bluuebunny/crossref_metadata_2025_split huggingface dataset:
A copper‐transporting P‐type ATPase found in the thylakoid membrane of the cyanobacterium Synechococcus species PCC7942
Authors: Kanamaru Kengo , Kashiwagi Seiji , Mizuno Takeshi 
Published: October 2006

SummaryP‐type ATPases constitute a large family of cation pumps that play crucial physiological roles in many organisms, including bacteria, plants and mammals. They are postulated to play important roles in a variety of environmental adaptation systems. Recently, we cloned two distinct putative P‐type ATPase genes (pacS and pacL) from a photosynthetic cyanobacterium, Synechococcus species PCC7942. in this study, one of the gene products (named PacS) was found to possess a putative metal‐binding motif (Gly‐Met‐X‐Cys‐X‐X‐Cys) in its N‐terminal portion. Thus we supposed that this ATPase may function as a metal pump. Indeed, the results of Northern blotting analysis showed that pacS‐mRNA specifically increases upon addition of copper or silver to the growth medium. The results of Western blotting analysis confirmed the view that PacS accumulates in copper‐treated Synechococcus cells. Thus we concluded that the expression of PacS ATPase is regulated in response to the change in concentration of external metals, namely copper and silver. Consistent with this, an insertional inactivation mutant of pacS exhibited hypersensitivity in terms of growth to these potentially toxic metals, it was also revealed that PacS was mainly located in the thylakoid membrane, in which the photosynthetic reactions take place. This P‐type ATPase in the thylakoid membrane is implicated as a copper‐transporting system that may be involved in copper‐homeostasis crucial to the photosynthetic thylakoid function.
Identification of a second oligopeptide transport system in Bacillus subtilis and determination of its role in sporulation
Authors: Koide Akiko , Hoch James A. 
Published: October 2006

SummarySporulation in Bacillus subtilis depends on an intact oligopeptide transport system, the Opp system. Mutants in opp sporulate poorly but second‐site revertants can be found that restore sporulation and pep‐tide transport. These second‐site mutations were found in a second oligopeptide transport system, app, in which the peptide‐binding protein, AppA, is mutant owing to a frame‐shift mutation, and the revertants restore the original frame. The AppA mutation is present in the 168 strain of B. subtilis. The app operon consists of five genes in the order appD‐appF‐appA‐appB‐appC, with the locus designations corresponding to their homologue in the opp operon. Homology between the app and opp proteins ranges from 54% identity for AppF and OppF, to 22% identity for AppA and OppA. Both the App and Opp permease systems can transport tetra‐ and pentapeptides, but tripeptides are not transported by the App system. Strains of the genotype app+opp− are resistant to the tripeptide antibiotic bialaphos. The repaired App system can substitute completely for the Opp system in both sporulation and competence for genetic transformation. The pheno‐types raised some speculation about the subunit configuration of the Opp system.
EGTA induces the synthesis in Escherichia coli of three proteins that cross‐react with calmodulin antibodies
Authors: Laoudj Dalila , Andersen Catherine L. , Bras Ana , Goldberg Martin , Jacq Annick , Holland I. Barry 
Published: October 2006

Summary Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1‐D and 2‐D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat‐ or SDS‐treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of calmodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross‐reacted with polyclonal and monoclonal antibodies to eukaryote calmodulins or calerythrin, a heat‐resistant Ca2+‐binding protein from Saccharo‐polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+.
A dominant mutation in the bacteriophage lambda S gene causes premature lysis and an absolute defective plating phenotype
Authors: Johnson‐Boaz Rebecca , Chang Chung‐Yu , Young Ry 
Published: October 2006

SummaryThe S and R genes of the bacteriophage λ are required for lysis of the host. R encodes ‘endolysin’, a soluble transglycosylase which accumulates in the cytoplasm during late protein synthesis. S encodes a ‘holin’, a small membrane protein which, at a precisely scheduled time, terminates the vegetative cycle by forming a lethal lesion in the membrane through which gpR gains access to the peptidoglycan. A missense allele of S, Ala52Gly, causes lysis to occur prematurely at about 19–20 min after induction of a lysogen, compared to 45min for the wild type. This allele has a severe plaque‐forming defect which appears to be entirely a consequence of the early lysis and resultant severe reduction in particle burst size. The early‐lysis phenotype is dominant and is aggravated, in terms of an even more reduced burst size, at both 30°C and 42°C. The mutation maps in the middle of a putative membrane‐spanning helical domain of S, near the sites of other S− mutations with recessive non‐lytic phenotypes. The mutation has no effect on S‐protein accumulation or on the ratio of S107 and S105 products in the membrane. The mutation appears to affect the intrinsic timing function by which the S protein controls the lysis schedule.
Domain structure and conserved epitopes of Sfb protein, the fibronectin‐binding adhesin of Streptococcus pyogenes
Authors: Talay Susanne R. , Valentin‐Weigand Peter , Timmis Kenneth N. , Chhatwal Gursharan S. 
Published: October 2006

Summary Streptococcus pyogenes expresses a fibronectin‐binding surface protein (Sfb protein) which mediates adherence to human epithelial cells. The nucleotide sequence of the sfb gene was determined and the primary sequence of the Sfb protein was analysed. The protein consists of 638 amino acids and comprises five structurally distinct domains. The protein starts with an N‐terminal signal peptide followed by an aromatic domain. The central part of the protein is formed by four proline‐rich repeats which are flanked by non‐repetitive spacer sequences. A second repeat region, consisting of four repeats that are distinct from the proline repeats and have been shown to form the fibronectin‐binding domain, is located in the Cterminal part of the protein. The protein ends with a typical cell wall and membrane anchor region. Comparative sequence analysis of the N‐terminal aromatic domain revealed similarities with carbohydrate‐binding sites of other proteins. The proline repeat region of the Sfb protein shares characteristic features with proline‐rich repeats of functionally distinct surface proteins from pathogenic Gram‐positive cocci. Immunoelectron microscopy revealed an even distribution of the fibronectin‐binding domain of Sfb protein on the surface of streptococcal cells. Analyses of 38 sfb genes originating from different S. pyogenes isolates revealed primary sequence variability in regions coding for the N‐termini of mature Sfb proteins, whereas sequences coding for the central and C‐terminal repeats were highly conserved. The repeat sequences are postulated to act as target sites for intragenic recombination events that result in variable numbers of repeats within the different sfb genes. A model of the Sfb protein is presented.
Vitamin B12 repression of the cob operon in Salmonella typhimurium: translational control of the cbiA gene
Authors: Richter‐Dahlfors A. A. , Ravnum S. , Andersson D. I. 
Published: October 2006

SummaryExpression of the cob operon is repressed by B12 via a post‐transcriptional control mechanism which requires sequence elements within the leader region of the mRNA and the first gene of the operon, the cbiA gene. Here we show that B12 repression of cbiA gene expression occurs at the level of translation initiation through sequestration of the ribosomal binding site (rbs) in an RNA hairpin. Analysis of mutations that destabilize or restabilize the secondary structure demonstrates that folding of the hairpin is essential for repression. The existence of the hairpin was confirmed by a secondary structure analysis of RNA from the wild type and three mutants. Deletions that remove the upstream part of the leader confer a drastic reduction in translation efficiency. This low‐level translation is caused by the hairpin, as indicated by the finding that suppressor mutations that destabilize the hairpin restore efficient translation. Thus, the native upstream RNA functions as a translation enhancer and acts to relieve the hairpin's inhibitory effect on translation initiation. The inhibitory effect of the hairpin was confirmed by a ribosomal toeprinting analysis. We propose that the translational control of the cbiA gene mediates repression of the entire cob operon.
The Salmonella typhimurium invasion genes invF and invG encode homologues of the AraC and PulD family of proteins
Authors: Kaniga Koné , Bossio Juan C. , Galán Jorge E. 
Published: October 2006

SummaryWe have identified two novel Salmonella typhimurium genes, invF and invG, which are required for the efficient entry of these organisms into cultured epithelial cells. invF and invG are located immediately upstream of invE, a previously identified gene also required for Salmonella entry. Non‐polar mutations in these genes rendered S. typhimurium severely deficient for entry into cultured epithelial cells. The nucleotide sequences of invF and invG indicated that these genes encode polypeptides with predicted molecular weights of 24373 and 62275, respectively. Proteins of similar sizes were observed when invF and invG were expressed in a bacteriophage T7 RNA polymerase‐based expression system. Comparison of the predicted sequence of InvF with translated sequences in the existing databases indicated that this protein is homologous to members of the AraC family of prokaryotic transcription regulators. However, mutations in invF did not significantly affect the expression of other members of the inv locus. InvG was found to be homologous to members of the PuID family of specialized translocases. This homology suggests that InvG may be necessary for the export of invasion‐related determinants or involved in the assembly of a supramolecular structure that promotes entry.
Mutational analysis of the arginine repressor of Escherichia coli
Authors: Tian Guoling , Maas Werner K. 
Published: October 2006

SummaryArginine biosynthesis in Escherichia coli is negatively regulated by a hexameric repressor protein, encoded by the gene argR and the corepressor arginine. By hydroxylamine mutagenesis two types of argR mutants were isolated and mapped. The first type is transdominant. In heterodiploids, these mutant polypeptides reduce the activity of the wild‐type repressor, presumably by forming heteropolymers. Four mutant repressor proteins were purified. Two of these map in the N‐terminal half of the protein. Gel retardation experiments showed that they bind poorly to DNA, but they could be precipitated by l‐arginine at the same concentration as the wild‐type repressor. The other two mutant repressors map in the C‐terminal half of the protein. They are poorly precipitated by L‐arginine and they bind poorly to DNA. In addition, one of these mutants appears to exist as a dimer. The second type of argR mutant repressor consists of super‐repressors. Such mutants behave as arginine auxotrophs as a result of hyper‐repression of arginine biosynthetic enzymes. They map at many locations throughout the argR gene. Three arginine super‐repressor proteins were purified, in comparison with the wild‐type repressor, two of them were shown to have a higher DNA‐binding affinity in the absence of bound arginine, while the third was shown to have a higher DNA‐binding affinity when bound to arginine.
Partial suppression of an Escherichia coli TonB transmembrane domain mutation (ΔV17) by a missense mutation in ExbB
Authors: Larsen Ray A. , Thomas Michael G. , Wood Gwendolyn E. , Postle Kathleen 
Published: October 2006

SummaryActive transport of vitamin B12 and Fe(III)‐siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane‐generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the amino‐terminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The ΔV17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonBΔV17. Molecular modeling suggested that the ΔV17 mutation caused a significant change in the predicted conserved face of the TonB amino‐terminal membrane anchor. TonBΔV17 was unable to achieve the 23 kDa proteinase K‐resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild‐type TonB also failed to achieve the proteinase K‐resistant configuration when ExbB was absent. Taken together these results suggested that the ΔV17 mutation interrupted productive TonB–ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K‐resistant form of TonBΔV17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonBΔV17 export. Unlike the tonBΔV17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild‐type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonBδV17, ExbBA39E pair resulted in a TonB with a greatly reduced half‐life (≅ 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.
Fluctuations in glycolytic mRNA levels during morphogenesis in Candida albicans reflect underlying changes in growth and are not a response to cellular dimorphism
Authors: Swoboda R. K. , Bertram G. , Delbrück S. , Ernst J. F. , Gow N. A. R. , Gooday G. W. , Brown A. J. P. 
Published: October 2006

SummaryThe levels of pyruvate kinase (PYK), alcohol dehydrogenase (ADH1), phosphoglycerate kinase (PGK1) and phosphoglycerate mutase (GPM1) mRNAs were measured during batch growth and during the yeast‐to‐hyphal transition in Candida albicans. The four mRNAs behaved in a similar fashion. PYK1, ADH1, PGK1 and GPM1 mRNA levels were shown to increase dramatically during the exponential growth phase of the yeast form, and then to decrease to relatively low levels in the stationary phase. The dimorphic transition was induced using two sets of conditions: (i) an increase in temperature (from 25°C to 37°C) combined with the addition of serum to the medium; and (ii) an increase in temperature (from 25°C to 37°C) and an increase in pH of the growth medium (from pH 4.5 to pH 6.5). Additional cultures were analysed to control for the addition of serum, and for changes in temperature or pH. Immediately following dilution of late‐exponential cells into fresh media the levels of all four glycolytic mRNAs decreased rapidly in contrast to the ACT1 mRNA control, the level of which increased under most conditions. The recovery of glycolytic mRNA levels depended on the culture conditions, but there was no direct correlation with the formation of germ tubes, with the addition of serum to the medium, the Increase in culture temperature, the medium pH, or the glucose concentration. This indicates that the changes in glycolytic gene expression that accompany the dimorphic transition in C. albicans reflect the underlying physiological status of the cells during morphogenesis and not alterations to cell shape.
The pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2
Authors: Fetherston Jacqueline D. , Perry Robert D. 
Published: October 2006

SummaryThe pigmentation (Pgm+) phenotype of Yersinia pestis includes a number of different characteristics which appear to be associated with a 102 kb segment of chromosomal DNA known as the pgm locus. In Y. pestis KIM6+, the pgm locus is flanked by direct copies of a repeated element that probably plays a role in the spontaneous deletion of this region. We have sequenced the ends of these elements and shown that they have features in common with bacterial insertion sequences, in addition we show that a clone, pSDR498, from the pgm locus of KIM6+ restores pesticin sensitivity and the iron‐regulated expression of three polypeptides, 240 kDa, 190 kDa, and 68 kDa in size, to Pgm− cells. In vitro transcription/translation assays and Escherichia coli minicells were used to analyse the products encoded by various subciones of pSDR498. Pesticin sensitivity mapped to a 5.9 kb fragment that encodes a 68 kDa protein derived from a 72 kDa precursor. Synthesis of the 190 kDa protein was restored by a 19.2 kb clone, indicating that the structural gene for this protein also resides within the pgm locus of Y. pestis KIM6+. Finally, a survey of our pgm− strains indicates that 97% have also deleted the sequences encoding the 190 kDa protein and pesticin sensitivity.
Molecular characterization of upstream regulatory sequences controlling the photoinduced expression of the albino‐3 gene of Neurospora crassa
Authors: Carattoli Alessandra , Cogoni Carlo , Morelli Glorgio , Macino Giuseppe 
Published: October 2006

SummaryIn the filamentous fungus Neurospora crassa the biosynthesis of carotenoids is regulated by blue light, principally through transcriptional activation of some key genes in the carotenogenic enzymatic pathway. Here we report the characterization of the photoinducible promoter of the albino‐3 (al‐3) gene, encoding GGPP synthase. We have modified the 5’non‐coding sequence of the cloned al‐3 gene by deletion and site‐directed mutagenesis, and we have tested the residual photoinducibility of the different constructs by transformation and subsequent analysis of gene expression in dark‐grown and light‐induced mycelia. The results indicate that a promoter region between positions −226 and −55 contains all the necessary information for blue light photoinduction. Multiple regulatory elements are involved in the regulated expression of the al‐3 gene. One (termed the APE element) is important for the light‐induction switch on of the gene and a second belongs to the CCAAT boxes family. The putative APE element is also found in the promoter of other N. crassa photoinducible genes.
Residue threonine‐149 of the Salmonella typhimurium CysB transcription activator: mutations causing constitutive expression of positively regulated genes of the cysteine regulon
Authors: Colyer Tracey E. , Kredich Nicholas M. 
Published: October 2006

SummaryIn both Salmonella typhimurium and Escherichia coli, CysB is a LysR family transcriptional activator, which regulates genes of the cysteine regulon. Transcription activation of cys genes also requires an inducer, W‐acetyl‐L‐serine, and cysB mutants that do not require Inducer are termed constitutive, i.e. cysBc. After finding that two independently isolated cysBc mutants are substituted at amino acid residue threonine‐149 (T149), we isolated the other 17 single‐amino‐acid substitutions by site‐directed mutagenesis. Of the 19 mutant alleles, 11 supported normal growth on sulphate, and nine of these were cysBc. Four other mutants were‘leaky’cysB+, and four were cysB−. Insertions of up to 14 amino acids were also tolerated at T149, and two of three such mutants were cysBc. An allele containing a TAG translation terminator at codon 149 had no detectable function in a δcysB strain, but gave a constitutive phenotype when introduced into either wild‐type S. typhimurium or the E. coll strain NK1, which contains a cysB− mutation in a predicted helix‐turn‐helix region that interferes with specific binding of CysB to DNA and with autoregulation ot cysB. The peptide encoded by the T149ter allele is proposed to interact with the wild‐type CysB peptide or with the NK1 mutant peptide to form hetero‐oligomers that do not require N‐acetyl‐L‐serine for cys gene activation.
The novel lectin‐like protein CHB1 is encoded by a chitin‐inducible Streptomyces olivaceoviridis gene and binds specifically to crystalline α‐chitin of fungi and other organisms
Authors: Schnellmann Jürgen , Zeltins Andris , Blaak Harald , Schrempf Hildgund 
Published: October 2006

SummaryThe chb1 gene, which encodes the unique lectin‐like α‐chitin‐binding protein CHB1 of Streptomyces olivaceoviridis, was cloned. Transformants of Streptomyces lividans harbouring the plasmid pCHB10 overproduced the extracellular CHB1 protein; the protein showed neither enzymatic nor antifungal activity. Biochemical analyses and immunofluorescence microscopy indicated that CHB1 binds strongly to α‐chitin, but neither to chitosan and β‐chitin, nor to various types of cellulose. Within hyphae of fungi, the relative location of crystalline chitin was visualized with fluor‐escein‐labelled CHB1. These studies suggest that the new protein could serve as a tool to identify α‐chitin within different organisms. The chb1 gene consists of a reading frame of 603 bp and its transcription occurred only if the Streptomyces strain was cultivated with chitin as the sole carbon source. The deduced mature CHB1 protein (18.7 kDa) shows no apparent similarity to any known protein. Within a region containing 100 residues of the deduced CHB1 protein, four tryptophan and two asparagine residues as well as one glycine and one cysteine residue were identified, the relative positions of which are analogous to those of several cellulose‐binding domains of bacterial glycohydrolases. The results of spectroscopical studies suggest a possible involvement of tryptophan residues in the interaction of CHB1 with α‐chitin.