bluuebunny/crossref_metadata_2025_split huggingface dataset:
A novel family of potentially mobile DNA elements encoding site‐specific gene‐integration functions: integrons
Authors: Stokes H. W. , Hall R. M. 
Published: October 2006

SummaryA family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3′ to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3′ and 5′ conserved regions of the DNA element have been determined. A 59‐base sequence element, found at the junctions of inserted DNA sequences and the conserved 3′ segment, is also present at this location in the R46 sequence. A copy of one half of this 59‐base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site‐specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5′ conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site‐specific integration functions (the Integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.
Functional analyses of Proteus mirabilis wild‐type and mutant RecBCD enzymes in Escherichia coli reveal a new mutant phenotype
Authors: Weichenhan D. , Wackernagel W. 
Published: October 2006

SummaryCloned into Escherichia coli the recB, recC and recD genes of Proteus mirabilis produce a recBCD enzyme (exoV) functional in recombination and DNA repair. The direction of transcription of recB, recC and recD, the sizes of the enzyme subunits, and their composition in the active enzyme are similar to that observed for the E. coli enzyme. In lambda crosses, the P. mirabilis enzyme has only about 40% of the Chi activity of the E. coli enzyme. The recBCD genes were also cloned from an exoV mutant of P. mirabilis which is u.v.‐sensitive and partly deficient in exoV. The defect was attributed to the recB gene by complementation studies. In a recBCD deletion strain of E. coli, the enzyme from the mutant produced 40% of conjugational recombinants and had retained about 25% of Chi activity. However, it did not restore normal DNA repair, cell viability or recombination in lambda crosses and P1 transduction. The new mutant phenotype is discussed in the light of the assumption that prokaryotic recBCD enzymes can promote recombination in a Chi‐dependent and a Chi‐independent manner.
A simple method for maximizing the yields of membrane and exported proteins expressed in Escherichia coli
Authors: Broome‐Smith J. K. , Bowler L. D. , Spratt B. G. 
Published: October 2006

SummaryThe feasibility of using a β‐lactamase fusion approach for maximizing the levels of periplasmic or membrane‐bound proteins expressed in Escherichia coli was investigated. The coding region for mature TEM β‐lactamase was fused after the signal peptide and amino‐terminal portion of the coding region of a weakly expressed periplasmic protein, PBP3*. The resultant plasmid was mutagenized and transformants expressing increased levels of ampicillin resistance were selected. The PBP3*gene of the unmutagenized I β‐lactamase fusion plasmid, and of two mutant derivatives encoding increased ampicillin resistance, were then reassembled and the latter constructs were found to express increased levels of PBP3*. The applications of a β‐lactamase fusion approach in monitoring and optimizing levels of extracytoplasmic gene products expressed in E. coli are considered.
Molecular cloning of the outJ gene involved in pectate lyase secretion by Erwinia chrysanthemi
Authors: Ji J. , Hugouvieux‐Cotte‐Pattat N. , Robert‐Baudouy J. 
Published: October 2006

SummaryThe phytopathogenic enterobacterium Erwinia chrysanthemi secretes a number of enzymes involved in plant‐tissue degradation, notably the five isoenzymes of pectate lyase. We have cloned a region involved in pectate lyase and cellulase secretion by complementation of non‐secretory outJ mutants of E. chrysanthemi strain 3937 using the RP4::miniMu plasmid pULB110. The cloned region maps near the ade‐22 marker on the E. chrysanthemi 3937 chromosome. An R‐prime containing a chromosomal DNA insert of about 30 kb was first obtained; subcloning into pBR325 permitted the isolation of a 4 kb Cial/Sspl fragment able to complement outJ mutations in E. chrysanthemi. The isolation of phoA fusions in this fragment allowed us to determine the direction of transcription of the encoding region, which extends over about 2.5 kb, and demonstrate that this region encodes exported protein(s). When the TnphoA insertions were transferred back into E. chrysanthemi chromosome, the recombined strains no longer secreted pectate lyases or cellulases. Identification of the products encoded by the Cial/Sspl fragment demonstrated that oufJ encodes an 83 kD polypeptide which is processed to an 81 kD polypeptide by cleavage of a signal sequence. The cloned DNA fragment did not endow Escherichia coli with the ability to secrete pectate lyases.
Molecular analysis of linear plasmid‐encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi
Authors: Bergström S. , Bundoc V. G. , Barbour A. G. 
Published: October 2006

SummaryThe ospA and ospB genes encode the major outer membrane proteins of the Lyme disease spirochaete Borrelia burgdorferi. The deduced translation products from the ospA and ospB genes were: (OspA) 273 amino acids long with a molecular weight of 29334, and (OspB) 296 amino acids long with a molecular weight of 31739. The two Osp proteins showed a great degree of sequence similarity indicating a recent evolutionary event. Molecular analysis and sequence comparison of OspA and OspB with other proteins revealed a sequence similarity to the signal peptides of prokaryotic lipoproteins. These are the first sequences from Borrelia and provide interesting data on the evolutionary relationship between spirochaetes and other species as well as providing potential for spirochaete diagnostics and vaccines.
Expanded linkage map of Erwinia chrysanthemi strain 3937
Authors: Hugouvieux‐Cotte‐Pattat N. , Reverchon S. , Robert‐Baudouy J. 
Published: October 2006

SummaryIn this paper we describe the chromosomal location of various loci in Erwinia chrysanthemi strain 3937. Auxotrophic markers were obtained by chemical mutagenesis, antibiotic resistances were isolated spontaneously and mutations in sugar utilization were obtained by means of Mu insertions. These markers were located on the genetic linkage map of strain 3937 by using a conjugative system mediated by RP4::mini‐Mu plasmids which permitted transfer of genetic material from any point of origin. The location of these markers was compared to that of previously located mutations. Many genes involved in pectinolysis were also located on the E. chrysanthemi 3937 map. These results permitted us to present a new genetic map containing 61 markers distributed over 34 widely scattered loci on the chromosome. Some pairs of markers giving high cotransfer frequencies were tested for cotransduction mediated by the generalized transducing phage phi‐EC2; nine cotransducing pairs were found. It appears that the chromosomal locations of many of these loci are quite different to those of the well‐known enterobacterium Escherichia coli but seem similar to those described for other E. chrysanthemi strains.
Molecular characterization of a surface protein antigen gene from serotype c Streptococcus mutans, implicated in dental caries
Authors: Okahashi N. , Sasakawa C. , Yoshikawa M. , Hamada S. , Koga T. 
Published: October 2006

SummaryThe complete nucleotide sequence of the gene for a cell‐surface protein antigen (PAc) of Streptococcus mutans MT8148 (serotype c) was determined. The pac gene consisted of 4695 bp and coded for a 170 773 D protein. The pac gene product contained a putative 38 amino acid signal peptide, resulting in a 166817D mature protein. A potential promoter sequence and a putative Shine‐Dalgarno sequence preceded the open reading frame. Two internal repeating amino acid sequences were present in the PAc. One repeating region located in the N‐terminal region was rich in alanine, and the other located in the central region was rich in proline. Southern blot analysis under the less stringent condition (allowing up to 35% base mismatch) revealed that the probe covering the prolinerich region hybridized to DNA preparations from strains of Streptococcus cricetus, Streptococcus sobrinus and Streptococcus downei as well as Streptococcus mutans.
Topology and function of the integral membrane protein conferring immunity to colicin A
Authors: Geli V. , Baty D. , Pattus F. , Lazdunski C. 
Published: October 2006

SummaryThe topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore‐forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N‐and C‐terminal regions were found to be directed towards the cytoplasm. Oligonucleotide‐directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.
Construction of chimaeric promoter regions by exchange of the upstream regulatory sequences from fdhF and nif genes
Authors: Birkmann A. , Hennecke H. , Bock A. 
Published: October 2006

SummaryHybrid 5′ regulatory regions were constructed in which the upstream activator sequence (UAS) and promoter of various nif genes were exchanged with the upstream regulatory sequence (URS) of the fdhF gene from Escherichia coli. They were analysed for their regulatory response under different growth conditions with the aid of fdhF′‐′lacZ or nif′‐′lacZ fusions. Placement of the UAS from the Bradyrhizobium japonicum nifH gene in front of the spacer (DNA region between URS and promoter) plus promoter from fdhF renders fdhF expression activatable by the Klebsiella pneumoniae NIFA protein, both under aerobic and anaerobic conditions. This excludes the possibility that the spacer of the fdhF 5’flanking region contains a site recognized by a putative oxygen‐ or nitrate‐responsive repressor. There was also considerable activation by NIFA of fdhF expression in a construct lacking the nifH UAS but containing the fdhF spacer plus promoter. Further experimental evidence suggests that this reflects a direct interaction between NIFA and RNA polymerase at the nfrA‐dependent promoter. A second set of hybrid constructs in which the URS from fdhF (E. coli) was placed In front of the nifD spacer plus promoter from B. Japonicum or in front of the K. pneumoniae nifH, nifU, nifB spacers and promoters, delivered inactive constructs in the case of the nifD, nifU and nifB genes. However, a nifH′‐′lacZ fusion preceded by its own spacer and promoter plus the foreign fdhF URS displayed all the regulatory characteristics of fdhF expression, i.e. anaerobic induction with formate and repression by oxygen and nitrate. Although it is not known why only one out of the four nif promoters could be activated by the fdhF URS, this result nevertheless demonstrates that the various regulatory stimuli affecting expression of fdhF in E. coli have their target at the upstream regulatory sequence.
The Azorhizobium caulinodans nitrogen‐fixation regulatory gene, nifA, is controlled by the cellular nitrogen and oxygen status
Authors: Ratet P. , Pawlowski K. , Schell J. , de Bruijn F. J. 
Published: October 2006

SummaryThe nucleotrde sequence of the Azorhizobium caulinodans ORS571 nlfA locus was determined and the deduced NifA amino acid sequence compared with that of NifA from other nitrogen‐fixing species. Highly conserved domains, including helix‐turn‐helix and ATP‐binding motifs, and specific conserved residues, such as a cluster of cysteines, were identified. The nifA 5’upstream region was found to contain DNA sequence motifs highly homologous to promoter elements involved in nifAlntr‐mediated control and a consensus element found in the 5’upstream region of the Bradyrhizobium japonicum 5‐aminolevulinic acid synthase (hemA) gene and of Escherichia coli genes activated during anaerobiosis via the fnr (fumarate nitrate reduction) control system. A nifA‐lac fusion was constructed using miniMu‐lac and its activity measured in different genetic backgrounds and under various physiological conditions (in culture and in planta.) NifA expression was found to be negatively autoregulated, repressed by rich nitrogen sources and high oxygen concentrations, and controlled (partially) by the ntrC gene, both in culture and in planta. DNA supercoiling was also implicated in nifA regulation, since DNA gyrase inhibitors severely repressed nifA‐lac expression.
Functional domains of bacteriophage Mu transposase: properties of C‐terminal deletions
Authors: Bétermier M. , Alazard R. , Lefrère V. , Chandler M. 
Published: October 2006

SummaryWe have generated a series of 3’deletions of a cloned copy of the bacteriophage Mu transposase (A) gene. The corresponding truncated proteins, expressed under the control of the λ Pl promoter, were analysed in vivo for their capacity to complement a superinfecting Mu4am phage, both for lytic growth and lysogeny, and for their effect on growth of wild‐type Mu following infection or induction of a lysogen. Using crude cell extracts, we have also examined binding properties of these proteins to the ends of Mu. The results allow us to further define regions of the protein important in replicative transposition, establishment of lysogeny and DNA binding.
Characterization of the virA virulence gene of the nopaline plasmid, pTiC58, of Agrobacterium tumefaciens
Authors: Morel P. , Powell B. S. , Rogowsky P. M. , Kado C. I. 
Published: October 2006

SummaryWe have determined the complete nucleotide sequence of a 4.8 kilobase fragment encompassing the virA locus of the nopaline‐type plasmid, pTiC58, of Agrobacterium tumefaciens. virA is composed of a single open reading frame of 2499 nucleotides, capable of encoding a protein of 91.3 kiloDaltons. A trpE::virA gene fusion was used to confirm the reading frame of virA. High nucleotide and amino acid sequence homologies were observed between pTiC58 virA and the virA sequences of three octopine‐type plasmids. Strong homologies in amino acid sequence were observed between pTiC58 VirA and seven bacterial proteins which control various regulons. Two hydrophobic domains within VirA are also consistent with a model in which VirA acts as a membrane‐bound sensor of plant signal molecules.
The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins
Authors: Barlow A. K. , Heckels J. E. , Clarke I. N. 
Published: October 2006

SummaryThe class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.
Phage‐conversion of cytotoxin production in Pseudomonas aeruginosa
Authors: Hayashi T. , Baba T. , Matsumoto H. , Terawaki Y. 
Published: October 2006

SummaryWe isolated a temperate phage which carried the cytotoxin gene (ctx) from a cytotoxin (CTX)‐producing Pseudomonas aeruginosa strain, PA158. The phage, φCTX, had a head with a hexagonal outline and a contractile tail with tail fibres. The phage genome was a linear double‐stranded 35.5kb DNA with single‐stranded cohesive ends (cos). The attP, cos and ctx genes were all located very close to one another within a 2.3kb segment on the phage genome in the order given (in the circular form). φCTX converted CTX non‐producing P. aeruginosa strains into CTX producers. A single copy of φCTX DNA was integrated at the same site on the host chromosome (attB) in every lysogen, including PA15B. However, the amount of CTX produced in these lysogens varied from strain to strain and was less than that in PA158.
The variable antigens Vmp7 and Vmp21 of the relapsing fever bacterium Borrelia hermsii are structurally analogous to the VSG proteins of the African trypanosome
Authors: Burman N. , Bergström S. , Restrepo B. I. , Barbour A. G. 
Published: October 2006

SummaryThe relapsing fever agent Borrelia hermsii avoids the host's immune response by the strategy of multiphasic antigenic variation. A given Borrelia cell can express one of a number of alleles for polymorphic outer‐membrane proteins, known as Vmp proteins. The genes for the variant‐specific Vmp proteins of serotypes 7 and 21 of B. hermsii strain HS1 were sequenced. The genes, which were designated vmp7 and vmp21, were obtained from populations of borreliae before and after a switch in serotypes from 7 to 21. The analysis showed that vmp7 and vmp21 are 77% identical in terms of their coding sequence. The deduced translation products of vmp7 and vmp21 are polypeptides of 369 (37.2kD) and 364 amino acids (37.1kD), respectively. Vmp7 and Vmp21 have sequence features of prokaryotic lipoproteins and are processed as such during expression in E. coli. The secondary structure predictions of the Vmp proteins reveals analogous structures to the VSG proteins of the African trypanosome.
A family of dispersed repeats in Mycobacterium leprae
Authors: Woods S. A. , Cole S. T. 
Published: October 2006

SummaryThe genome of the causative agent of leprosy, Mycobacterium leprae, contains at least 28 copies of a dispersed repetitive sequence, RLEP. From nucleotide sequence analysis it was clear that the RLEP element consists of a 545 bp central domain flanked by a 100bp left‐end and a 44bp right‐end, sometimes associated with a 47bp extension. The presence of the left and right ends is variable and this allowed three different RLEP configurations to be defined. When the polymerase chain reaction was used to study variation of the central region at least twelve different classes were detected, suggesting that no two RLEP sequences may be identical. Furthermore, they have few features in common with classical bacterial insertion sequences.
Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence?
Authors: Higgins C. F. , Hinton J. C. D. , Hulton C. S. J. , Owen‐Hughes T. , Pavitt G. D. , Seirafi A. 
Published: October 2006

SummaryThere has been a recent revival of interest in one of the most abundant Escherichia coli proteins, H1 (also called H‐NS). This protein was first identified many years ago as a major component of the bacterial nucleoid, and has been characterized biochemically by several groups. However, no clear function for the protein emerged from these studies. Our thinking has been transformed by recent findings which complement the biochemistry with genetic data. Several mutations, selected over many years by virtue of their diverse effects on gene expression, have turned out to be allelic and to fall within the structural gene for H1. Bringing together the genetics and the biochemistry has demonstrated that the whole is worth more than the sum of the parts! These findings have far‐reaching implications for the mechanisms by which gene expression is regulated and also, perhaps, for the control of bacterial virulence.
Bacillus subtilis levansucrase: amino acid substitutions at one site affect secretion efficiency and refolding kinetics mediated by metals
Authors: Petit‐Glatron M. F. , Monteil I. , Benyahia F. , Chambert R. 
Published: October 2006

SummaryStudies of the equilibrium between native and denatured forms of wild‐type levansucrase showed that the denatured form was predominant at 37°C and pH 7 in the absence of free metal. The shift to the native form was promoted by metal ions such as Fe3+ or Ca2+. This metal‐dependent refolding process was not observed in levansucrase variants bearing the amino acid substitution Gly‐366→Asp or Gly‐366→Val. These variants were only slightly secreted by Bacillus subtilis although their signal sequences were normally cleaved and their exocellular forms stable. In contrast, the Gly‐366→Ser variant was secreted at near‐normal levels and shared a part of the in vitro refolding properties of the wild‐type protein. These differential properties might be related to the ability of the altered region to form a β‐turn structure.We discuss the possible role of metal ions in the coupling of protein folding and secretion.
Specific binding of VirG to the vir box requires a C‐ terminal domain and exhibits a minimum concentration threshold
Authors: Powei B. S. , Kado C. I. 
Published: October 2006

SummaryThe positive regulatory protein VirG from the virulence region of the Ti plasmid of Agrobacterium tumefaciens was first demonstrated to possess DNA‐binding capabilities using chromatographically purified protein and in vitro assays (Powell et al., 1989). This paper is an extension of that research and presents evidence on the in vivo DNA‐binding properties of VirG using a transcription interference assay. VirG protein bound specifically to a vir box'response element and repressed transcription of a lacZ reporter gene, but increased transcription in the absence of a vir box. A biphasic response in specific DNA‐binding was observed upon Increasing virG expression, suggesting that specific binding was co‐operatively affected by protein concentration. Certain TrpE′ ‐ ′VirG hybrid proteins also bound the vir box, but required sequences distal to amino acid Arg‐118 of the VirG polypeptide. These data further localize a DNA‐binding domain within VirG, and support a modified model for the regulation of virulence genes in which trans‐ phosphorylation by the coregulator VirA functions to stabilize specific DNA‐binding by low concentrations of VirG, resulting in gene activation. Otherwise, at high concentrations, VirG promotes expression of the virulence regulon without assistance from VirA as was shown previously (Rogowsky et al., 1987).