bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of hypoxanthine and guanine as the co‐repressors for the purine regulon genes of Escherichia coli
Authors: Meng L. M. , Nygaard P. 
Published: October 2006

SummaryAddition of purine compounds to the growth medium of Escherichia coli and Salmonella typhimurium causes repressed synthesis of the purine biosynthetic enzymes. The repression is mediated through a regulatory protein, PurR. To identify the co‐repressor(s) of PurR, two approaches were used: (i) mutations were introduced into purine salvage genes and the effects of different purines on pur gene expression were determined; (ii) purine compounds which dictate the binding of the PurR protein to its operator DNA were resolved by gel retardation. Both the in vivo and the in vitro data indicated that guanine and hypoxanthine are co‐repressors. The toxic purine analogues 6‐mercaptopurjne and 6‐thioguanine also activated the binding of PurR to its operator DNA.
Cloning and characterization of mepA, the structural gene of the penicillin‐insensitive murein endopeptidase from Escherichia coli
Authors: Keck W. , Van Leeuwen A. M. , Huber M. , Goodell E. Wm. 
Published: October 2006

SummaryThe putative structural gene mepA of the penicillin‐insensitive murein endopeptidase from Escherichia coli was cloned and sequenced. N‐terminal sequence determination with the isolated endopeptidase protein showed that this enzyme is coded by the mepA gene and that it is synthesized initially with an N‐terminal signal peptide. No significant sequence homoiogy with the other (penicillin‐sensitive) murein endopeptidase (dacB) or any other protein was found. The precise chromosomal mapping position of mepA relative to two other genes, aroC and fabB, was shown to be 50.4 min. E. coli strains carrying multicopy plasmids with the mepA gene produced 5‐6‐fold more endopeptidase and secreted it into the periplasm, where it appeared to function normally in vivo since the release of ceil wall peptides into the medium increased in parallel. The transformed cells were, however, not unusually sensitive to penicillin and their murein had a normal degree of cross‐bridges.
Molecular analysis of the Haemophilus influenzae type b pilin gene
Authors: Langermann S. , Wright A. 
Published: October 2006

SummaryA Haemophilus influenzae DNA library was prepared in the vector lambda EMBL3, and recombinant phage were screened for the pilin gene (pil) using a synthetic oligonucleotide. Southern blot analysis of the positive clones revealed a 2.5kb Pstl/Pvul fragment that hybridized with the oligonucleotide probe. This fragment was subcloned into pBR322 and sequenced. The nucleotide sequence disclosed an open reading frame of 653 bases. The deduced amino acid sequence corresponded with the known amino acid sequence of the purified pilin protein. Primer extension analysis using total RNA from piliated H. influenzae cells delineated a start site for the gene, ‐10 and ‐35 promoter regions, and a ribosome‐binding site. No transcripts were seen with the RNA derived from a non‐piliated strain. Southern blots of DNA from a number of H. influenzae strains revealed homology with the pil structural gene. DNA from a non‐piliated strain of H. influenzae also hybridized with the pil probe. Transcriptional and translational studies were performed in Escherichia coli with plasmids containing: (i) the pil gene on the 2.5kb Pstl/Pvul fragment, (ii) the pil gene fused to the phoA gene, and (iii) the pil gene present on a 12.2 kb insert containing extensive H. influenzae DNA flanking the pil gene. The results suggest that the H. influenzae pil gene is expressed in Escherichia coli, but from a promoter other than the one used in H. influenzae.
The regulation of transcription of the gerA spore germination operon of Bacillus subtilis
Authors: Feavers I. M. , Foulkes J. , Setlow B. , Sun D. , Nicholson W. , Setlow P. , Moir A. 
Published: October 2006

SummaryThe gerA operon of Bacillus subtilis 168 comprises three genes concerned with the triggering of spore germination by l‐alanine and its analogues. The expression of this operon has been characterized using chromosomal lacZ fusions to the gerA promoter. The gerA promoter is switched on 2.5–3 hours after the initiation of sporulation, in parallel with glucose dehydrogenase. A high proportion of the gerA‐driven β‐galactosidase detected in sporulating cells is found in the mature spore; the gerA promoter is therefore active in the forespore compartment of the sporulating cell. The gerA promoter is not expressed in spo0, spoII or spoIIIA, B, E and G mutant backgrounds, but is expressed in spoIIICand D and in spoIV and V mutants. The in vivo transcriptional startpoint of the operon has been mapped by primer extension experiments; sequences upstream from this startpoint show significant homology with recognition sequences for RNA polymerase containing σG(EσG). The gerA operon was transcribed in vitro by EσG with a startpoint identical to that used in vivo, and expression of the gerA operon was rapidly induced in vegetative cells by induction of σG synthesis. These data indicate that the gerA operon is an additional member of the σG regulon, which includes a number of genes expressed in parallel only in the forespore compartment of sporulating B. subtilis cells.
Isolation of a secY homologue from Bacillus subtilis: evidence for a common protein export pathway in eubacteria
Authors: Suh J.‐W. , Boylan S. A. , Thomas S. M. , Dolan K. M. , Oliver D. B. , Price C. W. 
Published: October 2006

SummaryGenetic and biochemical studies have shown that the product of the Escherichia coli secY gene is an integral membrane protein with a central role in protein secretion. We found the Bacillus subtilis secY homologue within the spc‐alpha ribosomal protein operon at the same position occupied by E. coli secY. B. subtilis secY coded for a hypothetical product 41% identical to E. coli SecY, a protein thought to contain 10 membrane‐spanning segments and 11 hydrophilic regions, six of which are exposed to the cytoplasm and five to the periplasm. We predicted similar segments in B. subtilis SecY, and the primary sequences of the second and third cytoplasmic regions and the first, second, fourth, fifth, seventh, and tenth membrane segments were particularly conserved, sharing greater than 50% identity with E. coli SecY. We propose that the conserved cytoplasmic regions interact with similar cytoplasmic secretion factors in both organisms and that the conserved membrane‐spanning segments actively participate in protein export. Our results suggest that despite the evolutionary differences reflected in cell wall architecture, Gram‐negative and Gram‐positive bacteria possess a similar protein export apparatus.
The role of transformation in the variability of the Neisseria gonorrhoeae cell surface
Authors: Scocca J. J. 
Published: October 2006

SummaryThis review discusses the genetic basis for surface changes in Neisseria gonorrhoeae and the role of specific transformation reactions in producing them. Variation in the structure of pilin, the subunit of gonococcal pili, occurs by transformation‐mediated recombination of DNA segments in storage loci with the expression locus. These pilin loci have low recombination potential since their sequences contain only short uninterrupted identical sequences. The DNA within storage or silent loci are also relatively deficient in the short defined sequences which target DNA for efficient uptake and thus have relatively low affinity for the DNA transport system. Consequently, pilin‐encoding DNA segments constitute relatively poor substrates for the general transformation system of gonococci. These considerations suggest the existence of locus‐specific factors which increase the efficiency of genetic exchange between pilin loci. I raise the speculative hypothesis that one function of transformation‐mediated DNA entry is to provide a regulatory stimulus signalling the death of neighbouring gonococci. This regulatory shift might lead to production of factors which accelerate genetic reshuffling of pilin loci either by transformation per se using external DNA as donor, or via a recombinational process which utilizes internally derived DNA segments as donors. A signalling function for transforming DNA also clarifies several general properties of specific transformation reactions.
Characterization of the repertoire of hypervariable regions in the Protein II (opa) gene family of Neisseria gonorrhoeae
Authors: Connelly T. D. , Shaffer D. , Cannon J. G. 
Published: October 2006

SummaryP.11 outer membrane proteins of Neisseria gonorrhoeae are encoded by a family of closely related genes. Although the genes are highly conserved, major differences in sequence among them occur in two short regions, designated hypervariable regions 1 (HV1) and 2 (HV2). In this study, we determined the number and DNA sequence of the hypervariable regions in the P.11 genes of strain FA1090. The FA1090 chromosome contained at least eleven P.11 loci, having six different versions each of HV1 and HV2 among them. Southern blotting with HV‐specific oligonucleotides showed that each version was present in one to three copies, and that there were nine unique combinations of HV1 and HV2 in the P.M genes. Although each of the versions of HV1 or HV2 had a unique DNA sequence, there were some similarities among them, particularly when certain ones were compared. Restriction fragments containing only the HV regions were cloned into an expression vector to demonstrate that the epitopes recognized by a set of monoclonal antibodies specific for different FA1090 P.11 proteins were completely encoded by either HV1 or HV2.
Identification of regB, a gene required for optimal exotoxin A yields in Pseudomonas aeruginosa
Authors: Wick M. J. , Frank D. W. , Storey D. G. , Iglewski B. H. 
Published: October 2006

SummaryThe yield of exotoxin A from Pseudomonas aeruginosa has been shown to be strain‐dependent. Exotoxin A production requires the presence of the positive regulatory gene, regA. We cloned the regA genetic locus from the prototypical P. aeruginosa strain PAO1 and examined its ability to influence exotoxin A yields compared to the same region cloned from the hyper‐toxin‐producing strain, PA103. The P. aeruginosa regA mutant strain, PA103‐29, containing the PAO1 regA locus in trans produced approximately five to seven times less extracellular exotoxin A than PA103‐29 containing the regA locus cloned from the hyper‐toxigenic strain, PA103. Nucleotide sequence analysis of the PAO1 regA locus revealed several differences, the most striking of which was the absence of a second open reading frame that was present in the analogous PA103 DNA. In addition, an amino acid substitution was found at position 144 of RegA (Thr in PAO1 and Ala in PA103). Recombinant molecules were constructed to test the contribution of each of these changes in nucleotide sequence on extracellular exotoxin A yields. The amino acid substitution in the PAO1 RegA protein was found not to affect overall exotoxin A yields. In contrast, the presence of the second open reading frame immediately downstream of the PA103 regA gene was found to influence extracellular exotoxin A yields. This open reading frame encodes a gene which we call regB. Nucleotide sequence analysis indicates that regB is 228 nucleotides in length and encodes a protein of 7527 Daltons. Our data suggest that regB is required for optimal exotoxin A production and its absence in strain PAO1 partially accounts for the difference in yield of extracellular exotoxin A between P. aeruginosa strains PAO1 and PA103.
Differential gene expression during sporulation in Bacillus subtilis: structure and regulation of the spoIIID gene
Authors: Stevens C. M. , Errington J. 
Published: October 2006

SummaryThe gene spoIIID, which is essential for spore formation in Bacillus subtilis, was cloned and sequenced. It consists of one open reading frame which would encode a 93‐amino‐acid protein with a classic helix‐turn‐helix motif, characteristic of sequence‐specific DNA‐binding proteins. SpoIIID protein is a previously identified transcription factor, capable of altering the specificity of RNA polymerase containing sigma K in vitro (Kroos et al., 1989). The spoIIID83 mutation (by which the locus was originally identified), was sequenced and found to be a single base substitution in the ribosome binding site upstream of the spoIIID open reading frame. A transcriptional fusion to lacZ was constructed and used to examine the regulation of spoIIID. Expression of spoIIID occurred only during sporulation, beginning 1.5 to 2 hours after the initiation of sporulation. The dependence of spoIIID expression on other spo loci suggests that it is mother‐cell‐specific, and that it is transcribed by sigma E‐containing RNA polymerase.
The major subunit of Escherichia coli type 1 fimbriae is not required for D‐mannose‐specific adhesion
Authors: Klemm P. , Krogfelt K. A. , Hedegaard L. , Christiansen G. 
Published: October 2006

SummaryType 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D‐mannose‐containing structures. These fimbriae are hetero‐polymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FimH proteins. The FimH protein is uniquely responsible for the D‐mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D‐mannose‐specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D‐mannose‐binding phenotype. However, the deletion resulted in a 25‐fold reduction of the adhesive potential, as measured by binding to D‐mannose‐coated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesin is capable of being exposed on the bacterial surface without being an integral part of the fimbriae.
Two plasmids other than the nodulation plasmid are necessary for formation of nitrogen‐fixing nodules by Rhizobium leguminosarum
Authors: Hynes M. F. , McGregor N. F. 
Published: October 2006

SummaryA system which allows direct selection for curing of plasmids in Gram‐negative bacteria was used to generate derivatives of Rhizobium leguminosarum VF39 cured of each of six plasmids present in this strain. Phenotypes could be correlated with the absence of five of the six plasmids. The smallest plasmid, pRIeVF39a, carries genes for the production of a melanin‐like pigment as has been previously reported. Plasmid pRIeVF39d carries nodulation and nitrogen fixation genes. Curing of the plasmids pRleVF39c and pRleVF39e gave rise to strains which formed Fix− nodules on peas, lentils, and faba beans. The nodules formed by the strains cured of pRIeVF39c contained few, if any, bacteria. Analysis of washed cells by SDS‐PAGE showed that this strain is defective in lipopolysaccharide (LPS) production; the defect could be complemented by introducing plasmids from several other R. leguminosarum strains, and by the R. leguminosarum biovar phaseoli LPS gene clones pCos126 and pDel27. The nodules formed by the strain cured of pRleVF39e had a reduced symbiotic zone, an enlarged senescence zone, and an abundance of starch granules. This strain grew at a much slower rate than the wild type, was unable to grow on minimal medium, and no longer produced melanin. These defects could be complemented by at least one other Rhizobium plasmid, pRle336e, a plasmid of strain 336 which is distinct from the nodulation plasmid (pRle336c) and the plasmid (pRle336d) which could complement the LPS defect associated with the loss of pRleVF39c. This demonstrates that genes necessary for symbiosis can be carried on at least three different plasmids in R. leguminosarum.
The identification of the Escherichia coli fts Y gene product: an unusual protein
Authors: Gill O. R. , Salmond G. P. C. 
Published: October 2006

SummaryThe ftsYEX operon in Escherichia coli encodes three proteins, two of which (FtsE and FtsX) are known to be required for cell division. Although FtsE and FtsX have been identified using SDS‐PAGE, the FtsY protein has not. We have used in vitro insertion mutagenesis to identify FtsY as a 92kD polypeptide in maxicell experiments, although predictions from the DNA sequence estimated FtsY to be 54kD. Results suggest that this disparity could be due to the unusually high percentage of acidic residues within the protein. Complementation tests indicated the presence of a promoter within ftsY required for expression of ftsE and ftsX. The FtsY protein exhibits sequence homology with the SRα protein of eukaryotes which is involved in protein secretion. The essential nature of the ftsY gene was also demonstrated.
Developmental regulation of cell‐type‐enriched mRNAs in Dictyostelium discoideum
Authors: Corney A. J. , Richards A. J. , Phillpots T. , Hames B. D. 
Published: October 2006

SummaryWe describe sixteen new families of cDNA clones representing mRNAs that are expressed preferentially in either prespore or prestalk cells during developmentm of Dictyostelium discoideum and two new mRNAs that are expressed in a non‐cell‐type‐specific manner. None of the prespore‐enriched mRNAs are detectable in Dictyostelium cells until 13–15h of development but then they increase dramatically and peak at 18–22h. Upon dissociation of developing aggregates, all these mRNAs rapidly decay to low levels. In marked contrast to data presented for prespore genes by other workers, cyclic AMP either has no effect on the mRNA levels in dissociated cells or is only weakly effective in restoring normal expression. A prestalk‐enriched mRNA examined, 5G mRNA, is similarly expressed late in development but is also expressed in vegetative cells. The level of 5G mRNA is only moderately affected by cell disaggregation.
Serratia marcescens rpr gene sensitizes Escherichia coli wild‐type, xth, and nfo strains to methyl methanesulphonate
Authors: Murphy K. E. , Braymer H. D. 
Published: October 2006

SummaryIt is reported here that the rpr DNA repair gene of Serratia marcescens does not complement an Escherichia coli xth nfo AP endonuclease mutation for resistance to methyl methanesulphonate (MMS). Rather, rpr sensitized Escherichia coli wild‐type, xth, and nfo strains to MMS. Also, it was found that rpr could not complement a triple tag alkA recA mutation in E. coli, indicating that there are limits to rpr complementing capabilities. It was determined that rpr gene dosage was not a factor in recA complementation. MMS sensitization of an E. coli wild‐type strain, however, was directly related to rpr copy number. These data indicate that Rpr does not have an associated AP endonuclease activity, and that it is incapable of substituting for Tag I, Tag II, and RecA in a tag alkA recA background.
Genetic instability and hypervariability in Streptomyces ambofaciens: towards an understanding of a mechanism of genome plasticity
Authors: Leblond P. , Demuyter P. , Simonet J. M. , Decaris B. 
Published: October 2006

SummaryMany Streptomyces species exhibit a very high degree of genetic instability which is usually manifested as genomic rearrangements such as large deletions. In Streptomyces ambofaciens DSM40697, two levels of genetic instability were previously described: (i) a basic genetic instability similar to that reported for other strains, and (ii) hypervariability, a phenomenon that we believe to be a new aspect of instability closely associated with DNA amplification. A large DNA region undergoes deletions, amplifications and large genomic changes strictly associated with both aspects of genetic instability. The genetic and molecular analyses of the different aspects of genetic instability allow us to propose that they result from a cascade of molecular events and to investigate the relationships between genetic instability phenomena and genome fluidity.
Transposition of Tn916 to different sites in the chromosome of Neisseria meningitidis: a genetic tool for meningococcal mutagenesis
Authors: Kathariou S. , Stephens D. S. , Spellman P. , Morse S. A. 
Published: October 2006

SummaryThe difficulty in obtaining mutants in pathogenic Neisseria has limited the ability to genetically define determinants responsible for virulence as well as the ability to generate a genetic map. We show that the 16.5kb conjugative transposon Tn916 can be introduced into Neisseria meningitidis on the suicide vectors pAM120 and pAM170. After introduction, Tn916 transposed to different sites in the chromosome of recipient meningococci, apparently at random, and was stably incorporated. Following its integration into the meningococcal chromosome, Tn916 did not appear to readily express its conjugative and transpositional functions. However, chromosomal DNA from Tn916‐carrying meningococci could be used to transform other meningococcal strains to tetracycline resistance. These studies indicate that Tn916 may be an important tool for genetic analysis of N. meningitidis.
Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa
Authors: DeVault J. D. , Kimbara K. , Chakrabarty A. M. 
Published: October 2006

SummaryMortality among cystic fibrosis (CF) patients is most commonly attributed to pulmonary infection by mucoid, alginate‐producing Pseudomonas aeruginosa. The initial infecting P. aeruginosa are typically non‐mucoid; however, upon continued exposure to the CF lung environment, they become highly mucoid. The CF lung is an osmotically high environment because of the presence of substantial concentrations of electrolytes and dehydrated mucus. In this report we demonstrate that ethanol (a commonly used dehydrating agent) activates transcription from a critical alginate promoter, algD, and show that prolonged exposure to ethanol allows switching to the mucoid form. This activation appears to be dependent on DNA gyrase. Analysis of alginate gene activation, and the subsequent reversal of the activation process by bacterial DNA gyrase inhibitors, should aid the development of treatment strategies for CF patients infected with this organism.
The complete nucleotide sequence of the Pasteurella multocida toxin gene and evidence for a transcriptional repressor, TxaR
Authors: Petersen S. K. 
Published: October 2006

SummaryThe osteolytic toxin of Pasteurella multocida induces bone resorption in vivo and in vitro (Foged et al., 1988; Kimman et al., 1987). In this report the toxin‐encoding toxA gene is sequenced, and the deduced primary structure of the toxin shows a protein of 1285 amino acids, containing a striking homology to a metal‐binding motif. Evidence that expression of the toxA gene is repressed at a transcriptional level in Escherichia coli is presented. Repression could be abolished either by deletion of a region upstream of toxA, or by a putative frame‐shift mutation in the same region. The repressor protein encoded within this region was efficient in trans, and was named TxaR.
Notes High‐efficiency transformation system for the biocontrol agents, Trichoderma spp.
Authors: Herrera‐Estrella A. , Goldman G. H. , Montagu M. Van 
Published: October 2006

SummaryWe have developed an efficient transformation system based on the use of polyethylene glycol and CaCl2 for the biocontrol agents, Trichoderma spp. Trans formation was obtained with the plasmid pAN7‐1, carrying a bacterial hygromycin‐resistance gene as a selectable marker, under the control of Aspergillus nidulans heterologous expression signals. The system described here yielded 200‐800 transformants per μg of DNA. Transformants contained several copies of the plasmid integrated into their genome, apparently at the same site in the different transformants analysed. Stability of the transformants was achieved by inserting a 2.4kb homologous DNA fragment into pAN7‐1. Southern blot analysis indicated that integration in the stable transformants occurs through non‐homologous recombination.