bluuebunny/crossref_metadata_2025_split huggingface dataset:
Iron‐responsive gene expression in Pseudomonas fluorescens M114; cloning and characterization of a transcription‐activating factor, PbrA
Authors: Sexton Ray , Gill Paul R. , Callanan Michael J. , O'Sullivan Daniel J. , Dowling David N. , O'Gara Fergal 
Published: October 2006

SummaryIn response to iron limitation, Pseudomonas fluorescens M114 induces a number of genes including an iron‐scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ‐induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron‐regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron‐regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseuciobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the σ70 sigma factor family, including fecl required for expression of the ferric dicitrate outer‐membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.
Cold‐shock induction of a family of TIP1‐related proteins associated with the membrane in Saccharomyces cerevisiae
Authors: Kowalski Leslie R. Z. , Kondo Keiji , Inouye Masayori 
Published: October 2006

Summary TIP1 is the first known cold‐shock‐and heat‐shock‐induced gene in Saccharomyces cerevisiae. Here it is demonstrated that a TIP1 homologue, TIR1, which had been previously cloned as SRP1 (serine‐rich protein), is strongly induced by a downshift in growth temperature from 30 to 10°C. We further cloned TIR2, which is transcribed at a low basal level but is increased strongly by cold shock and, to a lesser extent, by heat shock. The predicted protein sequence of TIR2 demonstrates remarkable homology to T1R1 (72.2%) and is also homologous with TIP1 (49%). TIP1, TIR1 and TIR2 are rich in both serine and alanine residues and each contains serine‐rich tandem repeats. The proteins contain putative N‐terminal signal peptides as well as hydro‐phobic C‐terminal sequences, indicating that the proteins may be membrane bound. The predicted protein sequences are also consistent with extensive O‐mannosylation as well as glycosyl‐phosphatidyl inositol (GPI) membrane anchoring. Cell fractionation analysis as well as studies using a yeast strain that is conditionally deficient in glycosylation demonstrate that TIP1 is a heavily modified membrane‐associated protein. Single, double combinations and triple mutants were created and none demonstrated any obvious phenotype, indicating that this family of genes is not essential for normal growth.
The recA gene of Streptococcus pneumoniae is part of a competence‐induced operon and controls lysogenic induction
Authors: Martin Bernard , Garcia Pedro , Castanié Marie‐Pierre , Claverys Jean‐Pierre 
Published: October 2006

SummaryThe recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae has been further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation has been shown to confer both DNA repair (as judged from sensitivity to u.v. and methyl methane sulphonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several fold at the onset of competence for genetic transformation, increased expression was associated with the appearance of a recA‐specific transcript, approximately 5.7 kb long. This transcript indicated that recA is part of a competence‐inducible (cin) operon. The major (about 4.3 kb) transcript detected from non‐competent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amounts of the 5.7 kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addition, mitomycin C treatment of a recA lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.
Phage‐exclusion enzymes: a bonanza of biochemical and cell biology reagents?
Authors: Snyder Larry 
Published: October 2006

SummaryMany parasitic DNA elements including prophages and plasmids synthesize proteins that kill the cell after infection by other phages, thereby blocking the multiplication of the infecting phages and their spread to other nearby cells. The only known function of these proteins is to exclude the infecting phage, and therefore to protect their hosts, and thereby the DNA elements themselves, against phage contagion. Many of these exclusions have been studied extensively and some have long been used in molecular genetics, but their molecular basis was unknown. The most famous of the phage exclusions are those caused by the Rex proteins of λ prophage. The Rex exclusions are still not completely understood, but recent evidence has begun to lead to more specific models for their action. One of the Rex proteins, RexA, may be activated by a DNA‐protein complex, perhaps a recombination or replication intermediate, produced after phage infection. In the activated state, RexA may activate RexB, which has been proposed to be a membrane ion channel that allows the passage of monovalent cations, destroying the cellular membrane potential, and killing the cell. We now understand two other phage exclusions at the molecular level which use strategies that are remarkably similar to each other. The parasitic DNA elements responsible for the exclusions both constitutively synthesize enzymes that are inactive as synthesized by the DNA element but are activated after phage infection by a short peptide determinant encoded by the infecting phage. In the activated state, the enzymes cleave evolutionarily conserved components of the translation apparatus, in one case EF‐Tu, and in the other case tRNALys. Translation is blocked and development of the phage is arrested. A myriad of different phage‐exclusion systems are known to exist and many of these may also be specific for highly conserved cellular components, furnishing generally useful enzymes for biochemical and biomedical research.
Isolation and analysis of mutants of the dnaK operon of Bacillus subtilis
Authors: Schulz Anja , Tzschaschel Barbara , Schumann Wolfgang 
Published: October 2006

Summary Bacillus subtilis contains at least three classes of heat‐shock genes regulated by different mechanisms. We are studying class I heat‐shock genes encoded by the operons dnaK and groE. These two operons are both expressed from a vegetative promoter, and their regulation involves a novel heat‐shock element designated CIRCE. Here we show that induction of both operons results from enhanced synthesis of mRNA and is independent of de novo protein synthesis. To answer the question of whether dnaK is involved in the deregulation of the heat‐shock response as reported for Escherichia coli, two different insertion mutations were isolated within the tetracistronic dnaK operon (orf39–grpE–dnaK–dnaJ). In one mutant a cat cassette was inserted at the beginning of orf39. Transcriptional analysis revealed that this mutation abolished expression of the whole operon. In contrast, the basal level of groE mRNA was significantly increased at 37°C, followed by a prolonged delay in the shut off after temperature upshift. These data point to a crucial role for the orf39 gene in the regulation of class I heat‐shock genes. In the other mutant an internal 0.8 kb Bgl II fragment of dnaK was replaced by the cat cassette. In contrast to E. coli dnaK null mutants, the two B. subtilis dnaK operon mutants could grow within a temperature range from 16–52°C. At temperatures above 52°C, they failed to form colonies on agar plates, started to filament, and lost motility. Furthermore, the induction profile of the groE and dnaK operons was not impaired in the dnaK::cat mutant. The expression of the flagellin gene is influenced on both the transcriptional and post‐transcriptional level. By Northern‐blot analysis we previously showed that expression of the dnaK operon resulted in two mRNA species of 4.9 and 2.6 kb. Our mutational analysis suggests the formation of the 2.6 kb transcript as a processing product of the 4.9 kb species.
comK encodes the competence transcription factor, the key regulatory protein for competence development in Bacillus subtilis
Authors: van Sinderen Douwe , Luttinger Amy , Kong Liyun , Dubnau David , Venema Gerard , Hamoen Leendert 
Published: October 2006

Summary comK is a positive autoregulatory gene occupying a central position in the competence‐signal‐transduction network. All regulatory routes identified in this network converge at the level of comK expression. The ComK protein is required for the transcriptional induction of comK and the late competence genes, which specify morphogenetic and structural proteins necessary for construction of the DNA‐binding and uptake apparatus. In this report we demonstrate that ComK specifically binds to DNA fragments containing promoter and upstream sequences of the genes it affects (comC, comE, comF, comG and comK) Using portions of the region upstream of comC we show that the ComK‐binding sequences are essential for the expression of competence. Moreover, we demonstrate that the presence of ComK stimulates the expression of comF–lacZ and comG–lacZ translational fusions in vivo in Escherichia coli These results indicate that the gene product of comK is identical to the previously inferred competence transcription factor (CTF).
Regulation of succinate dehydrogenase (sdhCDAB) operon expression in Escherichia coli in response to carbon supply and anaerobiosis: role of ArcA and Fnr
Authors: Park Soon‐Jung , Tseng Ching‐Ping , Gunsalus Robert P. 
Published: October 2006

SummarySuccinate dehydrogenase (SDH) of Escherichia coli, the sole membrane‐bound enzyme of the tricarboxylic acid cycle, participates in the aerobic electron‐transport pathway to generate energy via oxidative phosphorylation reactions. Previous studies have established that succinate dehydrogenase (SDiH) synthesis is elevated by aerobiosis and supressed during growth with glucose. To examine how the sdhCDAB genes that encode SDH are regulated by changes in the environment, sdh–lacZ fusions were constructed and analysed in vivo following cell growth under a variety of alternative culture conditions. Expression of sdh–lacZ was highest under aerobic conditions and was decreased 10‐foid in the absence of oxygen. The fnr and arcA gene products are required for this oxygen control and each acts to repress sdhC–lacZ expression. Expression of sdh–lacZ also varied 10‐ to 14‐foid depending on the type of carbon substrate used or the medium richness. This control was shown to be independent of the crp and fruR gene products, and indicates that some other regulatory element exists in the ceil to adjust SDH enzyme levels accordingly. Iron and haem availability affected sdhC–lacZ expression by two‐ to threefold. Lastly, fold. Lastly, sdhC–lacZ expression was shown to vary with the cell growth rate during aerobic and anaerobic conditions.
Comparative characterization of the iga gene encoding IgA1 protease in Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae
Authors: Lomholt Hans , Poulsen Knud , Kilian Mogens 
Published: October 2006

SummaryCloning and sequencing of the IgA1 protease gene (iga) from Neisseria meningitidis strain HF13 showed an overall structure equivalent to iga genes from Neisseria gonorrhoeae and Haemophilus influenzae, although no region corresponding to the gonococcal α‐peptide was evident. An additional 18 N. meningitidis and 3 H. influenzae iga genes were amplified by the polymerase chain reaction technique and sequenced corresponding approximately to the N‐terminal half of the mature enzyme. Comparative analyses of a total of 29 iga genes showed that pathogenic Neisseria have iga genes with a significantly lower degree of heterogeneity than H. influenzae iga genes. Recombinational events indicated by mosaic‐like structures corresponding to those found among N. gonorrhoeae protease genes were detected among N. meningitidis iga genes. One region showed characteristic differences in sequence and length which correlated with each of the different cleavage specificities. Meningococci were extremely conserved in this region with no evidence of recombination between isolates of different cleavage specificities. Sequences further downstream showed no obvious relationship with enzyme cleavage type. This region consisted of conserved areas interspersed with highly variable areas. Amino acid sequence homologies in the variable regions of meningococci reflected the antigenic types defined by using polyclonal neutralizing antibodies.
Overexpression of the Shigella flexneri genes coding for DNA topoisomerase IV compensates for loss of DNA topoisomerase I: effect on virulence gene expression
Authors: McNairn Elizabeth , Bhriain Niamh Ni , Dorman Charles J. 
Published: October 2006

SummaryIntroducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E. coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression. E. coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase l by amplifying the genes coding for topoismerase IV. Unlike the E. coli topA mutants, derivatives of S. flexneri harbouring this topA allele did not appear to acquire any compensatory mutations. We investigated the possibility that this was due in part to an inability of the S. flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed. The S. flexneri genes encoding the alpha‐ and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids. These plasmids complemented well‐characterized Salmonella typhimurium temperature‐sensitive topoisomerase IV mutations, showing that the S. flexneri and S. typhimurium proteins are capable of combining to form active complexes. When the S. flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S. flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling. The same plasmid also partially restored S. flexneri virulence gene transcription. These data show that overexpression of the S. flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression. The fact that S. flexneri does not exploit topoisomerase IV gene amplification as E. coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.
Purification, characterization and mode of action of PdhR, the transcriptional repressor of the pdhR–aceEF–Ipd operon of Escherichia coli
Authors: Quail M. A. , Guest J. R. 
Published: October 2006

SummaryThe repressor of the pdhR–aceEF–lpd operon of Escherichia coli, PdhR, was amplified to 23% of total cell protein and purified to homogeneity by heparin–agarose and catlon‐exchange chromatography. The purified protein is a monomer (Mr 29300) which binds specifically to DNA fragments containing the pdh promoter (Ppdh) in the absence of pyruvate. The pdh operator was identified by DNase I footprinting as a region of hyphenated dyad symmetry, +11AATTGGTaagACCAATT+27, situated just downstream of the transcript start site. In vitro transcription from Ppdh was repressed > 1000‐fold by PdhR and this repression was antagonized in a concentration‐dependent manner by its co‐effector, pyruvate. Studies on RNA polymerase binding at Ppdh showed that RNA polymerase protects the ‐44 to +21 region in the absence of PdhR, but no RNA polymerase binding or protection upstream of +9 could be detected in the presence of PdhR. It is concluded that PdhR represses transcription by binding to an operator site centred at +19 such that effective binding of RNA polymerase is prevented.
Identification of an mRNA element promoting rate‐limiting cleavage of the polycistronic puf mRNA in Rhodobacter capsulatus by an enzyme to RNase E
Authors: Fritsch Jürgen , Rothfuchs Rüdiger , Rauhut Reinhard , Klug Gabrrele 
Published: October 2006

SummaryWe have identified an mRNA element that is involved in the initial cleavage of the pufBALMX mRNA species in Rhodobacter capsulatus. This endoribonuclease recognition site, the first to be identified in a bacterial species other than Escherichia coli, shows strong similarities to mRNA sequences cleaved by the endoribonuclease E in E. coli. The presence of an RNase E‐like enzyme in R capsulatus is further supported by in vitro cleavage of E. coli transcripts by R. capsulatus extracts at sites attributed to RNase E and by the cross‐reaction of a polypeptide from R capsulatus with antisera against E. coli RNase E. Our data provide evidence that mRNAs are degraded in different bacterial species by enzymes with similar recognition sequences and activities. We present a model that attributes the segmental differences in stability of the polycistronic puf transcript to a specific distribution of mRNA decay‐promoting and mRNA decay‐impeding elements.
F plasmid CcdB killer protein ccdB gene mutants coding for non‐cytotoxic proteins which retain their regulatory functions
Authors: Bahassi EI Mustapha , Salmon Michel A. , van Melderen Laurence , Bernard Philippe , Couturier Martine 
Published: October 2006

SummaryThe ccd locus of the F plasmid codes for two gene products, CcdA and CcdB, which contribute to the plasmid's high stability by post‐segregational killing of plasmid‐free bacteria. Like the quinolones, the CcdB protein is a poison of the DNA‐topoisomerase II complexes, while CcdA acts as an antidote against CcdB. in addition to these poison‐antipoison properties, the CcdA and CcdB proteins act together at transcription level to repress their own synthesis. In this work, we have isolated, in vivo., and characterized several non‐killer CcdB mutants. All missense mutations which inactivate CcdB killer activity are located in the region coding for the last three C‐terminal residues. However, the resulting mutant CcdB proteins retain their auto‐regulatory properties. We conclude that the last three C‐terminal residues of CcdB play a key role in poisoning but are not involved in repressor formation.
Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358: involvement of a transcriptional activator and of the Fur protein
Authors: Venturi Vittorio , Ottevanger Clemens , Bracke Madelon , Weisbeek Peter 
Published: October 2006

SummaryPseudobactin 358 is the yellow‐green fluorescent siderophore produced by Pseudomonas putida WCS358 in conditions of iron limitation. The genes encoding for siderophore biosynthesis are iron‐regulated at the transcriptional level. Previous work has shown that a positive regulator, PfrA, is absolutely required for the activation under iron‐limiting conditions of pseudobactin 358 biosynthesis. In this study we identified a set of Tn5 insertion mutants of strain WCS358 which lost the ability to activate an iron‐regulated siderophore promoter. These mutants no longer produced pseudobactin 358. Molecular analysis revealed that they carried a Tn5 insertion in a gene, designated pfrl (Pseudomonas ferric regulator), which codes for a protein (Pfrl) of 19.5kDa. Pfrl contains a putative helix‐turn‐helix motif typical of DNA‐binding proteins and has homology to two DNA‐binding transcriptional activators, Fecl from Escherichia coli and Pupl from P. putida. The proposed role of Pfrl in strain WCS358 is an activator protein regulating pseudobactin 358 biosynthesis under iron limitation. The pfrl promoter region contains a sequence which displays high identity to the Fur‐box consensus. This 19bp consensus sequence is recognized by Fur, an iron‐binding repressor protein found in many different bacteria. The E. coli Fur protein can bind to the pfrl promoter region, indicating that this activator gene is likely to be iron‐regulated by Fur. We also report the identification and characterization of the P. putida WCS358 fur gene. The Fur protein of strain WCS358 is structurally and functionally similar to all other cloned Fur proteins from other bacterial species.
Branched‐chain fatty acids: the case for a novel form of cell‐cell signalling during Myxococcus xanthus development
Authors: Downward John , Toal Douglas 
Published: October 2006

SummaryThe esg locus is required for the formation of muiti‐cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E‐signal) used in cell‐cell communication and that E‐signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched‐chain keto acid dehydrogenase. a multienzyme complex involved in branched‐chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched‐chain fatty acids found in this organism. M. xanthus fatty acids (including the branched‐chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development‐defective mutants. These and other results suggest a model in which the branched‐chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting‐body formation. This model proposes that one or more of the branched‐chain fatty acids that are released constitutes the E‐signal which must be transmitted between cells to complete M. xanthus development.