bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extracellular addition of a domain of HIV‐1 Vpr containing the amino acid sequence motif H(S/F)RIG causes cell membrane permeabilization and death
Authors: Macreadie Ian G. , Arunagiri Chinniah K. , Hewish Dean R. , White Jacinta F. , Azad A. A. 
Published: October 2006

SummaryVpr is a virion‐associated protein of human immuno‐deficiency virus type 1 (HIV‐1) whose function in acquired immune deficiency syndrome (AIDS) has been uncertain. We previously employed yeast as a model to examine the effects of Vpr on basic cellular functions; intracellular Vpr was shown to cause cell‐growth arrest and structural defects, and these effects were caused by a region of Vpr containing the sequence HFRIGCRHSRIG. Here we show that peptides containing the H(S/F)RIG amino acid sequence motif cause death when added externally to a variety of yeast including Saccharomyces cerevisiae, Kluyveromyces lactis, Candida glabrata, Candida albicans and Schizosaccharomyces pombe. Such peptides rapldly entered the cell from the time of addition, resulting in cell death. Elevated levels of ions, particularly magnesium and calcium ions, abrogated the cytotoxic effect by preventing the Vpr peptides from entering the cells. Extracellular Vpr found in the serum, or breakdown products of extracellular Vpr, may have similar effects to the Vpr peptides described here and could explain the death of uninfected by‐stander cells during AIDS.
Saccharomyces cerevisiae TEC1 is required for pseudohyphal growth
Authors: Gavrias Victoria , Andrianopoulos Alex , Gimeno Carlos J. , Timberlake William E. 
Published: October 2006

SummaryDiverse eukaryotic organisms share developmental transcription factors with homologous DNA‐binding domains. We showed that the developmental regulator AbaA, a member of the ATTS/TEA (AbaA, TEF‐1, TEC1, Scalloped/TEF‐1, TEC1, AbaA) class of transcription factors of the filamentous fungus Aspergillus nidulans, induces pseudohyphal development in the yeast Saccharomyces cerevisiae. The S. cerevisiae homologue of AbaA, TEC1p, is required for this morphological transition. We provide evidence that TEC1p functions in co‐operation with STE12p to induce pseudohyphal development.
Characterization of the distal promoter element of halobacteria in vivo using saturation mutagenesis and selection
Authors: Danner Stefan , Soppa Jörg 
Published: October 2006

SummaryThe sequence and spacing requirements of the archaeal‘distal promoter element’(DPE) were examined by randomizing positions ‐19 to ‐32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 414 entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high‐activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: ‐32 RGTWWWWRACYGSY ‐19 (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)‐long transcript, and very resistant clones contained both the 700 nt‐long transcript and a second, more abundant, 500 nt‐long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3’‐untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.
Bacterial signalling involving eukaryotic‐type protein kinases
Authors: Zhang Cheng‐Cai 
Published: October 2006

SummaryProtein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities. During the last few years, a number of genes that encode eukaryotic‐type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes. Although many of them have yet to be fully characterized, several studies indicate that eukaryotic‐type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism. A model based on the possible coupling between two‐component systems and eukaryotic‐type protein kinases is proposed to explain the function of eukaryotic‐type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast. These two groups of eukaryotes possess signal‐transduction pathways involving both two‐component systems and eukaryotic protein kinases.
Molecular biology of iron acquisition in Saccharomyces cerevisiae
Authors: Askwith Candice C. , de Silva Deepika , Kaplan Jerry 
Published: October 2006

SummaryIn recent years, significant advances have been made in our understanding of the mechanism and regulation of elemental iron transport in the eukaryote Saccharomyces cerevisiae. This organism employs two distinct iron‐transport systems, depending on the bioavailability of the metal. In iron‐replete environments, a low‐affinity transport system (Km=30μM) is used to acquire iron. This system may also be used to acquire other metals including cobalt and cadmium. When environmental iron is limiting, a high‐affinity (Km=0.15 μM) iron‐transport system is induced. Genetic studies in S. cerevisiae have identified multiple genes involved in both iron‐transport systems. Cell‐surface reductases, FRE1 and FRE2, provide ferrous iron for both systems. A non‐ATP‐dependent transmembrane transporter (FET4) has been identified as the main component of low‐affinity transport. One gene identified to date as part of the high‐affinity transport system is FET3, which shows high sequence and functional homology to multicopper oxidases. Accessory genes required for the functioning of this transport system include a plasma‐membrane copper transporter (CTR1), an intracellular copper transporter (CCC2), and a putative transcription factor (AFT1). The mechanism by which these genes act in concert to ensure iron accumulation in S. cerevisiae presents an intriguing picture, drawing parallels with observations made in the human system almost 40 years ago.
The sxa2‐dependent inactivation of the P‐factor mating pheromone in the fission yeast Schizosaccharomyces pombe
Authors: Ladds Graham , Rasmussen Erik Michael , Young Tom , Nielsen Olaf , Davey John 
Published: October 2006

SummaryHaploid cells of the fission yeast Schizosaccharomyces pombe exist in one of two mating types, referred to as M and P. Conjugation occurs between cells of opposite mating type and is controlled by the reciprocal action of diffusible pheromones. Loss of function of the sxa2 gene in M cells causes hypersensitivity to the P‐factor mating pheromone and a reduction in mating efficiency. Here we demonstrate the secretion of an sxa2‐dependent carboxypeptidase that inactivates P‐factor by removal of the C‐terminal leucine residue.
The bundle‐forming pili of enteropathogenic Escherichia coli: transcriptional regulation by environmental signals
Authors: Puente Jose Luis , Bieber David , Ramer Sandra W. , Murray William , Schoolnik Gary K. 
Published: October 2006

SummaryThe bundle‐forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for the development of circumscribed colonies of bacteria attached to the surfaces of cultured epithelial cells, a process termed the localized adherence (LA) phenotype. Similar lesions are evident in jejunal biopsies from EPEC‐infected children. BFP production is not constitutive, but instead occurs upon transfer of bacteria from nutrient broth to tissue culture media, indicating that the expression of BFP may be environmentally regulated. To learn more about how BFP protein expression is induced during epithelial‐cell adherence, bfpA‐cat transcriptional fusions and northern blot analyses were employed to monitor bfpA expression as a function of environmental signals and growth kinetics. bfpA expression was found to be regulated at the transcriptional level, and to require a separate locus on the EPEC adherence factor (EAF) plasmid. Expression occurred selectively during exponential‐growth phase and was greatest between 35 and 37°C, and in the presence of calcium. Ammonium (20 mM) significantly reduced bfpA mRNA and protein expression and the development of the LA phenotype. Analysis of the bfpA upstream sequence and identification of the transcription initiation site revealed a conventional σ70‐dependent promoter and an AT‐rich tract that might affect promoter activity. Taken together, these findings further support the pathogenic role of BFP
Involvement of N‐acyl‐l‐homoserine lactone autoinducers in controlling the multicellular behaviour of Serratia liquefaciens
Authors: Eberl Leo , Winson Michael K. , Sternberg Claus , Stewart Gordon S. A. B. , Christiansen Gunna , Chhabra Siri Ram , Bycroft Barrie , Williams Paul , Molin Søren , Givskov Michael 
Published: October 2006

SummarySeveral bacterial species possess the ability to differentiate into highly motile swarmer cells capable of rapid surface colonization. In Serratia liquefaciens, we demonstrate that initiation of swarmer‐cell differentiation involves diffusible signal molecules that are released into the growth medium. Using high‐performance liquid chromatography (HPLC), high resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy, we identified N‐butanoyl‐l‐homoserine lactone (BHL) and N‐hexanoyl‐l‐homoserine lactone (HHL) in cell‐free Serratia culture supernatants. BHL and HHL are present in a ratio of approximately 10:1 and their structures were unequivocally confirmed by chemical synthesis. The swrlswarmer initiation) gene, the predicted translation product of which exhibits substantial homology to the Luxl family of putative Nacyl homoserine lactone (AHL) synthases is responsible for directing synthesis of both BHL and HHL. In an swrl mutant, swarming motility is abolished but can be restored by the addition of an exogenous AHL. These results add swarming motility to the rapidly expanding list of phenotypes known to be controlled through quorum sensing.
Conserved amino acids in the N‐ and C‐terminal domains of integral membrane transporter FhuB define sites important for intra‐ and intermolecular interactions
Authors: Böhm Brigitte , Boschert Hartmut , Köster Wolfgang 
Published: October 2006

SummaryTransport of iron(III) hydroxamates across the inner membrane of Escherichia coli is mediated by a periplasmic binding protein‐dependent transport (PBT) mechanism. FhuB, the integral membrane component of the system, is composed of covalently linked halves (FhuB[N] and FhuB[C]) which still function when present as two distinct polypeptide chains. Our analysis of two uptake‐deficient FhuB derivatives provides evidence for a mechanistically novel type of functional complementation:‘domain displacement’ in the cytoplasmic membrane. Amino acid residues 60 and 426 in the FhuB polypeptide chain may define key positions that are important for FhuB[N]–FhuB[C] interaction. Furthermore, FhuB derivatives, altered in either one of their conserved regions ‐ typical of PBT related integral membrane proteins ‐ displayed a dominant negative effect on ferric hydroxamate transport. The experimental data suggest that the two functionally equivalent conserved regions in FhuB[N] and FhuB[C] are primarily involved in the interaction with another component of the transport system, probably FhuC.
Genetic organization and functional analysis of the otn DNA essential for cell‐wall polysaccharide synthesis in Vibrio cholerae O139
Authors: Bik Elisabeth M. , Bunschoten Annelies E. , Willems Rob J. L. , Chang Annie C. Y. , Mooi Frits R. 
Published: October 2006

SummaryIn 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDvco139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1‐acceptor DNA is probably located downstream of rfbDvco139. The DNA region between rfaDvco139 and rfbQRSvco139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell‐wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found inV. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide‐synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.
Protein engineering studies of A‐chain loop 47‐56 of Escherichia coli heat‐labile enterotoxin point to a prominent role of this loop for cytotoxicity
Authors: Feil Ingeborg K. , Reddy Raghava , de Haan Lolke , Merritt Ethan A. , van den Akker Focco , Storm Daniel R. , Hol Wim G. J. 
Published: October 2006

SummaryHeat‐labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240‐residue A chain and five 103‐residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP‐ribosylation of an arginine residue in the G protein, GSα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild‐type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single‐site substitutions have been made in the LT‐A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild‐type residues Thr‐50 and Val‐53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active‐site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine‐substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild‐type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild‐type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.
Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis
Authors: Sundin George W. , Shankar Sandeep , Chugani Sudha A. , Chopade B. A. , Kavanaugh‐Black Andrew , Chakrabarty A. M. 
Published: October 2006

SummaryWe report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2an6 algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild‐type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane‐associated fractions of P. aeruginosa. The cytosolic Ndk was non‐specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction
Evidence for trehalose‐6‐phosphate‐dependent and ‐independent mechanisms in the control of sugar influx into yeast glycolysis
Authors: Hohmann Stefan , Bell Walter , NevesA Maria Jose , Valckx Dirk , Thevelein Johan M. 
Published: October 2006

SummaryIn the yeast Saccharomyces cerevisíae, trehalose‐6‐phosphate (tre‐6‐P) synthase encoded by GGS1/TPS1, is not only involved in the production of trehalose but also in restriction of sugar influx into glycolysis in an unknown fashion; it is therefore essential for growth on glucose or fructose. In this work, we have deleted the TPS2 gene encoding tre‐6‐P phosphatase in a strain which displays very low levels of Ggs1/Tps1, as a result of the presence of the byp1‐3 allele of GGS1/TPS1. The byp1‐3 tps2Δ double mutant showed elevated tre‐6‐P levels along with improved growth and ethanol production, although the estimated concentrations of glycolytic metabolites indicated excessive sugar influx. In the wild‐type strain, the addition of glucose caused a rapid transient increase of tre‐6‐P. In tps2Δ mutant cells, which showed a high tre‐6‐P level before glucose addition, sugar influx into glycolysis appeared to be diminished. Furthermore, we have confirmed that tre‐6‐P inhibits the hexokinases in vitro. These data are consistent with restriction of sugar influx into glycolysis through inhibition of the hexokinases by tre‐6‐P during the switch to fermentative metabolism. During logarithmic growth on glucose the tre‐6‐P level in wild‐type cells was lower than that of the byp1‐3 tps2Δ. mutant. However, the latter strain arrested growth and ethanol production on glucose after about four generations. Hence, other mechanisms, which also depend on Ggs1/Tps1, appear to control sugar influx during growth on glucose. In addition, we provide evidence that the requirement for Ggs1/Tps1 for sporulation may be unrelated to its involvement in trehalose metabolism or in the system controlling glycolysis.
The nd7‐binding protein (Nbp) regulates the ndh gene of Escherichia coti in response to growth phase and is identical to Fis
Authors: Green J. , Anjum M. F. , Guest J. R. 
Published: October 2006

SummaryThe ndh gene that encodes the non‐proton‐translocating NADH dehydrogenase II of Escherichia coli is anaerobically repressed by FNR. However, in the absence of FNR, ndh expression is enhanced by anaerobic growth in media containing amino acids. Two potential regulatory proteins that may be associated with this activation have previously been detected, Arr (amino acid response regulator) and Nbp (ndh‐bind'mg protein). Studies with the heat‐stable Nbp have now shown that it is present in E. coli grown both aerobicaliy and anaerobically in rich and minimal media, indicating that it is not specifically associated with the anaerobic enhancement of ndh expression. The Nbp activity of aerobic cultures was maximal during exponential growth phase (when ndh promoter activity is minimal) but fell rapidly as cultures entered stationary phase and ndh expression increased. Protein purification and mutant studies have further shown that Nbp is identical to the Fis protein (factor for inversion stimulation). Three major and two minor Nbp (Fis)‐binding sites have been identified in the ndh promoter by gel retardation and DNase I footprinting. The major sites are centred at ‐123, 72 and +51, in decreasing order of binding affinity. At low concentrations, Nbp (Fis) increased transcription from the ndh promoter by up to 25%, whereas at higher concentrations it prevented RNA polymerase (RNAP) binding and open complex formation. Consequently, Nbp (Fis) can both activate and repress transcription from the ndh promoter. The results suggest that Nbp (Fis) serves to ensure that the energetically efficient proton‐translocating NADH dehydrogenase I is used in preference to the non‐proton translocating NADH dehydrogenase II during periods of rapid growth, by repressing expression of the ndh gene.
Molecular basis of intercellular adhesion in the biofilm‐forming Staphylococcus epidermidis
Authors: Heilmann Christine , Schweitzer Oliver , Gerke Christiane , Vanittanakom Nongnuch , Mack Dietrich , Götz Friedrich 
Published: October 2006

SummaryThe Staphylococcus epidermidis genes icaABC are involved in the synthesis of the polysaccharide intercellular adhesin (PIA), which is located mainly on the cell surface, as shown by immunofluorescence studies with PIA‐specific antiserum. PIA was shown to be a linear β‐1,6‐linked glucosaminoglycan composed of at least 130 2‐deoxy‐2‐amino‐D‐glucopyrano‐syl residues of which 80–85% are N‐acetylated, the rest being non‐N‐acetylated and positively charged. A transposon insertion in the icaABC gene cluster (ica, intercellular adhesion) led to the loss of several traits, such as the ability to form a biofilm on a polystyrene surface, cell aggregation, and PIA production. The mutant could be complemented by transformation with the IcaABC‐carrying plasmid pCN27. Transfer of pCN27 into the heterologous host Staphylococcus carnosus led to the formation of large cell aggregates, the formation of a biofilm on a glass surface, and PIA expression. The nucleotide sequence of icaABC suggests that the three genes are organized in an operon and that they are co‐transcribed from the mapped ica A promoter. Ica A contains four potential transmembrane helices, indicative of a membrane location. The deduced Ica A sequence shows similarity to those of polysaccharide‐polymerizing enzymes, the most pronounced being with a Rhizobium meliloti N‐acetylglucosaminyltransferase involved in lipo‐chitin biosynthesis (22.5% overall identity and 37.4% overall similarity). This similarity suggests that Ica A has N‐acetylglucosaminyltransferase activity in the formation