bluuebunny/crossref_metadata_2025_split huggingface dataset:
Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P‐type ATPase and a cation‐binding protein associated with copper transport
Authors: Ge Zhongming , Hiratsuka Koji , Taylor Diane E. 
Published: October 2006

SummaryA 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, oRF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611‐amino‐acid sequence of ORF1, a P‐type ATPase (designated hpCopA), had striking homology (29‐38%) with several bacterial P‐type ATPases and contained the potential functional domains conserved in P‐type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion‐transporting protein, and contains the heavy metal‐binding motif. Disruption of ORF1 with a chloramphenicol‐resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alternation of susceptibility to Ni2+, Cd2+ and Hg2+ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation‐transporting system which is associated with copper export out of the H. pylori cells.
Region II of the Haemophilus influenzae Type b capsulation locus as involved in serotype‐specific polysaccharide synthesis
Authors: Van Eldere Johan , Brophy Lisa , Loynds Barbara , Celts Patrick , Hancock Ian , Carman Stephen , Kroll J. Simon , Moxon E. Richard 
Published: October 2006

SummaryThe central (serotype‐specific) Region II of the Haemophilus influenzae Type b capsulation locus cap is 8.3 kb long and contains a cluster of four genes. We show that these genes, designated orf1 to orf4, are involved in the biosynthetic steps required for the formation of the Type b capsular polysaccharide and that orf1 probably encodes a CDP‐ribitolpyrophosphorylase. We present evidence that growth of polysaccharide chains takes place through the alternating addition of single sugar nucleotides.
Regulation of citrate‐dependent iron transport of Escherichia coli: FecR is required for transcription activation by Feel
Authors: Ochs Martina , Veitinger Sabine , Kim InSook , Weiz Dietrich , Angerer Annemarie , Braun Volkmar 
Published: October 2006

SummaryCitrate‐dependent Fe3+ transport into Escherichia coli K‐12 is induced by iron and citrate. The inducer is probably ferric dicitrate which does not have to be taken up into the cytoplasm to induce transcription of the fec transport genes. Two regulatory genes, fed and fecR, located upstream of the fecABCDE transport genes, are required for induction. We report that in vivo the chromosomally encoded Feel protein activates transcription of the fecA and fecB transport genes in response to ferric citrate and the FecR protein. Cells expressing chromosomally and plasmid‐encoded truncated FecR derivatives no longer responded to ferric citrate and expressed the fec transport genes constitutively. The smallest active FecR derivative contained 59 amino acid residues as compared to the 317 residues of wild‐type FecR. Constitutive induction was lower than induction of the FecR wild‐type strain by ferric citrate. It is concluded that the N‐terminal portion of FecR activates Feel and that the C‐terminal portion of FecR responds to ferric citrate. Transcription of the fee transport genes is positively regulated by Feel and FecR and negatively regulated by the Fe2+‐Fur repressor. Transcription activation and repression may occur independently of each other.
Reinitiation kinetics in eight dnaA(Ts) mutants of Escherichia coli: rifampicin‐resistant Initiation of chromosome replication
Authors: Hansen Flemming G. 
Published: October 2006

SummaryThe kinetics of reinitiation of chromosome replication of eight dnaA(Ts) mutants was investigated in an isogenic set of strains. Five mutants (167, 46, 601, 606 and 5) are classified as reversible, since they can reinitiate at 30 C without protein synthesis, whereas the other three (508, 205, 204) require protein synthesis. In the presence of protein synthesis, reversible mutants initiate one round of replication rapidly after a shift to 30δC, indicating that they contain active or renaturable DnaA protein. The dnaA508 and dnaA204 mutants also reinitiate chromosome replication rapidly, whereas reinitiation is delayed 15–20min in dnaA205. The dnaA508 and dnaA204 mutants might contain active DnaA protein just below the threshold level at 42δC and only require synthesis of small amounts of new DnaA protein before initiation at 30δC, whereas dnaA205 accumulates DnaA protein for some time at 30δC before reaching the initiation threshold. Three of the reversible mutants (5, 601, and 606) exhibited, in addition to the protein synthesis‐independent initiation capacity, an RNA synthesis‐independent initiation capacity. The thermal stability of these initiation capacities is the same as for mutant DnaA protein, strongly suggesting that mutant DnaA protein is responsible for both.
Initiation of chromosome replication after induction of DnaA protein synthesis in a dnaA(null) rhn mutant of Escherichia coli
Authors: Hansen Flemming G. , Atlung Tove 
Published: October 2006

SummaryThe kinetics of initiation of chromosome replication after induction of DnaA protein synthesis was studied in a dnaA(null) rnh mutant of Escherichia coli. DnaA protein synthesis was induced to different extents using the wild‐type dnaA gene controlled by a lac promoter. Initiation of chromosome replication from oriC, measured as an increase in origin to terminus ratio, took place at different times after addition of an inducer dependent on the DnaA protein synthesis rate. The first initiations always occurred when DnaA protein had accumulated approximately to the average wild‐type concentration (24 ng of DnaA protein per ml cells at OD450= 1.0) At a low DnaA protein accumulation rate one synchronous round of replication was obtained after 30min of induction. The initiation kinetics obtained when DnaA protein accumulated rapidly was complicated and indicated that other factors might also be involved.
Activation and repression of the yeast ARO3 gene by global transcription factors
Authors: Künzler Markus , Springer Christoph , Braus Gerhard H. 
Published: October 2006

SummaryThe ARO3 gene of Saccharomyces cerevisiae codes for the phenylalanine‐inhibited 3‐deoxy‐d‐arabino‐heptulosonate‐7‐phosphate synthase (EC 4.1.2.15) and is regulated by the general control system of amino acid biosynthesis through a single GCN4‐binding site in its promoter. A combined deletion and mutation analysis of the ARO3 promoter region in a δgcn4‐background revealed two additional regulatory systems involved in ARO3 transcription. The ARO3 gene is (i) activated through a sequence element which binds the multifunctional DNA‐binding protein ABF1 in vitro and (ii) repressed through an URS1 element, which binds the same protein in vitro as the URS1 element In the CAR1 promoter. Since both the ABF1‐binding site and the URS1 element represent cis‐acting elements of global transcription regulatory systems in yeast, the ARO3 gene is the first example of a GCN4‐regulated gene which is both activated and repressed by global transcription factors. Activation of the ARO3 gene through the ABF1‐binding site and repression through the URS1 element seem to be independent of each other and independent of activation by the GCN4 protein.
Plasmid evolution by acquisition of mobile gene cassettes: plasmid pIE723 contains the aadB gene cassette precisely inserted at a secondary site in the IncQ plasmid RSF1010
Authors: Recchia Gavin D. , Hall Ruth M. 
Published: October 2006

SummaryGene cassettes are mobile DNA elements which contain a specific recombination site, a 59‐base element, recognized by the site‐specific recombination system of integrons. Gene cassettes are normally found inserted at a unique site in an integron, downstream of a promoter which directs transcription of the cassette‐associated genes. However, insertion of a gene cassette into a secondary site in a plasmid which does not contain an integron is also formally possible. Sequence analysis of the aadB gene in pIE723, a plasmid closely related to the IncQ plasmid RSF1010, revealed the presence of the complete aadB cassette inserted at a secondary site downstream of a known RSF1010 promoter. The site of insertion of the aadB cassette in RSF1010 conformed to the consensus for secondary sites recognized by the integron integrase (int), and it is likely that the cassette was inserted via a single Int‐mediated recombination event between the 59‐base element of a free, circular aadB cassette and a secondary site in RSF1010. The cassette‐associated recombination site was inactivated by the insertion, and Int‐mediated excision of the aadB cassette from this non‐specific location was not detectable, indicating that the cassette is stably inserted. The movement of gene cassettes to secondary sites is likely to play an important role in the acquisition of new genes by bacterial and plasmid genomes.
Virulence studies on chromosomal α‐toxin and Θ‐toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of α‐toxin in Clostridium perfringens‐mediated gas gangrene
Authors: Awad Miilena M. , Bryant Amy E. , Stevens Dennis L. , Rood Julian I. 
Published: October 2006

SummaryThe pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin‐resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the‘chromosomal pfoA gene, which encodes an oxygen‐labile haemolysin known as Θ‐toxin or perfringolysin O. and in the chromosomal pic gene, which encodes the α‐toxin or phospholipase C. The resultant mutants failed to produce detectable Θ‐toxin or α‐toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild‐type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the pic mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of α‐toxin in gas gangrene or clostridial myonecrosis.
Differential expression of two closely related deoxyribonuclease genes, nucA and nucB, in Bacillus subtilis
Authors: van Sinderen Douwe , Kiewiet Rense , Venema Gerard 
Published: October 2006

SummaryDespite the lack of involvement of the competence‐specific, membrane‐associated deoxyribonuclease (DNase) in competence development, the expression of the gene encoding this protein, nucA, was shown to be dependent on the competence signal transduction pathway, and in particular on ComK, the competence transcription factor, which was shown to bind to the DNA region upstream of nucA. The expression of nucB, specifying an extracellular DNase, which was cloned on the basis of its homology to nucA, was shown to be sporulation‐specific and dependent on the gene products of spo0A and spollG, the latter constituting an operon responsible for the synthesis of the mother‐cell‐specific sigma factor σE. The observed differential expression of nucA and nucB demarcates the appearance of DNase activities which are either associated with the cytoplasmic membrane or secreted into the medium during different post‐exponential growth‐phase processes.
A recognition component of the ubiquitin system is required for peptide transport in Saccharomyces cerevisiae
Authors: Alagramam Kumar , Naider Fred , Becker Jeffrey M. 
Published: October 2006

SummaryPeptide transport in Saccharomyces cerevisiae is controlled by three genes: PTR1, PTR2, and PTR3. PTR1 was cloned and sequenced and found to be identical to UBR1, a gene previously described as encoding the recognition component of the N‐end‐rule pathway of the ubiquitin‐dependent proteolytic system. Independently derived ubr1 mutants, like ptr1 mutants, were unable to transport small peptides into ceils. Concomitantly, ptr1 mutants, like ubr1 mutants, were unable to degrade an engineered substrate of the N‐end‐rule pathway. Further, ptr1 mutants did not express PTR2, a gene encoding the integral membrane component required for peptide transport in S. cerevisiae. These results establish a physiological role for a protein previously known to be required for the degradation of N‐end‐rule substrates. Our findings show that peptide transport and the ubiquitin pathway—two dynamic phenomena universal to eukaryotic cells—share a common component, namely UBR1/PTR1.
The pho regulon of Shigella flexneri
Authors: Scholten Monica , Janssen Riny , Bogaarts Corné , van Strien Joost , Tommassen Jan 
Published: October 2006

SummaryGrowth of Escherichia coli K‐12 in low‐phosphate conditions results in the induction of the synthesis of many proteins, including the outer membrane porin PhoE, alkaline phosphatase, and the Pst system for the transport of phosphate (Pi). This response is controlled by a two‐component regulatory system of which PhoB and PhoR are the response‐regulator and the sensor/kinase, respectively. When Shigella flexneri was starved for Pi, neither PhoE nor alkaline phosphatase was produced. However, induction of the synthesis of the PstS protein was observed, indicating that S. flexneri contains a functional PhoB/PhoR regulatory system. Consistent with this notion, the introduction of the B. coli phoA gene in S. flexneri resulted in the induction of alkaline phosphatase synthesis under phosphate limitation. However, introduction of phoE on a plasmid did not lead to the expression of PhoE protein, indicating that S. flexneri PhoB does not recognize the phoE promoter region. The phoB gene was cloned and sequenced and in the deduced amino acid sequence two deviations from that of E. coli PhoB were detected. Site‐directed mutagenesis revealed that one of these deviations, i.e. Leu‐172, which is Arg in E. coli PhoB, is responsible for the lack of expression of the PhoE protein in S. flexneri.
Construction of recombinant neisserial Hsp60 proteins and mapping of antigenic domains
Authors: Pannekoek Yvonne , Dankert Jacob , van Putten Jos P. M. 
Published: October 2006

SummaryHere we report the cloning and expression, in Escherichia coli, of PCR‐amplified DNA encoding the 63‐kDa stress‐inducible protein of Neisseria gonorrhoeae strains VP1 and PiD2, Neisseria meningitidis 2996 and the commensal Neisseria flavescens. DNA sequence analysis revealed in all cases one open reading frame of 541‐544 amino acids corresponding to a protein of approximately 57 000 Da. The various neisserial proteins were >98% identical at the amino acid level and showed extensive homology with proteins belonging to the HspSO heat‐shock‐protein family. We constructed defined glutathione S‐transferase fusion polypeptides of the gonococcal Hsp60 homologue to locate antigenic domains on the recombinant protein. Variation in the immunoreactivity of two monoclonal antibodies recognizing a conserved and a neisseria‐unique antigenic Hsp60 determinant, respectively, could thus be deduced to result from single amino acid substitutions. Analysis of the antibody response in patients’sera demonstrated reactivity with the same fusion polypeptides in six out of nine sera, indicating that neisserial Hsp60 is expressed during the natural infection and that distinct domains on the protein are immunodominant in vivo.
Cloning and sequence analysis of cycH gene from Paracoccus denitrificans: the cycH gene product n required for assembly of all c‐type cytochromes, including cytochrome c1
Authors: Page M. Dudley , Ferguson Stuart J. 
Published: October 2006

SummaryA transposon Tn5 mutant of Paracoccus denitrificans, DP108, was incapable of anaerobic or methylotrophic growth and scored negative in the Nadi cytochrome c oxidase test. P. denitrificans DP108 grown aerobically on succinate or choline was devoid of soluble c‐type cytochromes and accumulated periplasmic apocytochrome C550, but the membrane‐bound holocytochromes c1 and C552 were present at 5‐10% of the levels observed in wild‐type ceils, DP108 genomic DNA flanking the site of Tn5 insertion was cloned by marker rescue and used to probe a P. denitrificans wild‐type DNA library. A hybridizing 3.05 kb Bam HI fragment capable of complementing the DP108 mutation was isolated and a 2.05 kb region of this was sequenced. One major open reading frame equivalent to 413 amino acids was identified, the predicted product of which was similar (33% identity, 55% similarity) to the predicted product of the cycH gene previously identified in Bradyrhizobium japonicum. Similarity of the two cycH gene products to the predicted products of two Escherichia coli genes, nrfG and yejP, was also detected. Significant differences between the phenotypes of P. denitrificans DPI08 and the B. japonicum cycH mutant C0X3, especially with respect to cytochrome c1 synthesis, suggest that the cycH gene product may be an assembly factor.
Sequence analysis of subunits of the membrane‐bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron‐carrying arm of a redox loop
Authors: Berks Ben C. , Page M. Dudley , Richardson David J. , Reilly Ann , Cavill Alan , Outen Fenno , Ferguson Stuart J. 
Published: October 2006

SummaryThree genes, narH, narJ and narl of the membrane‐bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (β, putative δ and γ) subunits of the two membrane‐bound nitrate reductases of the enteric bacterium Escherichia coli. AU iron‐sulphur cluster ligands proposed for the E. coliβ subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly α‐helical structure. Comparison of T. pantotropha Narl wilh the b‐haembinding integral membrane subunits of the E. coli enzymes allows assignment of His‐53, His‐63, His‐186 and His‐204 (T. pantotropha Narl numbering) as b‐haem axial ligands and the construction of a three‐dimensional model of this subunit. This model, in which the two b‐haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane‐bound uptake hydrogenases and membrane‐bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate‐reducing bacteria has structural resemblance to Narl.
Coevolution of RNA helix stability and Shine‐Dalgarno complementarity in a translational start region
Authors: Olsthoorn René C. L. , Zoog Stephen , van Duin Jan 
Published: October 2006

SummaryThe initiation legion of the coat‐protein gene of RNA bacteriophage MS2 adopts a well‐defined hairpin structure with the start codon occupying the loop position, while the Shine‐Dalgarno (SD) sequence is part of the stem. In a previous study, we introduced mutations in this hairpin that changed its thermo dynamic stability. The resulting phages evolved to regain the wild‐type stability by second‐site compensatory substitutions. Neither the original nor the suppressor mutations were in the SD region. In the present analysis, we have made changes in the SD region that shorten or extend its complementarity to the 3 end of 16S rRNA and monitored their evolution to a stable pseudorevertant species. Phages in which the SD complementarity was decreased evolved an initiator hairpin of lower stability than wild type while those in which the complementarity was extended evolved a hairpin with an increased stability. We conclude that weaker SD sequences still allow maximal translation if the secondary structure of the ribosome‐landing site is destabilized accordingly. Alternatively, translation‐initiation regions with a stronger secondary structure still allow maximal expression, if the SD complementarity is extended. These findings support a previously published model in which the SD Interaction helps the ribosome to melt the structure in a translation‐initiation region.
Characterization of RNA polymerse and two sigma‐factor genes from Mycobacterium smegmatis
Authors: Predich Mima , Doukhan Laurence , Nair Gopalan , Smith Issar 
Published: October 2006

SummaryA search for Mycobacterium smegmatis genes showing similarity to the conserved family encoding major Sigma factors in diverse prokaryotes has identified two such determinants. Both genes are expressed in exponentially growing cells, as judged by Western immunoassays. A series of chromatographic steps was used to purify M. smegmatis RNA polymerase holoenzyme and it was shown that its ability to initiate in vitro transcription with a heterologous Bacillus subtilis promoter is dependent on the presence of these Sigma factor(s). Reconstitution of specific in vitro transcription activity was obtained upon mixing of M. smegmatis core RNA polymerase with the major Sigma factor of Bacillus subtilis. We also demonstrated in vitro transcription of the M. smegmatis rrnB promoter by the M. smegmatis RNA polymerase. Significantly, highly active B. subtilis RNA polymerase holoenzyme was unable to transcribe this gene.
Structure‐function analysis of the vitamin B12 receptor of Escherichia coli by means of informational suppression
Authors: Hufton Simon E. , Ward Richard J. , Bunce Nigel A. C. , Armstrong John T. , Fletcher Anthony J. P. , Glass Robert E. 
Published: October 2006

SummaryWe describe a genetic analysis of the vitamin B12 receptor of Escherichia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site. We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene. Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5‐like bacteriophage BF23, and vitamin B12 (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor completely non‐functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz, Q62, Q150 and Q299 and the response to phage BF23). These data are summarized on a two‐dimensional folding model for the BtuB protein in the outer membrane (devised using both amphipathic β‐strand analysis and sequence conservation amongst the TonB‐dependent receptors). In addition, we report that the extreme C‐terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane‐spanning β‐sheet with residue L588 situated on its hydrophobic surface. Two of the C‐terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.