bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rho1p mutations specific for regulation of β(1→3)glucan synthesis and the order of assembly of the yeast cell wall
Authors: Roh Dong‐Hyun , Bowers Blair , Riezman Howard , Cabib Enrico 
Published: March 2003

SummaryIn the yeast Saccharomyces cerevisiae, the GTP‐binding protein Rho1 is required for β(1→3)glucan synthase activity, for activation of protein kinase C and the cell integrity pathway and for progression in G1, cell polarization and exocytosis. A genetic screen for cells that become permeabilized at non‐permissive temperature was used to isolate in vitro‐generated mutants of Rho1p. After undergoing a battery of tests, several of them appeared to be specifically defective in the β(1→3)glucan synthesis function of Rho1p. At the non‐permissive temperature (37°C), the mutants developed defects in the cell wall, especially at the tip of new buds. In the yeast cell wall, β(1→6)glucan is linked to both β(1→3)glucan and mannoprotein, as well as occasionally to chitin. We have used the rho1 mutants to study the order of assembly of the cell wall components. The incorporation of [14C]‐glucose into β(1→3)glucan at 37°C was decreased or abolished in the mutants. Concomitantly, a partial defect in the incorporation of label into cell wall mannoproteins and β(1→6)glucan was observed. In contrast, YW3458, an inhibitor of glycosylphosphatidylinositol anchor formation, prevented mannoprotein incorporation, whereas the β(1→3)–β(1→6)glucan complex was synthesized at almost normal levels. As β(1→3)glucan can be synthesized in vitro or in vivo independently, we conclude that the order of addition in vivo is β(1→3)glucan, β(1→6)glucan, mannoprotein. Previous observations indicate that chitin is the last component to be incorporated into the complex.
Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytridNeocallimastixsp.
Authors: Voncken Frank , Boxma Brigitte , Tjaden Joachim , Akhmanova Anna , Huynen Martijn , Verbeek Fons , Tielens Aloysius G. M. , Haferkamp Ilka , Neuhaus H. Ekkehard , Vogels Godfried , Veenhuis Marten , Hackstein Johannes H. P. 
Published: March 2003

SummaryA mitochondrial‐type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungusNeocallimastixsp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA inEscherichia coliconfers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP – similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier ofNeocallimastixsp. L2 belongs to the family of veritable mitochondrial‐type AACs. Hydrogenosome‐bearing anaerobic ciliates possess clearly distinct mitochondrial‐type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellateTrichomonas vaginalisand its homologue fromTrichomonas gallinaedo not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial‐type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.
Depression of Saccharomyces cerevisiae invasive growth on non‐glucose carbon sources requires the Snf1 kinase
Authors: Palecek Sean P. , Parikh Archita S. , Huh Joon H. , Kron Stephen J. 
Published: March 2003

SummaryHaploid Saccharomyces cerevisiae cells growing on media lacking glucose but containing high concentrations of carbon sources such as fructose, galactose, raffinose, and ethanol exhibit enhanced agar invasion. These carbon sources also promote diploid filamentous growth in response to nitrogen starvation. The enhanced invasive and filamentous growth phenotypes are suppressed by the addition of glucose to the media and require the Snf1 kinase. Mutations in the PGI1 and GND1 genes encoding carbon source utilization enzymes confer enhanced invasive growth that is unaffected by glucose but requires active Snf1. Carbon source does not modulate FLO11 flocculin expression, but enhanced polarized bud site selection is necessary for invasion on certain carbon sources. Interestingly, deletion of SNF1 blocks invasion without affecting bud site selection. Snf1 is also required for formation of spokes and hubs in multicellular mats. To examine glucose repression of invasive growth more broadly, we performed genome‐wide microarray expression analysis in wild‐type cells growing on glucose and galactose, and snf1Δ cells on galactose. SNF1 probably mediates glucose repression of multiple genes potentially involved in invasive and filamentous growth. FLO11‐independent cell‐cell attachment, cell wall integrity, and/or polarized growth are affected by carbon source metabolism. In addition, derepression of cell cycle genes and signalling via the cAMP–PKA pathway appears to depend upon SNF1 activity during growth on galactose.
The role of the G2 box, a conserved motif in the histidine kinase superfamily, in modulating the function of EnvZ
Authors: Zhu Yan , Inouye Masayori 
Published: March 2003

SummaryHistidine kinase EnvZ, a transmembrane osmotic sensor for Escherichia coli, is a bifunctional enzyme having OmpR (its cognate response regulator) kinase and phosphorylated OmpR (OmpR‐P) phosphatase activities. Its cytoplasmic domain consists of domain A responsible for dimerization of EnvZ, histidine phosphotransfer and phosphatase activities, and domain B responsible for ATP binding. Here, we have constructed a number of substitution mutations at the G2 box, one of the conserved motifs in domain B, and demonstrated that they influence the phosphatase activity of EnvZ over a wide range. The effects of ADP, a cofactor for the phosphatase activity, were found to be substantially different depending upon the mutations. The effects of these mutations were also examined in vivo using a chimeric Tar–EnvZ construct (Taz1‐1), and the results agreed with the in vitro data for the phosphatase and kinase activities for all mutations. Using Taz1‐1 carrying the T402A mutation, three independent intragenic suppressor mutations (T235M, S269L and E276K) were isolated, and all were found in domain A. Together, the present results demonstrate for the first time that domain A and domain B are functionally co‐ordinated and topologi‐cally arranged in a specific manner. The G2 box may modulate the interaction between these two domains in response to extracellular osmolarity.
A third envelope stress signal transduction pathway in Escherichia coli
Authors: Raffa Robert G. , Raivio Tracy L. 
Published: March 2003

Summary Escherichia coli
uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The σ
E
and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The σ
E
pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the
spy
gene as a new Cpx regulon member of unknown function. Interestingly, induction of
spy
expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx‐regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of
spy
in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx‐regulated genes, however,
baeR cpxR
double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in
E. coli that induces expression of a distinct set of adaptive genes.
High resolution contact probing of the Lrp‐like DNA‐binding protein Ss‐Lrp from the hyperthermoacidophilic crenarchaeote Sulfolobus solfataricus P2
Authors: Enoru‐Eta Julius , Gigot Daniel , Glansdorff Nicolas , Charlier Daniel 
Published: March 2003

SummarySs‐Lrp, from Sulfolobus solfataricus, is an archaeal homologue of the global bacterial regulator Lrp (Leucine‐responsive regulatory protein), which out of all genome‐encoded proteins is most similar to Escherichia coli Lrp (E‐value of 5.6 e−14). The recombinant protein has been purified as a 68 kDa homotetramer. The specific binding of Ss‐Lrp to its own control region is suggestive of negative autoregulation. A high resolution contact map of Ss‐Lrp binding was established by DNase I and hydroxyl radical footprinting, small non‐intercalating groove‐specific ligand‐binding interference, and various base‐specific premodification and base removal binding interference techniques. We show that Ss‐Lrp binds one face of the DNA helix and establishes the most salient contacts with two major groove segments and the intervening minor groove, in a region that overlaps the TATA‐box and BRE promoter elements. Therefore, Ss‐Lrp most likely exerts autoregulation by preventing promoter recognition by TBP and TFB. Moreover, the results demonstrate profound Ss‐Lrp induced structural alterations of sequence stretches flanking the core contact site, and reveal that the deformability of these regions significantly contributes to binding selectivity.
High‐frequency conjugative transfer of antibiotic resistance genes to Yersinia pestis in the flea midgut
Authors: Hinnebusch B. Joseph , Rosso Marie‐Laure , Schwan Tom G. , Carniel Elisabeth 
Published: March 2003

SummaryThe acquisition of foreign DNA by horizontal transfer from unrelated organisms is a major source of variation leading to new strains of bacterial pathogens. The extent to which this occurs varies widely, due in part to lifestyle factors that determine exposure to potential donors. Yersinia pestis, the plague bacillus, infects normally sterile sites in its mammalian host, but forms dense aggregates in the non‐sterile digestive tract of its flea vector to produce a transmissible infection. Here we show that unrelated co‐infecting bacteria in the flea midgut are readily incorporated into these aggregates, and that this close physical contact leads to high‐frequency conjugative genetic exchange. Transfer of an antibiotic resistance plasmid from an Escherichia coli donor to Y. pestis occurred in the flea midgut at a frequency of 10−3 after only 3 days of co‐infection, and after 4 weeks 95% of co‐infected fleas contained an average of 103 antibiotic‐resistant Y. pestis transconjugants. Thus, transit in its arthropod vector exposes Y. pestis to favourable conditions for efficient genetic exchange with microbial flora of the flea gut. Horizontal gene transfer in the flea may be the source of antibiotic‐resistant Y. pestis strains recently isolated from plague patients in Madagascar.
Cell cycle‐dependent expression of an essential SMC‐like protein and dynamic chromosome localization in the archaeon Halobacterium salinarum
Authors: Herrmann Ute , Soppa Jörg 
Published: March 2003

SummaryThe genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily. Two proteins form a novel subfamily and are named ‘SMC‐like proteins of H. salinarum’ (Sph1 and Sph2). Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells. A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures. It allowed us for the first time to study cell cycle‐dependent transcription in an archaeon. The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation. The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle. The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction. These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression. A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures. After an initial dispersed localization, the nucleoid is condensed at mid‐cell. Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions. All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts. The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g. DNA repair, similar to eukaryotic Rad18 proteins.
The segregation of the Escherichia coli origin and terminus of replication
Authors: Li Yongfang , Sergueev Kirill , Austin Stuart 
Published: March 2003

Summary Escherichia coli chromosome replication forks are tethered to the cell centre. Two opposing models describe how the chromosomes segregate. In the extrusion‐capture model, newly replicated DNA is fed bi‐directionally from the forks toward the cell poles, forming new chromosomes in each cell half. Starting with the origins, chromosomal regions segregate away from their sisters progressively as they are replicated. The termini segregate last. In the sister chromosome cohesion model, replication produces sister chromosomes that are paired along much of their length. The origins and most other chromosomal regions remain paired until late in the replication cycle, and all segregate together. We use a combination of microscopy and flow cytometry to determine the relationship of origin and terminus segregation to the cell cycle. Origin segregation frequently follows closely after initiation, in strong support of the extrusion‐capture model. The spatial disposition of the origin and terminus sequences also fits this model. Terminus segregation occurs extremely late in the cell cycle as the daughter cells separate. As the septum begins to invaginate, the termini of the completed sister chromosomes are transiently held apart at the cell centre, on opposite sides of the cell. This may facilitate the resolution of topological linkages between the chromosomes.
In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures
Authors: Gur Eyal , Biran Dvora , Gazit Ehud , Ron Eliora Z. 
Published: March 2003

SummaryThe formation of protein aggregates is associated with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temperatures in minimal media. Homoserine trans‐succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temperatures higher than 44°C in vitro. Above this temperature, we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the soluble fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non‐permissive temperatures. This is the first demonstration of the physiological role of aggregation of a specific protein in the growth of wild‐type bacteria.
Many of the genes required for mating in Saccharomyces cerevisiae are also required for mating in Candida albicans
Authors: Magee B. B. , Legrand Melanie , Alarco Anne‐Marie , Raymond Martine , Magee P. T. 
Published: March 2003

Summary Candida albicans is the single, most frequently isolated human fungal pathogen. As with most fungal pathogens, the factors which contribute to pathogenesis in C. albicans are not known, despite more than a decade of molecular genetic analysis. Candida albicans was thought to be asexual until the discovery of the MTL loci homologous to the mating type (MAT) loci in Saccharomyces cerevisiae led to the demonstration that mating is possible. Using Candida albicans mutants in genes likely to be involved in mating, we analysed the process to determine its similarity to mating in Saccharomyces cerevisiae. We examined disruptions of three of the genes in the MAPK pathway which is involved in filamentous growth in both S. cerevisiae and C. albicans and is known to control pheromone response in the former fungus. Disruptions in HST7 and CPH1 blocked mating in both MTLa and MTLα strains, whereas disruptions in STE20 had no effect. A disruption in KEX2, a gene involved in processing the S. cerevisiae pheromone Mfα, prevented mating in MTLα but not MTLa cells, whereas a disruption in HST6, the orthologue of the STE6 gene which encodes an ABC transporter responsible for secretion of the Mfa pheromone, prevented mating in MTLa but not in MTLα cells. Disruption of two cell wall genes, ALS1 and INT1, had no effect on mating, even though ALS1 was identified by similarity to the S. cerevisiae sexual agglutinin, SAG1. The results reveal that these two diverged yeasts show a surprising similarity in their mating processes.
Lithium and valproate decrease the membrane phosphatidylinositol/phosphatidylcholine ratio
Authors: Ding Daobin , Greenberg Miriam L. 
Published: March 2003

SummaryLithium and valproate, two structurally different anti‐bipolar drugs, cause decreased intracellular inositol in the yeast Saccharomyces cerevisiae and an in‐crease in expression of a structural (INO1) and a regulatory (INO2) gene for phospholipid synthesis that responds to inositol depletion (Vaden, D., Ding, D., Peterson, B., and Greenberg, M.L., 2001, J Biol Chem 276: 15466–15471). We report here that both drugs decrease the relative rate of membrane phosphatidylinositol synthesis and, to a lesser but still significant degree, the steady state relative phosphatidylinositol composition. In addition, both drugs increase the rate of phosphatidylcholine (PC) synthesis. Finally, valproate, but not lithium, increases expression of phosphatidylcholine pathway genes CHO1 and OPI3. The overall effect on membrane phospholipid composition is a reduction in the phosphatidylinositol/phosphatidylcholine ratio by both drugs. Because maintenance of the appropriate phosphatidylinositol/phosphatidylcholine ratio is required for secretory vesicle formation, a decrease in this ratio may have far‐reaching implications for understanding the therapeutic mechanisms of action of these drugs.
Osmoregulation in Lactococcus lactis: BusR, a transcriptional repressor of the glycine betaine uptake system BusA
Authors: Romeo Yves , Obis David , Bouvier Jean , Guillot Alain , Fourçans Aude , Bouvier Isabelle , Gutierrez Claude , Mistou Michel‐Yves 
Published: March 2003

SummaryThe busA (opuA) locus of Lactococcus lactis encodes a glycine betaine uptake system. Transcription of busA is osmotically inducible and its induction after an osmotic stress is reduced in the presence of glycine betaine. Using a genetic screen in CLG802, an Escherichia coli strain carrying a lacZ transcriptional fusion expressed under the control of the busA promoter, we isolated a genomic fragment from the L. lactis subsp. cremoris strain MG1363, which represses transcription from busAp. The cloned locus responsible for this repression was identified as a gene present upstream from the busA operon, encoding a putative DNA binding protein. This gene was named busR. Electrophoretic mobility shift and footprinting experiments showed that BusR is able to bind a site that overlaps the busA promoter. Overexpression of busR in L. lactis reduced expression of busA. Its disruption led to increased and essentially constitutive transcription of busA at low osmolarity. Therefore, BusR is a major actor of the osmotic regulation of busA in L. lactis.
Expression of cnf1 by Escherichia coli J96 involves a large upstream DNA region including the hlyCABD operon, and is regulated by the RfaH protein
Authors: Landraud Luce , Gibert Maryse , Popoff Michel R. , Boquet Patrice , Gauthier Michel 
Published: March 2003

SummaryExamination of 55 clinical isolates of uropathogenic Escherichia coli producing the CNF1 toxin demonstrated that the cnf1 gene is systematically associated with a hly operon via a highly conserved hlyD‐cnf1 intergenic region (igs, 943 bp) as shown in the J96 UPEC strain. We examined if this association could reflect a co‐regulation of the production of these toxins. Translation of cnf1 from an immediately upstream promoter has been shown to be controlled by means of an anti‐Shine–Dalgarno sequence present in the cnf1 coding sequence [fold‐back inhibition (cnf1 fbi)]. The cnf1 fbi was not regulated by elements present in the igs. An RNA covering the full hlyD sequence, the igs and extending on the cnf1 gene, was then detected in the J96 strain. This RNA could be part of a HlyCABD mRNA. Transcription of the haemolysin operon requires RfaH antitermination activity. Inactivation of rfaH in J96 resulted in a 100‐fold reduction of the CNF1 content of bacteria. The production of CNF1 from a plasmidic igscnf1 DNA was not sensitive to RfaH, indicating that this factor acted on cnf1 transcription via the hly promoter. This way the cnf1 fbi mechanism might be overcome by transcription of cnf1 from the haemolysin promoter and antitermination by RfaH. This constitutes a novel system of bacterial virulence factors co‐regulation.
Characterization of Cu,Zn superoxide dismutase (SOD1) gene knock‐out mutant of Cryptococcus neoformans var. gattii: role in biology and virulence
Authors: Narasipura Srinivas D. , Ault Jeffrey G. , Behr Melissa J. , Chaturvedi Vishnu , Chaturvedi Sudha 
Published: March 2003

SummaryThe pathogenic yeast Cryptococcus neoformans (Cn) var. gattii causes meningoencephalitis in healthy individuals, unlike the better known Cn varieties grubii and neoformans, which are common in immunocompromised individuals. The virulence determinants and mechanisms of host predilection are poorly defined for var. gattii. The present study focused on the characterization of a Cu,Zn superoxide dismutase (SOD1) gene knock‐out mutant constructed by developing a DNA transformation system. The sod1 mutant was highly sensitive to the redox cycling agent menadione, and showed fragmentation of the large vacuole in the cytoplasm, but no other defects were seen in growth, capsule synthesis, mating, sporulation, stationary phase survival or auxotrophies for sulphur‐containing amino acids. The sod1 mutant was markedly attenuated in virulence in a mouse model, and it was significantly susceptible to in vitro killing by human neutrophils (PMNs). The deletion of SOD1 also resulted in defects in the expression of a number of virulence factors, i.e. laccase, urease and phospholipase. Complementation of the sod1 mutant with SOD1 resulted in recovery of virulence factor expression and menadione resistance, and in restoration of virulence. Overall, these results suggest that the antioxidant function of Cu,Zn SOD is critical for the pathogenesis of the fungus, but is dispensable in its saprobic life. This report constitutes the first instance in which superoxide dismutase has been directly implicated in the virulence of a fungal pathogen.