bluuebunny/crossref_metadata_2025 huggingface dataset:
Breeding system and demography shape population genetic structure across ecological and climatic zones in the African freshwater snail,Bulinus forskalii(Gastropoda, Pulmonata), intermediate host for schistosomes
Authors: GOW J. L. , NOBLE L. R. , ROLLINSON D. , MIMPFOUNDI R. , JONES C. S.
Published: December 2004

The role of breeding system and population bottlenecks in shaping the distribution of neutral genetic variation among populations inhabiting patchily distributed, ephemeral water bodies was examined for the hermaphroditic freshwater snailBulinus forskalii, intermediate host for the medically important trematodeSchistosoma guineensis. Levels of genetic variation at 11 microsatellite loci were assessed for 600 individuals sampled from 19 populations that span three ecological and climatic zones (ecozones) in Cameroon, West Africa. Significant heterozygote deficiencies and linkage disequilibria indicated very high selfing rates in these populations. Despite this and the large genetic differentiation detected between populations, high levels of genetic variation were harboured within these populations. The high level of gene flow inferred from assignment tests may be responsible for this pattern. Indeed, metapopulation dynamics, including high levels of gene flow as well as extinction/contraction and recolonization events, are invoked to account for the observed population structuring, which was not a consequence of isolation‐by‐distance. BecauseB. forskaliipopulations inhabiting the northern, Sahelian area are subject to more pronounced annual cycles of drought and flood than the southern equatorial ones, they were expected to be subject to population bottlenecks of increased frequency and severity and, therefore, show reduced genetic variability and elevated population differentiation. Contrary to predictions, the populations inhabiting the most northerly ecozone exhibited higher genetic diversity and lower genetic differentiation than those in the most southerly one, suggesting that elevated gene flow in this region is counteracting genetic drift.
Phylogeography recapitulates topography: very fine‐scale local endemism of a saproxylic ‘giant’ springtail at Tallaganda in the Great Dividing Range of south–east Australia
Authors: GARRICK R. C. , SANDS C. J. , ROWELL D. M. , TAIT N. N. , GREENSLADE P. , SUNNUCKS P.
Published: December 2004

Comparative phylogeography can reveal processes and historical events that shape the biodiversity of species and communities. As part of a comparative research program, the phylogeography of a new, endemic Australian genus and species of log‐dependent (saproxylic) collembola was investigated using mitochondrial sequences, allozymes and anonymous single‐copy nuclear markers. We found the genetic structure of the species corresponds with five a priori microbiogeographical regions, with population subdivision at various depths owing to palaeoclimatic influences. Closely related mtDNA haplotypes are codistributed within a single region or occur in adjacent regions, nuclear allele frequencies are more similar among more proximate populations, and interpopulation migration is rare. Based on mtDNA divergence, a late Miocene–late Pliocene coalescence is likely. The present‐day distribution of genetic diversity seems to have been impacted by three major climatic events: Pliocene cooling and drying (2.5–7 million years before present, Mybp), early Pleistocene wet‐dry oscillations (c. 1.2 Mybp) and the more recent glacial‐interglacial cycles that have characterized the latter part of the Quaternary (< 0.4 Mybp).
Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana
Authors: JACKSON M. W. , STINCHCOMBE J. R. , KORVES T. M. , SCHMITT J.
Published: December 2004

Cold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C‐repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.
How to track and assess genotyping errors in population genetics studies
Authors: BONIN A. , BELLEMAIN E. , BRONKEN EIDESEN P. , POMPANON F. , BROCHMANN C. , TABERLET P.
Published: December 2004

Genotyping errors occur when the genotype determined after molecular analysis does not correspond to the real genotype of the individual under consideration. Virtually every genetic data set includes some erroneous genotypes, but genotyping errors remain a taboo subject in population genetics, even though they might greatly bias the final conclusions, especially for studies based on individual identification. Here, we consider four case studies representing a large variety of population genetics investigations differing in their sampling strategies (noninvasive or traditional), in the type of organism studied (plant or animal) and the molecular markers used [microsatellites or amplified fragment length polymorphisms (AFLPs)]. In these data sets, the estimated genotyping error rate ranges from 0.8% for microsatellite loci from bear tissues to 2.6% for AFLP loci from dwarf birch leaves. Main sources of errors were allelic dropouts for microsatellites and differences in peak intensities for AFLPs, but in both cases human factors were non‐negligible error generators. Therefore, tracking genotyping errors and identifying their causes are necessary to clean up the data sets and validate the final results according to the precision required. In addition, we propose the outline of a protocol designed to limit and quantify genotyping errors at each step of the genotyping process. In particular, we recommend (i) several efficient precautions to prevent contaminations and technical artefacts; (ii) systematic use of blind samples and automation; (iii) experience and rigor for laboratory work and scoring; and (iv) systematic reporting of the error rate in population genetics studies.
Mitochondrial gene diversity in the common vole Microtus arvalis shaped by historical divergence and local adaptations
Authors: FINK SABINE , EXCOFFIER LAURENT , HECKEL GERALD
Published: December 2004

The phylogeography of the common vole (Microtus arvalis) was examined by analysing mitochondrial DNA (mtDNA) sequence variation in 1044 base pairs (bp) of the cytochrome b (cytb) gene and in 322 bp of the control region (ctr) among 106 individuals from 58 locations. The geographical distribution of four previously recognized cytb evolutionary lineages in Europe was refined and a new lineage was found in southern Germany. All lineages were distributed allopatrically, except in one sample that was probably located in a contact zone. The occurrence of several lineages in the Alps is in keeping with their recent recolonization from distinct sources. The translation of 84 cytb DNA sequences produced 33 distinct proteins with relationships that differed from those of the DNA haplotypes, suggesting that the mtDNA lineages did not diverge in response to selection. In comparison with M. agrestis, a neutrality test detected no overall evidence for selection in the cytb gene, but a closer examination of a structural model showed that evolutionarily conserved and functionally important positions were often affected. A new phylogeographical test of random accumulation of nonsynonymous mutations generated significant results in three lineages. We therefore conclude that the molecular diversity of cytb in M. arvalis is overall the result of the demographic history of the populations, but that there have been several episodes of local adaptation to peculiar environments.
JlpA, a novel surface‐exposed lipoprotein specific to Campylobacter jejuni, mediates adherence to host epithelial cells
Authors: Jin Songmu , Joe Angela , Lynett Jennifer , Hani Eric Kurt , Sherman Philip , Chan Voon Loong
Published: December 2004

A 1116 bp open reading frame (ORF), designated jlpA, encoding a novel species‐specific lipoprotein of Campylobacter jejuni TGH9011, was identified from recombinant plasmid pHIP‐O. The jlpA gene encodes a polypeptide (JlpA) of 372 amino acid residues with a molecular mass of 42.3 kDa. JlpA contains a typical signal peptide and lipoprotein processing site at the N‐terminus. The presence of a lipid moiety on the JlpA molecule was confirmed by the incorporation of [3H]‐palmitic acid. Immunoblotting analysis of cell surface extracts prepared using glycine–acid buffer (pH 2.2) and proteinase K digestion of whole cells indicated that JlpA is a surface‐exposed lipoprotein in C. jejuni. JlpA is loosely associated with the cell surface, as it is easily extracted from the C. jejuni outer membrane by detergents, such as sarcosyl and Triton X‐100. JlpA is released to the culture medium, and its concentration increases in a time‐dependent fashion. The adherence of both insertion and deletion mutants of jlpA to HEp‐2 epithelial cells was reduced compared with that of parental C. jejuni TGH9011. Adherence of C. jejuni to HEp‐2 cells was inhibited in a dose‐dependent manner when the bacterium was preincubated with anti‐GST–JlpA antibodies or when HEp‐2 cells were preincubated with JlpA protein. A ligand‐binding immunoblotting assay showed that JlpA binds to HEp‐2 cells, which suggests that JlpA is C. jejuni adhesin.
The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons
Authors: Opel Michael L. , Arfin Stuart M. , Hatfield G. Wesley
Published: December 2004

Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription‐induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co‐ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling‐dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.
What makes an Escherichia coli promoter σS dependent? Role of the −13/−14 nucleotide promoter positions and region 2.5 of σS
Authors: Becker Gisela , Hengge‐Aronis Regine
Published: December 2004

The σS and σ70 subunits of Escherichia coli RNA polymerase recognize very similar promoter sequences. Therefore, many promoters can be activated by both holoenzymes in vitro. The same promoters, however, often exhibit distinct sigma factor selectivity in vivo. It has been shown that high salt conditions, reduced negative supercoiling and the formation of complex nucleoprotein structures in a promoter region can contribute to or even generate σS selectivity. Here, we characterize the first positively acting σS‐selective feature in the promoter sequence itself. Using the σS‐dependent csiD promoter as a model system, we demonstrate that C and T at the −13 and −14 positions, respectively, result in strongest expression. We provide allele‐specific suppression data indicating that these nucleotides are contacted by K173 in region 2.5 of σS. In contrast, σ70, which features a glutamate at the corresponding position (E458), as well as the σS(K173E) variant, exhibit a preference for a G(−13). C(−13) is highly conserved in σS‐dependent promoters, and additional data with the osmY promoter demonstrate that the K173/C(−13) interaction is of general importance. In conclusion, our data demonstrate an important role for region 2.5 in σS in transcription initiation. Moreover, we propose a consensus sequence for a σS‐selective promoter and discuss its emergence and functional properties from an evolutionary point of view.
A novel mechanism controls anaerobic and catabolite regulation of the Escherichia coli tdc operon
Authors: Sawers Gary
Published: December 2004

The tdc operon is subject to CRP‐controlled catabolite repression. Expression of the operon is also induced anaerobically, although this regulation does not rely on direct control by either FNR or ArcA. Recently, the anaerobic expression of the tdc operon was found to be fortuitously induced in the presence of glucose by a heterologous gene isolated from the Gram‐positive anaerobe Clostridium butyricum. The gene, termed tcbC, encoded a histone‐like protein of 14.5 kDa. Using tdc–lacZ fusions, it was shown that TcbC did not activate tdc expression by functionally replacing any of the operon regulators. In vitro transcription analyses with RNA polymerase and CRP revealed that faithful CRP‐dependent transcription initiation occurred only on supercoiled templates. No specific, CRP‐dependent transcription initiation was observed on relaxed or linear DNA templates. Surprisingly, purified His‐tagged TcbC activated transcription from a relaxed, circular template, but not from supercoiled or linear templates. Examination of the CRP binding site of the tdc promoter revealed that it was located 43.5 bp upstream of the transcription initiation site. Repositioning of the CRP site at −41.5 bp abolished activation by the TcbC protein and allowed CRP‐dependent transcription to occur on linear, relaxed and supercoiled templates. TcbC bound DNA non‐specifically; however, in topoisomerase I relaxation assays, it was demonstrated that TcbC imposed torsional constraints on negatively supercoiled DNA, which influenced the ability of the enzyme to relax the topoisomers. Taken together, these results strongly suggest that TcbC activates transcription of tdc by altering the local topological status of the tdc promoter and that, in the wild‐type tdc promoter, the CRP binding site is misaligned to allow transcription to occur only under optimal conditions. Indeed, in vivo transcription analyses revealed that repositioning of the CRP binding site to −41.5 bp resulted in high‐level, CRP‐dependent transcription, even under catabolite‐repressing conditions, and that transcription was no longer influenced by TcbC. Remarkably, however, anaerobic regulation of the mutant promoter was retained. This indicates that the other tdc regulators, TdcA and TdcR, govern anaerobic transcription activation by CRP.
Natural chaperonin of the hyperthermophilic archaeum, Thermococcus strain KS‐1: a hetero‐oligomeric chaperonin with variable subunit composition
Authors: Yoshida Takao , Ideno Akira , Hiyamuta Shuichi , Yohda Masafumi , Maruyama Tadashi
Published: December 2004

To study the difference in expression of the chaperonin α‐ and β‐subunits in Thermococcus strain KS‐1 (T. KS‐1), we measured their intracellular contents at various growth temperatures using subunit‐specific antibodies. The β‐subunit was significantly more abundant with increasing temperature (maximum at 93°C), whereas the α‐subunit was not. Native PAGE with Western blot analysis indicated that the natural chaperonins in the crude extracts of T. KS‐1 cells grown between 65°C and 95°C migrate as single bands with different mobility. The recombinant α‐ and β‐subunit homo‐oligomers migrated differently from each other and from natural chaperonins. Immunoprecipitation also showed that the natural chaperonin was the hetero‐oligomer. These results indicate that chaperonin in T. KS‐1 formed a hetero‐oligomer with variable subunit composition, and that the β‐subunit may be adapted to a higher temperature than the α‐subunit. T. KS‐1 probably changes its chaperonin subunit composition to acclimatize to the ambient temperature.
Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human malaria parasite Plasmodium falciparum
Authors: Wang Ping , Wang Qi , Aspinall Tanya V. , Sims Paul F. G. , Hyde John E.
Published: December 2004

Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosynthetic pathway converts GTP to polyglutamated derivatives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfection system adapted to studying this pathway, we investigated modulation of dihydropteroate synthase (DHPS) activity on parasite phenotypes. Dihydropteroate synthase incorporates p‐aminobenzoate (pABA) into dihydropteroate, the precursor of dihydrofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full‐length but mutated at two key residues and having < 10% of normal activity were viable in folate‐supplemented medium. Metabolic labelling showed that these parasites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfadoxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo. However, we found no evidence of a significant role for DHPS in folate salvage. Moreover, when biosynthesis was compromised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.
Transcription activation at the Escherichia coli melAB promoter: interactions of MelR with its DNA target site and with domain 4 of the RNA polymerase σ subunit
Authors: Grainger David C. , Webster Christine L. , Belyaeva Tamara A. , Hyde Eva I. , Busby Stephen J. W.
Published: December 2004

Activation of transcription initiation at the Escherichia coli melAB promoter is dependent on MelR, a transcription factor belonging to the AraC family. MelR binds to 18 bp target sites using two helix–turn–helix (HTH) motifs that are both located in its C‐terminal domain. The melAB promoter contains four target sites for MelR. Previously, we showed that occupation of two of these sites, centred at positions −42.5 and −62.5 upstream of the melAB transcription start point, is sufficient for activation. We showed that MelR binds as a direct repeat to these sites, and we proposed a model to describe how the two HTH motifs are positioned. Here, we have used suppression genetics to confirm this model and to show that MelR residue 273, which is in HTH 2, interacts with basepair 13 of each target site. As our model for DNA‐bound MelR suggests that HTH 2 must be adjacent to the melAB promoter −35 element, we searched this part of MelR for amino acid side‐chains that might be able to interact with σ. We describe genetic evidence to show that MelR residue 261 is close to residues 596 and 599 of the RNA polymerase σ70 subunit, and that they can interact. Similarly, MelR residue 265 is shown to be able to interact with residue 596 of σ70. In the final part of the work, we describe experiments in which the MelR binding site at position −42.5 was improved. We show that this is detrimental to MelR‐dependent transcription activation because bound MelR is mispositioned so that it is unable to make ‘correct’ interactions with σ.
Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes
Authors: Zenewicz Lauren A. , Skinner Jason A. , Goldfine Howard , Shen Hao
Published: December 2004

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T‐cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose‐dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non‐pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine‐preferring phospholipase C (PC‐PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC‐PLC synergized with LLO for the induction of FasL expression. FasL‐expressing cells induced by L. monocytogenes were capable of killing Fas‐expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC‐PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.
MicroReview: The bacterial Sm‐like protein Hfq: a key player in RNA transactions
Authors: Valentin‐Hansen Poul , Eriksen Maiken , Udesen Christina
Published: December 2004

The conserved RNA‐binding protein Hfq, originally discovered in Escherichia coli as a host factor for Qβ replicase, has emerged as a pleiotropic regulator that modulates the stability or the translation of an increasing number of mRNAs. During the past 5 years, Hfq‐mediated control has been an area of increasing focus because the protein has been linked to the action of many versatile RNA‐based regulators that use basepairing interactions to regulate the expression of target mRNAs. The recent findings that Hfq assists in bimolecular RNA–RNA interactions and is similar structurally and functionally to eukaryotic Sm proteins have further fuelled interest in this important post‐transcriptional regulator. Here, we summarize the history of Hfq and highlight results that have led to an important gain in insight into the physiology, biochemistry and evolution of Hfq and its homologues.
MreB, the cell shape‐determining bacterial actin homologue, co‐ordinates cell wall morphogenesis in Caulobacter crescentus
Authors: Figge Rainer M. , Divakaruni Arun V. , Gober James W.
Published: December 2004

The bacterial actin homologue, MreB, is required for the maintenance of a rod‐shaped cell and has been shown to form spirals that traverse along the longitudinal axis of Bacillus subtilis and Escherichia coli cells. The depletion of MreB in Caulobacter crescentus resulted in lemon‐shaped cells that possessed defects in the integrity of the cell wall. MreB localization appeared as bands or spirals that encircled the cell along its entire length and switched to a mid‐cell location at a time that coincided with the initiation of cell division. The formation of smaller MreB spirals or bands at the mid‐cell was dependent on the presence on the cytokinetic protein, FtsZ. Penicillin‐binding protein 2 (PBP2) also formed band‐like structures perpendicular to the cell periphery that resembled, and depended upon, MreB localization. PBP2 co‐immunoprecipitated with several other penicillin‐binding proteins, suggesting that these proteins are in association in Caulobacter cells. We hypothesize that MreB filaments function as a cytoskeleton that serves as an organizer or tracking device for the PBP2–peptidoglycan biosynthesis complex.
Re‐replication from non‐sequesterable origins generates three‐nucleoid cells which divide asymmetrically
Authors: Bach Trond , Skarstad Kirsten
Published: December 2004

In rapidly growing Escherichia coli cells replication cycles overlap and initiation occurs at multiple replication origins (oriCs). All origins within a cell are initiated essentially in synchrony and only once per cell cycle. Immediate re‐initiation of new origins is avoided by sequestration, a mechanism dependent on the SeqA protein and Dam methylation of GATC sites in oriC. Here, GATC sites in oriC were changed to GTTC. This reduced the sequestration to essentially the level found in SeqA‐less cells. The mutant origins underwent re‐initiation, showing that the GATC sites in oriC are required for sequestration. Each re‐initiation eventually gave rise to a cell containing an extra nucleoid. The three‐nucleoid cells displayed one asymmetrically placed FtsZ‐ring and divided into a two‐nucleoid cell and a one‐nucleoid cell. The three nucleoid‐cells thus divided into three daughters by two consecutive divisions. The results show that extra rounds of replication cause extra daughter cells to be formed prematurely. The fairly normal mutant growth rate and size distribution show, however, that premature rounds of replication, chromosome segregation, and cell division are flexibly accommodated by the existing cell cycle controls.
SCP1, a 356 023 bp linear plasmid adapted to the ecology and developmental biology of its host, Streptomyces coelicolor A3(2)
Authors: Bentley S. D. , Brown S. , Murphy L. D. , Harris D. E. , Quail M. A. , Parkhill J. , Barrell B. G. , McCormick J. R. , Santamaria R. I. , Losick R. , Yamasaki M. , Kinashi H. , Chen C. W. , Chandra G. , Jakimowicz D. , Kieser H. M. , Kieser T. , Chater K. F.
Published: December 2004

The sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365 023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma‐butyrolactone signalling system, members of the actinomycete‐specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTA‐containing genes is part of a large bldA‐dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB‐deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.
The large resolvase TnpX is the only transposon‐encoded protein required for transposition of the Tn4451/3family of integrative mobilizable elements
Authors: Lyras Dena , Adams Vicki , Lucet Isabelle , Rood Julian I.
Published: December 2004

Chloramphenicol resistance inClostridium perfringensandClostridium difficileis often encoded bycatPgenes located within the 6.3 kb integrative mobilizable elements Tn4451and Tn4453respectively. This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element. Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate. In this study,in vivodeletion analysis of the transposon‐encodedtnpVandtnpYgenes showed that they are not essential for excision or integration of this group of elements. Similar studies ontnpWsuggested either that this gene is not essential for these functions or that TnpW does not function when providedin trans. Development and use of anin vivoinsertion assay showed that TnpX is the only transposon‐encoded protein required for the integration reaction. Subsequently, a TnpXLEH6protein was purified and shown to catalyse excisionin vitroin the absence of any other protein and preferentially to excise a supercoiled DNA substrate. In summary, these studies have shown that TnpX is the only transposon protein requiredin vivoandin vitrofor the excision process and that, like excision, integration also occurs by a serine recombinase‐mediated site‐specific recombination mechanism.
Post‐transcriptional regulation of bacterial motility by aconitase proteins
Authors: Tang Yue , Guest John R. , Artymiuk Peter J. , Read Robert C. , Green Jeffrey
Published: December 2004

Escherichia coli and Bacillus subtilis aconitases can act as iron and oxidative stress‐responsive post‐transcriptional regulators. Here, it is shown that a Salmonella enterica serovar Typhimurium LT2 acnB mutant exhibits impaired binding to the surface of J774 macrophage‐like cells. Proteomic analyses were used to investigate further the binding defect of the acnB mutant. These revealed that the levels of the flagellum protein FliC were much lower for the acnB mutant. This strain was correspondingly less motile and possessed fewer flagella than either the parental strain or the acnA and acnAB mutants. The acnB lesion did not alter fliC transcription, nor did apo‐AcnB select the fliC transcript from a library of S. enterica transcripts; thus, the effect of AcnB on FliC is indirect. Evidence is presented to show that apo‐AcnB regulates FliC synthesis via interaction with the ftsH transcript to decrease the intracellular levels of FtsH. The lower levels of FtsH protease activity then influence σ32, DnaK and, ultimately, FliC production.
Upregulation of the Hsp104 chaperone at physiological temperature during recovery from thermal insult
Authors: Seppä Laura , Hänninen Anna‐Liisa , Makarow Marja
Published: December 2004

Thermal insult at 50°C causes protein denaturation in yeast, but the cells survive if preconditioned at 37°C. Survival depends on refolding of heat‐denatured proteins. Refolding of cytoplasmic proteins requires Hsp104, the expression of which increases several‐fold upon shift of the cells from physiological temperature 24°C to 37°C. We describe here a novel type of regulation of Hsp104, designated delayed upregulation (DUR). When Saccharomyces cerevisiae cells grown at 24°C, preconditioned at 37°C and treated briefly at 50°C were shifted back to 24°C, Hsp104 expression was negligible for 1 h, but increased then to a three to nine times higher level than that detected after growth at 24°C, returning to normal after 5 h. A heat shock element (HSE) of the upstream sequence of HSP104 was necessary and sufficient for DUR, whereas stress response elements (STRE) were dispensable. Destruction of HSE plus all three STREs abolished Hsp104 expression, resulting in cell death after thermal insult. Deletion of MSN2/4, encoding transcription factors driving STRE‐dependent gene expression, decreased DUR. Deletion of HOG1, encoding a heat‐responsive and osmosensitive mitogen‐activated protein kinase implicated to be functionally connected to Msn2/4p, abolished DUR. We suggest that DUR was regulated via HSE, required Hog1p and involved Msn2/4p‐regulated gene products.
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

Type 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

The 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

Protein 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

The 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

Double‐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

The 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

Non‐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

Certain 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

The 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.