bluuebunny/crossref_metadata_2025_split huggingface dataset:
The bacterial condensin/cohesin‐like protein complex acts in DNA repair and regulation of gene expression
Authors: Dervyn Etienne , Noirot‐Gros Marie‐Françoise , Mervelet Peggy , McGovern Steven , Ehrlich S. Dusko , Polard Patrice , Noirot Philippe 
Published: December 2004

SummaryStructural maintenance of chromosome (SMC) and the SMC‐interacting kleisin protein families have key functions in the chromosome organization of most organisms. Here, we report that the Bacillus subtilis kleisin, ScpA, can form a ternary complex with the SMC and ScpB proteins in a yeast tri‐hybrid assay, supporting the notion of a bacterial cohesin/condensin‐like complex. Furthermore, ScpA interacts in two‐hybrid assays with the AddAB complex, essential for recombinational repair, with DegS, a two‐component sensor kinase, as well as with other potential transcription regulators. Point mutations in scpA allowing growth under conditions not permissive for the spcA null and not affecting chromosome condensation were isolated. Among these mutations, some affected DNA repair and gene regulation, thus separating ScpA functions in these two pathways from its functions in chromosome condensation and segregation. Some separation‐of‐function mutations in scpA caused a deficiency in the repair of mitomycin C DNA lesions that was suppressed by increasing the intracellular dosage of the interacting AddAB complex. Another mutation in scpA deregulated the expression of genes encoding degradative enzymes that are known to be controlled by the interacting DegS kinase. We propose that the SMC–ScpA–ScpB complex could: (i) recruit the AddAB helicase/nuclease to act in post‐replicative repair; and (ii) form a complex with the DegS sensor kinase that inhibits its kinase activity. Moreover, our results indicate that the role of cohesin and condensin complexes in DNA repair and gene regulation is evolutionary conserved.
Mycobacterium tuberculosis ECF sigma factor sigC is required for lethality in mice and for the conditional expression of a defined gene set
Authors: Sun Ronggai , Converse Paul J. , Ko Chiew , Tyagi Sandeep , Morrison Norman E. , Bishai William R. 
Published: December 2004

SummaryBacterial alternative RNA polymerase sigma factors are key global adaptive response regulators with a likely role in Mycobacterium tuberculosis pathogenesis. We constructed a mutant lacking the sigma factor gene, sigC, by allelic exchange, in the virulent CDC1551 strain of M. tuberculosis and compared the resulting mutant with the isogenic wild‐type strain and complemented mutant strain. In vitro, compared to the wild‐type and complemented strains, the mutant was found to have similar ability to survive in both murine bone marrow‐derived macrophages and activated J774 macrophages. In time‐to‐death experiments in the mouse model, the ΔsigC mutant was significantly attenuated, causing no death in infected mice whereas the wild‐type and complemented strains caused 100% mortality within 235 days after aerosol infection with a median time to death of 170 days. Mouse organ bacterial burdens indicated that the mutant proliferated and persisted at the same level as the wild‐type and complemented strains in lung tissue and was able to persist in mice without causing death for > 300 days. A complete genomic microarray study demonstrated that SigC modulates the expression of several key virulence‐associated genes including hspX, senX3 and mtrA, encoding the α‐crystallin homologue, a two‐component sensor kinase and a two‐component response regulator respectively. Altered expression of a subset of these genes was confirmed by quantitative RT‐PCR analysis. Analysis of genes modulated by SigC also revealed a putative consensus DNA recognition sequence for SigC of SSSAAT–N16−20–CGTSSS (S = C or G). Promoter recognition for one of these genes was confirmed by in vitro transcription analysis after purification of recombinant SigC and reconstitution of an EσC RNA polymerase holoenzyme. These data indicate that the M. tuberculosis transcription factor SigC governs expression of an important M. tuberculosis regulon and is essential for lethality in mice, but is not required for bacterial survival in this species. These observations place the ΔsigC mutant in a class of M. tuberculosis mutants which persist in tissues but are attenuated in their ability to elicit lethal immunopathology.
In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA‐dependent and ‐independent promoters that are differentially activated by GTP
Authors: Luo Qin , Rauch Marcus , K. Marr Alexandra , Müller‐Altrock Stefanie , Goebel Werner 
Published: December 2004

SummaryMost known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA. Using our recently established in vitro transcription system, we have studied the PrfA‐dependent promoter (PinlC) regulating the expression of the small, secreted internalin C. PrfA‐dependent and PrfA‐independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same −10 box. Although the PrfA‐dependent transcription requires, as expected, the PrfA‐box, PrfA‐independent transcription depends on a −35 box located directly downstream of the PrfA‐box. PrfA‐independent transcription starts at A, 7 bp downstream of the common −10 box (A7), and is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the −35 box. PrfA‐dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the −10 box can also be used. The −35 box of the PrfA‐independent promoter can be functionally inactivated without affecting PrfA‐dependent transcription as long as the distance between the PrfA‐box and the −10 box remains fixed to 22 (or 23) bp. Vice versa, the PrfA‐box can be deleted without affecting PrfA‐independent transcription from PinlC, which is no longer inhibited by PrfA. The PrfA‐dependent transcription initiation needs, in contrast to the PrfA‐independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP. Overlapping PrfA‐dependent and PrfA‐independent promoter activity was also demonstrated for the mpl promoter (Pmpl). Again, PrfA‐dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA‐independent transcription at low GTP. Here too, the PrfA‐dependent and the PrfA‐independent promoters share the same −10 box characteristic of SigA‐loaded RNA polymerase. High GTP concentration also appears to be necessary for transcription initiation at other PrfA‐dependent promoters (Phly, PactA) but not at the PrfA‐independent promoter PinlC‐m8.
Control of Cre recombination by regulatory elements from Xer recombination systems
Authors: Gourlay Sarah C. , Colloms Sean D. 
Published: December 2004

SummarySite‐specific recombination by the Cre recombinase takes place at a simple DNA site (loxP), requires no additional proteins and gives topologically simple recombination products. In contrast, cer and psi sites for Xer recombination contain ≈ 150 bp of accessory sequences, require accessory proteins PepA, ArgR and ArcA, and the products are specifically linked to form a four‐noded catenane. Here, we use hybrid sites consisting of accessory sequences of cer or psi fused to loxP to probe the function of accessory proteins in site‐specific recombination. We show that PepA instructs Cre to produce four‐noded catenane, but is not required for recombination at these hybrid sites. Mutants of Cre that require PepA and accessory sequences for efficient recombination were selected. PepA‐dependent Cre gave products with a specific topology and displayed resolution selectivity. Our results reveal that PepA acts autonomously in the synapsis of psi and cer accessory sequences and is the main architectural element responsible for intertwining accessory site DNA. We suggest that accessory proteins can activate recombinases simply by synapsing the regulatory DNA sequences, thus bringing the recombination sites together with a specific geometry. This may occur without the need for protein–protein interactions between accessory proteins and the recombinases.
The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells
Authors: Schmid Michael C. , Schulein Ralf , Dehio Michaela , Denecker Geertrui , Carena Ilaria , Dehio Christoph 
Published: December 2004

Summary Bartonella henselae is an arthropod‐borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required  to  establish  intraerythrocytic  infection  in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated  with  the  interaction  of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor κB‐dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB‐independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.
Identification of a sporozoite‐specific member of the Toxoplasma SAG superfamily via genetic complementation
Authors: Radke Jay R. , Gubbels Marc‐Jan , Jerome Maria E. , Radke Joshua B. , Striepen Boris , White Michael W. 
Published: December 2004

Summary Toxoplasma gondii sporozoites possess an array of stage‐specific antigens that are localized to the membrane and internal cellular space, as well as secreted into the primary parasitophorous vacuole. Specific labelling of viable sporozoites excysted from oocysts reveals a complex admixture of surface proteins partially shared with tachyzoites. SAG1, SRS3 and SAG3 were detected on sporozoites as well as numerous minor antigens. In contrast, tachyzoite SAG2A and B were completely absent whereas a dominant 25 kDa protein was unique to the sporozoite surface. The sporozoite gene encoding this protein was identified in tachyzoites genetically complemented with a sporozoite cDNA library and cloned via site‐specific recombination into a bacterial shuttle vector. The sporozoite cDNA identified in these experiments encoded a protein with conserved structural features of the prototypical T. gondii SAG1 (P30) and shared sequence identity with surface proteins from Sarcocystis spp. This new member of the SAG superfamily was designated SporoSAG. Expression of SporoSAG in tachyzoites conferred enhanced invasion on transgenic parasites suggesting a role for this protein in oocyst/sporozoite transmission to susceptible hosts.
The Plasmodium falciparum clag9 gene encodes a rhoptry protein that is transferred to the host erythrocyte upon invasion
Authors: Ling Irene T. , Florens Laurence , Dluzewski Anton R. , Kaneko Osamu , Grainger Munira , Yim Lim Brian Y. S. , Tsuboi Takafumi , Hopkins John M. , Johnson Jeffrey R. , Torii Motomi , Bannister Lawrence H. , Yates John R. , Holder Anthony A. , Mattei Denise 
Published: December 2004

SummaryThe first gene characterizing the clag (cytoadherence linked asexual gene) family of Plasmodium falciparum was identified on chromosome 9. The protein product (Clag9) was implicated in cytoadhesion, the binding of infected erythrocytes to host endothelial cells, but little information on the biochemical characteristics of this protein is available. Other genes related to clag9 have been identified on different chromosomes. These genes encode similar amino acid sequences, but clag9 shows least conservation. Clag9 was detected in schizonts, merozoites and ring‐stage parasites after protease digestion and peptide analysis by mass spectrometry. Using antisera raised against unique regions of Clag9 and against RhopH2, a component of the RhopH high‐molecular‐mass protein complex of merozoites, immunofluorescence co‐localized the two proteins to the apical region of merozoites. Immunoelectron microscopy co‐localized Clag9 and RhopH2 exclusively to the basal bulb region of rhoptries rather than to their apical ducts. The same Clag9‐specific antibodies bound the RhopH complex, and the protein was detected in the complex purified by antibodies to RhopH2. Clag9 protein was also shown to be present in ring‐stage parasites, carried through from the previous cycle with the RhopH complex, in a location identical to that of RhopH2. Transcription of the clag9 gene was shown to occur at the same time as the genes for other members of the RhopH complex, rhoph2 and 3. The results indicate that Clag9 is part of the RhopH complex and suggest that, within this complex, the protein previously designated RhopH1 is composed of more than one protein product of the clag gene family. The results cast doubt on a direct role for Clag9 in cytoadhesion; we suggest that the primary role of the RhopH complex is in remodelling the infected red blood cell after invasion by the merozoite. The complex may have multiple functions dependent on its exact composition, which may include, with respect to Clag9, a contribution to the mechanism of cytoadhesion.
Allelic polymorphisms in apical membrane antigen‐1 are responsible for evasion of antibody‐mediated inhibition in Plasmodium falciparum
Authors: Healer Julie , Murphy Vince , Hodder Anthony N. , Masciantonio Rosella , Gemmill Alan W. , Anders Robin F. , Cowman Alan F. , Batchelor Adrian 
Published: December 2004

SummaryApical membrane antigen‐1 (AMA‐1) is a target of antibodies that inhibit invasion of Plasmodium falciparum into human erythrocytes and is a candidate for inclusion in a malaria vaccine. We have identified a line of P. falciparum (W2mef) less susceptible to anti‐AMA1 antibodies raised to the protein from a heterologous parasite line (3D7). We have constructed transgenic P. falciparum expressing heterologous AMA‐1 alleles. In vitro invasion assays show that these transgenic parasites differ from parental lines in susceptibility to inhibitory antibodies, providing direct evidence that sequence polymorphisms within AMA‐1 are responsible for evasion of immune responses that inhibit parasite invasion. We also generated a parasite line that would express a chimeric AMA‐1 protein, in which highly polymorphic residues within domain 1 were exchanged. Inhibition assays suggest that these residues are not sufficient for inhibition by invasion‐blocking antibodies. This study is the first to use P. falciparum allelic exchange to examine the relationship between genetic diversity and susceptibility to protective antibodies. The findings have important implications for the development of an AMA‐1‐based malaria vaccine.
Differential roles of multiple signal peptidases in the virulence of Listeria monocytogenes
Authors: Bonnemain Claire , Raynaud Catherine , Réglier‐Poupet Hélène , Dubail Iharilalao , Frehel Claude , Lety Marie‐Annick , Berche Patrick , Charbit Alain 
Published: December 2004

SummaryMost bacteria contain one type I signal peptidase (Spase I) for cleavage of signal peptides from exported and secreted proteins. Here, we identified a locus encoding three contiguous Spase I genes in the genome of Listeria monocytogenes. The deduced Sip proteins (denoted SipX, SipY and SipZ) are significantly similar to SipS and SipT, the major SPase I proteins of Bacillus subtilis (38% to 44% peptidic identity). We studied the role of these multiple signal peptidases in bacterial pathogenicity by constructing a series of single‐ and double‐chromosomal knock‐out mutants. Inactivation of sipX did not affect intracellular multiplication of L. monocytogenes but significantly reduced bacterial virulence (∼ 100‐fold). Inactivation of sipZ impaired the secretion of phospholipase C (PC‐PLC) and listeriolysin O (LLO), restricted intracellular multiplication and almost abolished virulence (LD50 of 108.3), inactivation of sipY had no detectable effects. Most importantly, a mutant expressing only SipX was impaired in intracellular survival and strongly attenuated in the mouse (LD50 of 107.2), whereas, a mutant expressing only SipZ behaved like wild‐type EGD in all the assays performed. The data establish that SipX and SipZ perform distinct functions in bacterial pathogenicity and that SipZ is the major Spase I of L. monocytogenes. This work constitutes the first report on the differential role of multiple Spases I in a pathogenic bacterium and suggests a possible post‐translational control mechanism of virulence factors expression.
A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles
Authors: Tjaden Joachim , Haferkamp Ilka , Boxma Brigitte , Tielens Aloysius G. M. , Huynen Martijn , Hackstein Johannes H. P. 
Published: December 2004

SummaryThe evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP‐generating cellular organelles. Hydrogenosomes, i.e. ATP‐ and hydrogen‐generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial‐type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial‐type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.
Functional consequences of single:double ring transitions in chaperonins: life in the cold
Authors: Ferrer Manuel , Lünsdorf Heinrich , Chernikova Tatyana N. , Yakimov Michail , Timmis Kenneth N. , Golyshin Peter N. 
Published: December 2004

SummaryThe cpn60 and cpn10 genes from psychrophilic bacterium, Oleispira antarctica RB8, showed a positive effect in Escherichia coli growth at low temperature, shifting its theoretical minimal growth temperature from +7.5°C to −13.7°C [Ferrer, M., Chernikova, T.N., Yakimov, M., Golyshin, P.N., and Timmis, K.N. (2003) Nature Biotechnol 21: 1266–1267]. To provide experimental support for this finding, Cpn60 and 10 were overproduced in E. coli and purified to apparent homogeneity. Recombinant O.Cpn60 was identical to the native protein based on tetradecameric structure, and it dissociates during native PAGE. Gel filtration and native PAGE revealed that, in vivo and in vitro, (O.Cpn60)7 was the active oligomer at 4–10°C, whereas at > 10°C, this complex was converted to (O.Cpn60)14. The dissociation reduces the ATP consumption (energy‐saving mechanism) and increases the refolding capacity at low temperatures. In order for this transition to occur, we demonstrated that K468 and S471 may play a key role in conforming the more advantageous oligomeric state in O.Cpn60. We have proved this hypothesis by showing that single and double mutations in K468 and S471 for T and G, as in E.GroEL, produced a more stable double‐ring oligomer. The optimum temperature for ATPase and chaperone activity for the wild‐type chaperonin was 24–28°C and 4–18°C, whereas that for the mutants was 45–55°C and 14–36°C respectively. The temperature inducing unfolding (TM) increased from 45°C to more than 65°C. In contrast, a single ring mutant, O.Cpn60SR, with three amino acid substitutions (E461A, S463A and V464A) was as stable as the wild type but possessed refolding activity below 10°C. Above 10°C, this complex lost refolding capacity to the detriment of the double ring, which was not an efficient chaperone at 4°C as the single ring variant. We demonstrated that expression of O.Cpn60WT and O.Cpn60SR leads to a higher growth of E. coli at 4°C (µmax, 0.22 and 0.36 h−1 respectively), whereas at 10–15°C, only E. coli cells expressing O.Cpn60 or O.Cpn60DR grew better than parental cells (–cpn). These results clearly indicate that the single‐to‐double ring transition in Oleispira chaperonin is a wild‐type mechanism for its thermal acclimation. Although previous studies have also reported single‐to‐double ring transitions under many circumstances, this is the first clear indication that single‐ring chaperonins are necessary to support growth when the temperature falls from 37°C to 4°C.
Transcriptional and translational regulation of the marRAB multiple antibiotic resistance operon in Escherichia coli
Authors: Martin Robert G. , Rosner Judah L. 
Published: December 2004

SummaryThe marRAB multiple antibiotic resistance operon of Escherichia coli is autorepressed by MarR. MarR binds to two palindromic sequences in vitro: site I lies between and overlaps the −35 and −10 hexamers for RNA polymerase binding; site II lies between the transcription start site and the GTG initiation codon of marR. To assess the importance of these sites in vivo, the effects of mutant sites on transcription were analysed using fusions to lacZ in the presence and absence of wild‐type MarR. When both sites were wild type, transcription in the derepressed marR‐deleted strain was 19‐fold that of the wild‐type strain; when only site I or site II was wild type, this ratio was reduced to 4.3‐ and 2.6‐fold, respectively, showing that full repression requires both sites, but some repression can occur at one site independently of the other. Translational fusions of the wild‐type promoter to lacZ demonstrated that marR translation proceeds at only 4.5% of the transcription rate. Analysis of translational fusions with mutant leader sequences demonstrated that the principal reason for inefficient translation is a weak Shine–Dalgarno (SD) sequence, AGG(G). Although the SD sequence is located within the potential stem–loop structure of site II, no evidence for occlusion of the SD sequence was found in the wild‐type strain. However, a single basepair mutation that strengthens the stem–loop structure drastically reduced the translational efficiency. Substitution of ATG for GTG as the initiation codon increased translational efficiency by 50%. Increasing the 5 bp spacing between the SD sequence and the GTG codon by one to four bases reduced the translational efficiency by 50–75%. Inefficient translation of marR may help to sensitize the cell to environmental signals.