bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular cloning and characterization of a genetic region from Serratia marcescens involved in DNA repair
Authors: Murphy K. E. , Braymer H. D. 
Published: October 2006

SummaryWe report here the molecular isolation of a DNA fragment which encodes Tag‐like activity from the Gram‐negative bacterium Serratia marcescens. A recombinant plasmid encoding Tag‐like activity was isolated from a S. marcescens plasmid gene library by complementation of an Escherichia coli tag mutant, which is deficient in 3‐methyladenine DNA glycosylase I. The clone complements E. coli tag, recA, alkA, but not alkB, mutants for resistance to the DNA‐damaging agent methyl methanesulphonate (MMS). The coding region of the Tag activity, initially isolated on a 6.5kb BamHI fragment, was defined to a 1.8kb BglII‐Smal fragment. Labelling of plasmid‐encoded proteins using maxicells revealed that the 1.8kb fragment encodes two proteins of molecular weights 42000 and 16000. Data presented here suggest that the cloned fragment encodes a DNA repair protein(s) that has similar activity to the 3‐methyladenine DNA glycosylase I of E. coli.
Mutations that alter the helix‐turn‐helix region of the spollAC protein: a Bacillus subtilis sporulation‐specific sigma factor
Authors: Yudkin M. D. , Millonig J. H. , Appleby L. 
Published: October 2006

SummaryIt has previously been shown that spollAC561, a mutation that diminishes the incidence of sporulation by more than six orders of magnitude, alters the residue at position 13 of the helix‐turn‐helix region of the sporulation‐specific sigma factor encoded by spoll AC from valine to methionine (Yudkin, 1987b). We have now found that four spontaneous revertants, which sporulate at an incidence of 30‐60%, all contain transitions within the codon that was altered by spo‐561. The mutant methionine is replaced by isoleucine in two revertants, and by threonine in the other two.
Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa
Authors: Filloux A. , Bally M. , Murgier M. , Wretlind B. , Lazdunski A. 
Published: October 2006

SummaryPleiotropic mutations (xcp) affecting secretion of proteins in Pseudomonas aeruginosa have been previously characterized and mapped at 0 min, 55 min and 65 min. Genomic libraries of this organism have been constructed and the genes xcp‐5 and xcp‐54, located at the 55 min region, were cloned using the adjacent met allele as a marker, and complementation of xcp strains. From our linkage and cloning analysis, the most probable gene order in this region appears to be pyrD., xcp‐5/xcp‐54/met‐9011/oru‐314/trpF/leu‐10. Restriction mapping and transposon (Tn 1725) insertion mutagenesis demonstrated that: (i) the overall size of DNA necessary for xcp expression was 9 kb, (ii) the two loci are not adjacent on the chromosome, and (iii) the two loci are expressed independently. The xcp‐5 gene has been subcloned on a 4 kb Eco RI fragment.
Regulation of bacterial gene expression by metal–protein complexes
Authors: Hennecke H. 
Published: October 2006

SummaryMetal ions are essential cofactors in several transacting bacterial gene regulators. Upon binding of the metal, the receptor proteins act either as repressors of gene expression or, in other systems, as transcriptional activators. Other metal‐dependent regulatory proteins may function, directly or indirectly, as sensors of the cellular oxygen status, and may even be mediators in light‐responsive gene regulation.
Precursor/product antiport in bacteria
Authors: Poolman B. 
Published: October 2006

SummaryMany microorganisms metabolize their substrates (precursors) only partially and excrete the products of the metabolism into the medium. Although uptake of precursor and exit of product can proceed as two independent steps, there is increasing evidence that these processes are often linked and that transport is facilitated by a single antiport mechanism. Features of antiport mechanisms and advantages for the organism of catalysing precursor/product antiport will be illustrated by discussing a number of well‐characterized systems. Based on precursor‐product conversion stoichiometries, structural relatedness between precursors and products, and energetic and kinetic considerations, new examples of antiport systems will be proposed.
Molecular genetic analysis of an FNR‐dependent anaerobically inducible Escherichia coli promoter
Authors: Bell A. I. , Cole J. A. , Busby S. J. W. 
Published: October 2006

SummaryFrom the effects of 13 deletions and three linker‐scanner mutations at the Escherichia coli nirB promoter we have located sequences necessary for FNR‐dependent induction of activity by anaerobiosis and further nitrite‐dependent stimulation of expression. We describe a nirB promoter derivative that allows the cloning of ‘cassettes’ carrying different FNR‐binding sequences and experiments in which a number of point mutations were introduced into these sequences. FNR‐dependent stimulation of expression from the nirB promoter is critically dependent on the location of the FNR‐binding site, and deletion or insertion of one base pair is sufficient to disrupt promoter function. We have transferred a number of cassette FNR‐binding sequences from the nirB promoter to the unrelated melR promoter. The insertion of FNR‐binding sequences at the melR promoter is sufficient to confer fnr‐dependency on expression. However expression from these hybrid promoters is not as efficiently repressed during aerobic growth, suggesting that the function of bound FNR is dependent on the sequence context of the FNR‐binding sequence.
Generation of a cytotoxic T‐lymphocyte response using a Salmonella antigen‐delivery system
Authors: Flynn J. L. , Weiss W. R. , Norris K. A. , Seifert H. S. , Kumar S. , So M. 
Published: October 2006

SummaryWe have constructed a general‐use vector for the cloning and stable expression of foreign genes in the chromosome of attenuated Salmonella typhimurium. Using this chromosomal expression vector (CEV), we expressed the circumsporozoite (CS) gene of the mouse malaria Plasmodium yoelii in an aroA S. typhimurium strain. Mice immunized with CS‐expressing Salmonella recombinants mount a CS‐specific cytotoxic T‐lymphocyte (CTL) response. This is the first demonstration that attenuated Salmonella can elicit a specific CTL response to a foreign protein in mice. The ability to easily and stably express foreign genes from the Salmonella chromosome and the generation of specific CTL greatly expands the potential of Salmonella as an antigen‐delivery system.
Specific β‐lactam antibiotics inhibit secretion of lipo‐β‐lactamase in yeast
Authors: London A. , Pines O. 
Published: October 2006

SummaryThe β‐lactam antibiotic cloxacillin can inhibit secretion of prokaryotic lipo‐β‐lactamase into the periplasm of yeast. The results indicate that this phenomenon is specific with respect to both the antibiotic and the lipo‐β‐lactamase whose secretion is affected, strongly suggesting that this involves an interaction between the enzyme and its substrates. The effect of the antibiotic on secretion is reversible. With different β‐lactam antibiotics, the clearest difference is observed between type A and type S penicillins; the former exert a strong inhibition of secretion whereas the latter exhibit a weak effect or no effect at all. Type A penicillins have been previously shown to cause a conformational change in various β‐lactamases. Mature lipo‐β‐lactamase species in yeast were localized either to the periplasmic space or bound to the outer surface of the cytoplasmic membrane and thus exposed to periplasm. The results are consistent with the hypothesis that binding of cloxacillin to lipo‐β‐lactamase induces a conformation on the protein that is unfavourable for its release from the membrane.
CiS‐ and trans‐acting factors involved in centromere function in Saccharomyces cerevisiae
Authors: Murphy M. , Fitzgerald‐Hayes M. 
Published: October 2006

SummaryThe function of centromeric DNA in the yeast Saccharomyces cerevisiae has been studied in detail. Twelve of the sixteen S. cerevisiae centromeres have been sequenced to date, and a consensus sequence has been identified. This sequence consists of a central region 78 to 86 bp in length which is greater than 90% A+T, usually in runs of As and runs of Ts. The central region is flanked on one side by a highly conserved 8bp sequence and on the other side by a highly conserved 25 bp sequence which contains partial dyad symmetry around a central C/G base pair. Mutational analyses have been used to determine the importance of each subset of the consensus sequence to centromere function. A protein which binds to the 8 bp sequence and at least one that binds to the 25bp sequence have been identified. The roles of these proteins in centromere function in mitosis and meiosis are currently under investigation.
Escherichia coli cells resistant to the DNA gyrase inhibitor, ciprofloxacin, overproduce a 60 kD protein homologous to GroEL
Authors: Hallett P. , Mehlert A. , Maxwell A. 
Published: October 2006

SummaryUsing a variety of mutagenic methods, we have generated a series of ciprofloxacin‐resistant mutants derived from Escherichia coli strains which overproduce the DNA gyrase A protein. Many of these mutants are found to overexpress a 60 kD protein which is shown to be highly homologous in terms of N‐terminal a mi no acid sequence to the E. coli heat‐shock protein, GroEL. Other evidence confirms that the 60 kD protein is unrelated to DNA gyrase and is similar, but not identical, to GroEL.
Five genes at the 3′ end of the Klebsiella pneumoniae pulC operon are required for pullulanase secretion
Authors: Pugsley A. P. , Reyss I. 
Published: October 2006

SummaryThe nucleotide sequence of a 5082bp fragment of chromosomal DNA from Klebsiella pneumoniae strain UNF5023 is reported. The sequence includes the last four genes of an operon of genes specifically required for the secretion of the enzyme pullulanase. All four genes (puIL, puIM, puIN and puIO) are shown to be required for pullulanase secretion, as is a fifth gene (puIK) which extends beyond the 5′ end of the sequenced DNA. The products of the puIL, puIM, puIN and puIO genes (44kD, 18kD, 27kD and 24kD, respectively) are all predicted to have one or more hydrophobic domains typical of signal sequences and/or membrane anchors, and were all found mainly associated with the inner membranes of subfractionated cells in which the corresponding genes had been expressed from the bacteriophage T7 gene 10 promoter. The results of this study increase the number of genes which have been identified as required for pullulanase secretion to eight, in addition to genes coding for components of the general export pathway.
Multiple promoters control the regulation of the Pseudomonas aeruginosa regA gene
Authors: Storey D. G. , Frank D. W. , Farinha M. A. , Kropinski A. M. , Iglewski B. H. 
Published: October 2006

SummaryExpression studies utilizing the regA promoters, fused in tandem or separately to promoterless reporter genes, indicated that regA is transcribed from two promoters (P1 and P2). Both promoters can act independently. Expression from the P1 promoter is not affected by the iron content of the medium. Expression from the P2 promoter is tightly regulated by iron.
Mini‐Mu mediates deletion‐inversions in vivo by intra‐transposon transposition
Authors: Leach D. R. F. , Okely E. A. , Percy‐Robb M. I. 
Published: October 2006

SummaryWe have shown that a mini‐Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion‐inversion product, which has previously been observed in vitro, is formed by non‐replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini‐Mu can undergo non‐replicative reactions in vivo and that these generate a 5bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.
A lipopeptide‐encoding sequence upstream from the IysA gene of Pseudomonas aeruginosa
Authors: Jann A. , Cavard D. , Martin C. , Cami B. , Patte J.C. 
Published: October 2006

SummaryAn open reading frame (ORF) of 141 bp was observed upstream from the Pseudomonas aeruginosa lysA gene. The translation product of this ORF contains a signal peptide with a lipoprotein box, fle‐Ala‐Ala‐Cys, at the predicted signal peptidase cleavage site. The Escherichia coli phoA gene without its signal sequence was fused in frame to this ORF in a broad host‐range plasmid. The resulting construct expressed a hybrid protein exhibiting alkaline phosphatase activity in phoA mutants of both E. coli and P. aeruginosa. This indicates that the ORF encodes a peptide, part of which acts as an export signal. The hybrid peptide was identified by immunoblotting with alkaline phosphatase antiserum. The accumulation of a precursor form was observed when P. aeruginosa cells carrying this gene fusion on a plasmid were treated with globomycin. Moreover, the mature form could be labelled with 2‐[3H]‐glycerol, indicating that lipidic residues may be linked to the hybrid protein. Taken together, these results strongly suggest that the ORF encodes a lipopeptide. We propose that the gene is called IppL.
The arcB gene of Escherichia coli encodes a sensor‐regulator protein for anaerobic repression of the arc modulon
Authors: Iuchi S. , Matsuda Z. , Fujiwara T. , Lin E. C. C. 
Published: October 2006

SummaryThe arcA (dye) and arcB genes of Escherichia coli are responsible for anaerobic repression of target operons and regulons of aerobic function (the arc modulon). The amino acid sequence of ArcA (Dye) indicated that it is the regulator protein of a two‐component control system. Here we show that ArcB is a membrane sensor protein on the basis of its deduced amino acid sequence (778 residues), hydropathicity profile, and cellular distribution. On the carboxyl end of the ArcB sequence there is an additional domain showing homology with conserved regions of regulator proteins. Deletion into this domain destroyed ArcB function. ArcB conserved a histidine residue for autophosphorylation of the sensor proteins, and aspartic residues important for the regulator proteins.
Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli
Authors: White D. A. , Barlow A. K. , Clarke I. N. , Heckels J. E. 
Published: October 2006

SummaryThe entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2 kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1‐16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS‐PAGE. Western blotting with anti‐class 1 protein antibodies revealed constitutive expression of a protein of 41kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N‐terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno‐gold electron microscopy showed that the protective epitope recognized by a P1‐16 subtype‐specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells.Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein. Such constructs illustrate the potential use of recombinant DNA technology for the development of effective human vaccines against meningococcal infection.
Notes An inversion truncating the creA gene of Aspergillus niduians results in carbon catabolite derepression
Authors: Arst H. N. , Tollervey D. , Dowzer C. E. A. , Kelly J. M. 
Published: October 2006

SummaryThe creAd‐30 mutation leading to carbon catabolite derepression in Aspergillus nidulans is a pericentric inversion, having one breakpoint within the creA gene on the left arm of chromosome I and the other breakpoint between binG and yA on the right arm. The left‐arm breakpoint alters the creA transcript. The likelihood that the inversion truncates creA centrally strengthens a previous proposal that derepression is the phenotype of loss‐of‐function mutations in creA.
Regulatory functions of the three nodD genes of Rhizobium leguminosarum biovar phaseoli
Authors: Davis E. O. , Johnston A. W. B. 
Published: October 2006

SummaryThe three nodD genes of a strain of Rhizobium leguminosarum biovar phaseoli were cloned to study their effects on transcription of themselves and of the nodC genes of biovars phaseoli and viciae. Efficient transcription of nodD1 required nodD1 and was enhanced by exposure of the cells to bean exudate consistent with the presence of a nod‐box preceding the nolE‐nodD1 operon. Transcription of nodD2 and nodDZ was constitutive. nodC of R. leguminosarum biovar phaseoli was activated by each of the nodD genes of that biovar in the absence of inducers but expression was enhanced in cells grown with bean exudate or the flavonoids genistein or naringenin. A mutant of nodD2, tacking 60bp at its 3’end, activated nodC in the presence of inducer, but was defective in regulating certain of the nodD genes. The nodC gene of R. leguminosarum biovar viciae responded differently to the various nodD genes of R. leguminosarum biovar phaseoli than did the nodC of the latter biovar.
Developmental expression and characterization of the gene encoding spore coat protein SP60 in Dictyostelium discoideum
Authors: Widdowson D. C. C , Proffitt J. A. , Jagger P. S. , Richards A. J. , Hames B. D. 
Published: October 2006

SummaryThe complete coding sequence, upstream sequence and developmental expression of Dictyostelium discoideum AX2 spore coat protein gene SP60 is reported. The gene contains two exons, 154 bp and 1121 bp long, separated by a 119bp intron, and encodes a protein of 46925 molecular weight plus a 23‐amino‐acid hydrophobic leader sequence. The N‐terminus of the mature protein consists of four copies of a perfect hexapeptide repeat (GDWNNN). The central region is rich in cysteine residues, including four highly conserved cysteine‐rich repeats with homology to ‘EGF‐like’ repeats. The C‐terminus is aspartate‐rich and composed of multiple imperfect copies of a D(G/D)DYD repeat followed by several repeats of the tetrapeptide DNDW and derived sequences. A TATA box promoter motif juxtaposed to an oligo(dA) stretch lies 52 bp upstream of the main transcriptional start site of the gene. Six AC‐rich boxes occur in the region ‐327 to ‐556, all of which contain the consensus sequence CACAC. Two GC‐rich boxes and a C‐rich element (TTACCCCA) are also present upstream. Another open reading frame is positioned a short distance downstream of the SP60 gene in the opposite transcriptional orientation. Expression of the SP60 gene ceases upon disaggregation to single cells and cannot be restored by high levels of extracellular cAMP either alone or in combination with conditioned medium factors.
Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100
Authors: Dempsey W. B. , Fee B. E. 
Published: October 2006

SummaryIntegration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to or/T and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF), as measured by chloramphenicol‐resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.
Structural analysis of the pehA gene and characterization of its protein product, endopolygalacturonase, of Erwinia carotovora subspecies carotovora
Authors: Saarilahti H. T. , Heino P. , Pakkanen R. , Kalkkinen N. , Palva I. , Palva E. T. 
Published: October 2006

SummaryA clone producing a potygalacturonase (EC 3.2.1.15) in Escherichia coli was isolated from a genomic library of Erwinia carotovora subspecies carotovora constructed in pUC18. The DNA segment carrying the corresponding structural gene, named pehA, contained an open reading frame (ORF) encoding a 402‐amino‐acid (aa) polypeptide with an Mr of 42849. In E. carotovora the polygalacturonase was synthesized with a 26‐aa cleavable signal peptide. The mature 376‐aa PehA had a calculated Mr of 40064 and a pi of 10.19. The pH optimum of the enzyme was about 5.5 and the temperature optimum was in the range 35–45 ° C. Analysis of the reaction products of poly‐galacturonic acid hydrolysis indicated that the PehA protein is an endopolygalacturonase. No similarity was observed between the aa sequences of PehA and other pectic enzymes of Erwinias. However, substantial similarity was detected within the C‐terminal portions of PehA and a previously described tomato polygalacturonase, suggesting that the bacterial and eukaryotic potygalacturonases may have a common origin.
Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II)
Authors: Fridén H. , Hederstedt L. 
Published: October 2006

SummaryCytochrome 5558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron‐sulphur protein (Ip) in succinate: quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis‐imidazole ligation of the haem. Apo‐cytochrome was found in the membrane fraction of haem‐deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His‐47 can be excluded as a haem ligand. The remaining His residues (at position s 13, 28, 70,113 and 155) a re located in or close to four predicted transmembrane segments. The Tyr‐28 and Tyr‐70 mutant proteins appeared to lack one of the two haems. Only the Tyr‐13 and Tyr‐47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr‐13 mutant cytochrome assembles into an enzymatically defective succinate: quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His‐28, His‐70 and His‐155 are probably haem axial ligands in a dihaem cytochrome b558. His‐70 and His‐155 may be ligands to the same haem.