bluuebunny/crossref_metadata_2025_split huggingface dataset:
The inner membrane subassembly of the enteropathogenic Escherichia coli bundle‐forming pilus machine
Authors: Crowther Lynette J. , Anantha Ravi P. , Donnenberg Michael S. 
Published: December 2004

SummaryType IV pili (Tfps) are filamentous surface appendages expressed by Gram‐negative microorganisms and play numerous roles in bacterial cell biology. Tfp biogenesis machineries are highly conserved and resemble protein secretion and DNA uptake systems. Although components of Tfp biogenesis systems have been identified, it is not known how they interact to form these machineries. Using the bundle‐forming pilus (BFP) of enteropathogenic Escherichia coli as a model Tfp system, we provide evidence of a cytoplasmic membrane subassembly of the Tfp assembly machine composed of putative cytoplasmic nucleotide‐binding and cytoplasmic membrane proteins. A combination of genetic, biochemical and biophysical approaches revealed interactions among putative cytoplasmic nucleotide‐binding proteins BfpD and BfpF and cytoplasmic membrane proteins BfpC and BfpE of the BFP biogenesis machine. The polytopic membrane protein BfpE appears to be a central component of this subassembly as it interacts with BfpC, BfpD and BfpF. We report that BFP biogenesis probably requires interactions among BfpC, BfpD and BfpE, whereas BFP retraction requires interaction of the PilT‐like putative ATPase BfpF with a conserved domain of BfpE. BfpE is the first protein that is not a member of the PilT family to be implicated in Tfp retraction. Furthermore, we found that the putative ATPases BfpD and BfpF play antagonistic roles in BFP biogenesis and retraction, respectively, by interacting with distinct domains of the BFP biogenesis machine.
Mode of action of the TyrR protein: repression and activation of the tyrP promoter of Escherichia coli
Authors: Yang Ji , Hwang Jung Shan , Camakaris Helen , Irawaty Windy , Ishihama Akira , Pittard James 
Published: December 2004

SummaryThe tyrP gene of Escherichia coli encodes a tyrosine specific transporter. Its synthesis is repressed by tyrosine but is activated by phenylalanine and to a lesser extent by tryptophan. Both of these effects are mediated by the TyrR protein when it binds to one or both of its cognate binding sites (TyrR boxes) which encompass nucleotides − 30 to − 75. Activation in the presence of phenylalanine or tryptophan involves a dimer binding to the upstream box and interacting with the α subunit (αCTD) of RNA polymerase (RNAP). Repression in the presence of tyrosine involves a hexamer binding to both TyrR boxes. The molecular basis for this repression has been studied in vitro. Whereas initial gel shift experiments fail to show the exclusion of RNAP from the promoter region when TyrR hexamer is bound, a DNase I analysis of slices from the gel shows that in the presence of TyrR, RNAP now binds to a previously unrecognized upstream promoter. Although this upstream promoter is bound strongly by RNAP and forms an open complex on linear DNA templates, it fails to form an open complex on supercoiled templates in vitro and is unable to initiate transcription in vivo. A subsequent gel shift assay using a tyrP fragment which eliminates the upstream RNAP binding site confirms conclusively that, in the presence of tyrosine and ATP, the TyrR protein prevents RNAP from binding to the tyrP promoter. In vitro studies have also been carried out in the presence of TyrR protein and phenylalanine. Binding of TyrR protein to the upstream TyrR box in the presence of phenylalanine is shown to increase the affinity of RNAP for the promoter and stimulate open complex formation at the − 10 region of the tyrP promoter. This observation coupled with the results from mutational analysis supports the proposal that TyrR‐phenylalanine activates tyrP transcription by stimulating the onset of open complex formation.
Getting out: protein traffic in prokaryotes
Authors: Pugsley Anthony P. , Francetic Olivera , Driessen Arnold J. , Lorenzo Victor de 
Published: December 2004

SummaryProtein secretion systems in prokaryotes are increasingly shifting from being considered as experimental models for ‘more complex’ processes (i.e. eukaryotes) to being a major source of key biological questions in their own right. The pathways by which proteins move between compartments or insert into membranes in prokaryotic cells are certainly less numerous than in eukaryotes (though not dramatically so). However, the quality and complexity of bacterial protein targeting systems indicate that virtually all mechanistic problems associated with protein traffic were solved very efficiently well before eukaryotes appeared on the Earth crust. Indeed, recent studies have both increased the number of known prokaryotic protein traffic systems and indicated new layers of complexity for those that were already well characterized. This report describes some recent developments in bacterial protein traffic that were presented at two meetings in the autumn of 2003.
GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation
Authors: Banerjee Manidipa , Copp Jeremy , Vuga Danka , Marino Michael , Chapman Tara , Van Der Geer Peter , Ghosh Partho 
Published: December 2004

SummaryThe Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met. Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond. In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N‐terminal LRR region and C‐terminal GW domains. The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans. Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor‐mediated mode, as opposed to the ligand‐mediated mode observed for the LRR fragment. Surprisingly, we find that gC1q‐R, a host protein implicated in InlB‐mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q‐R is unnecessary for bacterial invasion. Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone‐like mimicry of HGF.
RecG helicase promotes DNA double‐strand break repair
Authors: Meddows Tom R. , Savory Andrew P. , Lloyd Robert G. 
Published: December 2004

SummaryDouble‐strand breaks pose a major threat to the genome and must be repaired accurately if structural and functional integrity are to be preserved. This is usually achieved via homologous recombination, which enables the ends of a broken DNA molecule to engage an intact duplex and prime synthesis of the DNA needed for repair. In Escherichia coli, repair relies on the RecBCD and RecA proteins, the combined ability of which to initiate recombination and form joint‐molecule intermediates is well understood. To shed light on subsequent events, we exploited the I‐SceI homing endonuclease of yeast to make breaks at I‐SceI cleavage sites engineered into the chromosome. We show that survival depends on RecA and RecBCD, and that subsequent events can proceed via either of two pathways, one dependent on the RuvABC Holliday junction resolvase and the other on RecG helicase. Both pathways rely on PriA, presumably to facilitate DNA replication. We discuss the possibility that classical Holliday junctions may not be essential intermediates in repair and consider alternative pathways for RecG‐dependent separation of joint molecules formed by RecA.
Oxidative stress triggers thiol oxidation in the glyceraldehyde‐3‐phosphate dehydrogenase of Staphylococcus aureus
Authors: Weber Harald , Engelmann Susanne , Becher Dörte , Hecker Michael 
Published: December 2004

SummaryThe high‐resolution two‐dimensional protein gel electrophoresis technique combined with matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) was used to analyse the oxidative stress response in Staphylococcus aureus COL. Exponentially growing cells were supplemented with 100 mM H2O2 leading to a growth arrest lasting 30 min. The comparison of the two‐dimensional pattern of cytoplasmic protein extracts of stressed and unstressed cells revealed only a few changes in the protein synthesis profile. However, the isoelectric points of Gap (glyceraldehyde‐3‐phosphate dehydrogenase), AhpC (alkylhydroperoxide reductase) and MvaS (HMG‐CoA‐synthase) changed strikingly. For analysis of the modification of Gap, tandem hybrid mass spectrometry (Q‐Star) was used. The observed pI shift resulted from the oxidation to sulphonic acid of cysteine 151, which is crucial for catalytic activity. A drop in ATP and a complete inactivation of Gap was accompanied by the growth arrest. About 30 min after the addition of H2O2, the damaged Gap was still present, but a new protein spot at the original location became visible, representing the newly synthesized enzyme that is active again. This is accompanied by the restoration of Gap enzyme activity, ATP levels and recovery of growth. There is a strong correlation between growth, ATP level and Gap activity under oxidative stress conditions, indicating that the H2O2‐triggered Gap inactivation might be one reason for growth arrest under these conditions. Our data indicate that the damaged Gap protein was not repaired.
Disruption of the peroxisomal citrate synthase CshA affects cell growth and multicellular development in Dictyostelium discoideum
Authors: Huang Ying‐Chieh , Chen Yi‐Hsing , Lo San‐Ren , Liu Chia‐I , Wang Cheng‐Wei , Chang Wen‐Tsan 
Published: December 2004

SummaryNon‐mitochondrial citrate synthase catalyses citrate synthesis in the glyoxylate cycle in gluconeogenesis. Screening Dictyostelium discoideum mutants generated by insertional mutagenesis isolated a poor‐growing mutant that displayed aberrant developmental morphology on bacterial lawns. Axenically grown mutants developed normally and formed mature fruiting bodies on buffered agar. The affected locus encoded a novel protein (CshA) that was homologous to glyoxysomal citrate synthase. cshA was expressed maximally during vegetative growth and gradually decreased through subsequent developmental stages. An in vitro citrate synthase assay revealed that cshA disruption resulted in a 50% reduction in enzyme activity, implicating CshA as an active citrate synthase. The amino‐terminus of CshA was found to have an atypical mitochondrial targeting signal, instead containing a unique nonapeptide sequence (RINILANHL) that was homologous to the conserved peroxisomal targeting signal 2 (PTS2). CshA protein was shown to be localized in the peroxisomes, and the RINILANHL sequence only efficiently targeted the peroxisomal green fluorescent protein. The growth defect of cshA– cells was associated with the impairment of phagocytosis and fluid‐phase endocytosis, independent from cytokinesis. Disrupted multicellular development on bacterial lawns resulted from the abnormal susceptibility to the environmental conditions, perhaps because of citrate insufficiency. Taken together, these results provide new insights into the function of peroxisomal citrate synthase in cell growth and multicellular development.
Novel yeast killer toxins provoke S‐phase arrest and DNA damage checkpoint activation‡
Authors: Klassen Roland , Teichert Sabine , Meinhardt Friedhelm 
Published: December 2004

SummaryCertain strains of Pichia acaciae and Wingea robertsiae (synonym Debaryomyces robertsiae) harbour extranuclear genetic elements that confer a killer phenotype to their host. Such killer plasmids (pPac1‐2 of P. acaciae and pWR1A of W. robertsiae) were sequenced and compared with the zymocin encoding pGKL1 of Kluyveromyces lactis. Both new elements were found to be closely related to each other, but they are only partly similar to pGKL1. As for the latter, they encode functions mediating binding of the toxin to the target cell's chitin and a hydrophobic region potentially involved in uptake of a toxin subunit by target cells. Consistently, mutations affecting the target cell's major chitin synthase (Chs3) protect it from toxin action. Heterologous intracellular expression of respective open reading frames identified cell cycle‐arresting toxin subunits deviating structurally from the likewise imported γ‐subunit of the K. lactis zymocin. Accordingly, toxicity of both P. acaciae and Wingea toxins was shown to be independent of RNA polymerase II Elongator, which is indispensable for zymocin action. Thus, P. acaciae and Wingea toxins differ in their mode of action from the G1‐arresting zymocin. Fluorescence‐activated cell sorting analysis and determination of budding indices have proved that such novel toxins mediate cell cycle arrest post‐G1 during the S phase. Concomitantly, the DNA damage checkpoint kinase Rad53 is phosphorylated. As a mutant carrying the checkpoint‐deficient allele rad53‐11 displays toxin hypersensitivity, damage checkpoint activation apparently contributes to coping with toxin stress, rather than being functionally implemented in toxin action.
The folate pathway is a target for resistance to the drug para‐aminosalicylic acid (PAS) in mycobacteria
Authors: Rengarajan Jyothi , Sassetti Christopher M. , Naroditskaya Vera , Sloutsky Alexander , Bloom Barry R. , Rubin Eric J. 
Published: December 2004

SummaryThe increasing rate of multidrug‐resistant tuberculosis has led to more use of second‐line antibiotics such as para‐aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS‐resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS‐resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.
Buffering of stable RNA promoter activity against DNA relaxation requires a far upstream sequence
Authors: Rochman Mark , Blot Nico , Dyachenko Marina , Glaser Gad , Travers Andrew , Muskhelishvili Georgi 
Published: December 2004

SummaryThe stable RNA promoters of Escherichia coli are exquisitely sensitive to variations in the superhelical density of DNA. Previously, we have shown that binding of the DNA architectural protein FIS at the upstream activating sequences (UASs) of stable RNA promoters prevents the transcription complexes from inactivation induced by changes in the supercoiling level of DNA. Here, we identify a strong FIS binding site 89 bp upstream of the previously described cluster of FIS binding sites located between positions −64 and −150 in the rrnA P1 UAS. Binding of FIS to this ‘far upstream sequence’ allows the recruitment of additional FIS molecules to the region. We demonstrate that, upon DNA relaxation, the maintenance of promoter activity requires, in addition to UAS, the presence of the far upstream sequence. The far upstream sequence shows no effect in the absence of an intact cluster. This requirement for the integrity of the region encompassing the far upstream sequence and the UAS cluster is correlated with the in vitro modulation of binding of FIS to UAS and interaction of RNA polymerase with the UP element and the region around the transcriptional start point. Our results suggest that, at the rrnA P1 promoter, the entire region comprising the UAS and the far upstream sequence is involved in the assembly of the transcription initiation complex. We propose that the extensive engagement of upstream DNA in this nucleoprotein complex locally compensates for the lack of torsional strain in relaxed DNA, thus increasing the resistance of the promoter to global DNA relaxation.
A novel anti‐anti‐activator mechanism regulates expression of the Pseudomonas aeruginosa type III secretion system
Authors: Dasgupta Nandini , Lykken Guinevere L. , Wolfgang Matthew C. , Yahr Timothy L. 
Published: December 2004

SummaryExpression of the Pseudomonas aeruginosa type III secretion system (TTSS) is coupled to the secretion status of the cells. Environmental signals such as calcium depletion activate the type III secretion channel and, as a consequence, type III gene transcription is derepressed. Two proteins, ExsA and ExsD, were shown previously to play a role in coupling transcription to secretion. ExsA is an activator of TTSS gene transcription, and ExsD is an anti‐activator of ExsA. In the absence of environmental secretion cues, ExsD binds ExsA and inhibits transcription. Here, we describe the characterization of ExsC as an anti‐anti‐activator of TTSS expression. Transcription of the TTSS is repressed in an exsC mutant and is derepressed upon ExsC overexpression. The dependence on exsC for transcription is relieved in the absence of exsD, suggesting that ExsC and ExsD function together to regulate transcription. Consistent with this idea, ExsC interacts with ExsD in bacterial two‐hybrid and co‐purification assays. We propose a model in which the anti‐anti‐activator (ExsC) binds to and sequesters the anti‐activator (ExsD) under low Ca2+ conditions, freeing ExsA and allowing for transcription of the TTSS. The P. aeruginosa system represents the first example of an anti‐activator/anti‐anti‐activator pair controlling transcription of a TTSS.
Mimicry and the evolution of premating isolation in Heliconius melpomene Linnaeus
Authors: Jiggins C. D. , Estrada C. , Rodrigues A. 
Published: December 2004

Abstract
Ecological divergence can cause speciation if adaptive traits have pleiotropic effects on mate choice. In Heliconius butterflies, mimetic patterns play a role in mate detection between sister species, as well as signalling to predators. Here we show that male butterflies from four recently diverged parapatric populations of Heliconius melpomene are more likely to approach and court their own colour patterns as compared with those of other races. A few exceptions, where males were more attracted to patterns other than their own, suggest that some mimetic patterns are sub-optimal in mate choice. Genotype frequencies in hybrid zones between races of H. melpomene suggest that mating is random, so reinforcement is unlikely to have played a role in intra-specific divergence. In summary, co-evolved divergence of colour pattern and mate preference occurs rapidly and is likely the first step in Heliconius speciation.
Social group size, potential sperm competition and reproductive investment in a hermaphroditic leech, Helobdella papillornata (Euhirudinea: Glossiphoniidae)
Authors: Tan G. N. , Govedich F. R. , Burd M. 
Published: December 2004

Abstract
Social group size may affect the potential for sperm competition, and this in turn may favour ontogenetic adjustments in testicular mass according to the likely requirements for sperm and spermatophore production. In a number of comparative analyses of testis mass among vertebrate species that differ in mating system or social organization, increasing potential for sperm competition is associated with larger testis size. Intraspecific phenotypic plasticity should be able to produce the same pattern if social group size is heterogenous and reflects differing degrees of average sperm competition, but this intraspecific effect is less well studied. We tested the effect of social groups on both male and female investment in the simultaneously hermaphroditic leech, Helobdella papillornata. Leeches were placed in groups of one, two, four or eight. Sexual investment at the onset of reproductive maturity was quantified as the total testisac volume for male function and total egg volume for female function. We found that testisac volume (statistically adjusted for body size) showed a significant increase with increasing group size. Total egg volume (also adjusted for body size) was unaffected by group size. Our findings indicate adaptive developmental plasticity in male gonad investment in response to the potential for sperm competition.
Parasitism, host immune defence and dispersal
Authors: Møller A. P. , Martín-Vivaldi M. , Soler J. J. 
Published: December 2004

Abstract
Host-parasite interactions have been hypothesized to affect the evolution of dispersal by providing a possibility for hosts to escape debilitating parasites, and by influencing the level of local adaptation. We used a comparative approach to investigate the relationship between a component of host immune function (which reflects the evolutionary history of parasite-induced natural selection) and dispersal in birds. We used a sample of 46 species of birds for which we had obtained field estimates of T-cell response for nestlings, mainly from our own field studies in Denmark and Spain. Bird species with longer natal, but not with longer breeding dispersal distances had a stronger mean T-cell-mediated immune response in nestlings than species with short dispersal distances. That was also the case when controlling for the potentially confounding effect of migration from breeding to wintering area, which is known from previous studies to be positively associated with dispersal distance. These relationships held even when controlling for similarity among species because of common ancestry. Avian hosts with a larger number of different breeding habitats had weaker mean T-cell-mediated immune responses than habitat specialists. This relationship held even when controlling for similarity among species because of common ancestry. Therefore, T-cell-mediated immunity is an important predictor of evolutionary changes in dispersal ability among common European birds.
Evolutionary dynamics of a B chromosome invasion in island populations of the grasshopper Eyprepocnemis plorans
Authors: Riera L. , Petitpierre E. , Juan C. , Cabrero J. , Camacho J. P. M. 
Published: December 2004

Abstract
Four natural populations of the grasshopper Eyprepocnemis plorans in the Mallorca island were analysed for several years revealing the recent invasion of the B1 chromosome from the south-west part of the island (Palma region) towards the north and to the east. In only 10 years, the mean number of Bs in the northern population at Pollença increased from 0.053 to 0.692. Therefore, B chromosome invasion seems to be very rapid and has recently arrived to the north of the island. The south-west (close to Palma) is the most likely point at which B invasion started in the Mallorca Island. Finally, the number of B chromosomes was significantly associated to an increase in chiasma frequency (and thus recombination) in A chromosomes.
Nonlinear relationships and phylogenetically independent contrasts
Authors: Quader S. , Isvaran K. , Hale R. E. , Miner B. G. , Seavy N. E. 
Published: December 2004

AbstractThe method of phylogenetically independent contrasts is commonly used for exploring cross-taxon relationships between traits. Here we show that this phylogenetic comparative method (PCM) can fail to detect correlated evolution when the underlying relationship between traits is nonlinear. Simulations indicate that statistical power can be dramatically reduced when independent contrasts analysis is used on nonlinear relationships. We also reanalyse a published data set and demonstrate that ignoring nonlinearity can affect biological inferences. We suggest that researchers consider the shape of the relationship between traits when using independent contrasts analysis. Alternative PCMs may be more appropriate if data cannot be transformed to meet assumptions of linearity.