bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the Yersinia enterocolitica enterotoxin Yst gene. Influence of growth phase, temperature, osmolarity, pH and bacterial host factors
Authors: Mikulskis Alvydas V. , Delor Isabelle , Thi Vinh Ha , Cornelis Guy R. 
Published: October 2006

SummaryThe chromosome of Yersinia enterocolitica encodes an enterotoxin called Yst. We analysed transcription of chromosomal yst′–luxAB and plasmid‐borne yst′–lacZ operon fusions and we observed that regulation of yst expression occurs at transcriptional level. In a wild‐type strain, yst was transcribed from at least two major promoters, yst transcription reached a maximum at the entry to the stationary phase and significantly varied in different Y. enterocolitica strains. In some strains, it gradually decreased during the course of our work, suggesting the existence of a mechanism switching the expression of yst to a silent state. Changes in the status of bacterial host factors rather than modifications in the yst gene are responsible for this silencing. Negative regulator YmoA participates in yst silencing and temperature regulation of yst YmoA was also required for proper growth‐phase regulation of yst, although it is not the only factor involved in this regulation. Physicochemical parameters of the environment play an important role in yst transcription. In usual culture media (e.g. tryptic soy broth), the enterotoxin gene was transcribed only at temperatures below 30°C, which argued against the role of Yst in a prolonged diarrhoea at body temperatures. However, yst transcription could be induced at 37°C by increasing osmolarity and pH to the values normally present in the ileum lumen. This finding reconciles the observations concerning yst expression in a host environment and in bacterial cultures, thus supporting the idea that enterotoxin Yst is a virulence factor of Y. enterocolitica.
Cloning and disruption of the gene encoding an extracellular metalloprotease of Aspergillus fumigatus
Authors: Jaton‐Ogay Katia , Paris Sophie , Huerre Michel , Quadroni Manfredo , Falchetto Rocco , Togni Giuseppe , Latgé Jean‐Paul , Monod Michel 
Published: October 2006

Summary Aspergillus fumigatus secretes a serine alkaline protease (ALP) and a metalloprotease (MEP) when the fungus is cultivated in the presence of collagen as sole nitrogen and carbon source. The gene encoding ALP was isolated and characterized previously. We report here the cloning and the sequencing of the gene encoding MEP. Genomic and cDNA clones were isolated from A. fumigatus libraries using synthetic oligonucleotides as probes. Stretches of the deduced amino acid sequence were found to be in agreement with the N‐terminal amino acid sequence of MEP and with internal peptide sequences. The amino acid sequence of the enzyme contains a putative active‐site sequence HEYTH homologous to the active site of other bacterial and eukaryotic zinc metalloproteases. Sequence analysis reveals that MEP has a pre‐proregion consisting of 245 amino acid residues preceding the 388 amino acid residues of the mature region (molecular mass of 42 kDa). An alp mep mutant, deficient in proteolytic activity at neutral pH in vitro, was constructed and tested for pathogenicity in a murine model. No difference in pathogenicity was observed between the wild‐type strain and the alp mep double mutant, suggesting that ALP and MEP are not essential for the invasion of the lung tissues by A. fumigatus.
Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C‐125
Authors: Hamamoto Tetsuo , Hashimoto Michizane , Hino Motohiro , Kitada Makio , Seto Yasuyuki , Kudo Toshiaki , Horikoshi Koki 
Published: October 2006

SummaryAn alkali‐sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C‐125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1–4). By sub‐cloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly‐393 to Arg of the putative 0RF1 product, which was deduced to be an 804‐amino‐acid polypeptide with a molecular weight of 89 070. The N‐terminal part of the putative ORF1 product showed amino acid similarity to those of the chain‐5 products of eukaryotic NADH quinine oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (δψ)‐driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ anti‐porter system is important in the alkalophily of alkalo‐philic microorganisms.
Ribosomal protein methylation in Escherichia coli: the gene prmA, encoding the ribosomal protein L11 methyltransferase, is dispensable
Authors: Vanet Anne , Plumbridge Jacqueline A. , Guérin Marie‐France , Alix Jean‐Hervé 
Published: October 2006

SummaryThe prmA gene, located at 72 min on the Escherichia coli chromosome, is the genetic determinant of ribosomal protein L11‐methyltransferase activity. Mutations at this locus, prmA1 and prmA3, result in a severely undermethylated form of L11. No effect, other than the lack of methyl groups on L11, has been ascribed to these mutations. DNA sequence analysis of the mutant alleles prmA1 and prmA3 detected point mutations near the C‐terminus of the protein and plasmids overproducing the wild‐type and the two mutant proteins have been constructed.The wild‐type PrmA protein could be crosslinked to its radiolabelled substrate, S‐adenosyl‐L‐methionine (SAM), by u.v. irradiation indicating that it is the gene for the methyltransferase rather than a regulatory protein. One of the mutant proteins, PrmA3, was also weakly crosslinked to SAM. Both mutant enzymes when expressed from the overproducing plasmids were capable of catalysing the incorporation of 3H‐labelled methyl groups from SAM to L11 in vitro. This confirmed the observation that the mutant proteins possess significant residual activity which could account for their lack of growth phenotype. However, a strain carrying an in vitro‐constructed null mutation of the prmA gene, transferred to the E. coli chromosome by homologous recombination, was perfectly viable.
The site‐specific recombination system regulating expression of the Type 1 fimbrial subunit gene of Escherichia coli is sensitive to changes in DNA supercoiling
Authors: Dove Simon L. , Dorman Charles J. 
Published: October 2006

SummaryWe have studied the effect of altering the in vivo level of DNA supercoiling on the phase‐variable expression of the Escherichia coli fimA gene. Transcription from the fimA promoter was unaffected by changes in DNA supercoiling whether caused by the introduction of a topA::7n10 mutation or by inhibition of DNA gyrase with the antibiotic novobiocin. However, inversion of the fimA promoter fragment was altered in response to perturbation of DNA supercoiling. Specifically, inactivation of topA reduced the rate of promoter fragment inversion in both the ON‐to‐OFF and the OFF‐to‐ON directions. This effect correlated with the loss of functional topA and not with the global level of DNA supercoiling. Inhibition of DNA gyrase introduced a bias in favour of the OFF‐to‐ON inversion; the ON‐to‐OFF inversion was affected only slightly. Changes in expression of fimB, the gene coding for the recombinase that catalyses fimA promoter fragment inversion in the strains used In this study, did not correlate with effects on fimA phase variation: we found that transcription of fimB was inhibited by loss of functional topA and was enhanced by inhibition of DNA gyrase in a manner that correlated well with the global level of in vivo DNA supercoiling. A model is presented to account for the effects of lost topoisomerase function on fimA gene expression.
The gene encoding the periplasmic cyclophilin homologue, PPlase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP–CRP complex and negatively regulated by the CytR repressor
Authors: Nørregaard‐Madsen Mads , Myglnd Bente , Pedersen Rolf , Valentin‐Hansen Poui , Søgaard‐Andersen Lotte 
Published: October 2006

SummaryThe rot gene in Escherichia coli encodes PPlase A, a periplasmic peptldyl‐prolyl cis‐trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA‐binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP–CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP–CRP to two sites separated by 53 bp. Moreover, one of the cAMP–CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP–CRP/DNA nucleoprotein complex consisting of the two cAMP–CRP molecules and CytR bound in between. The two regulators bind co‐operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP–CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo‐ and deoxyribonucleoside catabolism or transport.
Nucleotide sequence, organization and expression of rdgA and rdgB genes that regulate pectin lyase production in the plant pathogenic bacterium Erwinia carotovora subsp. cartovora in response to DNA‐damaging agents
Authors: Liu Yang , Chatterjee Asita , Chatterjee Arun K. 
Published: October 2006

SummaryIn most soft‐rotting Erwinia spp., including E. carotovora sub sp. carotovora strain 71 (Ecc71), production of the plant cell wall degrading enzyme pectin lyase (PnI) is activated by DNA‐damaging agents such as mitomycin C (MC). Induction of PnI production in Ecc71 requires a functional recA gene and the rdg locus DNA sequencing and RNA analyses revealed that the rdg locus contains two regulatory genes, rdgA and rdgB, in separate transcriptional units. There is high homology between RdgA and repressers of lambdoid phages, specially φ80. RdgB, however, has significant homology with transcriptional activators of Mu phage. Both RdgA and RdgB are also predicted to possess helix‐turn‐helix motifs. By replacing the rdgB promoter with the IPTG‐inducible tac promoter, we have determined that rdgB by itself can activate PnI production in Escherichia coli. However, deletion analysis of rdg+ DNA indicated that, when driven by their native promoters, functions of both rdgA and rdgB are required for the induction of pnIA expression by MC treatment. While rdgB transcription occurs only after MC treatment, a substantial level of rdgA mRNA is detected in the absence of MC treatment. Moreover, upon induction with MC, a new rdgA mRNA species, initiated from a different start site, is produced at a high level. Thus, the two closely linked rdgA and rdgB genes, required for the regulation of PnI production, are expressed differently in Ecc71.
The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site‐specific recombinease
Authors: Höfte Monica , Dong Qinghan , Kourambas Sophia , Krishnapillai Viji , Sherratt David , Mergeay Max 
Published: October 2006

SummaryPyoverdin production by Pseudomonas aeruginosa strain 7NSK2 was induced by Zn(II) in the presence of iron. A mutant was isolated in which Zn(II) no longer induced pyoverdin production. The sss gene which was inactivated in this mutant was cloned and sequenced. Its protein sequence showed 50% identity to the XerC protein of Escherichia coli, which is a member of the lambda integrase family of site‐specific recombinases. An open reading frame was found upstream of sss whose protein sequence showed strong identity to DapF, the diaminopimelate epimerase. In E. coli, xerC is part of a multicistronic unit that also contains dapF. The sss gene of P. aeruginosa could restore site‐specific recombination at cer in an E. coii xerC mutant and the E. coii xerC gene could complement a genomic sss mutation in P. aeruginosa.
Expression of the genes coding for the Escherichia coli integration host factor are controlled by growth phase, rpoS, ppGpp and by autoregulation
Authors: Aviv Michal , Giladi Hilla , Schreiber Gideon , Oppenheim Amos B. , Glaser Gad 
Published: October 2006

SummaryTranscriptional control of the himA and the himD/hip genes coding for the two subunits of the integration host factor (IHF) was investigated. The promoters for the two genes were identified by the use of primer extension and S1 analysis. Expression from both promoters was found to increase as the cells enter stationary phase. Mutation in rpoS, known to be induced upon entry to stationary phase, dramatically reduced the growth‐phase response of the himA P4 promoter but had only a small effect on the induction of the himD/hip promoter. The increased activity of both promoters required the presence of the rel4 and spoT genes, suggesting that ppGpp plays a major role in the response to stationary phase. An artificial increase in ppGpp in exponentially growing cells induced a rapid increase in himA P4 and himD/hip mRNA levels. Experiments with a mutant defective in rpoS showed that the response of the himA P4 promoter to high ppGpp levels was greatly reduced while that of himD/hip was only slightly affected. Therefore, it seems that different mechanisms involving RpoS and ppGpp regulate the growth‐phase response of the two promoters. We propose that the effect of ppGpp on himA P4 is mediated via RpoS whereas the himD/hip promoter is affected by ppGpp independently of RpoS.Expression of the himD/hip and himA genes was found to be subject to negative autoregulation. IHF‐binding sites, implicated in autoregulation, were found to overlap both the himD/hip and himA P4 promoters. An additional IHF‐binding site was found upstream of the himD/hip promoter. AM three sites show low binding affinity to IHF suggesting that autoregulation can take place only after sufficiently high levels of IHF accumulate in the cell.
Control of translation by mRNA secondary structure: the importance of the kinetics of structure formation
Authors: Ma Charles K. , Koslesnikow Tassia , Rayner John C. , Simons Elizabeth L. , Yim Harry , Simons Robert W. 
Published: October 2006

SummaryRNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS 10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosomebinding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The signficance of these observations for the RNA folding problem in general is discussed.
PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a cis‐acting sequence upstream of the pilin gene promoter
Authors: Jin Shouguang , Ishimoto Karyn S. , Lory Stephen 
Published: October 2006

SummaryThe PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two‐component sensor–regulator family. PilR was overproduced by fusing pilR to the gene for the maltose‐binding protein (malE), yielding a MalE–PilR hybrid protein. The plasmid with the malE–pilR fusion, when introduced into a non‐piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE‐PilR protein was purified from Escherichia coli and used in a series of DNA‐binding studies. A specific pilin promoter‐binding activity of MalE‐PilR was observed in a gef retardation assay. Subsequent DNase I footprinting analysis revealed a 40bp PilR‐binding site located at the −120 to −80 region, relative to the transcriptional start site of the pilin gene. This PilR‐binding region consists of a nine‐base sequence and three consensus sequences of 5‐(N)4–6C/GTGTC‐3′, in a tandem array in which the first 7–9 bp are bound by the PilR on the non‐Goding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds of sequences complementary to the two middle consensus sequences of the non‐coding strand. A sequence similar to the NifA recognition site (5‐TGT‐(N)11‐ACA‐3′) is also found within the PilR‐binding region. Deletion analysis and disruption of the individual consensus PilR‐binding sequences by site‐directed mutagenesis revealed that all four PilRbinding sites are absolutely required for the PilS/PilR‐mediated pilin gene expression. The presence of four PilR‐binding repeat sequences suggests that PilR protein may bind co‐operatively or as a multimer.
Immunobiology of purified recombinant outer membrane porin protein I of Neisseria gonorrhoeae
Authors: Elkins Christopher , Barkley Katherine B. , Carbonetti Nicholas H. , Coimbre Alex J. , Sparling P. Frederick 
Published: October 2006

SummaryGonococcal porins (Por) from strains FA19 (Por‐1, serogroup A), MS11 (Por‐2, serogroup B) and FA6434 (Por‐5, a hybrid porin containing epitopes from both serogroups), were expressed in Escherichia coli and purified under non‐denaturing conditions. Porins were inserted into liposomes, and they were bound by monoclonal antibodies which bind native Por and intact gonococci, but not denatured Por. All three recombinant porins (rPor) were highly immunogenic in rabbits without additional adjuvant. The rPor antisera were specific for Por by Western blotting and whole‐cell radioimmunoprecipitation and were broadly cross‐reactive within serogroups. Post‐immune, but not pre‐immune, sera bound to intact gonococci, induced deposition of complement components C3 and C9 onto gonococcal membranes and increased association with and activation of human neutrophils. Gonococci were not killed in bactericidal assays, and there was no phagocytic killing with gonococci opsonized with recombinant antisera. Lack of killing in bactericidal assays was not caused by the presence of blocking antibodies to the outermembrane protein Rmp.
Partial purification of the rat erythrocyte receptor for the channel‐forming toxin aerolysin and reconstitution into planar lipid bilayers
Authors: Gruber Hermann J. , Wilmsen Hans U. , Cowell Simon , Schindler Hansgeorg , Buckley J. Thomas 
Published: October 2006

SummaryThe cytolytic toxin aerolysin binds to a receptor on the surface of eukaryotic cells. Murine erythrocytes are among the most sensitive to the toxin. Here we describe the detergent solubilization and partial purification of the receptor from rat erythrocytes. We show that it can be successfully incorporated into planar lipid bilayers, greatly decreasing the concentration of aerolysin required to form channels. Exploiting the ability of the receptor to bind aerolysin after SDS electrophoresis and blotting, we obtain evidence that it is a 47 kDa glycoprotein that is sensitive to proteases and N‐glycosidase. It may correspond to CHIP28, the water channel of the human erythrocyte.
Biosynthesis and functional role of haem O and haem A
Authors: Mogi Tatsushi , Saiki Keitarou , Anraku Yasuhiro 
Published: October 2006

SummaryHaem O and/or haem A are specifically synthesized for the haem‐copper respiratory oxidases. A 17‐carbon hydroxyethylfarnesyl chain at the pyrrole ring A of the haems seems essential for catalytic functions at the oxygen‐reduction site. The discovery of haem O in the cytochrome bo complex from Escherichia coli was a breakthrough in the studies on haem A biosynthesis. Molecular biological and biochemical studies in the past three years demonstrated that the cyoE/ctaB/COX10 genes are indispensable for functional expression of the terminal oxidases and encode a novel enzyme haem O synthase (protohaem IX farnesyltransferase). It has recently been suggested that the ctaA gene adjacent to the ctaB‐ctaCDEF gene cluster in Bacillus subtilis encodes haem A synthase (haem O monooxygenase). In this article, we review current knowledge of the genes for haem O and haem A biosyntheses, the location and regulation of haem O synthase, the possible enzymatic mechanism of farnesyl transfer to haem B and the possible roles of the farnesylated haems.
The amino‐terminal domain of the P‐pilus adhesin determines receptor specificity
Authors: Haslam David B. , Borén Thomas , Falk Per , Liver Dag , Chou Amy , Xu Zheng , Normark Staffan 
Published: October 2006

SummaryPyelonephritic isolates of Escherichia coli commonly express P‐pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P‐pili have been defined, based on varying specificity for galabiose‐containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo‐terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo‐terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino‐terminus of the PapG adhesin and is independent of the complex pilus architecture.