bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Aspergillus fumigatus alkaline protease mutant constructed by gene disruption is deficient in extracellular elastase activity
Authors: Tang C. M. , Cohen J. , Holden D. W. 
Published: October 2006

SummaryInvasive pulmonary aspergillosis, usually caused by Aspergillus fumigatus, is a life‐threatening condition of immunosuppressed patients. We have created a mutant strain of this fungus that lacks an extracellular alkaline protease (AFAlp). This was accomplished by transformation of A. fumigatus with a plasmid containing a selectable marker for hygromycin B resistance, and a 504 bp segment of the AFAlp gene, obtained by polymerase‐chain‐reaction‐based amplification of A. fumigatus genomic DNA. Approximately 25% of transformants resulted from disruption of the AFAlp gene. SDS‐polyacrylamide gel etectrophoresis of proteins from the culture filtrate of a strain carrying the AFAlp gene disruption showed that it lacked a major protein of 33 kDa. Furthermore, in contrast to the culture filtrate from wild‐type cells, the mutant had undetectable activity on azocollagen and elastin‐Congo red, over a broad pH range. This shows that AFAlp accounts for most, if not all, of the extracellular elastinolytic activity of A. fumigatus, and that the mutant strain will be useful in assessing the role of AFAlp in pathogenicity.
Phylogeny of LCR‐1 and OXA‐5 with class A and class D β‐lactamases
Authors: Couture France , Lachapelle Jean , Levesque Roger C. 
Published: October 2006

SummaryThe nucleotide sequences of blaLCR‐1 and blaOXA‐5β‐lactamase genes have been determined. Polypeptide products of 260 and 267 amino acids with estimated molecular masses of 27 120 Da and 27387 Da were obtained for the mature form of LCR‐1 and OXA‐5 proteins. A progressive alignment was used to evaluate the extent of identity between LCR‐1 and OXA‐5 with 29 other β‐lactamase amino acid sequences. The data showed that both belong to class D. We identified amino acids conserved in 24 positions for class A β‐lactamases and in 28 positions for five class D enzymes. The structural similarities between class A and class D β‐lactamases are more extensive than indicated by earlier biochemical studies with overall 16% identity between both classes. From the alignment, dendograms were constructed with a distance‐matrix and parsimony methods which defined three major groups of proteins subdivided into clusters giving insight on β‐lactamase phylogeny and evolution.
Occurrence of a Photosystem II polypeptide in non‐photosynthetic membranes of cyanobacteria
Authors: Smith Dawn , Bendall Derek S. , Howe Christopher J. 
Published: October 2006

SummaryCytoplasmic and thylakoid membranes have been purified from the cyanobacteria Anacystis nidulans R2 and Phormidium laminosum by sucrose density gradient centrifugation. Probing of Western blots of proteins from these purified membrane fractions with antibodies directed against the 33 kDa polypeptide of Photosystem II from pea indicates that this protein is present in both the thylakoid and cytoplasmic membranes, rather than just the thylakoid membranes. This has been confirmed by immunogold labelling of cells. Oxygen evolution assays have been used to show that the 33 kDa polypeptide is not assembled into a functional Photosystem II complex in the cytoplasmic membranes. This may be due to the absence of other Photosystem II components.
A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae
Authors: Yu Jun , Webb Helen , Hirst Timothy R. 
Published: October 2006

SummaryA strain of Vibrio cholerae, which had been engineered to express high levels of the non‐toxic B subunit (EtxB) of Escherichia coli heat‐labile enterotoxin, was subjected to transposon (TnphoA) mutagenesis. Two chromosomal TnphoA insertion mutations of the strain were isolated that showed a severe defect in the amount of EtxB produced. The loci disrupted by TnphoA in the two mutant derivatives were cloned and sequenced, and this revealed that the transposon had inserted at different sites in the same gene. The open reading frame of the gene predicts a 200‐amino‐acid exported protein, with a Cys–X–X–Cys motif characteristic of thioredoxin, protein disulphide isomerase, and DsbA (a periplasmic protein required for disulphide bond formation In E. coli). The V. cholerae protein exhibited 40% identity with the DsbA protein of E. coli, including 90% identity in the region of the active‐site motif. Introduction of a plasmid encoding E. coli DsbA into the V. cholerae TnphoA derivatives was found to restore enterotoxin formation, whilst expression of Etx or EtxB in a dsbA mutant of E. coli confirmed that DsbA is required for enterotoxin formation in E. coli. These results suggest that, since each EtxB subunit contains a single intramolecular disulphide bond, a transient intermolecular interaction with DsbA occurs during toxin subunit folding which catalyses formation of the disulphide in vivo.
Isolation of phosphorylation‐deficient mutants of the Rhizobium Meliloti two‐component regulatory protein, FixJ
Authors: Weinstein Michael , Lois Augusto F. , Monson Ellen K. , Ditta Gary S. , Helinski Donald R. 
Published: October 2006

Summary Rhizobium meliloti FixL and FixJ are members of a symbiotically essential two‐component system that regulates nitrogen‐fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of the nifA and fixK genes by activating transcription of the nifA and fixK promoters (p‐nifA and p‐fixK, respectively). In this study, we examined the relationship between the in vivo activity of FixJ as a transcriptional regulator and its ability to be phosphorylated in vitro by the sensor FixL. FixJ mutants were isolated that showed decreased activity on p‐nifA in Escherichia coli. Most of the FixJ mutant proteins also showed decreased activity on the fixK promoter. These mutants were analysed in R. meliloti for activity on p‐nifA during vegetative growth, where similarities and differences were observed when compared with their phenotypes in E. coli. Three mutants showing significantly less activity in R. meliloti were examined for symbiotic activity in planta and were found to be ineffective. When these three mutant FixJ proteins were examined in vitro for their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings are discussed in relation to signal transduction in the FixLJ system.
Molecular characterization of pcp, the structural gene encoding the pyrrolidone carboxylyl peptidase from Streptococcus pyogenes
Authors: Cleuziat P. , Awadé A. , Robert‐Baudouy J. 
Published: October 2006

SummaryThis paper describes the cloning of a gene (pep) coding for pyrrolidone carboxylyl peptidase (PYRase), an enzyme which selectively removes N‐terminal pyroglutamic acid residues from polypeptldes. This gene was isolated from Streptococcus pyogenes by construction of a gene library with a bacteriophage λ‐derived cosmid‐Escherichia coli host system. Nucleotide sequence determination of a 1.3 kb restriction fragment revealed a 645 bp open reading frame encoding a 215‐amino‐acid product of Mr, 23 135 consistent with the 26 kDa polypeptide obtained from in vivo overexpression in E. coll. Southern hybridization confirmed that pep is a single‐copy gene on the S. pyogenes chromosome. 5’and 3’endpoint mapping of the 0.7 kb specific transcript observed by Northern analysis permitted the identification of transcriptional initiation and termination signals. Structural features of the pep gene product from S. pyogenes are discussed and compared with that from Bacillus subtilis. The lack of sequence identity with any other known protein or nucleotide sequence suggests that this enzyme belongs to a new class of peptidase.
Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1Tyr)
Authors: Hromockyj Alexander E. , Tucker Stephanie C. , Maurelli Anthony T. 
Published: October 2006

Summary virR is the central regulatory locus required for coordinate temperature‐regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI‐Accl DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique Hpal restriction site resulted in a loss of VirR+ activity. The vir R ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone‐like protein, H‐NS. Based on the predicted amino acid sequence of E. coli H‐NS, only a single conservative base‐pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNATyr. These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.
Cloning, nucleotide sequence determination and expression of the genes encoding cytochrome P‐450soy and ferredoxinsoy (soyB) from Streptomyces griseus
Authors: Trower M. K. , Lenstra R. , Omer C. , Buchholz S. E. , Sariaslani F. S. 
Published: October 2006

SummaryXenobiotic transformation by Streptomyces griseus (ATCC13273) is catalysed by a cytochrome P‐450, designated cytochrome P‐450soy A DNA segment carrying the structural gene encoding P‐450soy (soyC) was cloned using an oligonucleotide probe constructed from the protein sequence of a tryptic peptide. Following DNA sequencing the deduced amino acid sequence of P‐450soy was compared with that for P‐450cam, revealing conservation of important structural components including the haem pocket. Expression of the cloned soyC gene product was demonstrated in Streptomyces lividans by reduced CO:difference spectral analysis and Western blotting. Downstream of soyC, a gene encoding a putative [3Fe‐4S] ferredoxin (soyB), named ferredoxinsoy, was identified.
Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene
Authors: Merino Enrique , Balbás Paulina , Recillas Felix , Becerril Baltazar , Valle Fernando , Bolivar Francisco 
Published: October 2006

SummaryQuantitative analysis of specific pac mRNA and a lacZ fusion to the 5’‐terminal region of the pac gene demonstrated that both phenylacetic acid induction and catabolite repression by glucose are involved, at the transcriptional level, in the regulation of the pac gene. The studies presented here suggest that this regulation is also present in Escherichia coli transformed strains in which the pac gene was not originally present. Analysis of the nucleotide sequence of the 5′‐terminal region of this gene, with a statistical algorithm, confirms that the putative promoter previously proposed by our group is the most feasible within this region. We demonstrate that penicillin acylase activity can confer on E. coli the ability to use penicillin G as a metabolic substrate, by detaching the phenylacetic group which can be used as a carbon source. Based on these data, the regulation properties of the pac gene studied in this work, and the specificity profile of the penicillin acylase enzyme we suggest a role for it in E. coli as a scavenger enzyme for phenylacetylated compounds.
Osmostress‐induced changes in yeast gene expression
Authors: Varela Joäo C. S. , van Beekvelt Catelijne , Planta Rudi J. , Mager Willem H. 
Published: October 2006

SummaryWhen Saccharomyces cerevisiae cells are exposed to high concentrations of NaCI, they show reduced viability, methionine uptake and protein biosynthesis. Cells can acquire tolerance against a severe salt shock (up to 1.4 M NaCI) by a previous treatment with 0.7 M NaCI, but not by a previous heat shock. Two‐dimensional analysis of [3H]‐leucine‐labelled proteins from salt‐shocked cells (0.7 M NaCt) revealed the elevated rate of synthesis of nine proteins, among which were the heat‐shock proteins hsp12 and hsp26. Northern analysis using gene‐specific probes confirmed the identity of the latter proteins and, in addition, demonstrated the induction of glycerol‐3‐phos‐phate dehydrogenase gene expression. The synthesis of the same set of proteins is induced or enhanced upon exposure of cells to 0.8 M sucrose, although not as dramatically as in an iso‐osmolar NaCI concentration (0.7 M).
Horizontal gene transfer of the Escherichia coli pap and prs pili operons as a mechanism for the development of tissue‐specific adhesive properties
Authors: Marklund Britt‐lnger , Tennent Jan M. , Garcia Elisa , Hamers Anja , Baga Monika , Lindberg Frederik , Gaastra Wim , Normark Staff an 
Published: October 2006

Summary Escherichia coli strains bind to Galα1‐4Gal‐containing glycolipids via P pili‐associated G‐adhesins. Three functional classes of adhesins with different binding specificities are encoded by conserved G‐alleles. We suggest that the Class I papG‐allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain. Closely related strains in the ECOR collection of natural E. coli isolates carry either a Class II or a Class III G‐adhesin. Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual paplprs genes have occurred. We propose that the retention and spread of pap prs DNA among E. coli is the result of selection pressure exerted by mammalian intestinal isoreceptors.
Identification of a novel large extrachromosomal DNA (LED) in the Trypanosomatidae
Authors: Wagner Wilma , So Magdalene 
Published: October 2006

SummaryWe have identified a novel 75 kbp large extrachromosomal DNA (LED) which is stably maintained during developmental conversion of Trypanosoma cruzi. It has a covalently closed circular conformation and is not derived from the kinetoplast network. In all T. cruzi strains analysed, LED contains 18S rRNA and spliced leader (sl) sequences. LED from the T. cruzi Y strain contains a minimum of 15 copies of the sl repeat arrayed in a head‐to‐tail configuration and 50 copies of a 196 bp repeat. LED is also present in Trypanosoma dionisii (subgenus Schizotrypanosoma) and in other members of the family Trypanosomatidae. LED from different T. cruzi strains and from other members of the Trypanosomatidae differ in their content of large ribosomal subunit rRNA sequences and the 196 bp repeat. The presence of LED in four evolutionarily distant trypanosomatid species suggests that it plays an important rote in the biology of these parasites.
The effects of 5′‐capping, 3′‐polyadenylation and leader composition upon the translation and stability of mRNA in a cell‐free extract derived from the yeast Saccharomyces cerevisiae
Authors: Gerstel Birgit , Tuite Mick F. , McCarthy John E. G. 
Published: October 2006

SummaryA new modular expression system was developed to direct the in vitro synthesis of defined transcripts that were used as templates for translation in yeast cell‐free extracts. The system was used to examine the influence of 5′‐capping, 3′‐polyadenylation and leader sequence upon the translation and stability of the synthetic Tn9 cat (chloramphenicol acetyl transferase), yeast PGK (phosphoglycerate kinase) and yeast HSP26 (heat‐shock protein 26) mRNAs. The addition of a methylated cap (m7Gppp) or of a poly(A) tail enhanced translation and stabilized the mRNA. The dependence of translation upon capping was reduced in the presence of the HSP26 leader sequence. This may indicate the existence of a translational mechanism that enhances cap‐independent translation. The enhancement of the translation and stability of mRNA was relatively insensitive to changes in the position of the poly(A) tail relative to the reading frame.
Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity
Authors: Drefus Lawrence A. , Urban Robert G. , Whipp Shannon C. , Slaughter Clive , Tachias Kathy , Kupersztoch Yankel M. 
Published: October 2006

SummaryThe methanol‐insolouble heat‐stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino‐terminal residue, Ser‐24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys‐33 and Cys‐71 and between Cys‐44 and Cys‐59. Oligonucleotide‐directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys‐44—Cys‐59 bond contains at least two amino acids (Arg‐52 and Asp‐53) required for STQ toxic activity.
Characterization of a Vibrio cholerae virulence factor homologous to the family of TonB‐dependent proteins
Authors: Goldberg Marcia B. , Boyko Stephanie A. , Butterton Joan R. , Stoebner Janice A. , Payne Shelley M. , Calderwood Stephen B. 
Published: October 2006

SummaryIrgA is an iron‐regulated virulence factor for infection in an animal model with classical Vibrio cholerae strain 0395. We detected gene sequences hybridizing to irgA at high stringency in clinical isolates in addition to 0395, including another classical strain of V. cholerae, three V. cholerae strains of the EI Tor biotype, three non‐O1 isolates of V. cholerae, and individual isolates of Vibrio parahaemolyticus, Vibrio fluvialis, and Vibrio alginolyticus. No hybridization to IrgA was seen with chromosomal DNA from Vibrio vulnificus or Aeromonas hydrophila. To verify that irgA is the structural gene for the major iron‐regulated outer membrane protein of V. cholerae, we determined the amino‐terminal sequence of this protein recovered after gel electrophoresis and demonstrated that it corresponds to the amino acid sequence of IrgA deduced from the nucleotide sequence. Gel electrophoresis showed that two EI Tor strains of V. cholerae had a major iron‐regulated outer membrane protein identical in size and appearance to IrgA in strain 0395, consistent with the findings of DNA hybridization. We have previously suggested that IrgA might be the outer membrane receptor for the V. cholerae siderophore, vibriobactin. Biological data presented here, however, show that a mutation in IrgA had no effect on the transport of vibriobactin and produced no defect in the utilization of iron from ferrichrome, ferric citrate, haemin or haemoglobin. The complete deduced amino acid sequence of IrgA demonstrated homology to the entire class of Escherichia coli TonB‐dependent proteins, particularly Cir. Unlike the situation with Cir, however, we were unable to demonstrate a role for IrgA as a receptor for catechol‐substituted cephalosporins. The role of IrgA in the pathogenesis of V. cholerae infection, its function as an outer membrane receptor, and its potential interaction with a TonB‐like protein in V. cholerae remain to be determined.
DNA curving and bending in protein–DNA recognition
Authors: Harrington Rodney E. 
Published: October 2006

SummaryMost biological events are regulated at the molecular level by site‐specific associations between specialized proteins and DNA. These associations may bring distal regions of the genome into functional contact or may lead to the formation of large multisubunit complexes capable of regulating highly site‐specific transactional events. It is now believed that sequence‐specific protein–DNA recognition and the ability of certain proteins to compete for multiple binding sites is regulated at several levels by the local structure and conformation of the binding partners. These encompass the micro structure of DNA, including its curvature, bending and flexing as well as conformational lability in the DNA‐binding domains of the proteins. Possible mechanisms for binding specificity are discussed in the context of specific nucleoprotein systems with particular emphasis given to the roles of DNA conformations in these interactions.
Construction of chimaeric genes for mapping a surface‐exposed epitope on the pilus of non‐typable Haemophilus influenzae strain M37
Authors: Palmer Katherine L. , Munson Robert S. 
Published: October 2006

SummaryA murine monoclonal antibody (mAb), designated 3H12, reacts with a surface‐exposed conformational epitope on the pilus of non‐typable Haemophilus influenzae strain M37. This antibody does not recognize the related pilus from H. influenzae type b, strain MinnA. Although mAb 3H12 does not recognize strain M37 pilin on Western blots, mAb 3H12 recognizes the recombinant M37 pilin protein expressed by Escherichia coli. In order to map the epitope recognized by mAb 3H12, we constructed a series of chimaeric genes. The chimaeric genes were expressed in E. coli and the chimaeric proteins characterized with respect to their reactivity with mAb 3H12. Residues between 37 and 100 of the M37 pilin protein are essential for the expression of the mAb 3H12 epitope. Residues in the carboxyl half of the M37 protein enhance the reactivity of mAb 3H12 when expressed in the presence of residues 37–100. Construction of chimaeric genes may provide a general methodology for mapping of conformational epitopes expressed by one of a related pair of proteins.
Mapping the cAMP receptor protein contact site on the α subunit of Escherichia coli RNA polymerase
Authors: Zou Chao , Fujita Nobuyuki , Igarashi Kazuhiko , Ishihama Akira 
Published: October 2006

SummaryThe C‐terminal region (amino acid residues 236–329) of the Escherichia coli RNA polymerase α subunit carries the contact site I for positive transcription factors. For detailed mapping of the contact site for the cAMP receptor protein (CRP), we made a library of mutant rpoA by polymerase chain reaction (PCR) mutagenesis, such that each should carry a single mutation on average and exclusively in the C‐terminal half of the rpoA gene, and then screened this library for mutants with decreased expression of the lacZ gene. Reconstituted holoenzyme containing the mutant a subunits transcribed galP1 but not lacP1 in vitro in the presence of cAMP–CRP. DNA sequence determination of several ‘Lac‐’ mutant rpoA genes revealed that all had mutations clustered within a short segment near the C‐terminus of α between amino acid residues 265 and 270. A cluster of contact sites appear to exist within the contact site I region, each comprising of about five amino acids and responding in molecular communication with a different transcription factor(s).