bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication restart in gyrB Escherichia coli mutants
Authors: Grompone Gianfranco , Ehrlich S. Dusko , Michel Bénédicte
Published: December 2004

Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co‐lethal in the presence of a ΔtopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA‐dependent primosome assembly, directly on the in‐activated replication forks, without the formation of a recombination intermediate.
Dicarboxylic amino acids and glycine‐betaine regulate chaperone‐mediated protein‐disaggregation under stress
Authors: Diamant Sophia , Rosenthal David , Azem Abdussalam , Eliahu Noa , Ben‐Zvi Anat Peres , Goloubinoff Pierre
Published: December 2004

Active protein‐disaggregation by a chaperone network composed of ClpB and DnaK + DnaJ + GrpE is essential for the recovery of stress‐induced protein aggregates in vitro and in Escherichia coli cells. K‐glutamate and glycine‐betaine (betaine) naturally accumulate in salt‐stressed cells. In addition to providing thermo‐protection to native proteins, we found that these osmolytes can strongly and specifically activate ClpB, resulting in an increased efficiency of chaperone‐mediated protein disaggregation. Moreover, factors that inhibited the chaperone network by impairing the stability of the ClpB oligomer, such as natural polyamines, dilution, or high salt, were efficiently counteracted by K‐glutamate or betaine. The combined protective, counter‐negative and net activatory effects of K‐glutamate and betaine, allowed protein disaggregation and refolding under heat‐shock temperatures that otherwise cause protein aggregation in vitro and in the cell. Mesophilic organisms may thus benefit from a thermotolerant osmolyte‐activated chaperone mechanism that can actively rescue protein aggregates, correctly refold and maintain them in a native state under heat‐shock conditions.
The purified Shigella IpaB and Salmonella SipB translocators share biochemical properties and membrane topology
Authors: Hume Peter J. , McGhie Emma J. , Hayward Richard D. , Koronakis Vassilis
Published: December 2004

An essential early event in Shigella and Salmonella pathogenesis is invasion of non‐phagocytic intestinal epithelial cells. Pathogen entry is triggered by the delivery of multiple bacterial effector proteins into target mammalian cells. The Shigella invasion plasmid antigen B (IpaB), which inserts into the host plasma membrane, is required for effector delivery and invasion. To investigate the biochemical properties and membrane topology of IpaB, we purified the native full‐length protein following expression in laboratory Escherichia coli. Purified IpaB assembled into trimers via an N‐terminal domain predicted to form a trimeric coiled‐coil, and is predominantly α‐helical. Upon lipid interaction, two transmembrane domains (residues 313–333 and 399–419) penetrate the bilayer, allowing the intervening hydrophilic region (334–398) to cross the membrane. Purified IpaB integrated into model, erythrocyte and mammalian cell membranes without disrupting bilayer integrity, and induced liposome fusion in vitro. An IpaB‐derived 162 residue α‐helical polypeptide (IpaB418−580) is a potent inhibitor of IpaB‐directed liposome fusion in vitro and blocked Shigella entry into cultured mammalian cells at 10−8 M. It is also a heterologous inhibitor of Salmonella invasion protein B (SipB) activity and Salmonella entry. In contrast, IpaB418−580 failed to prevent the contact‐dependent haemolytic activity of Shigella. These findings question the proposed direct link between contact‐dependent haemolysis and Shigella entry, and demonstrate that IpaB and SipB share biochemical properties and membrane topology, consistent with a conserved mode of action during cell entry.
Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast
Authors: El Alami Mohamed , Messenguy Francine , Scherens Bart , Dubois Evelyne
Published: December 2004

In Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cell's response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82Δ or kcs1Δ cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR‐regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine‐responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1‐dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS‐box proteins against degradation.
A MAP kinase cascade composed of cell type specific and non‐specific elements controls mating and differentiation of the fungal pathogen Cryptococcus neoformans
Authors: Davidson Robert C. , Nichols Connie B. , Cox Gary M. , Perfect John R. , Heitman Joseph
Published: December 2004

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle in which the α allele of the mating type locus is linked to virulence and haploid differentiation. Here we analysed a conserved MAP kinase cascade composed of mating‐type specific (Ste11α, Ste12α) and non‐specific (Ste7, Cpk1) elements. Gene disruption experiments demonstrate that this specialized MAP kinase pathway is required for both mating and cell type‐specific differentiation but not for virulence. The Ste11α, Ste7 and Cpk1 kinases were found to act as a co‐ordinate signalling module, whereas the Ste12α transcription factor functions with a redundant partner or in a branched or parallel signalling pathway. Our studies illustrate how MAP kinase cascades can be constructed from cell type‐specific and non‐specific components, yielding pathways that contribute to cell type‐specific patterns of signalling and differentiation.
Ty3 requires yeast La homologous protein for wild‐type frequencies of transposition
Authors: Aye Michael , Sandmeyer Suzanne B.
Published: December 2004

The Saccharomyces cerevisiae retrovirus‐like element Ty3 inserts specifically into the initiation sites of genes transcribed by RNA polymerase III (pol III). A strain with a disruption of LHP1, which encodes the homologue of autoantigen La protein, was recovered in a screen for mutants defective in Ty3 transposition. Transposition into a target composed of divergent tRNA genes was decreased eightfold. In lhp1 mutants, Ty3 polyproteins were produced at wild‐type levels, assembled into virus‐like particles (VLPs) and processed efficiently. The amount of cDNA associated with these particles was about half the amount in a wild‐type control at early times, but approached the wild‐type level after 48 h of induction. Ty3 integration was examined at two genomic tRNA gene families and two plasmid‐borne tRNA promoters. Integration was significantly decreased at one of the tRNA gene families, but was only slightly decreased at the second tRNA gene family. These findings suggest that Lhp1p contributes to Ty3 cDNA synthesis, but might also act at a target‐specific step, such as integration.
Downregulation of the heat shock response is independent of DnaK and σ32 levels in Caulobacter crescentus
Authors: Da Silva Antonio C.A. , Simão Rita C.G. , Susin Michelle F. , Baldini Regina L. , Avedissian Marcelo , Gomes Suely L.
Published: December 2004

Expression of heat shock genes in Gram‐negative proteobacteria is positively modulated by the transcriptional regulator RpoH, the σ32 subunit of RNA polymerase (RNAP). In this study we investigated the chaperones DnaK/DnaJ and GroES/GroEL as possible modulators of the heat response in Caulobacter crescentus. We have shown that cells overexpressing DnaK show poor induction of heat shock protein (HSP) synthesis, even though σ32 levels present a normal transient increase upon heat stress. On the other hand, depletion of DnaK led to higher levels of σ32 and increased transcription of HSP genes, at normal growth temperature. In contrast, changes in the amount of GroES/EL had little effect on σ32 levels and HSP gene transcription. Despite the strong effect of DnaK levels on the induction phase of the heat shock response, downregulation of HSP synthesis was not affected by changes in the amount this chaperone. Thus, we propose that competition between σ32 and σ73, the major sigma factor, for the core RNAP could be the most important factor controlling the shut‐off of HSP synthesis during recovery phase. In agreement with this hypothesis, we have shown that expression of σ73 gene is heat shock inducible.
Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance
Authors: Land Kirkwood M. , Delgadillo‐Correa Maria G. , Tachezy Jan , Vanacova Stepanka , Hsieh Christine L. , Sutak Robert , Johnson Patricia J.
Published: December 2004

Ferredoxin, Fd, is often deficient in metronidazole‐resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by ∼2.6 and ∼2.0 kBp of the Fd 5′ and 3′ flanking regions (pKO‐FD‐NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO‐FD‐NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.
Receptor conformational changes enhance methylesterase activity during chemotaxis by Bacillus subtilis
Authors: Bunn Michael W. , Ordal George W.
Published: December 2004

Addition and removal of the attractant asparagine causes methanol formation as a consequence of methylation and demethylation of conserved glutamate residues in the Bacillus subtilis chemotaxis receptor McpB C‐terminal domain. We found that methanol was released on both addition and removal of asparagine even when the response regulator domain of CheB was removed (to produce CheB(141−357)). Thus, in undergoing the transition from unbound receptor to ligand‐bound adapted receptor, the receptor must pass through a state of heightened susceptibility to demethylation by CheB that is independent of phosphorylation. The same result occurred when the aspartate phosphorylation site of CheB, Asp54, had been mutated to an asparagine residue, provided the enzyme was sufficiently induced. However, no methanol release was observed for an active site point mutant, cheB(S173C), in response to addition or removal of asparagine even when induced. Finally, methanol release was observed only for attractant addition in a mutant background lacking the coupling proteins, CheW and CheV, provided CheB(141−357) was present. Thus, on attractant addition, methanol must arise from a transient conformation of the receptor C‐terminal domain that is an intrinsic property of the receptor; on attractant removal, however, methanol must arise from a different transient conformation, one dependent on the presence of coupling proteins.
Lack of O‐antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica
Authors: Kukkonen Maini , Suomalainen Marjo , Kyllönen Päivi , Lähteenmäki Kaarina , Lång Hannu , Virkola Ritva , Helander Ilkka M. , Holst Otto , Korhonen Timo K.
Published: December 2004

The O‐antigen of lipopolysaccharide (LPS) is a virulence factor in enterobacterial infections, and the advantage of its genetic loss in the lethal pathogen Yersinia pestis has remained unresolved. Y. pestis and Salmonella enterica express β‐barrel surface proteases of the omptin family that activate human plasminogen. Plasminogen activation is central in pathogenesis of plague but has not, however, been found to be important in diarrhoeal disease. We observed that the presence of O‐antigen repeats on wild‐type or recombinant S. enterica, Yersinia pseudotuberculosis or Escherichia coli prevents plasminogen activation by PgtE of S. enterica and Pla of Y. pestis; the O‐antigen did not affect incorporation of the omptins into the bacterial outer membrane. Purified His6‐Pla was successfully reconstituted with rough LPS but remained inactive after reconstitution with smooth LPS. Expression of smooth LPS prevented Pla‐mediated adhesion of recombinant E. coli to basement membrane as well as invasion into human endothelial cells. Similarly, the presence of an O‐antigen prevented PgtE‐mediated bacterial adhesion to basement membrane. Substitution of Arg‐138 and Arg‐171 of the motif for protein binding to lipid A 4′‐phosphate abolished proteolytic activity but not membrane translocation of PgtE, indicating dependence of omptin activity on a specific interaction with lipid A. The results suggest that Pla and PgtE require LPS for activity and that the O‐antigen sterically prevents recognition of large‐molecular‐weight substrates. Loss of O‐antigen facilitates Pla functions and invasiveness of Y. pestis; on the other hand, smooth LPS renders plasminogen activator cryptic in S. enterica.
Genetics of capsule O‐acetylation in serogroup C, W‐135 and Y meningococci
Authors: Claus Heike , Borrow Ray , Achtman Mark , Morelli Giovanna , Kantelberg Carmen , Longworth Emma , Frosch Matthias , Vogel Ulrich
Published: December 2004

Capsular polysaccharides of serogroup C, W‐135 and Y meningococci were previously reported to be O‐acetylated at the sialic acid residues. There is evidence that O‐acetylation affects the immunogenicity of polysaccharide vaccines. We identified genes indispensable for O‐acetylation of serogroup C, W‐135 and Y meningococci downstream of the capsule synthesis genes siaA–D. The genes were co‐transcribed with the sia operon as shown by reverse transcription polymerase chain reaction analysis. The putative capsular polysaccharide O‐acetyltransferases were designated OatC and OatWY. The protein OatWY of serogroups W‐135 and Y showed sequence homologies to members of the NodL–LacA–CysE family of bacterial acetyltransferases, whereas no sequence homology with any known protein in the different databases was found for the serogroup C protein OatC. In serogroup W‐135 and Y meningococci, several clonal lineages either lacked OatWY or OatWY was inactivated by insertion of IS1301. For serogroup C meningococci, we observed in vitro phase variation of O‐acetylation, which resulted from slipped‐strand mispairing in homopolymeric tracts. This finding explains the observation of naturally occurring de‐O‐acetylated serogroup C meningococci. Our report is the first description of sequences of sialic acid O‐acetyltransferase genes that have not been cloned from either other bacterial or mammalian organisms.
Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum
Authors: Otto Grant P. , Wu Mary Y. , Clarke Margaret , Lu Hao , Anderson O. Roger , Hilbi Hubert , Shuman Howard A. , Kessin Richard H.
Published: December 2004

The Gram‐negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free‐living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum (ER) and mitochondria and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy‐inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. As L. pneumophila replicates in Dictyostelium discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1, apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild‐type D. discoideum. Furthermore, a green fluorescent protein (GFP)‐tagged marker of autophagosomes, Apg8, does not systematically co‐localize with DsRed‐labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum.
Cross‐species hybridization of a Borrelia burgdorferi DNA array reveals infection‐ and culture‐associated genes of the unsequenced genome of the relapsing fever agent Borrelia hermsii
Authors: Zhong Jianmin , Barbour Alan G.
Published: December 2004

The known genome sequence of Borrelia burgdorferi, an agent of Lyme borreliosis, was used to study the genetic content and gene expression in B. hermsii, another spirochete pathogen and a cause of relapsing fever. Cross‐species hybridization of a DNA array representing 1628 open reading frames (ORF) of B. burgdorferi with genomic DNA of B. hermsii indicated that the latter organism has at least 81% of the chromosomal genes and 43% of the plasmid genes of B. burgdorferi. We then carried out quantitative hybridization of the arrays with multiple replicates of cDNA produced from B. hermsii cells growing in the blood of infected mice or in culture medium that was adjusted to the same pH, temperature and a spirochete density as infected blood. Of 642 B. burgdorferi ORFs hybridized by all replicates under both conditions, 12 (1.9%) demonstrated differential expression by a regularized t‐test and stringent criteria. BBP07 and BBG30, two plasmid‐borne ORFs with the greatest measurable difference in expression between in vivo and in vitro conditions, putatively encode proteins of unknown function. Orthologues of BBP07 in B. hermsii were identified, and increased expression in infected mice was demonstrated by quantitative reverse‐transcriptase polymerase chain reaction.
Several distinct localization patterns for penicillin‐binding proteins in Bacillus subtilis
Authors: Scheffers Dirk‐Jan , Jones Laura J. F. , Errington Jeffery
Published: December 2004

Bacterial cell shape is determined by a rigid external cell wall. In most non‐coccoid bacteria, this shape is also determined by an internal cytoskeleton formed by the actin homologues MreB and/or Mbl. To gain further insights into the topological control of cell wall synthesis in bacteria, we have constructed green fluorescent protein (GFP) fusions to all 11 penicillin‐binding proteins (PBPs) expressed during vegetative growth of Bacillus subtilis. The localization of these fusions was studied in a wild‐type background as well as in strains deficient in FtsZ, MreB or Mbl. PBP3 and PBP4a localized specifically to the lateral wall, in distinct foci, whereas PBP1 and PBP2b localized specifically to the septum. All other PBPs localized to both the septum and the lateral cell wall, sometimes with irregular distribution along the lateral wall or a preference for the septum. This suggests that cell wall synthesis is not dispersed but occurs at specific places along the lateral cell wall. The results implicate PBP3, PBP5 and PBP4a, and possibly PBP4, in lateral wall growth. Localization of PBPs to the septum was found to be dependent on FtsZ, but the GFP–PBP fluorescence patterns were not detectably altered in the absence of MreB or Mbl.
The RNA degradosome and poly(A) polymerase of Escherichia coli are required in vivo for the degradation of small mRNA decay intermediates containing REP‐stabilizers
Authors: Khemici Vanessa , Carpousis Agamemnon J.
Published: December 2004

In Escherichia coli, REP‐stabilizers are structural elements in polycistronic messages that protect 5′‐proximal cistrons from 3′→5′ exonucleolytic degradation. The stabilization of a protected cistron can be an important determinant in the level of gene expression. Our results suggest that RNase E, an endoribonuclease, initiates the degradation of REP‐stabilized mRNA. However, subsequent degradation of mRNA fragments containing a REP‐stabilizer poses a special challenge to the mRNA degradation machinery. Two enzymes, the DEAD‐box RNA helicase, RhlB and poly(A) polymerase (PAP) are required to facilitate the degradation of REP‐stabilizers by polynucleotide phosphorylase (PNPase). This is the first in vivo evidence that these enzymes are required for the degradation of REP‐stabilizers. Furthermore, our results show that REP degradation by RhlB and PNPase requires their association with RNase E as components of the RNA degradosome, thus providing the first in vivo evidence that this ribonucleolytic multienzyme complex is involved in the degradation of structured mRNA fragments.
A novel integrative and conjugative element (ICE) of Escherichia coli: the putative progenitor of the Yersinia high‐pathogenicity island
Authors: Schubert Sören , Dufke Severin , Sorsa Johanna , Heesemann Jürgen
Published: December 2004

Diversification of bacterial species and pathotypes is largely caused by horizontal transfer of diverse DNA elements such as plasmids, phages and genomic islands (e.g. pathogenicity islands, PAIs). A PAI called high‐pathogenicity island (HPI) carrying genes involved in siderophore‐mediated iron acquisition (yersiniabactin system) has previously been identified in Yersinia pestis, Y. pseudotuberculosis and Y. enterocolitica IB strains, and has been characterized as an essential virulence factor in these species. Strikingly, an orthologous HPI is a widely distributed virulence determinant among Escherichia coli and other Enterobacteriaceae which cause extraintestinal infections. Here we report on the HPI of E. coli strain ECOR31 which is distinct from all other HPIs described to date because the ECOR31 HPI comprises an additional 35 kb fragment at the right border compared to the HPI of other E. coli and Yersinia species. This part encodes for both a functional mating pair formation system and a DNA‐processing region related to plasmid CloDF13 of Enterobacter cloacae. Upon induction of the P4‐like integrase, the entire HPI of ECOR31 is precisely excised and circularised. The HPI of ECOR31 presented here resembles integrative and conjugative elements termed ICE. It may represent the progenitor of the HPI found in Y. pestis and E. coli, revealing a missing link in the horizontal transfer of an element that contributes to microbial pathogenicity upon acquisition.
A HU‐like gene mutation in Rhizobium leguminosarum bv. viciae affects the expression of nodulation genes
Authors: Li Qiang , Feng Jie , Hu Hai‐Liang , Chen Xiao‐Chun , Li Feng‐Qing , Hong Guo‐Fan
Published: December 2004

NodD is the major regulator of nod genes expression in rhizobia. Previously, a HU‐like protein in Rhizobium leguminosarum bv. viciae has been identified to bind specifically with nod promoters and be involved in in vitro nodD transcription, but its in vivo function remained unknown. In this work we have cloned and sequenced the R. leguminosarum bv. viciae gene, named hurL, for this HU‐like protein. Using the E. coli‐expressed HurL proteins, we proved that HurL had high affinity to several nod promoters and showed a stimulation effect on in vitro nodD transcription at appropriate concentration. The R. leguminosarum bv. viciae hurL gene was mutated by insertion of a kanamycin resistance cassette. The obtained hurL mutant strain M704 exhibited poor growth under free‐living conditions and failed to induce nodules on Pisum sativum cv. Frisson and Vicia hirsuta. Further studies of NodD production and nod genes‐lacZ fusions expression in the hurL mutant revealed that inactivation of hurL led to severe impairment in the nodD expression, repression in the inducible expression of nodA and nodF, and slight enhancement in the expression of px2, a gene identified earlier in this lab. These results suggested that hurL might be required for maintaining the normal expression of nod genes in R. leguminosarum bv. viciae.
R174 of Escherichia coli FtsZ is involved in membrane interaction and protofilament bundling, and is essential for cell division
Authors: Koppelman Cecile‐Marie , Aarsman Mirjam E. G. , Postmus Jarne , Pas Evelien , Muijsers Anton O. , Scheffers Dirk‐Jan , Nanninga Nanne , Den Blaauwen Tanneke
Published: December 2004

We investigated the interaction between FtsZ and the cytoplasmic membrane using inside‐out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane‐bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ‐R174D was fused to green fluorescent protein (GFP). FtsZ‐R174D‐GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non‐permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild‐type FtsZ. FtsZ‐R174D could also localize in an FtsZ depletion strain. However, in contrast to wild‐type FtsZ, FtsZ‐R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ‐R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl2. This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.
The high‐resolution functional map of bacteriophage SPP1 portal protein
Authors: Isidro Anabela , Santos Mário A. , Henriques Adriano O. , Tavares Paulo
Published: December 2004

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl‐terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high‐resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.
Deletion analyses of the peptidoglycan‐associated lipoprotein Pal reveals three independent binding sequences including a TolA box
Authors: Cascales Eric , Lloubès Roland
Published: December 2004

The Tol–Pal system of the Escherichia coli cell envelope is composed of five proteins. TolQ, TolR and TolA form a complex in the inner membrane, whereas TolB is a periplasmic protein interacting with Pal, the peptidoglycan‐associated lipoprotein anchored to the outer membrane. This system is required for outer membrane integrity and has been shown to form a trans‐envelope bridge linking inner and outer membranes. The TolA–Pal interaction plays an important role in the function of this system and has been found to depend on the proton motive force and the TolQ and TolR proteins. The Pal lipoprotein interacts with many components, such as TolA, TolB, OmpA, the major lipoprotein and the murein layer. In this study, six pal deletions were constructed. The analyses of the resulting Pal protein functions and interactions defined an N‐terminal region of 40 residues, which can be deleted without any cell‐damaging effect, and three independent regions required for its interaction with TolA, OmpA and TolB or the peptidoglycan. The analyses of the integrity of the cells producing the various Pal lipoproteins revealed strong outer membrane destabilization only when binding regions were deleted. Furthermore, a conserved polypeptide sequence located downstream of the peptidoglycan binding motif of Pal was required for the TolA–Pal interaction and for the maintenance of outer membrane stability.
A search for amino acid substitutions that universally activate response regulators
Authors: Smith Jenny G. , Latiolais Jamie A. , Guanga Gerald P. , Pennington J. Daniel , Silversmith Ruth E. , Bourret Robert B.
Published: December 2004

Two‐component regulatory systems, typically composed of a sensor kinase to detect a stimulus and a response regulator to execute a response, are widely used by microorganisms for signal transduction. Response regulators exhibit a high degree of structural similarity and undergo analogous activating conformational changes upon phosphorylation. The activity of particular response regulators can be increased by specific amino acid substitutions, which either prolong the lifetime or mimic key features of the phosphorylated state. We probed the universality of response regulator activation by amino acid substitution. Thirty‐six mutations that activate 11 different response regulators were identified from the literature. To determine whether the activated phenotypes would be retained in the context of a different response regulator, we recreated 51 analogous amino acid substitutions at corresponding positions of CheY. About 55% of the tested substitutions completely or partially inactivated CheY, ≈ 30% were phenotypically silent, and ≈ 15% activated CheY. Three previously uncharacterized activated CheY mutants were found. The 94NS (and presumably 94NT) substitutions resulted in resistance to CheZ‐mediated dephosphorylation. The 113AP substitution led to enhanced autophosphorylation and may increase the fraction of non‐phosphorylated CheY molecules that populate the activated conformation. The locations of activating substitutions on the response regulator three‐dimensional structure are generally consistent with current understanding of the activation mechanism. The best candidates for potentially universal activating substitutions of response regulators identified in this study were 13DK and 113AP.
Signal quenching, detoxification and mineralization of vir gene‐inducing phenolics by the VirH2 protein of Agrobacterium tumefaciens
Authors: Brencic Anja , Eberhard Anatol , Winans Stephen C.
Published: December 2004

Plant tumorigenesis by Agrobacterium tumefaciens requires approximately 20 Vir proteins, transcription of which is induced by a family of phenolic compounds released from plant wound sites. One Vir protein, VirH2, plays a role in the metabolism of at least one phenolic inducer inasmuch as it converts ferulic acid, a potent vir gene inducer, to the non‐inducer caffeate by O‐demethylation of a methoxyl group. Here, we tested VirH2‐dependent O‐demethylation of 16 other vir‐inducing phenolics, and detected this activity for each compound. However, O‐demethylation rates differed enormously, with the strongest vir gene inducers such as acetosyringone being demethylated extremely slowly. Compounds containing two methoxyl groups were demethylated at both positions. In general, phenolic inducers were more toxic than their demethylated counterparts. A virH2 mutant was more sensitive than the wild type to growth inhibition by virtually all phenolic inducers tested, indicating that VirH2 detoxifies these compounds. VirH2 also played a role in mineralization of some phenolics. It converted vanillate to protocatechuate, which was then mineralized via the β‐ketoadipate pathway. Vanillyl alcohol and vanillin were also mineralized after being oxidized to vanillate. All three compounds served as sole sources of carbon, whereas the remaining 13 compounds did not.
Analysis of HutP‐dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N‐terminus of HutP in binding of HutP to the antiterminator RNA
Authors: Oda Masanao , Kobayashi Nobuhiro , Fujita Masaya , Miyazaki Yuusuke , Sadaie Yoshito , Kurusu Yasurou , Nishikawa Satoshi
Published: December 2004

We investigated HutP‐dependent transcription antitermination of the Bacillus subtilis hut operon. In vitro transcription assays with the B. subtilisσA‐containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine. Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo. Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator. The in vivo HutP antiterminator activity was reduced by the mutations in the N‐terminal region of HutP. The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro. A 25‐mer peptide consisting of amino acid residues 2–26 of HutP bound to the antiterminator RNA. These results indicated that the N‐terminus of HutP is involved in binding of HutP to the antiterminator RNA.
A Gα subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans
Authors: Latijnhouwers Maita , Ligterink Wilco , Vleeshouwers Vivianne G.A.A. , van West Pieter , Govers Francine
Published: December 2004

The heterotrimeric G‐protein pathway is a ubiquitous eukaryotic signalling module that is known to regulate growth and differentiation in many plant pathogens. We previously identified Pigpa1, a gene encoding a G‐protein α subunit from the potato late blight pathogen Phytophthora infestans. P. infestans belongs to the class oomycetes, a group of organisms in which signal transduction processes have not yet been studied at the molecular level. To elucidate the function of Pigpa1, PiGPA1‐deficient mutants were obtained by homology‐dependent gene silencing. The Pigpa1‐silenced mutants produced zoospores that turned six to eight times more frequently, causing them to swim only short distances compared with wild type. Attraction to the surface, a phenomenon known as negative geotaxis, was impaired in the mutant zoospores, as well as autoaggregation and chemotaxis towards glutamic and aspartic acid. Zoospore production was reduced by 20–45% in different Pigpa1‐silenced mutants. Transformants expressing constitutively active forms of PiGPA1, containing amino acid substitutions (R177H and Q203L), showed no obvious phenotypic differences from the wild‐type strain. Infection efficiencies on potato leaves ranged from 3% to 14% in the Pigpa1‐silenced mutants, compared with 77% in wild type, showing that virulence is severely impaired. The results prove that PiGPA1 is crucial for zoospore motility and for pathogenicity in an important oomycete plant pathogen.
Escherichia coliprereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA
Authors: Ryan Valorie T. , Grimwade Julia E. , Camara Johanna E. , Crooke Elliott , Leonard Alan C.
Published: December 2004

Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre‐RC) that unwind theEscherichia colichromosome's origin of replication,oriC. Loss of either Fis or IHF perturbs synchronous initiation fromoriCcopies in rapidly growingE. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiledoriCtemplates. Low levels of Fis inhibitedoriCunwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites onoriC. This behaviourin vitrois analogous to observed assembly of pre‐RC in synchronizedE. coli. We propose that as new DnaA is synthesized inE. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.
Identification of competence pheromone responsive genes in Streptococcus pneumoniae by use of DNA microarrays
Authors: Peterson Scott N. , Sung Chang Kyoo , Cline Robin , Desai Bhushan V. , Snesrud Erik C. , Luo Ping , Walling Jennifer , Li Haiying , Mintz Michelle , Tsegaye Getahun , Burr Patrick C. , Do Yu , Ahn Susie , Gilbert Joseph , Fleischmann Robert D. , Morrison Donald A.
Published: December 2004

Natural genetic transformation in Streptococcus pneumoniae is controlled in part by a quorum‐sensing system mediated by a peptide pheromone called competence‐stimulating peptide (CSP), which acts to coordinate transient activation of genes required for competence. To characterize the transcriptional response and regulatory events occurring when cells are exposed to competence pheromone, we constructed DNA microarrays and analysed the temporal expression profiles of 1817 among the 2129 unique predicted open reading frames present in the S. pneumoniae TIGR4 genome (84%). After CSP stimulation, responsive genes exhibited four temporally distinct expression profiles: early, late and delayed gene induction, and gene repression. At least eight early genes participate in competence regulation including comX, which encodes an alternative sigma factor. Late genes were dependent on ComX for CSP‐induced expression, many playing important roles in transformation. Genes in the delayed class (third temporal wave) appear to be stress related. Genes repressed during the CSP response include ribosomal protein loci and other genes involved in protein synthesis. This study increased the number of identified CSP‐responsive genes from approximately 40 to 188. Given the relatively large number of induced genes (6% of the genome), it was of interest to determine which genes provide functions essential to transformation. Many of the induced loci were subjected to gene disruption mutagenesis, allowing us to establish that among 124 CSP‐inducible genes, 67 were individually dispensable for transformation, whereas 23 were required for transformation.
Differential transcriptome profiling identifies Plasmodium genes encoding pre‐erythrocytic stage‐specific proteins
Authors: Kaiser Karine , Matuschewski Kai , Camargo Nelly , Ross Jessica , Kappe Stefan H. I.
Published: December 2004

Invasive sporozoite and merozoite stages of malaria parasites that infect mammals enter and subsequently reside in hepatocytes and red blood cells respectively. Each invasive stage may exhibit unique adaptations that allow it to interact with and survive in its distinct host cell environment, and these adaptations are likely to be controlled by differential gene expression. We used suppression subtractive hybridization (SSH) of Plasmodium yoelii salivary gland sporozoites versus merozoites to identify stage‐specific pre‐erythrocytic transcripts. Sequencing of the SSH library and matching the cDNA sequences to the P. yoelii genome yielded 25 redundantly tagged genes including the only two previously characterized sporozoite‐specific genes encoding the circumsporozoite protein (CSP) and thrombospondin‐related anonymous protein (TRAP). Twelve novel genes encode predicted proteins with signal peptides, indicating that they enter the secretory pathway of the sporozoite. We show that one novel protein bearing a thrombospondin type 1 repeat (TSR) exhibits an expression pattern that suggests localization in the sporozoite secretory rhoptry organelles. In addition, we identified a group of four genes encoding putative low‐molecular‐mass proteins. Two proteins in this group exhibit an expression pattern similar to TRAP, and thus possibly localize in the sporozoite secretory micronemes. Proteins encoded by the differentially expressed genes identified here probably mediate specific interactions of the sporozoite with the mosquito vector salivary glands or the mammalian host hepatocyte and are not used during merozoite–red blood cell interactions.
Smt3/SUMO and Ubc9 are required for efficient APC/C‐mediated proteolysis in budding yeast
Authors: Dieckhoff Patrick , Bolte Melanie , Sancak Yasemin , Braus Gerhard H. , Irniger Stefan
Published: December 2004

Ubiquitin‐mediated proteolysis triggered by the anaphase‐promoting complex/cyclosome (APC/C) is essential for sister chromatid separation and the mitotic exit. Like ubiquitylation, protein modification with the small ubiquitin‐related modifier SUMO appears to be important during mitosis, because yeast cells impaired in the SUMO‐conjugating enzyme Ubc9 were found to be blocked in mitosis and defective in cyclin degradation. Here, we analysed the role of SUMOylation in the metaphase/anaphase transition and in APC/C‐mediated proteolysis in Saccharomyces cerevisiae. We show that cells depleted of Ubc9 or Smt3, the yeast SUMO protein, mostly arrested with undivided nuclei and with high levels of securin Pds1. This metaphase block was partially relieved by a deletion of PDS1. The absence of Ubc9 or Smt3 also resulted in defects in chromosome segregation. Temperature‐sensitive ubc9‐2 mutants were delayed in proteolysis of Pds1 and of cyclin Clb2 during mitosis. The requirement of SUMOylation for APC/C‐mediated degradation was tested more directly in G1‐arrested cells. Both ubc9‐2 and smt3‐331 mutants were defective in efficient degradation of Pds1 and mitotic cyclins, whereas proteolysis of unstable proteins that are not APC/C substrates was unaffected. We conclude that SUMOylation is needed for efficient proteolysis mediated by APC/C in budding yeast.
Excretion and uptake of cadaverine by CadB and its physiological functions in Escherichia coli
Authors: Soksawatmaekhin Waraporn , Kuraishi Aiko , Sakata Kaori , Kashiwagi Keiko , Igarashi Kazuei
Published: December 2004

The functions of the putative cadaverine transport protein CadB were studied in Escherichia coli. CadB had both cadaverine uptake activity, dependent on proton motive force, and cadaverine excretion activity, acting as a cadaverine‐lysine antiporter. The Km values for uptake and excretion of cadaverine were 20.8 and 303 µM respectively. Both cadaverine uptake and cadaverine‐lysine antiporter activities of CadB were functional in cells. Cell growth of a polyamine‐requiring mutant was stimulated slightly at neutral pH by the cadaverine uptake activity and greatly at acidic pH by the cadaverine‐lysine antiporter activity. At acidic pH, the operon containing cadB and cadA, encoding lysine decarboxylase, was induced in the presence of lysine. This caused neutralization of the extracellular medium and made possible the production of CO2 and cadaverine and aminopropylcadaverine instead of putrescine and spermidine. The induction of the cadBA operon also generated a proton motive force. When the cadBA operon was not induced, the expression of the speF–potE operon, encoding inducible ornithine decarboxylase and a putrescine‐ornithine antiporter, was increased. The results indicate that the cadBA operon plays important roles in cellular regulation at acidic pH.
Effect of extrinsic denervation on muscarinic neurotransmission in the canine ileocolonic region
Authors: Leelakusolvong S. , Bharucha A. E. , Sarr M. G. , Hammond P. I. , Brimijoin S. , Phillips S. F.
Published: December 2004

Abstract  To explore the hypothesis that denervation hypersensitivity increases ileocolonic motor activity after extrinsic denervation, we compared muscarinic neurotransmission in canine ileocolonic loops that were isolated and either extrinsically innervated or extrinsically denervated. We recorded ileal, ileocolonic sphincter (ICS) and colonic pressures, and colonic tone, compliance and relaxation during ileal distention. Muscarinic effects were probed by neostigmine, and minimally effective doses of muscarinic receptor antagonists. Denervation augmented ileal, ICS and colonic contractile activity; colonic high‐amplitude propagating contractions (HAPCs) were also augmented; colonic relaxation during ileal distention was abolished. Neostigmine induced HAPCs in both loop preparations. Pirenzipine (M1 antagonist) reduced ileal contractile activity in all loops and reduced colonic relaxation during ileal distention in innervated loops. Pirenzipine also reduced colonic tone and colonic HAPCs, more in denervated loops. Darifenacin (M3 antagonist) reduced ileocolonic contractile activity and tone more than did AF‐DX 116 (M2 antagonist) in all loops. Cholinergic receptor subtypes modulate different facets of ileocolonic motor activity in the canine ileocolonic region. Increased sensitivity at M1 muscarinic receptors may partly account for the effects of extrinsic denervation.