bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neisseria gonorrhoeae acquires mutations in analogous regions of gyrA and parC in fluoroquinolone‐resistant isolates
Authors: Belland R. J. , Morrison S. G. , Ison C. , Huang W. M. 
Published: October 2006

Summary Neisseria gonorrhoeae homologues of gyrA and parC have been identified using hybridization probes generated from conserved regions of diverse gyrA genes. These genes have been tentatively identified as gyrA and parC, based on predicted amino acid sequence homologies to known GyrA homologues from numerous bacterial species and to ParC from Escherichia coli and Salmonella typhimurium. The gyrA gene maps to a physical location distant from the gyrB locus on the gonococcal chromosome, which is similar to the situation found in E. coli. The parC gene is not closely linked (i.e. greater than 9 kb) to an identifiable parE gene in N. gonorrhoeae. The gonococcal GyrA is slightly larger than its E. coli homologue and contains several small insertions near the O‐terminus of the predicted open reading frame. A series of ciprofloxacin‐resistant mutants were selected by passage of N. gonorrhoeae on increasing concentrations of the antibiotic. Sequential passage resulted in the selection of isolates with minimum inhibitory concentrations approximately 10000‐fold higher than the parental strain. Mutations within gyrA resulted in low to moderate levels of resistance, while strains with high‐level resistance acquired analogous mutations in both gyrA and parC. Resistance mutations were readily transferred between N. gonorrhoeae strains by transformation. The frequencies of transformation, resulting in different levels of ciprofloxacin resistance, further support the notion that both gyrA and parC genes are invoived in the establishment of extreme levels of ciprofloxacin resistance.
Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis
Authors: Adams Lee F. , Mathewes Shannon , O'Hara Patrick , Petersen Annette , Gürtler Hanne 
Published: October 2006

SummaryNB176 is a Bacillus thuringiensis mutant derived by λ‐irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene‐bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn 1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by γ‐irradiation. Integration of additional copies of the cryIIIA gene into the native 90MDa plasmid of the wild‐type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.
Non‐congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci
Authors: Whatmore Adrian M. , Kapur Vivek , Sullivan Derek J. , Musser James M. , Kehoe Michael A. 
Published: October 2006

SummaryTo examine the molecular population genetics of the M protein family of Streptococcus pyogenes (group A Streptococcus), the 5′ regions of polymerase chain reaction‐amplified emm products from 79 M serotypes were sequenced and the phylogeny was compared to estimates of overall genetic relationships among strains determined by multilocus enzyme electrophoresis. Although the 5′emm sequences from several strains designated as distinct M types were identical or almost identical, the overall pattern is characterized by very extensive variation. The composition of distinct emm sequence clusters generally parallels the ability of strains to express serum opacity factor and in some cases historical associations of certain M types with acute rheumatic fever, but not with M types classified as nephritogenic. For many strains there is a lack of congruency between variation in 5′emm sequences and estimates of overall chromosomal relationships, which is undoubtedly due to horizontal transfer and recombination of emm sequences. The results of these studies provide insights into the nature and extent of emm sequence variation and describe how this variation ‘maps’ onto the population genetic structure of extant S. pyogenes lineages. The complexity of emm sequence and streptococcal cell lineage relationships revealed by this analysis has significant implications for understanding evolutionary events generating strain diversity and the epidemiology of S. pyogenes diseases.
NarL‐phosphate must bind to multiple upstream sites to activate transcription the narG promoter of Escherichia coli
Authors: Walker M. S. , DeMoss J. A. 
Published: October 2006

SummaryThe stimulation of Fnr‐dependent transcription from the narG promoter by NarL‐phosphate is known to require a cis‐acting sequence, the NarL box, located approximately 195 bp upstream from the transcription start site, and the interaction of integration host factor (IHF) with a binding site in the intervening region (positions −110 to −140) between the NarL box and the transcription start site. By gel retardation and DNase I protection studies, we have demonstrated that NarL‐phosphate, produced by the reaction of purified NarL with acetyl phosphate, specifically binds to a fragment derived from the upstream region of the narG promoter. The fragment was protected by NarL‐phosphate binding to two distinct regions. One was an extended sequence of approximately 40 bp surrounding the NarL box at −195; the second was located downstream from the IHF‐binding region and included a sequence extending from positions −80 to −120. Alteration by site‐directed mutagenesis of a putative inverted NarL box sequence identified within the downstream protected region in a plasmid containing a narG‐lacZ fusion eliminated the NarL‐phosphate‐mediated stimulation of transcription. NarL‐phosphate bound to the two regions independently from IHF binding and it bound to each site independently when the two sites were separated by cleavage of the promoter fragment. Stimulation of transcription from the narG promoter by NarL‐phosphate appears to result from the formation of a folded protein‐DNA structure created by the binding of NarL‐phosphate to multiple sites on either side of an IHF‐induced bend in the upstream region of the promoter.
afsR2: a previously undetected gene encoding a 63‐amino‐acid protein that stimulates antibiotic production in Streptomyces lividans
Authors: Vögtil Martin , Chang Poa‐Chun , Cohen Stanley N. 
Published: October 2006

SummaryEarlier work has shown that the afsR genetic locus promotes formation of the pigmented antibiotics actinorhadin and undecylprodiglosin in Streptomyces lividans and its close relative, Streptomyces coeiicolor. A protein designated as AfsR has been implicated in this activity. We report here the existence of a previously unknown gene, afsR2, which is separate from and adjacent to the AfsR‐encoding sequence and which, when present at high copy number, (i) stimulates transcription of biosynthetic and regulatory genes in the actinorhodin gene cluster (act), and (ii) stimulates the synthesis of undecyiprodigiosin., We show that the effects of afsR2 on actinorhodin synthesis are mediated through transcription of the actll‐0RF4 locus, which encodes a transcriptional activator of other genes in the act cluster. Analysis of the oloned afsR2 gene indicates that its activity is the result of the 63‐amino‐acid protein it specifies.
Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens
Authors: Pohlman Robert F. , Genetti Heather D. , Winans Stephen C. 
Published: October 2006

SummaryThe DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid. Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation. These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens TI plasmids. Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin. We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.
A protein complex mediating mRNA degradation in Escherichia coli
Authors: Py Béatrice , Causton Helen , Mudd Elisabeth A. , Higgins Christopher F. 
Published: October 2006

SummarymRNA degradation in Escherichia coli is mediated by a combination of exo‐ and endoribolucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease poly‐nucleotide phosphorylase (PNPase) and the endorlbo‐nuclease RNase E. An activity which impedes the processive activity of PNPase at stem‐loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional poly‐peptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co‐ordinated control of mRNA degradation.
Cell‐surface‐associated polypeptides CshA and CshB of high molecular mass are colonization determinants in the oral bacterium Streptococcus gordonii
Authors: McNab R. , Jenkinson H. F. , Loach D. M. , Tannock G. W. 
Published: October 2006

SummaryThe human oral bacterium Streptococcus gordonii expresses, on the cell surface, two antigenically related high‐molecular‐mass polypeptides denoted CshA and CshB, encoded by genes at separate chromosomal loci. The precursor form of CshA is composed of four distinct segments: (i) a 41‐amino‐acid residue leader peptide, (ii) W‐terminal 42–878 residues, (iii) residues 879–2417 comprising 13 repeat blocks of 101 amino acid residues and three shorter blocks, and (iv) a C‐terminal anchor domain similar to those present in some other Gram‐positive bacterial cell‐wall polypeptides. Insertional mutations within cshA reduced both cell‐surface hydrophobicity and ability to adhere to oral Actinomyces naeslundii. Insertional mutations in cshB had less effect on hydrophobicity and coadherence. However, expression of both polypeptides was found to be necessary for streptococci to colonize the murine oral cavity.
The Legionella pneumophila icm locus: a set of genes required for intracellular multiplication in human macrophages
Authors: Brand Bettina C. , Sadosky Alesia B. , Shuman Howard A. 
Published: October 2006

Summary Legionella pneumophila, the causative agent of Legionnaires’ disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular lifestyle is the ability of the organism to replicate within a specialized phagosome which does not fuse with Iysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host‐cell killing are unable to prevent phagosome–Iysosome fusion. In a previous study, a 12kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome‐Iysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea‐pigs. In order to gain information about how L. pneumophila prevents phagosome‐Iysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1–2.4kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have Inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.
Altered intracellular targeting properties associated with mutations in the Legionella pneumophila dotA gene
Authors: Berger Karen H. , Merriam Jennifer J. , Isberg Ralph R. 
Published: October 2006

Summary Legionella pneumophila dot mutations cause defects in intracellular targeting of the microorganism within cultured macrophages. Each of the previously characterized dot mutations was shown to be complemented by a single open reading frame designated dotA. The defects caused by the mutations appear to be due to disrupted function of the predicted 1048‐amino‐acid residue DotA protein, and not by polarity effects on a downstream gene. Complementation studies indicated that the product of the dotA53 mutation results in a partially functional DotA protein, consistent with a stable N‐terminal fragment having biological activity.
Isolation of motile and non‐motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells
Authors: Yao Ruijin , Burr Don H. , Doig Peter , Trust Trevor J. , Niu Haiying , Guerry Patricia 
Published: October 2006

SummaryA method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin‐resistant Insertional mutants of C. jejuni 81–176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18‐200‐fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2–32, which is non‐adherent and non‐invasive, has an insertion of the kanamycin‐resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non‐invasive mutants K2–37 and K2–55 are phenotypically paralysed, i.e. they have a full‐length flagellar filament but are non‐motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2–37 and K2–55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90 977 with no significant homology to known proteins. Site‐specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants.
Amoebapores, a family of membranolytic peptides from cytoplasmic granules of Entamoeba histolytica: isolation, primary structure, and pore bacterial cytoplasmic membranes
Authors: Leippe Matthias , Andrä Jörg , Nickel Rose , Tannich Egbert , Muller‐Eberhard Hans J. 
Published: October 2006

SummaryThree peptides with pore‐forming activity were isolated from the cytoplasmic granules of pathogenic Entamoeba histolytica by acidic extraction, gel filtration and reversed‐phase high‐performance liquid chromatography. Partial amino acid sequence analysis of the three active peptides revealed that the most abundant of them was amoebapore and the other two were isoforms thereof. Cloning and sequencing of genomic DNA resolved the amino acid sequence of the two newly recognized peptides. The three peptides designated amoebapores A, B and C were found to have the same molecular size but to differ markedly in their primary structure, although all six cysteine residues are conserved. Despite sequence divergence, structural implications predict for the three peptides a similar amphipathic α‐helical conformation stabilized by disulphide bonds. All three isoforms exhibit pore‐forming activity toward lipid vesicles, but they differ in their kinetics. They also are capable of perturbing the integrity of bacterial cytoplasmic membranes and thereby kill Gram‐positive bacteria. The amoebapores represent a distinct family of membrane‐active peptides that may function intracellularly as antimicrobial agents but may also confer cytolytic activity on the parasite.
Signal‐sensing mechanisms of the putative osmosensor KdpD in Escherichia coli
Authors: Sugiura Akemi , Hirokawa Kozo , Nakashima Kyoko , Mizurto Takeshi 
Published: October 2006

SummaryThe KdpD protein is a membrane‐located sensory kinase (or signal transducer) critically involved in the regulation of the kdpABC operon that is responsible for a high‐affinity transport system in Escherichia coli. In this study, a set of KdpD mutants, each resulting in a single amino acid substitution around the membrane‐spanning regions of KdpD, was isolated. Amino acid substitutions in these KdpD mutants were located non‐randomly, particularly within the C‐terminal half of the membrane‐spanning regions. This set of KdpD mutants exhibited altered transmembrane‐signalling properties in response to external K+ and other stimuli. In particular, these mutants were found to be insensitive, if not completely, to the K+ signal. However, they were able to respond to other stimuli such as high‐salt stress, as in the wild type. Therefore, in contrast to the wild type, the cells carrying these mutations exhibited high levels of the steady‐state expression of kdp, regardless of external K+, provided that high concentrations of ionic solutes were supplemented to the cultures. More interestingly, the set of KdpD mutants could also respond to high concentrations of external non‐ionic solutes such as sucrose and D‐arabinose, thereby increasing substantially the steady‐state expression of kdp in response to the medium osmolarity. Furthermore, it was found that certain chemicals, ethanol, chlorpromazine and procaine, could function as effectors for the KdpD mutants at relatively low concentrations in the media. Based on these findings, we have examined the primary signal(s) that regulates the function of KdpD. We propose here that KdpD can be considered to be an environmental sensor that exhibits sensing mechanisms in response to both the level of K+ and the physico‐chemical state of the cytoplasmic membrane.
Helicobacter pylori hspA‐hspB heat‐shock gene cluster: nucleotide sequence, expression, putative function and immunogenicity
Authors: Suerbaum Sebastian , Thiberge Jean‐Michel , Kansau Imad , Ferrero Richard L. , Labigne Agnès 
Published: October 2006

SummaryAll Helicobacter pylori isolates synthesize a 54 kDa immunodominant protein that was reported to be associated with the nickel‐dependent urease of H. pylori. This protein was recently recognized as a homologue of the heat‐shock protein of the GroEL class. The gene encoding the GroEL‐like protein of H. pylori (HspB) was cloned (plLL689) and was shown to belong to a bicistronic operon including the hspA and hspB genes. In Escherichia coli. the constitutive expression of the hspA and hspB genes was initiated from a promoter located within an IS5 insertion element that mapped upstream to the two open reading frames (ORFs). IS5 was absent from the H. pylori genome, and was thus acquired during the cosmid cloning process. hspA and hspB encoded polypeptides of 118 and 545 amino acid residues, corresponding to calculated molecular masses of 13.0 and 58.2 kDa, respectively. Amino acid sequence comparison studies revealed that, although H. pylori HspA and HspB proteins were highly similar to their bacterial homologues, the H. pylori HspA featured a striking motif at the C‐terminus. This unique motif consists of a series of cysteine and histidine residues resembling a nickel‐binding domain, which is not present in any of the other bacterial GroES homologues so far characterized. When the plLL689 recombinant plasmid was introduced together with the H. pylori urease gene cluster (plLL763) into an E. coli host strain, an increase of urease activity was observed. This suggested a close interaction between the HspA and HspB proteins and the urease enzyme, and a possible role for HspA in ihe chelation of nickel ions. The genes encoding each of the HspA and HspB polypeptides were cloned, expressed independently as proteins fused to the maltose‐binding protein (WIBP) and purified in large scale. The MBP‐HspA and MBP‐HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H. pylori‐infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic as proteins fused to the maltose‐binding protein (WIBP) and purified in large scale. The MBP‐HspA and WlBP‐HspB fusion proteins were shown to retain their antigenic properties. Both HspA and HspB represent antigens that are specifically recognized by the sera from H, py/or/‐infected patients. Whereas HspB was known to be immunogenic in humans, this is the first demonstration that HspA per se is also immunogenic.
The dipeptide permease of Escherichia coli closely resembles other bacterial transport systems and shows growth‐phase‐dependent expression
Authors: Abouhamad Walid N. , Manson Michael D. 
Published: October 2006

SummaryThe dipeptide permease (Dpp) of Escherichia coli transports peptides consisting of two or three L‐amino acids. The periplasmic dipeptide‐binding protein (DBP), encoded by the dppA gene, also serves as a chemoreceptor. We sequenced the dpp locus, which comprises an operon of five genes, dppABCDE. Its organization is the same as the oligopeptide permease (opp) operon of Salmonella typhimurium and the spo0K operon of Bacillus subtilis. The dpp genes are also closely related to the hbpA gene, which encodes a haem‐hinding lipoprotein, and four other genes in an unlinked operon of unknown function in Haemophilus influenzae. Each Dpp protein has an Opp, Spo0K and H. influenzae homologue. Transcription of the dpp operon initiates 165 bases upstream of the predicted dppA start codon. The start site for transcription is preceded by potential −35 and −10 regions of a σ70 promoter. During exponential growth in Luria‐Bertani (LB) broth, the level of dpp mRNA increases in two steps, one between A590 0.2 and 0.4 and one between A590 0.7 and 1.0. The 310 nucleotides between dppA and dppB include a RIP (repetitive IHF‐binding palindromic) element, whose deletion from a multi‐copy plasmid causes fivefold and 10‐fold reductions in the levels of upstream and downstream dpp mRNA, respectively.