bluuebunny/crossref_metadata_2025_split huggingface dataset:
Longus: a long pilus ultrastructure produced by human enterotoxigenic Escherichia coli
Authors: Girón Jorge A. , Levine Myron M. , Kaper James B. 
Published: October 2006

SummaryEnterotoxigenic Escherichia coli (ETEC) causes an acute cholera‐like diarrhoea in both humans and animals. We describe a new pilus termed longus produced by ETEC, which can extend for over 20 microns from the cell surface. Longus is composed of a repeating subunit of 22 kDa and its NH2‐terminal amino acid sequence revealed homology with the toxin‐coregulated pilus of Vibrio cholerae, the bundle‐forming pilus of enteropathogenic E. coli and type IV pilins of some Gram‐negative bacterial pathogens. The longus structural gene (IgA) is encoded in a large plasmid and was cloned in a 5 kb fragment, which proved to be sufficient for pilus production and assembly in E. coli K‐12. The presence of IngA was restricted to human ETEC strains. In contrast to other ETEC pili, IngA was widely distributed among ETEC strains independent of their geographical origin, serotype, toxin production, or other pili antigens expressed. Longus is a new member of the type IV pili family, which may represent a highly conserved intestinal colonization factor of ETEC. Common antigenic determinants exist among longus and their pilin subunits, produced by heterologous ETEC. Longus could be significant in the immuno‐prophylaxis of diarrhoeal disease caused by ETEC, especially against those strains in which no colonization factors have been identified and that produce heat‐stable toxin only.
Expression of the nmpC gene of Escherichia coli K‐12 is modulated by external pH. Identification of cis‐acting regulatory sequences involved in this regulation
Authors: Coll J. L. , Heyde M. , Portalier R. 
Published: October 2006

SummaryUsing a set of gene fusions generated with TnphoA, we previously identified the phmA locus, whose expression is modulated as a function of external pH (pHo). The phmA::phoA fusion was cloned and sequenced and the phmA locus was identified with the nmpC gene. This gene lies within the defective lambdoid prophage qsr′ and NmpC is an outer membrane protein which functions as a porin. We demonstrated that nmpC is sensitive to catabotite repression and dependent on the CRP—cAMP complex. However, cAMP is not a signal for the pHo‐dependent expression of nmpC. By generating step deletions in the sequence 5′ to the nmpC coding region, we identified a DNA region in position —345 to —127 which is involved in nmpC repression, mainly during growth at acid pHo. Four regions with strong homologies and a very well‐conserved organization of the functional sequence were found in the nmpC and ompF promoters. We propose that the negative regulation of nmpC during growth at low pHo might involve DNA looping of the nmpC promoter.
The pdhR–aceEF–lpd operon of Escherichia coli expresses the pyruvate dehydrogenase complex
Authors: Quail M. A. , Haydon D. J. , Guest J. R. 
Published: October 2006

SummaryTranscript mapping and studies with lacZ translational fusions have shown that the pdhR gene (formerly genA) is the proximal gene of the pdhR–aceE–aceF–Ipd operon encoding the pyruvate dehydrogenase (PDH) complex of Escherichia coli. A pdhR–lpd read‐through transcript (7.4 kb) initiating at the pyruvate‐inducible pdh promoter, and a smaller lpd transcript (1.7 kb) initiating at the independent lpd promoter, were identified. Evidence showing that the pdhR gene product negatively regulates the synthesis of the PdhR protein and the PDH complex via the pdh promoter was obtained, with pyruvate (or a derivative) serving as the putative inducing coeffector. The partially purified PdhR protein was also found to specifically retard and protect DNA fragments containing the pdh promoter region. The pdh promoter was not strongly controlled by ArcA, FNR or CRP.
Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β‐lactamase
Authors: Palzkill Timothy , Le Quyen‐Quyen , Venkatachalam K. V. , LaRocco Mark , Ocera Hermes 
Published: October 2006

SummaryIn order to understand how TEM‐1 β‐lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161–170 region includes a portion of an omega loop structure, which is involved in the formation of the active‐site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended‐spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild‐type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161‐170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100‐fold increase in ceftazidime resistance were identified. Characterization of altered β‐lactamase enzymes indicated that while their catalytic efficiency (Kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild‐type TEM‐1 β‐lactamase.
Modulated expression of promoters containing upstream curved DNA sequences by the Escherichia coli nucleoid protein H‐NS
Authors: Zuber F. , Kotlarz D. , Rimsky S. , Buc H. 
Published: October 2006

SummaryReplacement of the CRP‐binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H‐NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H‐NS may repress promoters both at short and long distances from a favoured binding site are discussed.
The porA gene in serogroup A meningococci: evolutionary stability and mechanism of genetic variation
Authors: Suker Janet , Feavers Ian M. , Achtman Mark , Morelli Giovanna , Wang Jian‐Fu , Maiden Martin C.J. 
Published: October 2006

SummaryMolecular analyses were applied to the genes encoding variants of the serosubtyping antigen, the class 1outer membrane protein (PorA), from 55 serogroup A Neisseria meningitidis strains. These genes were evolutionarily stable and exhibited a limited range of genetic variation, primarily generated by recombination. Translation of the gene sequences revealed a total of 19 distinct amino acid sequences in the variable regions of the protein, 6 of which were not recognized by currently available serosubtyping monoclonal antibodies. Knowledge of these aminoacid sequences permitted a rational re‐assignment of serosubtype names. Comparison of the complete genes with porA gene sequences from serogroup B and C meningococci showed that serogroup A possessed a limited number of the possible porA genes from a globally distributed gene pool. Each serogroup A subgroup was characterized by one of four porA gene types, probably acquired upon subgroup divergence, which was stable over periods of decades and during epidemiological spread. Comparison with other variable genes (pil and iga) indicated that the three alleles were independently assorted within the subgroup, suggesting that their gene types were older than the subgroups in which they occurred.
Deregulation of temperature‐dependent transcription of the invasion regulatory gene, virB, in Shigella by rho mutation
Authors: Tobe Toru , Yoshikawa Masanosuke , Sasakawa Chihiro 
Published: October 2006

SummaryExpression of the virB gene, the transcriptional regulator for the invasion genes encoded by the large plasmid of Shigella flexneri, is temperature‐regulated. virB transcription is under the control of VirF and H‐NS, which act as positive and negative regulators, respectively, and is highly responsive to changes in DNA superhelicity. To further investigate the molecular mechanisms underlying the thermoregulation of virB transcription, a mutant which expressed an invasion phenotype at both 30° C and 37° C was isolated using miniTn10‐Kan (mini KAN) random insertion mutagenesis. The insertion site was mapped to the rho gene, and resulted in the addition of 11 amino acids to the C‐terminus of the Rho protein. Consequently, decreased transcription termination activity at a p‐dependent terminator, λtL1, was observed, in the rho mutant, both the transcription of virB and expression of invasion genes were activated at 30°C and were less responsive to changes in temperature. The deregulation of virB expression by the mutation was dependent upon the virB promoler, since the effects of the mutation on virB transcription were abolished when its promoler region was replaced by the tac promoler. Temperature‐responsive changes in DNA topology, as determined by linking numbers of a reporter plasmid, showed that changes in DNA superhelicity in the rho mutant were smaller than that in the wild type. Furthermore, when the mutant was grown in medium containing novobiocin, an inhibitor of DNA gyrase, virB transcription at 30° C as well as at 37°C was greatly diminished. These results indicated that Rho protein could have a profound effect on topological temperature‐dependent changes in DNA structure, thus contributing to thermoregulation of VirS transcription.
Genetic analysis of the Helicobacter pylori vacuoiating cytotoxin: structural similarities with the IgA protease type of exported protein
Authors: Schmitt Wolfgang , Haas Rainer 
Published: October 2006

SummaryThe human gastric bacterial pathogen Helicobacter pylori has been implicated in type B gastritis, peptic ulceration and gastric adenocarcinoma. Here we report on the cloning and genetic characterization of an H. pylori gene named vacA, which encodes the vacuolating cytotoxin VacA, a novel type of antigenic bacterial toxin that induces the formation of intracellular vacuoles in epithelial cells. The vacuolating cytotoxin activity is expressed by a subset of clinical isolates (Vac+), all of which produce the 87kDa cytotoxin antigen, but strains which produce neither the activity nor the cytotoxin protein (Vac−) also carry the gene, Isogenic H. pylori mutants in vacA generated by transposon shuttle mutagenesis produce neither the VacA antigen nor a vacuolating activity in a cell culture model. The vacA gene itself encodes a precursor protein of 139.6 kDa consisting of a 33‐amino acid signal sequence, the 87 kDa cytotoxin and a 50 kDa C‐termlnal domain with features typical of a bacterial outer membrane protein. The VacA precursor shows no significant primary sequence homology with any previously reported protein, but its structural organization closely resembles the IgA protease‐type of exoprotein produced by pathogenic Neisseriae and Haemophilus species. Our current data support a model for secretion of the cytotoxin through the two bacterial membranes which involves the 50 kDa domain for outer membrane translocation with subsequent proteolytic cleavage and release of the mature 87 kDa cytotoxin into the extracellular environment.
DNA‐binding proteins as site‐specific nucleases
Authors: Pan Clark Q. , Landgraf Rail , Sigman David S. 
Published: October 2006

SummaryDNA‐binding proteins can be converted into site‐specific nucleases by linking them to the chemical nuclease 1,10‐phenanthroline‐copper. This can be readily accomplished by converting a minor groove‐proximal amino acid to a cysteine residue using site‐directed mutagenesis and then chemically modifying the sulphydryl group with 5‐iodoacetamido‐1,10‐ phenanthroline‐copper. These chimeric scission reagents can be used as rare cutters to analyse chromosomal DNA, to test predictions based on high‐resolution nuclear magnetic resonance and X‐ray crystal structures, and to locate binding sites of proteins within genomes.
Acquisition and rearrangement of sequence motifs in the evolution of bacteriophage tail fibres
Authors: Sandmeler Heinrich 
Published: October 2006

SummaryMolecular analysis reveals a surprising sharing of short gene segments among a variety of large double‐stranded DNA bacteriophages of enteric bacteria. Ancestral genomes from otherwise unrelated phages, including λ Mu, P1, P2 and T4, must have exchanged parts of their tail‐fibre genes, Individual genes appear as mosaics with parts derived from a common gene pool. Therefore, horizontal gene transfer emerges as a major factor in the evolution of a specific part of phage genomes. Current concepts of homologous recombination cannot account for the formation of such chimeric genes and the recombinational mechanisms responsible are not known. However, recombination sites for DNA invertases and recombination site‐like sequences are present at the boundaries of gene segments conferring the specificity for the host receptor. This, together with the properties of the DNA inversion mechanism, suggests that these site‐specific recombination enzymes could be responsible for the exchange of host‐range determinants.
CooC and CooD are required for assembly of CS1 pili
Authors: Froehlich Barbara J. , Karakashian Alexander , Meisen Lawrence R. , Wakefield Jeffrey C. , Scott June R. 
Published: October 2006

SummaryMany strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces. These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple Identical protein subunits encoded by cooA. We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA. Following cooD Is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E. coli K‐12 to synthesize CS1 pili. Complementation analysis of mutants cloned in E. coli K‐12 and constructed in an ETEC‐derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.
Cellobiohydrolase A (CbhA) from the cellulolytic bacterium Cellulomonas fimi is a β‐1,4‐exoceilobiohydrolase analogous to Trichoderma reesei CBH II
Authors: Meinke Andreas , Gilkes Neil R. , Kwan Emily , Kilburn Douglas G. , Warren R. Antony J. , Miller Robert C. 
Published: October 2006

SummaryThe gene cbhA from the cellulolytic bacterium Cellulomonas fimi encodes a protein of 872 amino acids designated cellobiohydrolase A (CbhA). Mature CbhA contains 832 amino acid residues and has a predicted molecular mass of 85 349 Da. It is composed of five domains: an N‐terminal catalytic domain, three repeated sequences of 95 amino acids, and a C‐terminal cellulose‐binding domain typical of other C. fimi glycanases. The structure and enzymatic activities of the CbhA cataiytic domain are closely related to those of CBH ll, an exocelloblohydrolase in the glycosyl hydrolase family B from the fungus Trichoderma reesel. CbhA is the first such enzyme to be characterized in bacteria. The data support the proposal that extended loops around the active site distinguish exohydrolases from endohydrolases in this enzyme family.
Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) ‘minicircle’: roles of open reading frames and origin of tandem insertions
Authors: Smokvina Tamara , Henderson Duncan J. , Melton Rachel E. , Brolle Dirk‐F , Kieser Tobias , Hopwood David A. 
Published: October 2006

SummaryIS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20‐fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild‐type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co‐transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.
Regulation of the xylanase‐encoding xlnA gene of Aspergilius tubigensis
Authors: de Graaff Leo K. , van den Broeck Hetty C. , van Ooijen Albert J. J. , Visser Jaap 
Published: October 2006

SummaryA gene encoding an endo‐1,4‐β‐xylanase from Aspergillus tubigensis was cloned by oligonucleotide screening using oligonucleotides derived from amino acid sequence data obtained from the purified protein. The isolated gene was functional as it could be expressed in the very closely related fungus Aspergillus niger. The xylanase encoded by this gene is synthesized as a protein of 211 amino acids. After cleavage of the presumed prepropeptide this results in a mature protein of 184 amino acids with a molecular weight of 19 kDa and an isoelectric point of 3.6. The regulatory region of the xlnA gene was studied with respect to the response to xylan induction and carbon catabolite repression. By deletion analysis of the 5′ upstream region of the gene a 158bp region involved in the xylan specific induction was identified. To study this regulatory element a reporter system for transcriptional activating sequences was developed that is based on the A. niger glucose oxidase‐encoding gene. From the results with this reporter system it is concluded that this 158bp fragment not only contains the information required for induction of transcription but that it also plays a role in carbon catabolite repression of the xlnA gene. The region directly upstream of this fragment contains four potential CREA target sites; deletion of this region leads to an increase in the level of transcription. These results suggest that carbon catabolite repression of the xinA gene is controlled at two levels, directly by repression of xlnA gene transcription and indirectly by repression of the expression of a transcriptional activator. This type of mechanism would be similar to the double lock mechanism for the regulation of gene expression of alcA in Aspergillus nidulans. The reporter system was also used to study the regulation of expression via the functions located on this fragment in A. niger and in A. niduians. Essentially the same pattern of regulation was found in both of these hosts. Therefore, regulation of xylanase gene expression is basically conserved in ail three aspergilli.
Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitumβ‐lactamase genes cloned from a natural isolate and a high‐level β‐lactamase producer
Authors: Timm J. , Perilli M. G. , Duez C. , Trias J. , Orefici G. , Fattorini L. , Amicosante G. , Oratore A. , Joris B. , Frére J. M. , Pugsley A. P. , Gicquel B. 
Published: October 2006

SummaryThe gene encoding a class A β‐lactamase was cloned from a natural isolate of Mycobacterium fortuitum (blaF) and from a high‐level amoxicillin‐resistant mutant that produces large amounts of β‐lactamase (blaF). The nucleotide sequences of the two genes differ at 11 positions, including two in the region upstream from the coding sequence. Gene fusions to Escherichia coli lacZ and transcription and expression analysis of the cloned genes in Mycobacterium smegmatis indicated that high‐level production of the β‐lactamase in the mutant is mainly or wholly due to a single base pair difference in the promoter. These analyses also showed that transcription and translation start at the same position. A comparison of the amino acid sequence of BlaF, as predicted from the nucleotide sequence, with the determined N‐terminal amino acid sequence indicated the presence of a typical signal peptide. The fusion of blaF (or biaF) to the E. coli gene phoA resulted in the production of BlaF‐PhoA hybrid proteins that had alkaline phosphatase activity. These results demonstrate that phoA can be used as a reporter gene for studying protein export in mycobacteria.
A two‐component signal‐transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae
Authors: Guenzi Eric , Gasc Anne‐Marie , Sicard Michel A. , Hakenbeck Regine 
Published: October 2006

SummaryPenicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin‐binding protein (PBP) variants with decreased affinities for β‐lactam antibiotics. Cefotaxime‐resistant laboratory mutants, selected after several steps on increasing concentrations of this β‐lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal‐transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N‐terminal extracelluiar sensor part, and an intracelluiar C‐terminal domain with the conserved His‐226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime‐resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime‐resistant mutants were each located in the C‐terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal‐transducing system cia is involved in early steps of competence regulation.
Frequency of deletion formation decreases exponentially with distance between short direct repeats
Authors: Chédin F. , Dervyn E. , Dervyn R. , Ehrlich S. D. , Noirot P. 
Published: October 2006

SummaryThe effect of distance between 18 bp direct repeats on deletion formation has been examined in Bacillus subtilis. The deletion frequency decreased exponentially by more than 1000‐fold as the distance increased from 33 to 2313 bp. This decrease occurred in two distinct phases, which may be determined by DNA‐duplex flexibility. A similar relationship between deletion formation and distance was observed in a θ‐replicating plasmid and in the chromosome, indicating that this relationship might have a general validity.