bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic footprint of the ToxR‐binding site in the promoter for cholera toxin
Authors: Pfau James D. , Taylor Ronald K. 
Published: October 2006

SummaryThe transmembrane DNA‐binding protein, ToxR, of Vibrio cholerae is a global transcriptional regulator of virulence gene expression. ToxR has been shown to interact with promoter regions upstream of both the ctxAB operon encoding cholera toxin, and the regulatory gene toxT. Deletion analysis has shown that a repeated sequence, TTTTGAT, is required for ToxR binding and activation of the ctxAB promoter. However, this sequence is not found upstream of the toxT promoter. Genetic selections using P22 challenge phages were used to define sites within the promoter for ctxAB which are critical for ToxR‐DNA interactions. Single‐base‐pair changes and deletion mutations that impair ToxR binding cluster within two regions: ‐57 to ‐69 within two of three tandem TTTTGAT sequences; and from ‐39 to ‐47, between the repeat sequences; and the ‐35 region of the promoter. ToxR does not bind to a synthetic target that has three tandem repeats which lack a flanking upstream and downstream sequence. These results suggest that the ToxR‐binding site lies immediately upstream of the ‐35 position of the ctx promoter, and that the affinity of ToxR binding to this site is influenced by the repeat sequences.
Phage presentation
Authors: Hill H. Rachael , Stockley Peter G. 
Published: October 2006

SummaryThere has recently been great interest in the use of the filamentous bacteriophage fd as a vehicle for the display of peptides and proteins. Phage libraries displaying random peptides up to 38 amino acids in length can be used (i) to select for ligands able to bind specific target molecules; (ii) to mimic non‐proteinaceous ligands; and (iii) as a tool to map epitopes recognized by antibodies. The display of proteins or their functional domains provides a system for the analysis of structure‐function relationships, and the potential to generate proteins with altered binding characteristics or novel catalytic properties. The display of short immunogenic determinants on fusion phage may provide a basis for the development of novel peptide vaccines, whilst the expression of libraries of antibody fragments may provide a method to by‐pass hybridoma technology in the generation of monoclonal antibodies.
The sodium‐driven polar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar expression
Authors: Kawagishi Ikuro , Imagawa Miho , Imae Yasuo , McCarter Linda , Homma Michio 
Published: October 2006

SummaryCertain marine Vibrio species swim in sea water, propelled by a polar flagellum, and swarm over surfaces using numerous lateral flagella. The polar and the lateral flagellar motors are powered by sodium‐ and proton‐motive forces, respectively. The lateral flagella are produced in media of high viscosity, and the relevant viscosity sensor is the polar flagellum. The cell might monitor either the rotation rate of the flagellar motor or the mechanical force applied against the flagellum. To test these possibilities, we examined the effects of amiloride and its derivatives, which inhibit the rotation of the sodium‐driven motor, on lateral flagellar gene (Iaf) expression in Vibrio parahaemolyticus. Phenamil, an amiloride analogue that inhibits swimming at micromolar concentrations, induced Iaf transcription in media devoid of viscous agents in a dose‐dependent manner. The relationship between the average swimming speed and Iaf induction in the presence of various concentrations of phenamil was very similar to that observed when viscosity was changed. These results indicate that marine Vibrio sense a decrease in the rotation rate of (or the sodium influx through) the polar flagellar motor as a trigger for Iaf induction. Alternative mechanisms for Iaf induction are also discussed.
Participation of the molecular chaperone DnaK in intracellular growth of Brucella suis within U937‐derived phagocytes
Authors: Köhler Stephan , Teyssier Jacques , Cloeckaert Axel , Rouot Bruno , Liautard Jean‐Pierre 
Published: October 2006

SummaryIn the intracellular bacterium Brucella suis, the molecular chaperone DnaK was induced under heat‐shock conditions and at low pH. Insertional inactivation of dnaK and dnaJ within the dnaK/J locus led to the conclusion that DnaK, but not DnaJ, was required for growth at 37°C in vitro. Viability of the dnaK null mutant was also greatly affected at low pH. Under conditions allowing intracellular multiplication, the infection of U937‐derived phagocytes resulted in long‐lasting DnaK induction in the wild‐type bacteria. In infection experiments performed with both mutants at the reduced temperature of 30°C, the dnaK mutant of B. suis survived but failed to multiply within U937 cells, whereas the wild‐type strain and the dnaJ mutant multiplied normally. Complementation of the dnaK mutant with the cloned dnaK gene restored growth at 37°C, increased resistance to acid pH, and increased intracellular multiplication. This is the first report of the effects of dnaK inactivation in a pathogenic species, and of the temperature‐independent contribution of DnaK to intracellular multiplication of the pathogen B. suis.
Alternate promoters direct stress‐induced transcription of the Bacillus subtilis clpC operon
Authors: Krüger Elke , Msadek Tarek , Hecker Michael 
Published: October 2006

Summary clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co‐transcribed and encode stress‐inducible proteins. Two promoters (PA and PB) were mapped upstream of the first gene. PA resembles promoters recognized by the vegetative RNA polymerase EσA. The other promoter (PB) was shown to be dependent on σB, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σB‐dependent manner. This is the first evidence that σB in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ32 in Escherichia coli, indicating a new function of σB‐dependent general stress proteins. PB deviated from the consensus sequence of σB promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σB‐dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σA‐like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σA‐like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress‐induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.
Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli
Authors: Palmer Tracy , Santini Claire‐Lise , Iobbi‐Nivol Chantal , Eaves Deborah J. , Boxer David H. , Giordano Gerard 
Published: October 2006

SummaryThe Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl2, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over‐expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.
An environmentally regulated pilus‐like appendage involved inCampylobacterpathogenesis
Authors: Doig Peter , Yao Ruijin , Burr Donald H. , Guerry Patricia , Trust Trevor J. 
Published: October 2006

SummaryExamination of strains ofCampylobacter jejuni, Campylobacter coli, andCampylobacter fetusby electron microscopy revealed that they produced peritrichous pilus‐like appendages when the bacteria were grown in the presence of bile salts. Various bile‐salt supplements were used and it was found that deoxycholate and chenodeoxycholic acid caused a significant enhancement of pilus production and resulted in a highly aggregative phenotype. Morphologically, the pili were between 4 and 7 nm in width and were greater than 1 μm in length. A gene, termedpspA, which encodes a predicted protein resembling protease IV ofEscherichia coli, was identified inC. jejunistrain 81–176. A site‐specific insertional mutation within this gene resulted in the loss of pilus synthesis as determined by electron microscopy. Insertions upstream and downstream of the gene had no effect on pilus production. The non‐piliated mutant of strain 81–176 showed no reduction in adherence to or invasion of INT 407 cellsin vitro. However, this mutant, while still possessing the ability to colonize ferrets, caused significantly reduced disease symptoms in this animal model.
Control of bacterial chemotaxis
Authors: Eisenbach Michael 
Published: October 2006

SummaryBacterial chemotaxis, which has been extensively studied for three decades, is the most prominent model system for signal transduction in bacteria. Chemotaxis is achieved by regulating the direction of flagellar rotation. The regulation is carried out by the chemotaxis protein, CheY. This protein is activated by a stimulus‐dependent phosphorylation mediated by an autophosphorylatable kinase (CheA) whose activity is controlled by chemoreceptors. Upon phosphorylation, CheY dissociates from its kinase, binds to the switch at the base of the flagellar motor, and changes the motor rotation from the default direction (counter‐clockwise) to clockwise. Phosphorylation may also be involved in terminating the response. Phosphorylated CheY binds to the phosphatase CheZ and modulates its oligomeric state and thereby its dephosphorylating activity. Thus CheY phosphorylation appears to be involved in controlling both the excitation and adaptation mechanisms of bacterial chemotaxis. Additional control sites might be involved in bacterial chemotaxis, e.g. lateral control at the receptor level, control at the motor level, or control by metabolites that link central metabolism with chemotaxis.
Sequence specificity for DNA binding by Escherichia coli SoxS and Rob proteins
Authors: Li Ziyi , Demple Bruce 
Published: October 2006

Summary SoxS is a transcriptional activator of oxidative stress genes in Escherichia coli. SoxS in vitro binds the promoters of soxRS‐regulated genes such as micF, zwf, nfo and sodA, forms multiple protein‐DNA complexes, and recruits RNA polymerase to the promoters. E. coli Rob protein, with an N‐terminus 55% identical to SoxS, was initially identified by its binding to the oriC replication origin, but Rob in vitro binds some of the same promoters as SoxS and in vivo activates some SoxS‐regulated genes. In this work we show that the multiple complexes with SoxS arise from the presence at least two independent binding sites in each of the micF and zwf promoters. SoxS and Rob each form only a single complex with a 20 bp DNA oligonucleotide corresponding to the region immediately upstream of the ‐35 element of the micF promoter. Methylation interference identified several conserved purine residues required for binding to micF and five other SoxS‐binding sites. Together with binding studies using mutated oligonucleotides and published DNase I footprinting data, this information was used to form a consensus for SoxS sequence specificity: AN2GCAYN7CWA (where N is any base, Y is a pyrimi‐dine, and W is A or T). The sequence requirements for Rob binding differed somewhat from those of SoxS. Using the SoxS‐binding consensus, several genes potentially regulated by soxRS were identified in an E. coli genomic database; some of these genes have functions that might contribute to cellular resistance to oxidative stress.
The leucine‐responsive regulatory protein (Lrp) acts as a specific repressor for σs‐dependent transcription of the Escherichia coli aidB gene
Authors: Landini Paolo , Hajec Laurel I. , Nguyen Lam H. , Burgess Richard R. , Volkert Michael R. 
Published: October 2006

Summary The product of the Escherichia coli aidB gene is homologous to human isovaleryl‐coenzyme A dehydrogenase (IVD), an enzyme involved in the breakdown of the amino acid leucine. The aidB gene is not expressed constitutively, but its transcription is induced via distinct mechanisms in response to: (i) exposure to alkylating agents; (ii) acetate at a slightly acidic pH; and (iii) anoxia. Induction by alkylating agents is mediated by the transcriptional activator Ada, in its methylated form (meAda); the other forms of induction are Ada independent and require σs, the alternative σs factor mainly expressed during the stationary phase of bacterial growth. In this report we show that, in the absence of any transcriptional factor, aidB is efficiently transcribed in vitro by the σs, but not by the σ70, form of RNA polymerase holoenzyme. In the presence of meAda, levels of transcription by both forms of RNA polymerase are significantly increased. However, σs ‐dependent transcription of aidB is inhibited both in vitro and in vivo by binding of the transcriptional regulator Lrp (leucine responsive protein) to the aidB promoter region (PaidB)‐ Lrp acts as a specific repressor for σs ‐dependent transcription of aidB. Leucine counteracts Lrp binding to PaidB as does binding to PaidB of meAda, which causes Lrp to dissociate from the promoter. The physiological significance of aidB transcription regulation is discussed.
The Ustilago maydis virally encoded KP1 killer toxin
Authors: Park Chung‐Mo , Banerjee Nanditta , Koltin Yigal , Bruenn Jeremy A. 
Published: October 2006

SummarySome strains of the plant‐pathogenic fungus Ustilago maydis secrete toxins (killer toxins) that are lethal to susceptible strains of the same fungus. There are three well‐characterized killer toxins in U. maydis–KP1, KP4, and KP6–which are secreted by the P1, P4, and P6 subtypes, respectively. These killer toxins are small polypeptides encoded by segments of an endogenous, persistent double‐stranded RNA (dsRNA) virus in each U. maydis subtype. In P4 and P6, the M2 dsRNA segment encodes the toxin. In this work, the KP1 killer toxin was purified for internal amino acid sequence analysis, and P1M2 was identified as the KP1 toxin‐encoding segment by sequence analysis of cDNA clones. The KP1 toxin is a monomer with a predicted molecular weight of 13.4kDa and does not have extensive sequence similarity with other viral anti‐fungal toxins. The P1M2 segment is different from the P4 and P6 toxin‐encoding dsRNA segments in that the 3’non‐coding region of its plus strand has no sequence homology to the 3’ends of the plus strands of P1M1, P4M2, or P6M2.
Role of nodl and nodj in lipo‐chitooligosaccharide secretion in Azorhizobium caulinodans and Escherichia coli
Authors: Fernández‐López Manuel , D'Haeze Wim , Mergaert Peter , Verplancke Christa , Prome Jean‐Claude , Van Montagu Marc , Holsters Marcelle 
Published: October 2006

SummaryLipo‐chitooligosaccharide (LCO) Nod factors are produced and secreted by rhizobia and trigger nodule development in leguminous host plants. The products of the bacterial nodlJ genes are related to transporters of capsular polysaccharides and were proposed to be involved in LCO transport. We have studied nodlJ of Azorhizobium caulinodans ORS571 by analysis of cell‐associated and secreted radioactively labelled Nod factors in wild‐type ORS571, a nodJ mutant and a complemented strain. Secretion was strongly reduced in the nodJ mutant, and restored to wild‐type levels after complementation. Constructs were made for expression of combinations of different nod genes in Escherichia coli DH5a. The strain DH5α(pUCNABCSU) synthesized LCOs, but they were only secreted when a plasmid containing both nodl and nodJ was supplied in trans, nodi or nodJ alone was not sufficient. In E. coli as well as in Azorhizobium, the nod/J‐encoded transporter showed a specificity for more hydrophilic LCOs.
A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus
Authors: Zheng Su , Haselkorn Robert 
Published: October 2006

Summary A mutant of Rhodobacter capsulatus was identified in which an operon encoding a binding‐protein‐dependent transporter was interrupted by Tn5 transposition. Cloning and sequence analysis of the wild‐type operon revealed a four‐gene cluster with similarities to genes encoding periplasmic binding proteins (BztA), integral membrane proteins (BztB and BztC), and ATP‐binding proteins (BztD). To assess the function of this putative binding‐protein‐dependent transport system, a mutant was constructed in which most of the bztABCD operon was deleted and replaced by an antibiotic‐resistance marker. The deletion mutant grew more slowly than the wild type in NH‐free medium supplemented by glutamate, glutamine, aspartate or asparagine; it was resistant to toxic analogues of Glu, Asp, and Asn at concentrations that inhibited growth of the wild type; and it was defective in the uptake of Glu, Gin, and Asp. A complementing plasmid containing the wild‐type copy of bztABCD was able to rescue all the mutant phenotypes. Taken together, these results indicate that the proteins encoded by bztABCD are active in the uptake of Glu, Gin, Asp, and Asn. In addition, competition experiments, in which the ability of each of the four amino acids to compete for the transport of one another was examined, demonstrated that all four substrates share at least one component of this transport system.
Random initiation of replication of plasmids P1 and F (oriS) when integrated into the Escherichia coli chromosome
Authors: Ellasson Åsa , Bernander Rolf , Nordström Kurt 
Published: October 2006

Summary We have constructed intP1 and intFs strains of Escherichia coli in which the basic replicons of either plasmid P1 or plasmid F (oriS) were integrated into an inactivated oriC, such that chromosome replication is controlled by the integrated plasmid replicon. In this study, we have further analysed these strains, and density‐shift experiments revealed that chromosome replication occurred randomly during the cell cycle. Flow‐cytometry analyses of exponentially growing populations supported this conclusion, and also showed that the DNA/mass ratio of the strains decreased with increasing growth rate. Flow cytometry of exponentially growing cultures treated with rifampicin demonstrated that initiation of replication was uncoordinated in cells containing multiple replication origins.
PNPase modulates RNase II expression in Escherichia coli: implications for mRNA decay and cell metabolism
Authors: Zilhão Rita , Cairrão Fátima , Régnier Philippe , Arraiano Cecília M. 
Published: October 2006

Summary PNPase and RNase II are the key regulatory exo‐nucleases controlling mRNA decay in Escherichia coli The rnb transcripts were found to proceed through the terminator and PNPase was found to be involved in the 3’to 5’degradation of rnb mRNA. Analysis of these longer 3’termini revealed that they are located in UA‐rich regions. Comparison of single and double mutants suggested that PNPase and RNase II could have different roles in the degradation of these unstructured regions. We have shown that RNase II levels can vary over a fivefold range in haploid cells and that its expression depends on PNPase levels. PNPase‐deficient strains were found to have a 2–2.5‐fold increase in RNase M activity, while PNPase‐overproducing strains reduced the rnb message and RNase II levels. Conversely, the amount of PNPase in the rnb deletion strain was approximately twofold higher than that in the wild‐type strain. These observations suggest that the two main exonu‐cleases are inter‐regulated through a fine tuning mechanism. We discuss the implications of these results with regard to mRNA degradation and cell metabolism.
YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signalling associated with phagocytosis
Authors: Andersson Kerstin , Carballeira Nivia , Magnusson Karl‐Eric , Persson Cathrine , Stendahl Olle , Wolf‐Watz Hans , Fällman Maria 
Published: October 2006

SummaryThe PTPase YopH of Yersinia is essential to the ability of these bacteria to block phagocytosis. Wild‐type Yersinia pseudotuberculosis, but not the yopH mutant strain, resisted phagocytosis by J774 cells. Ingestion of a yopH mutant was dependent on tyrosine kinase activity. Transcomplementation with wild‐type yopH restored the anti‐phagocytic effect, whereas introduction of the gene encoding the catalytically inactive yopHC403A was without effect. The PTPase inhibitor orthovanadate impaired the anti‐phagocytic effect of the wild‐type strain, further demonstrating the importance of bacteria‐derived PTPase activity for this event. The ability to resist phagocytosis indicates that the effect of the bacterium is immediately exerted when it becomes associated with the phagocyte. Within 30 s after the onset of infection, wild‐type Y. pseudotuberculosis caused a YopH‐dependent dephosphorylation of phosphotyrosine proteins in J774 cells. Furthermore, interaction of the cells with phagocytosable strains led to a rapid and transient increase in tyrosine phosphorylation of paxillin and some other proteins, an event dependent on the presence of the bacterial surface‐located protein invasin. Co‐infection with the phagocytosable strain and the wild‐type strain abolished the induction of tyrosine phosphorylation. Taken together, the present findings demonstrate an immediate YopH‐mediated dephosphorylation of macrophage phosphotyrosine proteins, suggesting that this PTPase acts by preventing early phagocytosis‐linked signalling in the phagocyte.
Countertranscript‐driven attenuation system of the pAMβ1 repE gene
Authors: Le Chatelier E. , Ehrlich S. D. , Jannière L. 
Published: October 2006

Summary The plasmid‐encoded RepE protein is absolutely essential and rate‐limiting for replication of the promiscuous plasmid pAMβ1 originating from Enterococcus faecalis. We previously showed that the rep gene is transcribed from a promoter that is negatively regulated (10‐fold reduction) by the CopF repressor. In this report, we show that this transcription is decreased a further 10‐times by a countertranscript‐driven transcriptional attenuation system. Extensive mutagenesis revealed that this system operates by a mechanism similar to that previously described for the unrelated repC gene of plasmid pT181.