bluuebunny/crossref_metadata_2025_split huggingface dataset:
The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs
Authors: Paulsen Ian T. , Skurray Ronald A. , Tam Roland , Saier Milton H. , Turner Raymond J. , Weiner Joel H. , Goldberg Edward B. , Grinius Leonas L. 
Published: October 2006

SummaryThe sequenced members of a novel family of small, hydrophobic, bacterial multidrug‐resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic α‐helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.
Alteration of haem‐attachment and signal‐cleavage sites for Paracoccus denitrificans cytochrome c550 probes pathway of c‐type cytochrome biogenesis in Escherichia coli
Authors: Sambongi Yoshihiro , Stoll Raphael , Ferguson Stuart J. 
Published: October 2006

Summary Paracoccus denitrificans cytochrome c550 is expressed as a periplasmic holo‐protein in Escherichia coli; amino acid substitutions of cysteine residues in the haembinding motif (Cys‐X‐X‐Cys‐His), either together or singly, prevented covalent attachment of haem but not polypeptide translocation into the periplasm. When the three alanine residues at positions ‐3 to ‐1 in the native signal‐cleavage site were deleted, or alanine at ‐1 was changed to glutamine, signal cleavage was at alternative sites (after only ten residues in the latter case), but haem attachment still occurred. When the same three alanines were changed to Asp‐Glu‐Asp, a membrane‐associated apo product that had retained the complete signal sequence was detected. These and other results presented here indicate that (i) haem attachment is not required for the apo‐cytochrome c550 export to the periplasm; (ii) haem cannot attach to apocytochrome c550 when attached to the cytoplasmic membrane, suggesting that signal‐sequence cleavage precedes periplasmic haem attachment, which can occur at as few as six residues from the mature N‐terminus; and (iii) two cysteines are required for haem attachment, possibly because a disulphide bond is an intermediate. The gene for Saccharomyces cerevisiae mitochondrial iso‐1‐cytochrome c was expressed as a holo‐protein in E. coli when fused with the signal sequence plus the first 10 residues of the mature cytochrome c550, indicating that the E. coli cellular apparatus for the c‐type cytochrome biogenesis has a broad substrate specificity.
Identification of a second family of high‐molecular‐weight adhesion proteins expressed by non‐typable Haemophilus influenzae
Authors: Barenkamp Stephen J. , Geme Joseph W. St. 
Published: October 2006

SummaryWe previously reported that two surface‐exposed high‐molecular‐weight proteins, HMW1 and HMW2, expressed by a prototypic strain of non‐typable Haemophilus influenzae (NTHI), mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to 70–75% of NTHI strains. NTHI strains that lack HMW1/ HMW2‐like proteins remain capable of efficient attachment to cultured human epithelial cells, suggesting the existence of additional adhesion molecules. We reasoned that characterization of high‐molecular‐weight immunogenic proteins from an HMW1/HMW2‐deficient strain might identify additional adhesion proteins. A genomic library was prepared in λEMBL3 with chromosomal DNA from non‐typable Haemophilus strain 11, a strain that lacks HMW1/HMW2‐like proteins. The library was screened immunologically with convalescent serum from a child naturally infected with strain 11, and phage clones expressing high‐molecular‐weight recombinant proteins were identified by Western blot analysis. One clone was identified that expressed a protein with an apparent molecular mass greater than 200 kDa. Transformation of non‐adherent Escherichia coli strain DH5α with plasmids containing the genetic locus encoding this protein gave rise to E. colitransformants that adhered avidly to Chang conjunctival cells. Subcloning and mutagenesis studies localized the DNA conferring the adherence phenotype to a 4.8 kbp fragment, and nucleotide sequence analysis further localized the gene encoding the adhesion protein to a 3.3 kbp open reading frame predicted to encode a protein of 114kDa. The gene was designated hia for Haemophilus influenzae adhesin. Southern analysis revealed an hia homologue in 13 of 15 HMW1/HMW2‐deficient non‐typable H. influenzae strains. In contrast, the hia gene was not present in any of 23 non‐typable H. influenzae strains which expressed HMW1/HMW2‐like proteins. Identification of this second family of high‐molecular‐weight adhesion proteins suggests the possibility of developing vaccines based upon a combination of HMW1/HMW2‐like proteins and Hia‐like proteins which would be protective against disease caused by most or all non‐typable H. influenzae
Aperiplasmic protein (Skp) of Escherichia coli selectively binds a class of outer membrane proteins
Authors: Chen Robert , Henning Ulf 
Published: October 2006

Summary A search was performed for a periplasmic molecular chaperone which may assist outer membrane proteins of Escherichia coli on their way from the cytoplasmic to the outer membrane. Proteins of the periplasmic space were fractionated on an affinity column with sepharose‐bound outer membrane porin OmpF. A 17kDa polypeptide was the predominant protein retained by this column. The corresponding gene was found in a gene bank; it encodes the periplasmic protein Skp. The protein was isolated and it could be demonstrated that it bound outer membrane proteins, following SDS‐PAGE, with high selectivity. Among these were OmpA, OmpC, OmpF and the maltoporin LamB. The chromosomal skp gene was inactivated by a deletion causing removal of most of the signal peptide plus 107 residues of the 141‐residue mature protein. The mutant was viable but possessed much‐reduced concentrations of outer membrane proteins. This defect was fully restored by a plasmid‐borne skp gene which may serve as a periplasmic chaperone.
Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t
Authors: van Sinderen Douwe , Karsens Harma , Kok Jan , Terpstra Peter , Ruiters Marcel H. J. , Venema Gerard , Nauta Arjen 
Published: October 2006

Summary The temperate lactococcal bacteriophage r1t was isolated from its lysogenic host and its genome was subjected to nucleotide sequence analysis. The linear r1t genome is composed of 33 350 bp and was shown to possess 3′ staggered cohesive ends. Fifty open reading frames (ORFs) were identified which are, probably, organized in a life‐cycle‐specific manner. Nucleotide sequence comparisons, N‐terminal amino acid sequencing and functional analyses enabled the assignment of possible functions to a number of DNA sequences and ORFs. In this way, ORFs specifying regulatory proteins, proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and a dUTPase were putatively identified. One ORF seems to be contained within a self‐splicing group I intron. In addition, the bacteriophage att site required for site‐specific integration into the host chromosome was determined.
Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes
Authors: Quiocho Florante A. , Ledvina Polly S. 
Published: October 2006

SummaryCrystallographic structure refinement at very high resolutions of a dozen periplasmic receptors has revealed that, though they have different sizes (26 to 60kDa) and little sequence homology, they have high tertiary structure similarity. They consist of two distinct globular domains bisected by a cleft or groove wherein the ligand binds and is buried by a hinge‐bending motion between the two domains. Structural analysis also reveals how hydrogen‐bonding interactions can be tailored to a wide spectrum of specificity, ranging from the stringent specificity for phosphate and sulphate to the more loose specificity for peptides.
Cloning of a second gene encoding 6‐phosphofructo‐2‐kinase in yeast, and characterization of mutant strains without fructose‐2,6‐bisphosphate
Authors: Bolesm Eckhard , Göhlmann Hinrich W. H. , Zimmermann Friedrich K. 
Published: October 2006

SummaryWe have identified a new gene, PFK27, that encodes a second inducible 6‐phosphofructo‐2‐kinase in the yeast Saccharomyces cerevisiae. Sequencing shows an open reading frame of 397 amino acids and 45.3 kDa. Amino acid sequence comparisons with other bifunctional 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase isoenzymes of various organisms revealed similarities only to the kinase domains. Expression of PFK27 was induced severalfold by glucose and sucrose, but not by galactose or maltose, suggesting that sugar transport might be involved in triggering the induction signal. We have constructed a mutant strain devoid of any fructose‐2,6‐bisphosphate. The mutant strain grew well on several kinds and concentrations of carbon sources. The levels of hexose phosphates in the cells were increased, but flux rates for glucose utilization and ethanol production were similar to the wild‐type strain. However, after the transfer of the mutant cells from respiratory to fermentative growth conditions, growth, glucose consumption and ethanol production were delayed in a transition phase. Our results show that fructose‐2,6‐bisphosphate is an important effector in vivo of the 6‐phosphofructo‐1‐kinase/fructose‐1,6‐bisphospha‐tase enzyme pair, and is involved in the initiation of glycolysis during the transition to a fermentative mode of metabolism. Nevertheless, it can be effectively replaced by other effectors and regulatory mechanisms during growth on glucose.
The ATP‐binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3
Authors: Mahé Yannick , Parle‐McDermott Anne , Nourani Amine , Delahodde Agnès , Lamprecht Andrea , Kuchler Karl 
Published: October 2006

SummaryPleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP‐binding cassette (ABC) efflux pumps such as Pdr5 and Snq2. Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR. We show here that a pdr1–3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4‐nitroquinoline‐N‐oxide, a known substrate for Snq2 but not for Pdr5. Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10‐fold overexpressed in a pdr1–3 gain‐of‐function mutant strain, whereas Snq2 expression is severely reduced in a Δpdr1 deletion strain, and almost abolished in a Δpdr1Δpdr3 double disruptant when compared to the PDR1 strain. However, expression of the Ste6 a‐factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1–3 mutant cells and in strains carrying Δpdr1, Δpdr3, or Δpdr1Δpdr3 deletions. Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3. Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1–3 mutant strain results from overexpression of more than one ABC drug‐efflux pump.
Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of Listeria monocytogenes
Authors: Guzmán Carlos A. , Domann Eugen , Ronde Manfred , Bruder Dunja , Darji Ayub , Weiss Siegfried , Wehland Jürgen , Chakraborty Trinad , Timmis Kenneth N. 
Published: October 2006

SummaryInfection of a murine‐spleen dendritic cell line by Listeria monocytogenes was found to induce cell death through apoptosis. To characterize the bacterial product(s) involved in induction of apoptosis, dendritic cells were infected with the L. monocytogenes EGD strain and several isogenic mutants deficient in the production of individual listerial virulence factors. The ability to induce cellular apoptosis was retained by all mutants tested, except the prfA and Δhly mutants, both of which are unable to produce listeriolysin. Apoptosis was also induced by purified listeriolysin suggesting that this protein directly induces apoptosis. Purified recombinant listeriolysins rendered either weakly haemolytic by a C‐484 to S mutation, or non‐haemolytic by a W‐491 to A mutation exhibited little or no capacity to induce apoptosis, indicating that both activities are associated within the same protein region. Treatment with purified listeriolysin or L. monocytogenes infection also triggers apoptosis in explanted bone‐marrow dendritic cells. Thus invasion of dendritic cells by L. monocytogenes, which results in cell death, may play an important role in the pathogenesis of listerial infections by impairing immune responses, hindering bacterial clearance and promoting spread of the infection.
Vibrio parahaemolyticus FlaJ, a homologue of FliS, is required for production of a flagellin
Authors: Stewart Bonnie J. , McCarter Linda L. 
Published: October 2006

SummaryThe flaA locus of Vibrio parahaemolyticus encodes one of the four polar flagellin genes, the flagellum‐capping protein HAP2, and three additional flagellar genes. Sequence analysis downstream of the gene encoding HAP2 revealed the region to be similar to the fliD (HAP2) locus of Pseudomonas aeruginosa. The deduced protein sequences for the newly identified genes suggest that one protein belongs to the family of transcriptional regulatory proteins known to interact with σ54, one may be a rod component of the flagellum, and one resembles the FMS protein. fliS is an essential flagellar gene in many bacteria; however its function is not clear. The V. parahaemolyticus polar flaC flagellin gene was poorly expressed in Escherichia coli. Production of FlaC was stimulated by provision of the flaA locus in trans. Dissection of this locus revealed that the fliS‐like gene, flaJ, was required for increased expression of flaC. Stimulation by FlaJ occurred in E. coli mutants defective in either the master flagellar‐controlling operon or the gene encoding the flagellar σ28. Therefore the effect of FlaJ was not mediated through flagellar proteins. Nor was it mediated through σ54, for enhanced FlaC production was observed in mutants with defects in the gene encoding σ54.
Tandem repeats of the tetramer 5′‐CAAT‐3’present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae
Authors: High Nicola J. , Jennings Michael P. , Moxon E. Richard 
Published: October 2006

SummaryA novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase‐variable LPS structure expressed by Haemo philus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neis seria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)n encoded by multiple tandem repeats of 5′‐CAAT‐3′. This observation suggested that (SINQ)n might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5′‐CAAT‐3’repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine‐SDS‐PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)n is not a prerequisite for the enzymatic function of Lic2A and that the 5′‐CAAT‐3’repeats in lic2A function solely as a mechan ism for generating phase variation. This observation suggested that wide variation in the number of 5’‐CAAT‐3’repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5′‐CAAT‐3’repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)n indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.
A gene product related to Tral is required for the mobilization of Bacteroides mobilizable transposons and plasmids
Authors: Smith C. Jeffrey , Parker Anita C. 
Published: October 2006

SummaryThe antibiotic‐resistance transposon Tn4555 from Bacteroides can be transferred between strains by conjugation. The transposon is not self‐transmissible and must be mobilized by resident chromosomal tetracy‐cline‐resistance elements. In the present report, the mechanism of transfer was examined at the genetic level by deletion analysis and nucleotide sequencing of clones that conferred a transmissible phenotype on a non‐mobilizable plasmid. The results suggested that the product of mobATn was required for mobilization and it worked in concert with a cis‐acting oriT‐like sequence. This mechanism was compared with the mobilization system of a cryptic Bacteroides plasmid, pBl143, and the two systems were found to share a common transfer strategy. The mobA gene products from both genetic elements were related and they had limited homology to the broad group of mobilization proteins (relaxases) typified by Tral of RP4. Phylogenetic analysis of MobA and several other mobilization proteins from commensal gastrointestinal tract organisms suggested that they formed a new subgroup of the Tral superfamily. The mobilization regions of both Tn4555 and pBl143 were located on discrete segments of DNA within the parent genetic element. These segments were delineated by regions of secondary structure, suggesting that they could be defined mobilization cassettes.
Ste50p sustains mating pheromone‐induced signal transduction in the yeast Saccharomyces cerevisiae
Authors: Xu Gang , Jansen Gregor , Thomas David Y. , Hollenberg Cornelis P. , Rad Massoud Ramezani 
Published: October 2006

SummaryIn the yeast Saccharomyces cerevisiae, the hetero‐trimeric G protein transduces the mating pheromone signal from a cell‐surface receptor. Free Gβγ then activates a mitogen‐activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal‐transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone‐induced signal which leads cells to hormone‐induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone‐induced signal transduction, and overexpression of STE50 intensifies the pheromone‐induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α‐pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two‐hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.
A single substitution in the putative helix‐turn‐helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence
Authors: Sheehan Brian , Klarsfeld André , Ebright Richard , Cossart Pascale 
Published: October 2006

SummaryPrf A, the regulator of virulence‐gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP‐FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of Prf A predicted to contact DNA, i.e. Ser‐184 and Ser‐183. Substitution of Ser‐184 by Ala reduced DNA binding and virulence‐gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser‐183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target‐site recognition by PrfA and PrfA‐S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence‐gene activation, and virulence.
Interaction of Cody, a novel Bacillus subtillis DNA‐binding protein, with the dpp promoter region
Authors: Serror† Pascale , Sonenshein Abraham L. 
Published: October 2006

SummaryThe product of the codY gene is required for nutritional repression of the Bacillus subtilis dipeptide permease operon (dpp), an operon expressed at early stationary phase in nutrient‐rich medium. Though unrelated to any known DNA‐binding protein, Cody was shown to bind specifically to the dpp promoter region. DNase I footprinting experiments revealed that the Cody‐protected region encompasses the dpp transcription start site and overlaps with the region protected by another regulatory protein, AbrB. Cody and AbrB were found to compete, in vitro, for binding to the dpp promoter region. Binding of Cody was altered in mutants defective in nutritional regulation.
Characterization of an Escherichia coli mutant, feeA, displaying resistance to the calmodulin inhibitor 48/80 and reduced expression of the rare tRNA3Leu
Authors: Bouquin Nicolas , Chen Mao X. , Kim Sunghoon , Vannier Françoise , Bernard Sylvie , Holland I. Barry , Séror Simone J. 
Published: October 2006

SummaryWe previously described a mutation feeB1 conferring a temperature‐sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA3Leu recognizing CUA codons. We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30°C, and temperature‐sensitive, forming short filaments at 42°C. In the feeA mutant, tRNA3Leu expression (but not that of tRNA1Leu) was reduced approximately fivefold relative to the wild type. We previously showed that the synthesis of β‐galactosidase, which unusually requires the translation of 6‐CUA codons, was substantially reduced, particularly at 42°C, in feeB mutants. The feeA mutant also shows drastically reduced synthesis of β‐galactosidase at the non‐permissive temperature and reduced levels even at the permissive temperature. We also show that increased copy numbers of the abundant tRNA1Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation. This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA3Leu or the cellular amount of tRNA3Leu confer resistance to the drug 48/80, with concomitant inhibition of cell division at 42°C.