bluuebunny/crossref_metadata_2025_split huggingface dataset:
Topology of diphtheria toxin B fragment inserted in lipid vesicies
Authors: Cabiaux Veronique , Quertenmont Pierre , Conrath Karel , Brasseur Robert , Capiau Carina , Ruysschaert Jean‐Marie 
Published: October 2006

SummaryDiphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells In response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptides that were protected from digestion were separated by gel electrophoresls, transferred to a membrane and their N‐terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194–223, 266–375 and 429–528. The secondary structures of the peptides inserted in the membrane, determined by Fourier‐transformed infrared spectroscopy, were shown to be mostly α‐helices and β‐sheets (44% and 53%, respectively). On the basis of these data and the recently published X‐ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.
A new class of proteins regulating gene expression in enterobacteria
Authors: Mikulskis Alvldas V. , Cornetis Guy R. 
Published: October 2006

SummaryYmoA and Hha are highly similar bacterial proteins downregulating gene expression In Yersinia entero‐colitlca and Escherichia coli. respectively. The pheno‐type of ymoA mutants evokes that of mutants affected in some histone‐like proteins. This paper describes complementation of a ymoA mutation in Y. enterocolitica by the hha gene from E. coli. We show that YmoA and Hha are not only very similar proteins but that they are functionally Interchangeable. Genetic experiments indicate that Hha can also stimulate transposition events in vivo. By Southern Biol analysis we detected hha‐homologous genes at least in Citrobacter diversus, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Salmonella typhimurium. We suggest that both YmoA and Hha belong to a new family of proteins down regulating gene expression in different enterobacteria.
The citrulline biosynthetic operon, argC‐F, and a ribose transport operon, rbs, from Bacillus subtilis are negatively regulated by Spo0A
Authors: O'Reilly Mary , Woodson Karen , Dowds Barbara C. A. , Devine Kevin M. 
Published: October 2006

SummaryA method Is described here that can be used to identify operons whose expression Is controlled by any particular regulator protein. This method was used to Identify operons whose expression is negatively regulated by Spo0A in Bacillus subtilis. Twenty‐eight strains were identified, each of which contains an operon‐lacZ transcriptional fusion, negatively regulated, either directly or Indirectly, by Spo0A. In one of these strains (CSA8), the lacZ gene is fused to the argC‐F operon positioned at 100° on the B. subtilis chromosome. The regulated expression of this operon by Spo0A ∼ P is mediated indirectly through the transition state regulator AbrB and is manifest only during growth on solid medium. In a second strain (CSA15), the lacZ gene is fused to an operon encoding a transport system which displays features characteristic of the ABC group of transporters, and which has a very high level of identity to the ribose transport system from Escherichia coli Expression of the ribose transport operon is directed by a single SigA‐type promoter. Transcription from this promoter is repressed by the phosphorylated form of Spo0A during the late‐exponential/transition phase of the growth cycle and this control is not mediated through the transition‐state regulator, AbrB.
Growth phase and low pH affect the thermal regulation of the Yersinia enterocolitica inv gene
Authors: Pepe Jeffrey C. , Badger Juile L. , Miller Virginia L. 
Published: October 2006

SummaryThe inv gene encodes the protein invasin, which is the primary invasion factor for Yersinia enterocolitica in vitro and in vivo. Previous studies of Yersinia species have shown that inv expression and entry into mammalian cells are temperature regulated. Invasin production is reduced at the host temperature of 37°C as compared to production at ambient temperature; consequently, this study was initiated to determine whether other host environmental signals might induce inv expression at 37°C. An inv::phoA translational fusion was recombined on to the Y. enterocolitica chromosome by allelic exchange to monitor inv expression. Molecular characterization of expression of the wild‐type inv gene and the inv phoA fusion showed that invasin is not produced until early stationary phase in bacteria grown at 23°C. Y. enterocolitica grown at 37°C and pH 5.5 showed levels of inv expression comparable to those observed in bacteria grown at 23 C. An increase in Na+ ions caused a slight increase in expression at 37 C. However, expression at 37°C was unaffected by anaerobiosis, growth’medium, calcium levels, or iron levels. Additionally, Y. enterocolitica expressed invasin in Peyer's patches two days after being introduced intragastrically into BALB/c mice. These results suggest that invasin expression in K enterocolitica may remain elevated eariy during interaction with the intestinal epithelium, a site at which invasin was shown to be necessary.
Structure and heterologous expression of the Ustilago maydis viral toxin KP4
Authors: Park Chung‐Mo , Bruenn Jeremy A. , Ganesa Chandrashekar , Flurkey William F. , Bozarth Robert F. , Koltin Yigal 
Published: October 2006

SummaryKiller toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species, in the best‐characterized cases, they function by creating new pores in the ceil membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double‐stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double‐stranded RNA but differs from other known killer toxins in several respects: it has no N‐linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and h Is not processed by any of the known secretory protelnases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.
Genetic characterization of a Rhizobium meliloti lactose utilization locus
Authors: Jelesko John G. , Leigh John A. 
Published: October 2006

SummaryWe identified several linked genes of a lactose regulon in Rhizobium meliloti. These were lacZ, the structural gene for β‐galactosidase; lacR, the lactose repressor gene; and two genes encoding proteins of unknown function. lacW and lacX. Insertion mutants in lacW and lacZ belonged to a single genetic compiementation group, and lacW appeared to lie upstream of lacZ in an operon. Expression of lacZ, lacW and lacX was repressed by lacR, and expression of lacZ and lacW was derepressed by lactose. lacZ was not required for Induction of lacW by lactose, suggesting that lactose itself, rather than a processed form of lactose, may be the actual Inducer molecule. Expression of all three genes was repressed by succinate, and the lacR independence of this repression showed that inducer exciusion could not be the sole mechanism. This pattern of lac gene organization and regulation differs in several ways from that observed in enteric bacteria.
The mutL repair gene of Escherichia coli K‐12 forms a superoperon with a gene encoding a new cell‐wall amidase
Authors: Tsui Ho‐Ching Tiffany , Zhao Genshi , Feng Gang , Leung Hon‐Chlu Eastwood , Winkler Malcolm E. 
Published: October 2006

SummaryWe report a molecular genetic analysis of the region Immediately upstream from the Escherichia coli mutL DNA repair gene at 94.8 min. An open reading frame ending 9bp upstream from the start of mutL corresponds to a 48kDa polypeptide detected previously in minicells. The predicted amino acid sequence of this 48kDa polypeptide shows homology to the major N‐acetylmuramoyl‐L‐alanine amidase autolysin of Bacillus subtilis, a known amidase of Bacillus licheniformis, and the product of a Salmonella typhimurium gene that maps near SO min. Insertions in this upstream gene, which we named AmiB, or in mutL did not affect cell shape or viability; however, overexpression of the AmiB potypeptide caused ceil lysis, hypersensitivity to osmotic shock and treatment with water, and temporary autolysis by low levels of antibiotics, which are all consistent with AmiB acting as a cell‐wall hydrolase. Analysis of chromosomal transcription demonstrated that amiB forms a complex operon with mutL and two additional upstream genes. mutL transcripts also originated from an internal promoter, designated PmutL, located in amiB 312bp upstream from the translational start of mutL. Together, these results suggest that E. coli contains a second amidase possibly involved in cell‐wall hydrolysis, septation, or recycling, and that transcription of this amidase is directly linked to a gene central for DNA repair.
Protein prenylation in eukaryotic microorganisms: genetics, biology and biochemistry
Authors: Omer C. A. , Gibbs J. B. 
Published: October 2006

SummaryModrfication of proteins at C‐terminal cysteine residue(s) by the isoprenoids farnesyl (C15) and geranylgeranyl (C20) is essential for the biological function of a number of eukaryotic proteins including fungal mating factors and the small, GTP‐binding proteins of the Ras superfamily. Three distinct enzymes, conserved between yeast and mammals, have been identified that prenylate proteins: farnesyl protein transferase, geranylgeranyl protein transferase type I and geranylgeranyl protein transferase type II. Each prenyl protein transferase has its own protein substrate specificity. Much has been learned about the biology, genetics and biochemistry of protein prenylation and prenyl protein transferases through studies of eukaryotic microorganisms, particularly Saccharo‐myces cerevisiae. The functional Importance of protein prenylation was first demonstrated with fungal mating factors. The initial genetic analysis of prenyl protein transferases was in S. cerewisiae with the isolation and subsequent characterization of mutations in the RAM1, RAM2, CDC43 and BET2 genes, each of which encodes a prenyl protein transferase subunit. We review here these and other studies on protein prenylation in eukaryotic microbes and how they relate to and have contributed to our knowledge about protein prenylation in all eukaryotic cells.
Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus
Authors: McDevitt D. , Francois P. , Vaudaux P. , Foster T. J. 
Published: October 2006

SummaryFour mutants of Staphylococcus aureus strain Newman that were defective in the fibrinogen receptor (clumping factor) were isolated by transposon Tn917 mutagenesis. Southern hybridization analysis of the mutants identified transposon‐host DNA junction fragments, one of which was cloned and used to generate a probe to identify and clone the wild‐type clumping factor locus (clfA). The mutants failed to form clumps in soluble fibrinogen and adhered poorly to polymethylmethacrylate (PMMA) coverslips coated with fibrinogen. A single copy of the clfA gene, when introduced into the chromosome of the mutant strains, fuily compiemented the ciumping deficiency of these strains and restored the ability of these mutants to adhere to fibrinogen‐coated PMMA. in addition, the cloned clfA gene on a shuttle plasmid aiiowed the weakiy ciumping strain 8325‐4 to form clumps with the same avidity as the wild‐type strain Newman and also significantly enhanced the adherence of 8325‐4 strains. Thus the formation of clumps in soluble fibrinogen correlated with adherence of bacteria to solid‐phase fibrinogen. The clfA gene encodes a fibrinogen‐binding protein with an apparent molecular mass of c. 130 kDa. The amino acid sequence of the protein was deduced from the DNA sequence; it was predicted that a 896 residue protein (molecular mass 92 kDa) would be expressed. The putative ClfA protein has features that suggest that it is associated with the ceil surface. Furthermore it contains a novel 308 residue region comprising dipeptide repeats predominantly of Asp and Ser ending 28 residues upstream from the LPXTG motif common to wall‐associated proteins. Significant homology was found between the ClfA protein and the fibronectin‐binding proteins of S. S. aureus, particularly in the N‐and C‐termini.
The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites
Authors: Hayes Finbarr , Radnedge Lyndsay , Davis Michael A. , Austin Stuart J. 
Published: October 2006

SummaryThe plasmid‐partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent PI and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species‐specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA‐binding domain of the P7 auto‐repressor lies in the amino‐terminal end of the P7 ParA protein. This region includes a helix‐turn‐helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter‐operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.
Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections
Authors: Harth Günter , Mills Alea A. , Lin Teri , Araujo Fausto G. 
Published: October 2006

SummaryA ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin‐fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco‐protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi‐subunit vaccine against 7. cruzi.
Characterization of a 3‐dehydroquinase gene from Actinobacillus pleuropneumoniae with homology to the eukaryotic genes qa‐2 and QUTE
Authors: Lalonde G. , O'Hanley P. D. , Stacker B. A. D. , Denich K. T. 
Published: October 2006

SummaryA gene was cloned from Actinobacillus pleuropneumoniae strain 4074 by complementation of an aroD strain of Escherichia coli. The E. coli gene aroD codes for a 3‐dehydroquinase enzyme of type I, active in the aromatic biosynthesis pathway. The A. pleuropneumoniae gene, termed aroQ, displays no base or ami no acid sequence homoiogy to aroD of E. coli. It is instead homologous to the QUTE and qa‐2 genes, respectively of Aspergillus niduians and Neurospora crassa. These genes code for 3‐dehydroquinase enzymes of type ii, involved in the catabolism of quinic acid. The 1.6 kb fragment, which includes aroQ, carries four overlapping or adjacent open reading frames: a dapD gene; aroQ; one without homology to sequences in GenBank; and one with homology to the C‐terminal 40% of chlN of E. coli.
Characterization of the dTDP‐rhamnose biosynthetic genes encoded in the rfb iocus of Shigella flexneri
Authors: Macpherson Debbie F. , Manning Paul A. , Morona Renato 
Published: October 2006

SummaryThe nucleotide sequence of the proximal half of the rfb region of Shigella flexneri has been determined, and the genes encoding enzymes involved in the biosynthesis of dTDP‐rhamnose have been identified. These genes show strong homology to the rfb genes encoding dTDP‐rhamnose biosynthesis in Salmonella enterica serovar typhimurium (strain LT2) and S. enterica serovar anatum (strain M32) (Jiang et al., 1991; Wang et al., 1992). An open reading frame upstream of rfbB was also identified which encoded a protein having strong similarity with GalU, and has been designated galF. GalF has 92% amino acid sequence identity with an S. enterica LT2 gene, orf2X8, which is similarly situated upstream of rfbB (Jiang et al., 1991). The T7 expression system was utilized to identify proteins corresponding to those predicted from DNA sequence analysis. The similarity of the predicted proteins with proteins that are functionally identical or related, and with others of unknown function from the Yersinia enterocolitica O3 rfb region, and in the Escherichia coli K‐12 rff region are also described. We have re‐addressed the assignment of each gene of the dTDP‐rhamnose pathway with the known enzymes of the pathway, in particular rfbC and rfbD. A reporter plasmid to detect genes encoding enzymes of the dTDP‐rhamnose pathway is described. An analysis of the intergenic region between galF and rfbB has been made, and comparison with the same region from S. enterica LT2 discussed.
In vivo study of engineered G‐domain mutants of Escherichia coli translation initiation factor IF2
Authors: Laalami Soumaya , Timofeev Andrei V. , Putzer Harald , Leautey Josette , Grunberg‐Manago Marianne 
Published: October 2006

SummaryDuring the IF2‐catalysed formation of the 30S initiation complex, the GTP requirement and Its subsequent hydrolysis during 70S complex formation are considered to be essential for translation initiation in Escherichia coli. In order to clarify the role of certain amino acid residues believed to be crucial for the GTP hydrolytic activity of E. coli IF2, we have introduced seven single amino acid substitutions into its GTP‐binding site (Gly for Val‐400; Thr for Pro‐446; Gly, Glu, Gin for His‐448; and Asn, Glu for Asp‐501). These mutated IF2 proteins were expressed in vivo in physiological quantities and tested for their ability to maintain the growth of an E. coli strain from which the functional chromosomal copy of the infB gene has been deleted. Only one of the mutated proteins (Asp‐501 to Giu) was able to sustain cell viability and several displayed a dominant negative effect. These results emphasize that the amino acid residues we substituted are essential for the iF2 functions and demonstrate the importance of GTP hydrolysis in translation initiation. These findings are discussed in relation to a previously proposed theoretical model for the IF2 G‐domain.
The function of a ribosomal frameshifting signal from human immunodeficiency virus‐1 in Escherichia coli
Authors: Yelverton Elizabeth , Lindsley Dale , Yamauchi Phil , Gallant Jonathan A. 
Published: October 2006

SummaryA 15‐17 nucleotide sequence from the gag‐pol ribosome frameshift site of HIV‐1 directs analogous ribosomal frameshifting in Escherichia coli. Limitation for leucine, which is encoded precisely at the frameshift site, dramatically increased the frequency of leftward frameshifting. Limitation for phenylaianine or arginine, which are encoded just before and just after the frameshift, did not significantly affect frameshifting. Protein sequence analysis demonstrated the occurrence of two closeiy related frameshift mechanisms. In the first, ribosomes appear to bind leucyl‐tRNA at the frameshift site and then slip leftward. This is the 'simultaneous slippage’mechanism. In the second, ribosomes appear to slip before binding amlnoacyl‐tRNA, and then bind phenylaianyl‐tRNA, which is encoded in the left‐shifted reading frame. This mechanism is identicai to the‘overlapping reading’we have demonstrated at other bacterial frameshift sites. The HIV‐1 sequence is prone to frame‐shifting by both mechanisms in E. coli.
Nodulating ability of Rhizobium tropici is conditioned by a plasmid‐encoded citrate synthase
Authors: Pardo Marco A. , Lagunez Jaime , Miranda Juan , Martinez Esperanza 
Published: October 2006

Summary Rhizobium species elicit the formation of nitrogen‐fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen‐fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen‐fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl‐CoA and oxalo‐acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5‐mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino‐sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.
Characterization of the mutX gene of Streptococcus pneumoniae as a homologue of Escherichia coli mutT, and tentative definition of a catalytic domain of the dGTP pyrophosphohydroiases
Authors: Mejean Vincent , Salles Catherine , Bullions Linda C. , Bessman Maurice J. , Claverys Jean‐Pierre 
Published: October 2006

SummaryWe show that deletion of a gene of Streptococcus pneumoniae, which we call mutX, confers a mutator phenotype to resistance to streptomycin. Analysis of the DNA sequence changes that occurred in several streptomycin‐resistant mutants showed that mutations are unidirectional AT to CG transversions. The mutX gene is located immediately downstream of the previously identified ung gene and genetic evidence suggests that the two genes are coordinately regulated. Nucleotide sequence determination reveals that the mutX gene encodes a 17870 Da protein (154 residues) which exhibits significant homology with the MutT protein of Escherichia coli, a nucleoside tri‐phosphatase (dGTP pyrophosphohydrolase). The mutX gene complements the E coli mutT mutator phenotype when introduced on a plasmid. Site‐directed mutagenesis and analysis of nitrosoguanidine‐induced mutT mutants suggest that a small region of high homology between the two proteins (61% identity over 23 residues) is part of the catalytic site of the nucleoside triphosphatase. Computer searching for sequence homology to MutX uncovered a second E. coli protein, the product of orf17, a gene of unknown function located near the ruvC gene. The region of high homology between MutX and MutT is also conserved in this protein, which raises the interesting possibility that the orf17 gene plays some role in determining mutation rates in E. coli. Finally, a small set of proteins, including a family of virus‐encoded proteins and two evolutionarily conserved proteins encoded by an antisense transcript from the Xenopus laevis and human bFGF genes, were also found to harbour significant homology to this highly conserved region.