CPE into membranes.
ymoA, a Yersinia enterocolitica chromosomal gene modulating the expression of virulence functions
Authors: Cornells G. R. , Sluiters C. , Delor I. , Geib D. , Kaniga K. , de Rouvroit C. Lambert , Sory M.‐P. , Vanooteghem J.‐C. , Michiels T. 
Published: October 2006

SummaryThe virulence functions of Yersinia enterocolitica include the pYV‐encoded Yop proteins and YadA adhesin as well as the chromosome‐encoded enterotoxin, Yst. The yop and yadA genes form a temperature‐activated regulon controlled by the transcriptional activator VirF. Gene virF, also localized on pYV, is itself thermoinduced in the absence of other pYV genes. The enterotoxin ysf gene is silent in some collection strains including strain W22703. This paper describes two Tn5‐Tc1 chromosomal insertion mutants of W22703 transcribing virF, and hence the yop and yadA genes, at low temperature. These mutants also resumed their production of Yst, with its typical temperature dependence. Both mutations were insertions in the same gene called ymoA for‘Yersinia modulator’The cloned ymoA gene fully complemented the two mutations. Several properties of the mutants suggest that ymoA encodes a histone‐like protein. According to the nucleic acid sequence, the product of ymoA is an 8064 Da protein rich in aspartic acid (9%), glutamic acid (9%) and lysine (10.5%), but the predicted amino acid sequence shows no similarity with any described histone‐like protein. This work supports recent reports which propose a role for DNA topology and bacterial chromatin structure in thermoregulation of virulence functions.
A pyruvate‐stimulated adenylate cyclase has a sequence related to the fes/fps oncogenes and to eukaryotic cyclases
Authors: Peters E. P. , Wilderspin A. F. , Wood S. P. , Zvelebil M. J. J. M. , Sezer O. , Danchin A. 
Published: October 2006

SummaryThe pyruvate‐stimulated adenylate cyclase from Brevibacterium liquefaciens produces up to 450μM cyclic AMP in the culture medium when the bacterium is grown on glucose and alanine. In this paper we report the cloning, expression and sequencing of the gene for this enzyme. Residues were identified, within the C‐terminal domain, which are conserved in adenylate and guanylate cyclase sequences from eukaryotes and in the adenylate cyclase of the prokaryote Rhizobium meliloti. We have also identified a sequence of 30 residues near the N‐terminus of the protein which is homologous to part of the regulatory domain of the cellular homologues of the oncogenes fes and fps; this sequence is also present in the avian Fujinami sarcoma virus fps gene.
Escherichia coli chromosomal mutations that permit direct cloning of the Bacteroides fragiiis metallo‐β‐lactamase gene, ccrA
Authors: Rasmussen B. A. , Gluzman Y. , Tally F. P. 
Published: October 2006

SummaryThe class B, metallo‐β‐lactamase genes ccrA (carbapenem‐ and cephamycin resistance) from three Bacteroides fragilis isolates — QMCN3, QMCN4, and TAL3636 — were cloned and expressed in Escherichia coli. Cloning of the genes, by selecting for ampicillin resistance, was facilitated by two classes of Escherichia coli chromosomal mutations which resulted in at least a 5‐10‐fold increase in metallo‐β‐lactamase enzymatic activity. The observed increase in enzymatic activity is due to either increased translation of the ccrA gene or an effect on localization or stability of the protein. Comparison of the DNA sequences of the three ccrA genes revealed that their protein‐coding sequences shared greater than 97% DNA sequence identity. However, the 5’upstream sequence for the TAL3636 ccrA gene was unrelated to that of the other two genes.
Evidence for a common molecular origin of the capsule gene loci in Gram‐negative bacteria expressing group II capsular polysaccharides
Authors: Frosch M. , Edwards U. , Bousset K. , Krauße B. , Weisgerber C. 
Published: October 2006

SummaryCapsular polysaccharides of Gram‐negative bacteria contribute to a large extent to the pathogenicity of these organisms. We show here that the molecular organization of the capsule gene loci in different serogroups of Neisseria meningitidis is similar to that of Haemophilus influenzae and Escherichia coli. A common molecular origin of the mechanisms of encapsulation is indicated by strong homology of the genes involved in transport of the capsular polysaccharides to the cell surface in all these organisms. The proteins involved in capsular polysaccharide transport fit the characteristics of ABC (ATP‐binding cassette) transporters. Furthermore, by sequence comparison of the sialyltransferases of N. meningitidis B and E. coli K1, the capsule of which is composed of α2,8‐linked polyneuraminic acid, a significant degree of homology was observed, indicating that the capsular polysaccharide type itself has the same evolutionary origin in these two pathogens.
The hha gene modulates haemolysin expression in Escherichia coli
Authors: Nieto J. M. , Carmona M. , Bolland S. , Jubete Y. , de la Cruz F. , Juárez A. 
Published: October 2006

SummaryA mutation in the hha allele results in a large increase in the production of intracellular as well as extracellular haemolysin in Escherichia coli cells harbouring the haemolytic recombinant plasmid pANN202‐312. This single gene mutation was located between 490 and 491.6kb on the physical map of the E. coli chromosome. From the DNA sequence of hha a small polypeptide of 8629 Da was predicted and was expressed in minicells. The deduced polypeptide sequence did not show significant similarities to other characterized proteins related to the regulation of gene expression in E. coli, although it was shown that the hha mutation increases cyloplasmic synthesis of haemolysin.
TyrR protein of Escherichia coli and its role as repressor and activator
Authors: Pittard A. J. , Davidson B. E. 
Published: October 2006

SummaryThe TyrR protein regulates the expression of eight transcriptional units that comprise the TyrR regulon. In all but one case, regulation is by repression, while in two cases activation of expression can occur. Notwithstanding the fact that the TyrR protein contains an ATP‐binding domain and a helix‐turn‐helix DNA‐binding domain which are structurally homologous to domains of similar functions in proteins such as NifA, NtrC, DctD and XylR, it differs from them in a number of respects. It is not a part of a two‐protein component system and it lacks the amino‐terminal domain that is present on NtrC and DctD. It activates transcription from‘Eσ70’promoters but not from‘Eσ54’promoters. ATP binding seems to be essential for tyrosine‐mediated repression but not for activation. In addition, the activity of the TyrR protein is modulated by the binding of one or more of the aromatic amino acids. The consensus sequence for TyrR‐binding sites in DNA, referred to as TyrR boxes, is TGTAAAN6TTTACA. Tyrosine‐mediated repression occurs at operators containing a pair of adjacent boxes. These have unequal affinities for the TyrR protein. The box that overlaps the RNA polymerase binding site is only bound by TyrR in the presence of both ATP and tyrosine, and binding appears to involve co‐operativity between two TyrR protein dimers.In contrast, activation of expression by TyrR appears to require phenylalanine but not ATP. It occurs as a consequence of the binding of TyrR protein to a single upstream TyrR box, and appears to involve interactions between the bound TyrR molecule and a molecule of RNA polymerase bound to the promoter. The spacing between the box and the promoter is critical for effective activation.
Substitutions at a single amino acid residue in the nitrogen‐regulated activator protein NTRC differentially influence its activity in response to phosphorylation
Authors: Dixon R. , Eydmann T. , Henderson N. , Austin S. 
Published: October 2006

SummaryFour substitutions at serine residue 160 which increase the activity of the σ54‐dependent activator protein NTRC in the absence of NTRB have been analysed in detail. Mutagenesis of the putative phosphoacceptor site of NTRC and analysis of double mutants indicate that the positive control function of the S160W and S160C mutants is phosphorylation‐dependent, whereas the activity of the S160Y and S160F mutants is phosphorylation‐independent. This was confirmed with two purified mutant proteins in vitro. Occupancy of tandem NTRC‐binding sites upstream of the Klebsiella pneumoniae nifL promoter by S160W protein is also phosphorylation‐dependent in contrast to occupancy by S160F protein, confirming that both the DNA‐binding and activator functions of NTRC are influenced by phosphorylation. The S160W and S160C mutants are apparently more responsive than wild‐type protein to ‘cross‐talk’ by other members of the histidine protein kinase family but are less responsive to phosphorylation and dephosphorylation mediated by NTRB.
Characterization of the oriT region of the IncFV plasmid pED208
Authors: Di Laurenzio L. , Frost L. S. , Finlay B. B. , Paranchych W. 
Published: October 2006

SummaryDNA sequence analysis of a 2.2kb EcoRI‐Hin dIII fragment from pED208, the derepressed form of the IncFV plasmid Folac, revealed sequences highly homologous to the oriT region, traM, and traJ genes of other IncF plasm ids. The TraM protein was purified and immunoblots of fractionated cells containing pED208 or Folac showed that TraM was predominantly in the cytoplasm. Using DNA retardation assays and the DNase I footprinting technique, the TraM protein was found to bind to three large motifs in the oriT region: (I) an inverted repeat, (II) two direct repeats, and (III) the traM promoter region. These three footprint regions contained a HinfI‐like sequence (GANTC) that appeared 16 times, spaced 11–12bp (or multiples thereof) apart, suggesting that TraM protein binds in a complex manner over this entire region.
Site‐directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study
Authors: Wilmsen H. U. , Buckley J. T. , Pattus F. 
Published: October 2006

SummaryThe role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel‐forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water‐filled pore with an approximate diameter of 9.3 ± 0.4 Å.
Three in‐frame N‐terminally different proteins are produced from the repressor locus of the Streptomyces bacteriophage φC31
Authors: Smith M. C. M. , Owen C. E. 
Published: October 2006

SummaryThe sequence of the repressor locus, c, of the Streptomyces temperate phage, φC31, was shown previously to contain an open reading frame encoding a 74 kDa protein. Further analysis of the transcriptional and translational products of the c gene shows a more complex pattern of expression. A nest of three in‐frame N‐terminally different, C‐terminally identical proteins of 74, 54 and 42 kDa were found to be expressed from a corresponding nest of transcripts. The repressor proteins were produced in Escherichia coli and the 42 kDa protein was purified, verified by N‐terminal sequencing, and used to raise antibody. The antibody cross‐reacted in Western blots with the 74, 54 and 42 kDa proteins expressed in E. coli and Streptomyces lividans and from Streptomyces coelicolorφC31 lysogens. Analysis of transcription of the c gene by S1 mapping and primer extension showed that the nest of transcripts encoding the repressor protein were induced after heat treatment of the cts locus (Sinclair and Bibb, 1989; this paper). Correspondingly, all three of the repressor proteins were induced. In addition to a promoter, cp1, which lies upstream of the 74 kDa open reading frame, the c locus contained at least one internal promoter, cp2, which transcribes DNA encoding the 54 and 42 kDa proteins. Transcripts initiating from cp3 were observed in RNA preparations from S. lividans containing the c gene deleted for cp1 and cp2, but gene fusions using DNA which should contain any putative promoting activity from this region transcriptionally fused to the xylE gene showed very low levels of expression of catechol 2,3 dioxygenase in S. lividans. The 74 kDa protein was not necessary for super‐infection immunity. Data described here and current knowledge of the nature of other‘dual start’genes suggest a model for the regulation of lysis versus lysogeny in φC31.
Identification of Treponema pallidum subspecies pallidum genes encoding signal peptides and membrane‐spanning sequences using a novel alkaline phosphatase expression vector
Authors: Blanco D. R. , Giladi M. , Champion C. I. , Haake D. A. , Chikami G. K. , Miller J. N. , Lovett M. A. 
Published: October 2006

Summary Treponema pallidum subspecies pallidum is a pathogenic spirochaete for which there are no systems of genetic exchange. In order to provide a system for the identification of T. pallidum surface proteins and potential virulence factors, we have developed a novel expression vector which confers the utility of TnphoA transposition. The relevant features of this plasmid vector, termed pMG, include an inducible tac promoter, a polylinker with multiple cloning sites in three reading frames, and an alkaline phosphatase (AP) gene lacking the signal sequence‐encoding region. Library construction with Sau3A‐ digested T. pallidum genomic DNA resulted in the creation of functional T. pallidum‐AP fusion proteins. Analysis of fusion proteins and their corresponding DNA and deduced amino acid sequences demonstrated that they could be grouped into three categories: (i) those with signal peptides containing leader peptidase I cleavage sites, (ii) those with signal peptides containing leader peptidase II cleavage sites, and (iii) those with non‐cleavable hydrophobic membrane‐spanning sequences. Triton X‐114 detergent phase partitioning of individual T. pallidum‐AP fusions revealed several clones whose AP activity partitioned preferentially into the hydrophobic detergent phase. Several of these fusion proteins were subsequently shown to be acylated by Escherichia coli following [3H]‐palmitate labelling, indicating their lipoproteinaceous nature. DNA and amino acid sequence analysis of one acylated fusion protein, Tp75, confirmed the presence of a hydrophobic N‐terminal signal sequence containing a consensus
Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli
Authors: DeVault J. D. , Hendrickson W. , Kato J. , Chakrabarty A. M. 
Published: October 2006

SummaryThe environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein‐induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP‐CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non‐responsive to glucose repression. The involvement of c AM P‐CRP complex in the activation of the afgD promoter in E. coU has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP‐like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.
The hydrogenase structural operon in Rhodobacter capsulatus contains a third gene, hupM, necessary for the formation of a physiologically competent hydrogenase
Authors: Cauvin B. , Colbeau A. , Vignais P. M. 
Published: October 2006

SummaryThe hupM gene, previously called ORFX, found downstream from and contiguous with the structural hydrogenase genes hupS and hupL in Rhodobacter capsulatus, is shown here to form a single hupSLM transcription unit with the two other genes. The hupM gene was Inactivated by interposon mutagenesis. The two selected mutants, BCX1 and BCX2, which contained the kanamycin‐resistance gene in opposite orientation, still exhibited hydrogenase activity when assayed with the artificial electron acceptors benzyl‐viologen and methylene blue. However, the hydrogenase was not physiologically active in these mutants, which could not grow autotrophically and were unable to recycle electrons to nitrogenase or to respire on H2. The hupM gene starts nine base pairs downstream from the TGA stop codon of hupL gene, which encodes the large subunit of the [NiFe]hydrogenase of Rhodobacter capsulatus. The three contiguous genes hupS, hupL and hupM were subcloned downstream from the promoter of hupSL, either with the promoter in the correct orientation (plasmid pBC8) or with the promoter in the opposite orientation (plasmid pBC9), then the constructs were introduced into the mutant strains. Only plasmid pBC8 could restore the formation of a competent hydrogenase in mutants BCX1 and BCX2, indicating that the hupM gene is expressed only from the hupSL promoter.
Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence‐associated loci identified by Tn5 insertions
Authors: Okada N. , Sasakawa C. , Tobe T. , Talukder K. A. , Komatsu K. , Yoshikawa M. 
Published: October 2006

SummaryTo establish the molecular basis of the chromosomal virulence genes of Shigella flexneri 2a (YSH6000), a Notl restriction map of the chromosome was constructed by exploiting Notl‐linking clones, partial Notl digestion and DNA probes from various genes of Escherichia coli K‐12. The map revealed at least three local differences in the placements of genes between YSH6000 and E. coli K‐12. Using the additional Notl sites introduced by Tn5 insertion, nine virulence loci Identified previously by random Tn5 insertions were physically mapped on the chromosome. To demonstrate the versatility of the Notl map in direct assignment of the virulence loci tagged by Tn5 to a known genetic region in E. coli K‐12, the major class of avirulent mutants defective in the core structure of lipopolysaccharide (LPS) was examined for the sites of Tn5 insertions. The two Notl segments created by the Tn5 insertion in the Notl fragment were analysed by Southern blotting with two DNA probes for the 5′ and 3’flanking regions of the rfa region, and shown to hybridize separately with each of them, confirming the sites of Tn5 in the rfa locus. This approach will facilitate direct comparison of genetically mapped Tn5 insertion mutations of S. flexneri with genes physically determined in E. coli K‐12.
Separate promoters direct expression of phoAIII, a member of the Bacillus subtilis alkaline phosphatase multigene family, during phosphate starvation and sporulation
Authors: Chesnut R. S. , Bookstein C. , Hulett F. M. 
Published: October 2006

SummaryAlkaline phosphatase (APase) expression can be induced in Bacillus subtilis by phosphate starvation or by sporulation. We have recently shown that there are multiple APase structural genes contributing to the total alkaline phosphatase expression in B. subtilis. The expression of the alkaline phosphatase III gene (phoAIII) was analysed under both phosphate‐starvation induction and sporulation induction conditions. phoAIII is transcribed from two promoter regions, Pv and Ps The Pv promoter initiated transcription 37 bp before the translation initiation codon and was used to transcribe phoAIII during phosphate‐starvation induction in vegetative cells. The Ps promoter initiated transcription 119 bp before the translation initiation codon and was used during sporulation induction. Genes which have previously been shown to affect total vegetative APase, pho regulon genes phoP, phoR and phoS, affected expression of phoAIII during phosphate starvation. Genes known to affect expression of total sporulation APase, i.e. spoIIE, spoIIG and spoIIE, affected phoAIII expression during sporulation induction. Our data show that one member of the APase multigene family, phoAIII, contributes to the total APase expression both during phosphate‐starvation induction and sporulation induction, and that the mechanism of regulation includes two promoters, each requiring different regulatory genes.
Horizontal transfer of multiple penicillin‐binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae
Authors: Coffey T. J. , Dowson C. G. , Daniels M. , Zhou J. , Martin C. , Spratt B. G. , Musser J. M. 
Published: October 2006

SummaryMultiply antibiotic‐resistant serotype 23F isolates of Streptococcus pneumoniae are prevalent in Spain and have also been recovered recently in the United Kingdom and the United States. Analysis of populations of these isolates by multilocus enzyme electrophoresis, and restriction endonuclease cleavage electrophoretic profiling of penicillin‐binding protein (PBP) genes, has demonstrated that these isolates are a single clone (Muñoz et al., 1991). Here we report studies of non‐serotype 23F penicillin‐resistant pneumococci isolated in Spain and the United Kingdom. One of the isolates expressed serotype 19 capsule but was otherwise indistinguishable from the serotype 23F clone on the basis of multilocus enzyme electrophoresis, antibiotic resistance profiling, and restriction endonuclease patterns of genes encoding PBP1A, PBP2B and PBP2X, a result which suggests that horizontal transfer of capsular biosynthesis genes had occurred. These same techniques revealed that six other resistant isolates, all expressing serotype 9 polysaccharide capsule, represent a clone. Interestingly, the chromosomal lineage of this clone is not closely related to the 23F clone; however, the serotype 9 and 23F clones harbour apparently identical PBP1 A, ‐2B and ‐2X genes. To explain these data, we favour the interpretation that horizontal gene transfer in natural populations has distributed genes encoding altered forms of PBP1A, ‐2B and ‐2X to distinct evolutionary lineages of S. pneumoniae.