bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional analysis of the cya promoter of Bordetella pertussis
Authors: Goyard Sophie , Ullmann Agnes 
Published: October 2006

SummaryThe cyaA gene of Bordetella pertussis and of Bordetella bronchiseptica encodes a toxin which is a bifunctional protein exhibiting adenylate cyclase and haemolytic activities. In Bordetella, virulence factors are synthesized under the control of the bvg regulatory locus, in response to environmental signals. In Escherichia coli the cyaA gene is not expressed, nor is it activated by bvg indicating that the activation of cya by bvg is indirect. To characterize cis‐acting regulatory regions required for the activation of the cyaA gene we constructed cyaA–lacZY fusions containing progressive deletions in the promoter upstream region and isolated promoter mutations by chemical and site‐directed mutagenesis. Deletion analysis shows that a region extending from −569 to −136 bp upstream from the start site of transcription is required for transactivation by bvg, suggesting that multiple binding sites are involved in the activation of the cyaA promoter. No single or double mutations in the promoter upstream region were found which conferred inactive or bvg‐independent Cya phenotype. A double mutation in positions +10 and +13, relative to the transcription start site, rendered the promoter bvg‐independent and functional in E. coli. The constitutive mutations create a new transcription start site, 20 bp downstream from the witd‐type site, by providing new −10 and −35 elements recognized by RNA polymerase alone.
Initiation of mRNA decay in Bacillus subtilis
Authors: Di Mari John F. , Bechhofer David H. 
Published: October 2006

SummaryRibosome stalling in the leader region of ermC mRNA results in a 10–15‐fold increase in ermC mRNA half‐life in Bacillus subtilis. Fusion of the ermC 5′ regulatory region to several B. subtilis coding sequences resulted in induced stability of the fusion RNAs, showing that the ermC 5′ region acts as a general ‘5′ stabilizer’. RNA products of an ermC–lacZ transcriptional fusion were inducibly stable in the complete absence of translation and included a small RNA that is likely to be a decay product arising by blockage of a 3′‐to‐5′ exoribonuclease activity. Insertion of sequences that encode endonucleolytic cleavage sites into the ermC coding sequence resulted in cleavage products whose stability depended on the nature of their 5′ and 3′ ends. It can be concluded from this study that initiation of mRNA decay in B. subtilis generally occurs at or near the 5′ terminus.
A 55 kDa antigen of Pneumocystis carinii: analysis of the cellular immune response and characterization of the gene
Authors: Smulian A. G. , Theus S. A. , Denko N. , Walzer P. D. , Stringer J. R. 
Published: October 2006

SummaryRat‐derived Pneumocystis carinii contains a major antigen complex of 45–55 kDa. The fusion protein of a cDNA encoding the 3′ portion of the 55 kDa antigen, which had previously been shown to be recognized by serum antibodies of exposed subjects, was investigated for its ability to stimulate a cellular immune response. Rats exposed to P. carinii via the environment exhibited a vigorous proliferative response to the antigen whereas unexposed rats did not. The full‐length cDNA for a 55 kDa antigen was cloned and found to contain a 1245 bp open reading frame capable of encoding a 414‐amino‐acid peptide. The gene encoding this protein contained a single 39 bp intron and transcribed a 1.45 kb RNA message. The cloning and characterization of the 55 kDa antigen gene will allow production of the specific immunological reagents necessary to characterize this molecule and study its role in the biology and pathogenesis of P. carinii.
Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi
Authors: Py Béatrice , Chippaux Marc , Barras Frédéric 
Published: October 2006

SummaryExtracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so‐called Out general secretion pathway and, presumably, involves recognition of EGZ‐carried structural information by one or more of the Out proteins. Investigating the relationships between structure and secretability of EGZ was the purpose of the present work. EGZ is made of two independent domains, located at the N‐and C‐proximal sides, separated by a Ser/Thr‐rich region, which are responsible for catalysis and cellulose‐binding, respectively. The existence of a secretion region (‘targeting signal’) was investigated by studying the secretability of modified EGZ derivatives. These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide. Catalytic and/or cellulose‐binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins. In contrast, all of the proteins lost their secretability in E. chrysanthemi. This suggested that at least two secretion motifs existed, one lying within each functional domain. The role of the Ser/Thr‐rich linker region was subsequently tested. Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied. All three exhibited endoglucanase activity and cellulose‐binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction. In contrast, none was secreted by E. chrysanthemi. Collectively, our results with EGZ (i) suggest the occurrence of multiple secretion‐related sites either acting sequentially or forming a single three‐dimensional secretion signal, (ii) show that secretability is not determined by either one of the two functional domains alone, and (iii) reveal that the linker region plays a role in secretion. We propose that all EGZ derivatives were impaired in the recognition step, the nature of which is discussed.
TraM of plasmid R1 regulates its own expression
Authors: Schwab Margit , Reisenzein Helga , Högenauer Gregor 
Published: October 2006

SummaryRegulation of the traM gene, which encodes a factor essential for conjugation of resistance plasmid R1, was studied in vivo using translational gene fusions. tram″lacZ fusion constructs were transferred to the chromosome via the recombinant phage λRZ5. The level of β‐galactosidase expressed by the lysogens indicates that the traM promoters are very active. Expression of traM was diminished five‐ to sixfold when the single‐copy plasmids R1 or R1‐19 were present in trans. When recombinant plasmids carrying traM were present at higher copy numbers, traM expression was reduced as much as 45‐fold. The negative effect of R1 plasmids on traM expression in trans, which we interpret as autoregulation, was observed regardless of whether the plasmids were conjugatjvely repressed or derepressed. Site‐specific mutagenesis of the region encoding the N‐terminus of the TraM protein eliminated the autoregulative effect indicating that the N‐terminal amino acids of the protein are important to its DNA‐binding function. The autoregulatory behaviour of TraM is allele specific. R1‐ or P307‐encoded TraM molecules were found to recognize only the cognate DNA.
Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini‐replicon of pCU1 and the ori‐2 of F
Authors: Da Silva‐Tatley Fernanda M. , Steyn Lafras M. 
Published: October 2006

SummaryThe dominant, polA1‐independent replicon of pGSH500, repβ (1.8 kb), consists of a cis‐acting oriV region of 245 bp; a repB gene that is essential for autonomous replication and 18, 30 to 36 bp iterons which constitute the inc/cop region. The molecular organization of repβ resembles that of mini‐pCU1 (IncN). Furthermore, there is a 58% identity between the Rep proteins of these replicons. RepB also shows a 31% identity with RepE of mini‐F. In addition, an 80% identity over 200 bp was identified between the cis‐acting βoriV region and the equivalent region of ori‐2 (mini‐F). Replicons with deletions of repB could be complemented by Rep (pCU1) and RepE (mini‐F) in trans, supporting the hypothesis that repβ is a natural hybrid between a pCU1‐like and F‐like replicon.
Stationary‐phase production of the antibiotic actinorhodin in Streptomyces coelicolor A3(2) is transcriptionally regulated
Authors: Gramajo Hugo C. , Takano Eriko , Bibb Mervyn J. 
Published: October 2006

SummaryProduction of actinorhodin, a polyketide antibiotic made by Streptomyces coelicolor A3(2), normally occurs only in stationary‐phase cultures. S1 nuclease protection experiments showed that transcription of actII‐ORF4, the activator gene required for expression of the biosynthetic structural genes, increased dramatically during the transition from exponential to stationary phase. The increase in actII‐ORF4 expression was followed by transcription of the biosynthetic structural genes actIII and actVI‐ORF1, and by the production of actinorhodin. The presence of actII‐ORF4 on a multicopy plasmid resulted in enhanced levels of actII‐oRF4 mRNA, and transcription of actIII and actinorhodin production during exponential growth, suggesting that actinorhodin synthesis in rapidly growing cultures is normally limited only by the availability of enough of the activator protein. bldA, which encodes a tRNALeuUUA that is required for the efficient translation of a single UUA codon in the actII‐ORF4 mRNA, was transcribed throughout growth. Moreover, translational fusions of the 5prime; end of actII‐ORF4 that included the UUA codon to the ermE reporter gene demonstrated the presence of functional bldA tRNA in young, exponentially growing cultures and no increase in the efficiency of translation of UUA codons, relative to UUG codons, was observed during growth. The normal growth‐phase‐dependent production of actinorhodin in the liquid culture conditions used in these experiments appears to be mediated at the transcriptional level through activation of the actII‐ORF4 promoter.
Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon cataboiite repression in Aspergillus nidulans
Authors: Kulmburg P. , Mathieu M. , Dowzer C. , Kelly J. , Felenbok B. 
Published: October 2006

SummaryThe CREA repressor responsible for carbon catabolite repression in Aspergillus nidulans represses the transcription of the ethanol regulon. The N‐terminal part of the CREA protein encompassing the two zinc fingers (C2H2 class family) and an alanine‐rich region was expressed in Escherichia colias a fusion protein with giutathione‐S‐transferase. Our results show that CREA is a DNA‐binding protein able to bind to the promoters of both the specific trans‐acting gene, alcR, and of the structural gene, alcA, encoding the alcohol dehydrogenase I. DNase I protection foot‐printing experiments revealed several specific binding sites in the alcR and in the alcA promoters having the consensus sequence 5′‐G/CPyGGGG‐3′. The disruption of one of these CREA‐binding sites in the alcR promoter overlapping the induction target for the trans‐activator ALCR results in a partially derepressed alc phenotype and derepressed alcR transcription, showing that this binding site is functional in vivo. Our data suggest that CREA represses the ethanol regulon by a double lock mechanism repressing both the trans‐acting gene, alcR, and the structural gene, alc A.
NAD+ ‐dependent malic enzyme of Rhizobium meliloti is required for symbiotic nitrogen fixation
Authors: Driscoll Brian T. , Finan Turlough M. 
Published: October 2006

SummaryDEAE‐cellulose chromatography of extracts of free‐living Rhizobium meliloti cells revealed separate NAD+‐dependent and NADP+‐dependent malic enzyme activities. The NAD+ malic enzyme exhibited more activity with NAD+ as cofactor, but also showed some activity with NADP+. The NADP+ malic enzyme only showed activity when NADP+ was supplied as cofactor. Three independent transposon‐induced mutants of R. meliloti which lacked NADP+ malic enzyme activity (dme) but retained NADP+ malic enzyme activity were isolated. In an otherwise wild‐type background, the dme mutations did not alter the carbon utilization phenotype; however, nodules induced by these mutants failed to fix N2. Structurally, these nodules appeared to develop like wild‐type nodules up to the stage where N2‐fixation would normally begin. These results support the proposal that NAD+ malic enzyme, together with pyruvate dehydrogenase, functions in the generation of acetyl‐CoA required for TCA cycle function in N2‐fixing bacteroids which metabolize C4‐dicarboxylic acids supplied by the plant.
Positive transcriptional control of mry regulates virulence in the group A streptococcus
Authors: Okada Nobuhiko , Geist Robert T. , Caparon Michael G. 
Published: October 2006

SummaryTranscription of the antiphagocytic M protein in the group A streptococcus (Streptococcus pyogenes) is environmentally regulated in response to CO2 and requires Mry, a trans‐acting positive regulatory protein. We have examined the role of Mry in environmental regulation by analysing the factors that regulate expression of the gene that encodes Mry (mry). By employing a strategy that utilizes integrational plasmids, it was found that expression of mry requires the participation of DNA sequences that extend 473 base pairs upstream of the Mry coding region. Transcription of mry, as analysed in S1 nuclease protection assays, is initiated from two separate promoters located within this extended regulatory region. Construction and analysis of transcriptional fusions between the mry promoters and a promoterless chloramphenicol acetyltransferase gene demonstrated that mry is autoregulated and environmentally regulated in response to the level of CO2. These data suggest a model for the regulation of virulence in S. pyogenes where positive transcriptional control of mry in response to environmental stimuli regulates the expression of the M protein.
The Bacillus subtilis addAB genes are fully functional in Escherichia coli
Authors: Kooistra Jan , Haijema Bert Jan , Venema Gerard 
Published: October 2006

SummaryAn Escherichia coli recBCD deletion mutant was transformed with plasmids containing the Bacillus subtilis add genes. The transformants had relatively high ATP‐dependent exonuclease‐ and ATP‐dependent helicase activities, and their viability, the ability to repair u.v.‐damaged DNA and the recombination in conjugation were nearly completely restored. The B. subtilis Add enzyme did not show Chi‐activity in phage lambda recombination. The individual B. subtilis Add proteins were not able to form an enzymatically active complex with the E. coli RecB,C,D proteins, and they could not complement the recB,C,D deficiency.Evidence is presented that only two subunits are involved in the B. subtilis ATP‐dependent exonuclease. This is in contrast to E. coli in which the RecBCD enzyme consists of three subunits.
Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli
Authors: Bilge Sima S. , Apostol John M. , Fullner Karia Jean , Moseley Steve L. 
Published: October 2006

SummaryThe transcriptional organization of the gene cluster encoding the F1845 fimbrial adhesin of a diarrhoea‐associated Escherichia coli was investigated. Genes daaA to daaE were determined to constitute a single transcriptional unit under the control of the daaA promoter. The nucleotide sequence of daaA and that of an upstream open reading frame encoded on the opposite strand, designated daaF, were determined to share limited homology with the papB and papi genes of the P fimbrial adhesin, respectively. The 5′ termini of the daaF and daaABCDE transcripts were mapped by primer extension and nuclease protection analyses. The promoters for these transcripts were associated with potential regulatory sequences including two consensus leucine‐responsive regulatory protein (Lrp)‐binding sites which contained differentially methylated GATC sequences, a cAMP‐CRP‐binding site, and an integration host factor (IHF)‐binding site. Expression of the daa locus was determined to be dependent on Lrp, subject to catabolite repression, and dependent on IHF.
The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the Nif A promoter
Authors: Charlton Wayne , Cannon Wendy , Buck Martin 
Published: October 2006

SummaryThe nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed σ;54‐dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60‐200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase‐σ54 complex (Eσ54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between Eσ54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase‐σ54‐binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp.Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that Eσ54 forms a closed complex at DPE1 but not DPE2 and that bound Eσ54 closely approaches the −15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.
DNA sequencing and expression of the gene rnb encoding Escherichia coli ribonuclease II
Authors: Zilhão R. , Camelo L. , Arraiano C. M. 
Published: October 2006

SummaryThe Escherichia coli ribonuclease II (RNase II) is an exonuclease involved in mRNA degradation that hydrolyses single‐stranded polyribonucleotides processively in the 3′ to 5′ direction. Sequencing of a 2.2 kb Msel–Rsal fragment containing the rnb gene revealed an open reading frame of 1794 nucleotides that encodes a protein of 598 amino acid residues, whose calculated molecular mass is 67 583 Da. This value is in good agreement with that obtained by sodium dodecyl sulphate/ polyacrylamide gel electrophoresis of polypeptides synthesized by expression with the T7 RNA polymerase/promoter system. This system was also used to confirm the correct orientation of rnb. Translation initiation was confirmed by rnb–lacZ fusions. The mRNA start site was determined by S1 nuclease mapping. Two E. coli mutants harbouring different rnb alleles deficient in RNase II activity were complemented with the expressed fragment carrying the rnb gene.
Properties of FNR proteins substituted at each of the five cysteine residues
Authors: Green J. , Sharrocks A. D. , Green B. , Geisow M. , Guest J. R. 
Published: October 2006

SummaryFNR is a transcriptional regulator controlling the expression of a number of Escherichia coli genes in response to anoxia. It is structurally‐related to CRP (the cyclic AMP receptor protein) except for the presence of a cysteine‐rich N‐terminal extension, which may form part of an iron‐binding, redox‐sensing domain in FNR. Site‐directed substitution has previously shown that four of the cysteine residues (C20, C23, C29 and C122) are essential for FNR function, whereas the fifth (C16) is not. The FNR protein exists in two forms separable by non‐reducing SOS‐PAGE, and in studies with altered FNR proteins containing single substitutions at each of the five cysteine residues it was concluded that the faster‐migrating form (FNR(27)), possesses an intramolecular disulphide bond linking C122 to one of the cysteines near the N‐terminus. FNR(27)) was more abundant in aerobic cells but the physiological significance of this was not established. Footprint studies indicated that FNR proteins lacking essential cysteine residues are impaired in their ability to protect FNR sites in the ndh promoter. The non‐essential cysteine residue (C16) was identified as the source of the most reactive sulphydryl group and all of the inactive proteins exhibited different sulphydryl reactivities. The iron content of the C122A‐substituted protein was much reduced but those of the other proteins were less affected. Electrospray mass spectrometry confirmed the accuracy of the gene‐derived amino acid composition of FNR with a mutant protein and it showed that a fraction of the wild‐type protein may carry a 78 Da substituent which could not be removed with dithio‐threitol or β‐mercaptoethanol.
Multisensory activation of the phosphorelay initiating sporulation in Bacillus subtilis: identification and sequence of the protein kinase of the alternate pathway
Authors: Trach Kathleen A. , Hoch James A. 
Published: October 2006

SummaryThe phosphorelay is the signal‐transduction system recognizing and integrating environmental signals to initiate sporulation. The major signal input to the phosphorelay is an ATP‐dependent kinase, KinA, responsible for phosphorylating the Spo0F protein. Mutants lacking KinA, however, still sporulate, suggesting that other kinases can fulfil its role. In order to identify these kinases, genes for kinases were isolated by hybridization using a degenerate oligonucleotide probe designed for common regions of this class of kinases. A gene for a second kinase, KinB, was isolated which gave a sporulation negative phenotype when inactivated in a kinA background. The kinB locus was sequenced and found to be a small operon consisting of the kinB gene and another gene, kapB, transcribed from a single σ;A‐dependent promoter. Inactivation of either kinB or kapB in a kinA strain led to severe sporulation deficiency. The kinB gene coded for a 47774 Mr protein with the carboxyl half of this protein highly homologous to the same domain of KinA. The amino‐terminal domain of KinB was hydro‐phobic with six recognizable membrane‐spanning regions. The kapB gene coded for a moderately charged, probably soluble, protein of 14666 Mr, with no homology to any known protein. Genetic evidence suggests that KapB is required either for the function of KinB or for its expression. Although double mutants kinA kinB cannot sporulate and assume a stage 0 phenotype, the SpoA∼P‐dependent regulation of the abrB gene is normal in these strains, suggesting that low levels of SpoA∼P accumulate even in the absence of both kinases. This accumulation is dependent on functional spoOF and spoOB genes and its source is unknown. The KinA and KinB pathways are the only pathways capable of producing sufficient SpoOA∼P to allow initiation and completion of sporulation under laboratory conditions.
In vivo processing of an intron‐containing archael tRNA
Authors: Nieuwlandt Daniel T. , Carr Mary Beth , Daniels Charles J. 
Published: October 2006

Summary In vitro studies on the processing of halobacterial tRNA introns have led to the proposal that archaeal and eukaryotic tRNA intron endonucleases have distinctly different requirements for the recognition of pre‐tRNAs. Using a Haloferax volcanii in vivo expression vector we have examined the in vivo processing of modified forms of the halobacterial intron‐containing tRNATrp gene. As observed in vitro, changes in the exon–intron boundary structure of this pre‐tRNA block processing. Intron sequences, other than those at the exon–intron boundaries, are not essential for processing in vivo. We also show that conversion of the tryptophan anticodon to an opal suppressor anticodon is tolerated when the exon‐intron boundary structure is maintained.