bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular rearrangement of lactose plasmid DNA associated with high‐frequency transfer and cell aggregation in Lactococcus Iactis 712
Authors: Gasson Michael J. , Swindell Simon , Maeda Sumio , Dodd Helen M. 
Published: October 2006

SummaryHigh‐frequency conjugation of the lactose plasmid pLP712 is associated with a constitutive cell aggregation phenotype and is facilitated by cointegration with a sex factor. Analysis of 23 independently derived enlarged lactose plasmids revealed that the sex factor DNA present in cointegrates varied in size. This suggested that more than simple cointegration with a sex factor plasmid was involved. Further analysis led to the discovery of a chromosomally located sex factor that could excise and be lost or exist as labile plasmid DNA. Cointegration with this sex factor was shown to be promoted by transposition of a copy of ISSI present on the lactose plasmid, and models are presented to account for the complex and variable structures of the resulting enlarged lactose plasmids.
Ribosomal association of the yeast SAL4 (SUP45) gene product: implications for its role in translation fidelity and termination
Authors: Stansfield Ian , Grant Christopher M. , Akhmaloka  , Tuite Mick F. 
Published: October 2006

SummaryThe SAL4 gene of the yeast Saccharomyces cerevisiae encodes a novel translation factor (Sal4p) involved in maintaining translational fidelity. Using a polyclonal antibody raised against a Sal4p‐β‐galac‐tosidase fusion protein, Sal4p was shown to be almost exclusively associated with the ribosomal fraction. Even when the ribosomes were treated with 0.8 M KCl, only low levels of Sal4p were detected in the post‐ribosomal supernatant, suggesting a very strong affinity between Sal4p and the ribosome. Analysis of the distribution of Sal4p in the ribosomal population revealed that it was principally associated with 40S subunits, monosomes and polysomes. Incubation in high salt concentrations (0.8 M KCl) suggested that the affinity of Sal4p for the 40S subunit was lower than that for monosomes or polysomes. The Sal4p:ribosome association was only maintained when ribosomes were prepared in the presence of the translation elongation inhibitor cycloheximide; in uninhibited cells much lower levels of Sal4p were detectable in the ‘run‐off’ polysomes. In view of these data, and given the stoichiometry of Sal4p to individual ribosomal proteins (estimated at less than 1:20), we suggest that Sal4p plays an ancillary role in translation termination.
Tandem DctD‐binding sites of the Rhizobium meliloti dctA upstream activating sequence are essential for optimal function despite a 50‐ to 100‐fold difference in affinity for DctD
Authors: Ledebur H. , Nixon B. T. 
Published: October 2006

SummaryThe Rhizobium melilot genes dctB and dctD positively regulate the expression of dctA, which encodes a C4‐dicarboxylate transport protein. Here we characterize an element (UAS) located upstream of dctA that has tandem binding sites for the dctD gene product (DctD). At relatively low concentrations of active DctD, the element activated dctA transcription, but at relatively high concentrations of DctD it was inhibitory. The UAS failed to function when placed further upstream of dctA. Both DctD‐binding sites were required for optimal UAS function, despite a 50‐ to 100‐fold difference in binding affinities. Moving the promoter distal binding site 5 bp further upstream was functionally equivalent to its deletion. Based on these data, we hypothesize that the α54 ‐dependent activator DctD binds co‐operatively to the R. meliloti dctA UAS, and that occupancy of both sites is required for maximal activation of dctA.
The phosphorylation site of the Kdp‐ATPase of Escherichia coli: site‐directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit
Authors: Puppe Wolfram , Siebers Annette , Altendorf Karlheinz 
Published: October 2006

SummaryThe potassium‐translocating Kdp‐ATPase of Escherichia coli shares common functional properties with eukaryotic P‐type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp 307 in KdpB by Glu, Asn, Gin, Tyr, His, Ala or Ser by site‐directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K’ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp‐307 in KdpB is the phosphorylation site of the Kdp‐ATPase. In contrast, replacement of the close but non‐conserved Asp‐300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the Km for K+ of the ATPase activity has been increased 30‐fold compared with the wild‐type enzyme.
Functional analysis of the gas vesicle gene cluster of the halophilic archaeon Haloferax mediterranei defines the vac‐region boundary and suggests a regulatory role for the gvpD gene or its product
Authors: Englert Christoph , Wanner Gerhard , Pfeifer Felicitas 
Published: October 2006

SummaryA series of deletions introduced into the gvp gene cluster of Haloferax mediterranel, comprising 14 genes involved in gas vesicle synthesis (mc‐vac‐region), was investigated by transformation experiments. Gas vesicle production and the expression of the gvpA gene encoding the major gas vesicle protein, GvpA, was monitored in each Haloferax volcanii transformant. Whereas transformants containing the entire mc‐vac‐region produced gas vesicies (Vac+), various deletions in the region 5′ to gvpA (encompassing gvpD‐gvpM) or 3′ to gvpA (containing gvpC, gvpN and gvpO) revealed Vac− transformants. All these transformants expressed gvpA and contained the 8 kDa GvpA protein as shown by Western analysis. However, transformants containing the gvpA gene by itself indicated a lower level of GvpA than observed with each of the other transformants. None of these transformants containing deletion constructs assembled the GvpA protein into gas vesicles. In contrast, transformants containing a construct carrying a 918 bp deletion internal to gvpD exhibited a tremendous gas vesicle overproduction, suggesting a regulatory role for the gvpD gene or Its product. This is the first assignment of a functional role for one of the 13 halobacterial gvp genes found in addition to gvpA that are involved in the synthesis of this unique structure.
A reorganized Candida albicans DNA sequence promoting homologous non‐integrative genetic transformation
Authors: Herreros E. , Garcia‐Sáez M. I. , Nombela C. , Sánchez M. 
Published: October 2006

SummaryIn order to develop plasmids adequate for non‐integrative genetic transformation of Candida albicans, a DNA fragment of 15.3 kb was cloned from this organism on the basis of its capacity to convert the integrative Saccharomyces cerevisiae vector Ylp5 into a non‐integrative one. Southern hybridization analysis, carried out with a labelled DNA probe of 3.6 kb derived from the cloned fragment, showed that it consisted of C. albicans DNA, the hybridization pattern indicating that the corresponding sequences were homologous to several chromosomal regions. The size of the C. albicans DNA promoting autonomous replication In S. cerevisiae was substantially reduced by subcloning. A 5.1 kb subfragment, defined by BamHI and SalI restriction sites, retained autonomous replication sequences (ARS) functional in the heterologous S. cerevisiae system and in C. albicans, when inserted in plasmid constructions that carried a S. cerevisiae trichodermin‐resistance gene (tcm1) as selection marker. C. albicans transformants were both of the integrative and the non‐integrative type and the plasmids recovered from the latter very often carried a reorganized ARS, indicating that recombination of the inserted ARS DNA had occurred in the homologous host. Successive reorganizations of the ARS insert in C. albicans eventually led to a more stable and much smaller fragment of 687 bp that was subsequently recovered unchanged from transformants. Sequence analysis of the 687 bp fragment revealed four 11‐base blocks, rich in A+T, that carried the essential consensus sequence considered relevant for yeast ARS elements in addition to other features also described as characteristic of yeast replication origins.
Broad‐host‐range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O‐acetylated or sulphated
Authors: Price N. P. J. , RelicA B. , Talmont F. , Lewin A. , Promé D. , Pueppke S. G. , Maillet F. , Dénarié J. , Promé J.‐C. , Broughton W. J. 
Published: October 2006

Summary Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non‐legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod‐factors which are N‐acylated chitin pentamers carrying a variety of substituents. The terminal non‐reducing glucosamine is N‐acylated with vaccenic or palmitic acids, is N‐methylated, and carries varying numbers of carbamoyl groups. The reducing N‐acetyl‐glucosamine residue is substituted on position 6 with 2‐O‐methyl‐L‐fucose which may be acetylated or sulphated or non‐substituted. All three internal residues are N‐acetylated. At pico‐ to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non‐sulphated lipo‐oligosaccharide signals.
Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant
Authors: Homchampa P. , Strugnell R. A. , Adler B. 
Published: October 2006

SummaryThe aroA gene from Pasteurella multocida was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The nucleotide sequence of the P. multocida aroA gene indicated an open reading frame encoding a protein of 441 amino acids, which showed a high degree of homology with the amino acid sequences of various other bacterial AroA proteins. The cloned P. multocida aroA gene was inactivated by insertion of a kanamycin‐resistance gene and reintroduced by allelic exchange into the chromosome of P. multocida using the suicide vector pJM703.1. The P. multocida aroA mutant was highly attenuated in a mouse model. Mice immunized intraperitoneally with two doses of live P. multocida aroA mutant were completely protected against a lethal parental strain challenge.
Effect of lac repressor oligomerization on regulatory outcome
Authors: Chakerian Artemis E. , Matthews Kathleen S. 
Published: October 2006

SummaryRegulatory outcome in a bacterial operon depends on the interactions of all the components which influence mRNA production. Levels of mRNA can be altered profoundly by both negative and positive regulatory elements which modulate initiation of transcription. The occupancy of regulatory sites on the DNA by repressors and activators is determined not only by the affinity of these proteins for their cognate site(s) but also by the oligomeric state of the regulatory protein. The lac operon in Escherichia coli provides an excellent prototypic example of the influence of protein assembly on the transcriptional status of the associated structural genes. DNA loop formation is essential for maximal repression of the lac operon and is contingent upon the presence of multiple operator sites in the DNA and the ability of the repressor to self‐associate to form a bidentate tetramer. The stability of this looped complex is enhanced significantly by DNA supercoiling. Tetramer assembly from dimers apparently occurs via interactions of a‘leucine zipper’motif in the C‐terminal domain of the protein, and the tetramer is essential to formation of looped complexes. Furthermore, analysis of the DNA‐binding characteristics of dimeric mutants has established that the monomer‐dimer association and dimer‐DNA binding (monomer does not bind to DNA) are coupled equilibria. Thus, dimer assembly is essential for generating a DNA‐binding unit, and tetramer assembly is required for formation of the stable looped DNA structure that maximally represses mRNA synthesis. Protein‐protein interactions therefore play a pivotal role in the regulatory activities of the lac repressor and must be considered when analysing the activities of any oligomeric DNA‐binding protein.
Escherichia coli HIyT protein, a transcriptional activator of haemolysin synthesis and secretion, is encoded by the rfaH (sfrB) locus required for expression of sex factor and lipopolysaccharide genes
Authors: Bailey Marc J. A. , Koronakis Vassilis , Schmoll Thomas , Hughes Colin 
Published: October 2006

SummarySynthesis and secretion of the 110kDa haemolysin toxin of Escherichia coli and other pathogenic Gram‐negative bacteria are governed by the four genes of the hly operon. We have identified, by transposon mutagenesis, an E. coli cellular locus, hlyT, required for the synthesis and secretion of haemolysin encoded in trans by intact hly operons carrying the hly upstream regulatory region. Mutation of the hlyT locus specifically reduced the level of hlyA structural gene transcript 20‐100‐fold and thus markedly lowered both intracellular and extracellular levels of the HlyA protein. Genetic and structural analysis of the hlyT locus mapped it at co‐ordinate 3680 kbp (minute 87) on the chromosome adjacent to the fadBA operon, and identified it specifically as the rfaH (sfrB) locus which is required for transcription of the genes encoding synthesis of the sex pilus and also the lipopolysaccharide core for attachment of the O‐antigen of E. coli and Salmonella. Expression of the hly operon in the E. coli hlyT mutant was restored in trans by both the hlyT and rfaH genes, suggesting that the rfaH gene is an important activator of regulon structures that are central to the fertility and virulence of these pathogenic bacteria. DNA sequencing of the hlyT locus identifies the HlyT/RfaH transcriptional activator as a protein of 162 amino acids (Mr 18325) which shows no identity to characterized transcription factors.
A bifunctional xylanase encoded by the xynA gene of the rumen cellulolytic bacterium Ruminococcus flavefaciens 17 comprises two dissimilar domains linked by an asparagine/glutamine‐rich sequence
Authors: Zhang Jun‐Xian , Flint Harry J. 
Published: October 2006

SummaryThe nucleotide sequence of the xynA gene of Ruminococcus flavefaciens 17 was determined and found to consist of a 2862 bp open reading frame beginning with a TTG start codon. The predicted product, XYLA, consisted of distinct amino‐terminal (A) and carboxy terminal (C) domains (248 amino acids, including a putative signal sequence, and 332 amino acids, respectively) linked by a repetitive sequence (B, 374 amino acids) extraordinarily rich in asparagine (45%) and glutamine (26%) residues. Domains A and C were shown to be capable of expressing xylanase activity independently of each other when suitably truncated derivatives of the xynA coding region were expressed as lacZ fusions. The activities associated with the two domains were shown to differ with respect to the average size of hydrolysis products formed from oat‐spelt xylan, with domain C releasing relatively more xylose and domain A more xylo‐oligosaccharides. The amino acid sequence of domain A of XYLA closely resembled that of the Bacillus pumilus xynA enzyme (45% identical residues). On the other hand domain C showed significant similarity (33% to 40% identical residues) to a different group of bacterial xylanases and exoglucanases exemplified by the Caldocellum saccharolyticum xynA and celB products. The xynA product is, therefore, a bifunctional enzyme having two dissimilar catalytic domains capable of acting on xylan.
Analysis of the promoters and upstream sequences of nifA1 aud nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN
Authors: Foster‐Hartnett Dawn , Kranz Robert G. 
Published: October 2006

SummaryTranscription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. R capsulatus nifA1 and nifA2 encode identical NIFA proteins that activate transcription of nifHDK and other nif genes. In this study, we report that nifA1‐lacZ and nifA2‐lacZ fusions are repressed in the presence of NH3 and activated to similar levels under nitrogen‐deficient conditions. This nitrogen‐controlled activation was dependent on R. capsulatus ntrC (which encodes a transcriptional activator) but not rpoN (which encodes an RNA polymerase sigma factor). We have used primer extension analyses of nifA1, nifA2 and nifH and deletion analyses of nifA1 and nifA2 upstream regions to define likely promoters and cis upstream activation sequences required for nitrogen control of these genes. Primer extension mapping confirmed that ntrC but not rpoN is required for nifA1 and nifA2 activation, and that nifA1 and nifA2 do not possess typical RPON‐activated promoters.
Mobilization protein‐DNA binding and divergent transcription at the transfer origin of the Thiobacillus ferrooxidans pTF1 plasmid
Authors: Drolet M. , Lau P. C. K. 
Published: October 2006

SummaryThe possible interaction of the trans‐acting mobilization proteins, MobL and MobS, at the cognate origin of transfer (oriT) region of the Thiobacillus ferrooxidans plasmid pTF1 has been investigated. In gel retardation assays with crude protein extracts from overproducing strains, a truncated MobL (c. 28 kDa) as well as its native protein (42 kDa), but not the 11 kDa MobS protein, were found to bind specifically to a 42‐mer oligonucleotide which represents the transferred DNA strand of the minimal oriT fragment of pTF1. In vivo, the binding of MobL was studied by monitoring catechol 2,3‐dioxygenase (xylE) activities driven by promoters of the divergently transcribed mobL and mobS genes. The mob promoter sequences were found to resemble the Escherichia coliσ70‐dependent consensus promoter elements. The‘‐10’recognition sequences of mobL and one of the two mobS promoters overlap except for one base and they are positioned within the putative‘hairpin’structure in the minimal oriT sequence. In accordance with the twin supercoil‐domain model of Liu and Wang (1987) which suggests that transcription can generate local variations in DNA superhelicity, we propose a possible physiological role of DNA supercoiling in the transfer origin with reference to divergent transcription of mobL and mobS genes.
The Fis protein: it's not just for DNA inversion anymore
Authors: Finkel S. E. , Johnson R. C. 
Published: October 2006

SummaryHigher‐order nucleoprotein complexes are associated with many biological processes. In bacteria the formation of these macromolecular structures for DNA recombination, replication, and transcription often requires not only the participation of specific enzymes and co‐factors, but also a class of DNA‐binding proteins collectively known as‘nucleoid‐associated’or‘histone‐like’proteins. Examples of this class of proteins are HU, Integration Host Factor, H‐NS, and Fis. Fis was originally identified as the factor for inversion stimulation of the homologous Hin and Gin site‐specific DNA recombinases of Salmonella and phage Mu, respectively. This small, basic, DNA‐bending protein has recently been shown to function in many other reactions including phage λ site‐specific recombination, transcriptional activation of rRNA and tRNA operons, repression of its own synthesis, and oriC‐directed DNA replication. Cellular concentrations of Fis vary tremendously under different growth conditions which may have important regulatory implications for the physiological role of Fis in these different reactions. The X‐ray crystal structure of Fis has been determined and insights into its mode of DNA binding and mechanisms of action in these disparate systems are being made.
Role of the RNA polymerase α subunit in transcription activation
Authors: Ishihama Akira 
Published: October 2006

SummaryThe N‐terminal two‐thirds of the α subunit of Escherichia coli RNA polymerase plays an essential role in the initiation of subunit assembly, by gathering two large subunits, β and β', together into a coreenzyme complex. One group of RNA polymerase mutants deficient in response to transcription activation carries mutations in the C‐terminal region of the α subunit, indicating that the C‐terminal region of the a subunit is involved in protein‐protein contact in positive control of transcription. A set of activators (class I transcription factors) which make contact with this contact site I region on RNA polymerase a subunit bind in most cases to DNA upstream of the promoter ‐35 signal. Genetic fine mapping indicates that a cluster of subsites exists in the contact site I region, each interacting with a set of the class I factors and each consisting of a structure formed by only 5‐10 amino acid residues.
Characterization of the micro‐environment of Salmonella typhimurium–containing vacuoles within MDCK epithelial cells
Authors: Portillo F. Garcia–del , Foster John W. , Maguire Michael E. , Finlay B. Brett 
Published: October 2006

Summary Salmonella typhimurium has the capacity to enter into and multiply within epithelial cells. During the entire intracellular stage, bacteria are enclosed within a vacuole. To characterize the micro–environment of the bacteria–containing vacuoles, we have used a new method to measure the expression levels of several S. typhimurium genes in intracellular bacteria within Madin–Darby canine kidney (MDCK) epithelial cells. Our study was based on the determination of ß–galactosidase activity derived from lacZ transcriptional fusions using the highly sensitive substrate fluorescein–di–ß–D–galactoside (FDG). Expression of the iroA and mgtB genes (induced by Fe2+ and Mg2+ limitation respectively), and cadA (induced by pH 6.0 in the presence of lysine, with enhanced expression under anaerobiosis) were characterized at different post–infection times. High intracellular expression levels were detected for the iroA and mgtB genes, suggesting that the concentrations of free Fe2+ and Mg2+ in the vacuole may be low. cadA actitvity was detected only at early post–infection times (4 h), suggesting that the vacuole may have a mild–acidic pH, and oxygen and lysine present at this time. Globally, the results reported indicate that the use of a highly sensitive ß–galactosidase substrate can provide information about the micro–enviroment within which an intracellular pathogen, such as S. typhimurium, resides.