bluuebunny/crossref_metadata_2025_split huggingface dataset:
mRNA sequences influencing translation and the selection of AUG initiator codons in the yeast Saccharomyces cerevisiae
Authors: Yun Ding‐Fang , Laz Thomas M. , Clements John M. , Sherman Fred 
Published: October 2006

SummaryThe secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1‐Δ disruption, which stabilized mRNAs that had premature termination codons, resulting in wild‐type levels. The following major conclusions were reached by measuring β‐galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide‐long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position −3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A>G>C>U. Upstream out‐of‐frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (−3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.
Generation of Campylobacter fetus S‐layer protein diversity utilizes a single promoter on an invertible DNA segment
Authors: Dworkin Joel , Blaser Martin J. 
Published: October 2006

SummaryWild‐type strains of Campylobacter fetus contain a monomolecular array of surface layer proteins (SLPs) and vary the antigenicity of the predominant SLP expressed. Reciprocal recombination events among the eight genomic SLP gene cassettes, which encode 97‐ to 149 kDa SLPs, permit this variation. To explore whether SLP expression utilizes a single promoter, we created mutant bacterial strains using insertional mutagenesis by rescue of a marker from plasmids. Experimental analysis of the mutants created clearly indicates that SLP expression solely utilizes the single sapA promoter, and that for variation C. fetus uses a mechanism of DNA rearrangement involving inversion of a 6.2 kb segment of DNA containing this promoter. This DNA inversion positions the sapA promoter immediately upstream of one of two oppositely oriented SLP gene cassettes, leading to its expression. Additionally, a second mechanism of DNA rearrangement occurs to replace at least one of the two SLP gene cassettes bracketing the invertible element. As previously reported promoter inversions in prokaryotes, yeasts and viruses involve alternate expression of at most two structural genes, the ability of C. fetus to use this phenomenon to express one of multiple cassettes is novel.
Identification and characterization of a novel type of replication terminator with bidirectional activity on the Bacillus subtilis theta plasmid pLS20
Authors: Meijer Wilfried J. J. , Smith Mark , Wake R. Gerry , de Boer Arjo L. , Venema Gerard , Bron Sierd 
Published: October 2006

Summary We have sequenced and analysed a 3.1 kb fragment of the 55 kb endogenous Bacillus subtilis plasmid pLS20 containing its replication functions. Just outside the region required for autonomous replication, a segment of 18bp was identified as being almost identical to part of the major B. subtilis chromosomal replication terminator. Here, we demonstrate that this segment is part of a functional replication terminator. This newly identified element, designated Ter LS20, is the first replication terminator identified on a theta plasmid from a Gram‐positive bacterium. Ter LS20 is distinct from other known replication terminators in the sense that it is functional in both orientations. The region required for bipolar functionality of TerLS20 was delineated to a sequence of 29 bp, which is characterized by an imperfect dyad symmetry.
The Escherichia coli ribosomal protein S16 is an endonuclease
Authors: Oberto Jacques , Bonnefoy Eliette , Mouray Elisabeth , Pellegrini Olivier , Wikström P. Mikael , Rouvière‐Yaniv Josette 
Published: October 2006

Summary The histone‐like protein HU isolated from Escherichia coli exhibited, after several purification steps, a Mg2+‐dependent nuclease activity. We show here that this activity can be dissociated from HU by a denaturation‐renaturation step, and is due to a small fraction of ribosomal protein S16 co‐purifying with HU. S16 is an essential component of the 30S ribosomal particles. We have cloned, overproduced, and purified a histidine‐tagged S16 and shown that this protein is a DNA‐binding protein carrying a Mg2+‐Mn2+‐dependent endonuclease activity. This is an unexpected property for a ribosomal protein.
Tetrapac (tpc), a novel genotype of Neisseria gonorrhoeae affecting epithelial cell invasion, natural transformation competence and cell separation
Authors: Fussenegger Martin , Kahrs Andreas F. , Facius Dirk , Meyer Thomas F. 
Published: October 2006

Summary We characterized a novel mutant phenotype (tetrapac, tpc) of Neisseria gonorrhoeae (Ngo) associated with a distinctive rough‐colony morphology and bacterial growth in clusters of four. This phenotype, suggesting a defect in cell division, was isolated from a mutant library of Ngo MS11 generated with the phoA minitransposon TnMax4. The tpc mutant shows a 30% reduction in the overall murein hydrolase activity using Escherichia coli murein as substrate. Tetrapacs can be resolved by co‐cultivation with wild‐type Ngo, indicating that Tpc is a diffusible protein. Interestingly, Tpc is absolutely required for the natural transformation competence of piliated Ngo. Mutants in tpc grow normally, but show a ∼ 10‐fold reduction in their ability to invade human epithelial cells. The tpc sequence reveals an open reading frame of ∼1 kb encoding a protein (Tpc) of 37kDa. The primary gene product exhibits an N‐terminal leader sequence typical of lipoproteins, but palmitoylation of Tpc could not be demonstrated. The ribosomal binding site of tpc is immediately downstream of the translational stop codon of the folC gene coding for an enzyme involved in folic acid biosynthesis and one‐carbon metabolism. The tpc gene is probably co‐transcribed from the folC promoter and a promoter located within the folC gene. The latter promoter sequence shares significant homology with E. coli gearbox consensus promoters. All three mutant phenotypes, i.e. the cell separation defect, the transformation deficiency and the defect in cell invasion can be restored by complementation of the mutant with an intact tpc gene. To some extent the tcp phenotype is reminiscent of iap in Listeria, lytA in Streptococcus pneumoniae and lyt in Bacillus subtilis, all of which are considered to represent murein hydrolase defects.
Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation
Authors: Gentry Daniel R. , Cashel Michael 
Published: October 2006

SummaryThe spoT gene of Escherichia coli encodes a guanosine 3′,5′‐bis(diphosphate) 3′‐pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3′,5′‐bis(diphosphate) synthetase (designated PSII). To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid‐borne deletion mutations for their ability to complement chromosomal mutations defective in each activity. We found that a region containing the first 203 amino acids of the 702‐amino‐acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67–374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein. A ppGppase‐defective Δ1–58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild‐type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation. We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.
Integration of SecA protein into the Escherichia coli inner membrane is regulated by its amino‐terminal ATP‐binding domain
Authors: Rajapandi Thavamani , Oliver Donald 
Published: October 2006

SummarySecA protein, the ATPase promoting translocation of proteins across the Escherichia coli inner membrane, contains two ATP‐binding domains that differ greatly in their affinity for bound nucleotide. In order to define more precisely the location of the high‐affinity nucleotide‐binding site, oligonucleotide‐directed mutagenesis was used to introduce cysteine residues into the SecA sequence, and a cysteine‐specific cleavage reagent was employed to generate defined peptides of SecA protein after photocross‐linking with [α‐32P]‐ATP. This analysis revealed that the nucleotide was cross‐linked between amino acid residues 75 and 97 of SecA protein. The biochemical function of the high affinity ATP‐binding domain was explored by subcellular fractionation studies which demonstrated that SecA proteins defective in this region were found almost exclusively in their integral membrane form, while SecA proteins with defects in the low‐affinity ATP‐domain showed a normal distribution of cytosolic, peripheral and integral membrane forms. Interestingly, the SecA51(Ts) protein that has a Leu to Pro substitution at amino acid residue 43 bound ATP with high affinity, but its fractionation pattern and translocation ATPase activity were similar to those of proteins with defects in the high‐affinity ATP‐binding site. These results delimit more precisely the high‐affinity ATP‐binding domain of SecA, indicate the importance of the early amino‐terminal region of SecA protein in the functioning of this domain, and demonstrate the role of this domain in regulating penetration of SecA protein into the inner membrane. Our results lead to a simple model for the regulation of a cycle of SecA insertion into, and de‐insertion from, the inner membrane by the activity of the high‐affinity ATP‐binding domain.
Purification and characterization of a histone‐like protein from the Archaeal isolate AN1, a member of the Thermococcales
Authors: Ronimus Ron S. , Musgrave David R. 
Published: October 2006

SummaryWe have purified and characterized the histone‐like protein, termed HAN1, and an HAN1‐associated DNA‐binding protein (hDBP) from nucleoids of the hyper‐thermophilic Thermococcus‐like AN1. HAN1 is shown to be composed of two subunits, to be thermally stable and to compact DNA in a reversible manner. The N‐terminal sequence of HAN1 shares a high degree of homology with HMf, the histone‐like protein from Methanothermus fervidus. Consistent with this, the toroidal wrapping of DNA by HAN1 resembles that described for HMf. However, significant differences in both twist and writhe components of these complexes are indicated by the 12.0 bp helical repeat produced during hydroxyl radical footprinting with HAN1. Furthermore, the increased stability of HAN1: DNA complexes allows DNA to be protected from thermal denaturation and cleavage by the restriction enzyme TaqI at 65°C. The hDBP, which co‐purified with HAN1, is shown to represent a major portion of the acid‐washed nucleoid protein in AN1 and to enhance the mobility of DNA directly, yet decrease the mobility of HAN1:DNA complexes.
Complementation of deletion mutations in a cloned functional cluster of Erwinia chrysanthemi out genes with Erwinia carotovora out homologues reveals OutC and OutD as candidate gatekeepers of species‐specific secretion of proteins via the type II pathway
Authors: Lindeberg Magdalen , Salmond George P. C. , Collmer Alan 
Published: October 2006

SummaryThe type II or Sec‐dependent secretion system is used by diverse Gram‐negative bacteria for secretion of extracellular proteins. Of the 12–15 proteins involved in secretion, the requirement for many has not been demonstrated and little is known about their functions in the secretion process. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete extracellular pectate lyases (Pels) using the type II or Out pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed genes from the other species, suggesting the presence of species‐specific recognition factors in the Out systems of the two Erwinia species. We previously reported the isolation of a cosmid clone, pCPP2006, from E. chrysanthemi EC16, which enables Escherichia coli to secrete heterologously expressed E. chrysanthemi Pels. Sequencing in a region required for secretion revealed the presence of 12 genes, outC‐M and outO. We report here the construction of functionally non‐polar mutations in each gene in the outC‐M operon and outS and outB using a polAts strain of E. coli to facilitate homologous recombination between out genes carrying deletions and their wild‐type copies on pCPP2006. By testing for complementation of each deletion with wild‐type out genes from E. chrysanthemi EC16 and E. carotovora SCRI193 we have demonstrated that: (i) each out gene is required for secretion of E. chrysanthemi PelE from E. coli with the exception of outH; (ii) each mutation can be complemented by its homologue from E. carotovora, except for outC and outD; (iii) outC and outD from E. carotovora do not confer secretion of Pel1 on the E. chrysanthemi Out system; and (iv) Pel1 secretion can be conferred on the E. chrysanthemi Out system by the presence of outC‐M, S and B from E. carotovora. The data suggest that OutC and OutD are gatekeepers of the Out system involved in recognition of Pels targeted for secretion but that OutC and OutD from E. carotovora cannot be successfully assembled into the E. chrysanthemi Out system.
Multiple signalling pathways trigger the exquisite sensitivity of yeast gluconeogenic mRNAs to glucose
Authors: Yin Zhikang , Smith Rachel J. , Brown Alistair J. P. 
Published: October 2006

SummaryThe transcription of the yeast FBP1 and PCK1 genes, which encode the gluconeogenic enzymes fructose‐1,6‐bisphosphatase and phosphoenolpyruvate car‐boxykinase, is repressed by glucose. Here, we show that this repression is both very strong and exceptionally sensitive to glucose, being triggered by glucose at concentrations less than 0.005% (0.27 mM). This repression remains operative in yeast mutants carrying any one of the three hexose kinases, but is lost in a triple hxk1, hxk2, glk1 mutant. In addition, 2‐deoxyglucose can trigger the repression, but 6‐deoxy‐glucose cannot, suggesting that internalization and phosphorylation of the glucose is essential for repression to occur. While gluconeogenic gene transcription is subject to the Mig1p‐dependent pathway of glucose repression, the exquisite response to glucose is maintained in hxk2 and mig1 mutants, suggesting that this pathway is not essential for the response. The response can also be triggered by the addition of exogenous cAMP, suggesting that the Ras/cAMP pathway can mediate repression of the FPB1 and PCK1 mRNAs. However, the response is not dependent upon this pathway because it remains intact in Ras, adenyl cyclase and protein kinase A mutants. The data show that yeast cells can detect very low glucose concentrations in the environment, and suggest that several distinct signalling pathways operate to repress FPB1 and PCK1 transcription in the presence of glucose.
RAG3 gene and transcriptional regulation of the pyruvate decarboxylase gene in Kluyveromyces lactis
Authors: Prior C. , Tizzani L. , Fukuhara H. , Wésolowski‐Louvel M. 
Published: October 2006

SummaryThe RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3–1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.
Independent interaction of the acyltransferase HlyC with two maturation domains of the Escherichia coli toxin HlyA
Authors: Stanley Peter , Koronakis Vassilis , Hardie Kim , Hughes Colin 
Published: October 2006

SummaryThe apparently unique fatty acylation mechanism that underlies activation (maturation) of Escherichia coli haemolysin and related toxins is further clarified by investigation of the interaction of protoxin with the specific acyltransferase HlyC. Using deleted protoxin variants and protoxin peptides as substrates in an in vitro maturation reaction dependent upon HlyC and acyl‐acyl carrier protein, two independent HlyC recognition domains were identified on the 1024‐residue protoxin, proA, and they were shown to span the two target lysine residues K564 (KI) and K690 (KII) that are fatty acylated. Each domain required 15–30 amino acids for basal recognition and 50–80 amino acids for wild‐type acylation. The two domains (FAI and FAII) competed with each other in cis and in trans for HlyC. The affinity of FAI for HlyC is approximately four times greater than that of FAII resulting in an overall 80% acylation at KI and 20% acylation at KII in both whole toxin and peptide derivatives. No other proA sequences were required for toxin maturation, and excess Ca2+ prevented acylation of both lysines. The lack of primary sequence identity between FAI and FAll domains in proA and among corresponding sites on related protoxins currently precludes an explanation of the basis of HlyC recognition by proA.
Variability of gene order in different Helicobacter pylori strains contributes to genome diversity
Authors: Jiang Qin , Hiratsuka Koji , Taylor Diane E. 
Published: October 2006

SummaryConsiderable genomic microdiversity has been reported previously among Helicobacter pylori isolates. We have constructed genome maps of four unrelated H. pylori strains (NCTC11637, NCTC11639, UA802 and UA861) using pulsed‐field gel electrophoresis (PFGE) with Notl and Nrul, hybridization with extracted PFGE DNA fragments and probing with 17 gene probes. These strains of H. pylori were compared with a fifth unrelated H. pylori strain NCTC11638 mapped previously. Considerable diversity in gene arrangement was evident among the five H, pylori maps, and no consistent gene clustering was found. The association of only four genes, katA (catalase gene), vacA (vacuo‐lating cytotoxin gene), hpaA (a putative adhesin gene), and pfr (bacterial ferritin gene) were generally conserved within approximately the same 25% of the genome; however, the order of these genes also varied. Our study demonstrates that macrodiversity, i.e. variability in gene order, in addition to microdiversity, is a characteristic of the H. pylori genome.
Phase variation in Francisella tularensis affecting intracellular growth, lipopolysaccharide antigenicity and nitric oxide production
Authors: Cowley Siobhan C. , Myltseva Svetlana V. , Nano Francis E. 
Published: October 2006

SummaryMany microbial pathogens, such as Mycobacterium spp. and Salmonella spp., use macrophage intracellular growth or antigenic variation as mechanisms for avoiding the host immune system. In this work we present evidence to show that the intracellular pathogen Francisella tularensis uses phase variation to alter antigenicity and the host macrophage nitric oxide response simultaneously, thereby modulating its intracellular growth. The lipopolysaccharide (LPS) and lipid A of F. tularensis fails to stimulate production of significant levels of nitric oxide (NO) by rat macrophages. However, spontaneous variants of F. tularensis expressing an antigenically distinct LPS induce rat macrophages to produce increased levels of NO, thereby suppressing microbial intramacrophage growth. Similarly, lipid A isolated from these variants stimulates increased levels of NO production. A reverse phase shift can occur, which returns the LPS to the original antigenic form, reduces NO production, and restores intramacrophage growth. These findings represent the first demonstration of a phase‐variation phenomenon which modulates intracellular growth and an innate immune response. Furthermore, these results suggest that a microbial pathogen can exploit macrophage NO production for its own benefit, perhaps by prolonging the host‐pathogen association during the acute phase of disease or during the process of establishing a carrier state.
Generation of unmarked directed mutations in mycobacteria, using sucrose counter‐selectable suicide vectors
Authors: Pelicic Vladimir , Reyrat Jean‐Marc , Gicquel Brigitte 
Published: October 2006

Summary The expression of sacB, the Bacillus subtilis gene encoding levansucrase, is lethal to mycobacteria in the presence of 10% sucrose. In this study, we describe the use of sacB as a marker for positive selection of gene‐replacement events into Mycobacterium smegmatis. A sucrose counter‐selectable suicide plasmid was used to deliver an inactivated copy of the pyrF gene (pyrFKm) into the M. smegmatis genome. Only uracil auxotroph clones, resulting from replacement of the endogenous pyrF allele, survived in a one‐step selection on plates containing kanamycin and 10% sucrose. This demonstrated that selection on sucrose against the maintenance of the vector bearing the sacB gene is 100% efficient, enabling the positive selection of allelic‐exchange mutants. Two‐step selection is also feasible; it was used to construct unmarked pyrF mutants in which the gene was inactivated by a frameshift mutation. This method of generating unmarked, directed mutations is rapid and simple, making it a powerful tool for the genetic characterization of mycobacteria.