bluuebunny/crossref_metadata_2025 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

Structural 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

Bacterial 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

Most 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

Site‐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

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

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

The 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

Apical 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

Most 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

The 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

The 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

The 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.
Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation
Authors: Domain Francis , Houot Laetitia , Chauvat Franck , Cassier‐Chauvat Corinne
Published: December 2004

The cyanobacterial genes lexA, recA and ruvB were analysed in Synechocystis PCC6803, which is shown here to be more radiation resistant than the other unicellular model strain Synechococcus PCC7942. We found that cyanobacteria do not have an Escherichia coli‐type SOS regulon. The Synechocystis lexA and recA promoters were found to be strong and UV insensitive, unlike the ruvB promoter, which is weak and UV‐C inducible. Yet, lexA and recA are regulated by UV‐C, but the control is negative and occurs at the post‐transcriptional level. Two novel conserved elements were characterized in the lexA promoter: (i) an unusually long crucial box 5′‐TAAAATTTTGTATCTTTT‐3′ (−64, −47); and (ii) a negatively acting motif 5′‐TAT GAT‐3′ (−42, −37). These elements were not found in the recA promoter, which appeared to be unusually simple in harbouring only a single crucial element (i.e. the canonical −10 box). RuvB, operating in recombination‐dependent cellular processes, was found to be dispensable to cell growth, whereas LexA and RecA appeared to be critical to cell viability. Using DNA microarrays, we have identified 57 genes with expression that is altered, at least twofold, in response to LexA depletion. None of these genes is predicted to operate in DNA metabolism, arguing against the involvement of LexA in the regulation of DNA repair. Instead, most of the LexA‐responsive genes were known to be involved in carbon assimilation or controlled by carbon availability. Consistently, the growth of the LexA‐depleted strain was found to be strongly dependent on the availability of inorganic carbon.
Chimeras of P1‐type ATPases and their transcriptional regulators: contributions of a cytosolic amino‐terminal domain to metal specificity
Authors: Borrelly Gilles P. M. , Rondet Sabine A. M. , Tottey Stephen , Robinson Nigel J.
Published: December 2004

Zn2+‐responsive repressor ZiaR and Co2+‐responsive activator CoaR modulate production of P1‐type Zn2+‐ (ZiaA) and Co2+‐ (CoaT) ATPases respectively. What dictates metal selectivity? We show that Δ ziaΔcoa double mutants had similar Zn2+ resistance to Δzia single mutants and similar Co2+ resistance to Δcoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino‐terminal domain CoaTN with ZiaAN (in ziaR‐p ziaA‐ziaANcoaT) conferred Zn2+ resistance to ΔziaΔcoa, Zn2+ content was lowered and residual Co2+ resistance lost. Metal‐dependent molar absorptivity under anaerobic conditions revealed that purified ZiaAN binds Co2+ in a pseudotetrahedral two‐thiol site, and Co2+ was displaced by Zn2+. Thus, the amino‐terminal domain of ZiaA inverts the metals exported by zinc‐regulated CoaT from Co2+ to Zn2+, and this correlates simplistically with metal‐binding preferences; KZiaAN Zn2+ tighter than Co2+. However, Zn2+ did not bleach Cu+‐ZiaAN, and only Cu+ co‐migrated with ZiaAN after competitive binding versus Zn2+. Bacterial two‐hybrid assays that detected interaction between the Cu+‐metallochaperone Atx1 and the amino‐terminal domain of Cu+‐transporter PacSN detected no interaction with the analogous, deduced, ferredoxin‐fold subdomain of ZiaAN. Provided that there is no freely exchangeable cytosolic Cu+, restricted contact with the Cu+‐metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu+–ZiaAN complexes.
PhoP‐regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B
Authors: Shi Yixin , Cromie Michael J. , Hsu Fong‐Fu , Turk John , Groisman Eduardo A.
Published: December 2004

In Salmonella enterica, the PhoP–PhoQ two‐component system governs resistance to structurally different antimicrobial peptides including the alpha‐helical magainin 2, the β‐sheet defensins and the cyclic lipopeptide polymyxin B. To identify the PhoP‐regulated determinants mediating peptide resistance, we prepared a plasmid library from a phoP mutant, introduced it into a phoP mutant and selected for magainin‐resistant clones. One of the clones harboured the PhoP‐activated ugtL gene, deletion of which rendered Salmonella susceptible to magainin 2 and polymyxin B, but not defensin HNP‐1. We established that ugtL encodes an inner membrane protein that promotes the formation of monophosphorylated lipid A in the lipopolysaccharide. Inactivation of both ugtL and the regulatory gene pmrA, which controls lipid A modifications required for resistance to polymxyin B (but not to magainin 2) and is post‐transcriptionally activated by the PhoP–PhoQ system, resulted in a strain that was as susceptible to polymyxin B as a phoP mutant. The most frequently recovered clone harboured the yqjA gene, which we show is PhoP regulated and required for resistance to magainin 2 but not to polymyxin B or defensin HNP‐1. Our results indicate that different PhoP‐mediated modifications in lipid A are necessary for resistance to different antimicrobial peptides.
Targeted disruption of Plasmodium falciparum cysteine protease, falcipain 1, reduces oocyst production, not erythrocytic stage growth
Authors: Eksi Saliha , Czesny Beata , Greenbaum Doron C. , Bogyo Matthew , Williamson Kim C.
Published: December 2004

Cysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum, the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L‐like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion. Neither their expression nor their role in P. falciparum gametocytogenesis, which is required for malaria transmission, has been evaluated. In this study, RNA transcripts for the falcipain family members were identified as the parasite developed through all five stages of gametocytogenesis. Falcipain 1 transcript was upregulated in gametocytes, while levels of falcipain 2A/2B decreased in late‐stage gametocytes and gametes. To evaluate the function of falcipain 1, the gene was disrupted, and clones from independent transformations were isolated. The asexual growth of the falcipain 1 minus clones was not overtly affected, and they produced morphologically normal gametocytes and gametes. However, when falcipain 1 minus parasites were fed to a mosquito, oocyst production was reduced by 70–90%, suggesting an important role for falcipain 1 during parasite development in the mosquito midgut.
The human malaria parasite Plasmodium falciparum has distinct organelle‐specific lipoylation pathways
Authors: Wrenger Carsten , Müller Sylke
Published: December 2004

Lipoic acid is an essential cofactor of α‐keto acid dehydrogenase complexes (KADHCs). This study shows that Plasmodium falciparum possesses two distinct lipoylation pathways that are found in separate subcellular localizations. Lipoic acid synthesis comprising lipoic acid synthase and lipoyl‐ACP:protein N‐lipoyl transferase is present in the parasite's apicoplast, whereas the second pathway consisting of lipoic acid protein ligase is located in the parasite's mitochondrion. The two localizations were established by overexpressing green fluorescent protein fusions of the N‐terminal sequences of lipoic acid synthase and lipoic acid protein ligase in intraerythrocytic stages of P. falciparum. Northern and Western blot analyses revealed that the genes/proteins encoding lipoic acid synthase, lipoyl‐ACP:protein N‐lipoyl transferase and lipoic acid protein ligase are expressed maximally in the early and late stages of P. falciparum erythrocytic development. The functionality of the three proteins was proven by complementation of bacteria deficient in lipA and lipB. Our results show that P. falciparum possesses two independent pathways, with different locations, responsible for the post‐translational modification of KADHCs. Both pathways fundamentally differ from those in the human host. As KADHCs provide metabolites that are required for essential biosynthetic processes such as fatty acid biosynthesis and haem biosynthesis, the two lipoylation pathways of P. falciparum might be attractive therapeutic targets against malaria.
The impact of prophages on bacterial chromosomes
Authors: Canchaya Carlos , Fournous Ghislain , Brüssow Harald
Published: December 2004

Prophages were automatically localized in se‐quenced bacterial genomes by a simple semantic script leading to the identification of 190 prophages in 115 investigated genomes. The distribution of prophages with respect to presence or absence in a given bacterial species, the location and orientation of the prophages on the replichore was not homogeneous. In bacterial pathogens, prophages are particularly prominent. They frequently encoded virulence genes and were major contributors to the genetic individuality of the strains. However, some commensal and free‐living bacteria also showed prominent prophage contributions to the bacterial genomes. Lysogens containing multiple sequence‐related prophages can experience rearrangements of the bacterial genome across prophages, leading to prophages with new gene constellations. Transfer RNA genes are the preferred chromosomal integration sites, and a number of prophages also carry tRNA genes. Prophage integration into protein coding sequences can lead to either gene disruption or new proteins. The phage repressor, immunity and lysogenic conversion genes are frequently transcribed from the prophage. The expression of the latter is sometimes integrated into control circuits linking prophages, the lysogenic bacterium and its animal host. Prophages are apparently as easily acquired as they are lost from the bacterial chromosome. Fixation of prophage genes seems to be restricted to those with functions that have been co‐opted by the bacterial host.
Vibrio cholerae AphA uses a novel mechanism for virulence gene activation that involves interaction with the LysR‐type regulator AphB at the tcpPH promoter
Authors: Kovacikova Gabriela , Lin Wei , Skorupski Karen
Published: December 2004

AphA is required for expression of the Vibrio cholerae virulence cascade and for its regulation by quorum sensing. In order to activate transcription, AphA functions together with a second protein, the LysR‐type regulator AphB, at the tcpPH promoter. As AphA is a member of a new and largely uncharacterized regulator family, random mutagenesis was used to gain insights into how this protein activates transcription. As shown here, 17 amino acid substitutions were identified in AphA that reduced expression of the tcpPH promoter and prevented the protein from binding DNA. The amino acids involved in DNA recognition inferred from a dominant‐negative analysis were located throughout the N‐terminal domain from amino acids 18 to 67. This region of AphA has a conserved domain architecture similar to that of MarR, a multiple antibiotic resistance repressor. The analogous positions of the dominant‐negative mutations in AphA and MarR confirm that the DNA‐binding domains of these proteins are similar and indicate that AphA is a new member of the winged helix family of transcription factors. We also show that AphB is capable of rescuing two of the DNA binding‐defective AphA mutants, suggesting that the proteins interact directly on the DNA. Disruption of this interaction by insertion of half a helical turn between the two binding sites prevented AphB from rescuing the mutants and prevented the expression of the virulence cascade in a wild‐type background. These results provide a novel mechanism for the initiation of virulence gene expression at tcpPH.
Identification of mutator genes and mutational pathways in Escherichia coli using a multicopy cloning approach
Authors: Yang Hanjing , Wolff Erika , Kim Mandy , Diep Amy , Miller Jeffrey H.
Published: December 2004

We searched for genes that create mutator phenotypes when put on to a multicopy plasmid in Escherichia coli. In many cases, this will result in overexpression of the gene in question. We constructed a random shotgun library with E. coli genomic fragments between 3 and 5 kbp in length on a multicopy plasmid vector that was transformed into E. coli to screen for frameshift mutators. We identified a total of 115 independent genomic fragments that covered 17 regions on the E. coli chromosome. Further studies identified 12 genes not previously known as causing mutator phenotypes when overproduced. A striking finding is that overproduction of the multidrug resistance transcription regulator, EmrR, results in a large increase in frameshift and base substitution mutagenesis. This suggests a link between multidrug resistance and mutagenesis. Other identified genes include those encoding DNA helicases (UvrD, RecG, RecQ), truncated forms of the DNA mismatch repair protein (MutS) and a primosomal component (DnaT), a negative modulator of initiation of replication/GATC‐binding protein (SeqA), a stationary phase regulator AppY, a transcriptional regulator PaaX and three putative open reading frames, ycgW, yfjY and yjiD, encoding hypothetical proteins. In addition, we found three genes encoding proteins that were previously known to cause mutator effects under overexpression conditions: error‐prone polymerase IV (DinB), DNA methylase (Dam) and sigma S factor (RpoS). This genomic strategy offers an approach to identify novel mutator effects resulting from the multicopy cloning (MCC) of specific genes and therefore complementing the conventional gene inactivation approach to finding mutators.
A proteomic determination of cold adaptation in the Antarctic archaeon, Methanococcoides burtonii
Authors: Goodchild Amber , Saunders Neil F. W. , Ertan Haluk , Raftery Mark , Guilhaus Michael , Curmi Paul M. G. , Cavicchioli Ricardo
Published: December 2004

A global view of the biology of the cold‐adapted archaeon Methanococcoides burtonii was achieved using proteomics. Proteins specific to growth at 4°C versus Topt (23°C) were identified by mass spectrometry using the draft genome sequence of M. burtonii. mRNA levels were determined for all genes identified by proteomics, and specific enzyme assays confirmed the protein expression results. Key aspects of cold adaptation related to transcription, protein folding and metabolism, including specific roles for RNA polymerase subunit E, a response regulator and peptidyl prolyl cis/trans isomerase. Heat shock protein DnaK was expressed during growth at Topt, indicating that growth at ‘optimal’ temperatures was stressful for this cold‐adapted organism. Expression of trimethylamine methyltransferase involves contiguous translation of two open reading frames, which is likely to result from incorporation of pyrrolysine at an amber stop codon. Thermal regulation in M. burtonii is achieved through complex gene expression events involving gene clusters and operons, through to protein modifications.
Activation of pleiotropic drug resistance by the J‐protein and Hsp70‐related proteins, Zuo1 and Ssz1
Authors: Eisenman Helene C. , Craig Elizabeth A.
Published: December 2004

Ssz1 (Pdr13) and Zuo1, members of the Hsp70 and J‐protein molecular chaperone families, respectively, form a heterodimer and function on the ribosome with the Hsp70, Ssb, presumably assisting folding of newly synthesized polypeptides. As it has also been reported that Ssz1 induces pleiotropic drug resistance (PDR) when overexpressed, a possible role for Zuo1 in PDR was investigated. The C‐terminal domain of Zuo1, which is dispensable for Zuo1's chaperone function on the ribosome, is both necessary and sufficient for PDR induction by Zuo1. A single domain of Ssz1, the N‐terminal ATPase domain, is sufficient for PDR induction as well, indicating that Ssz1 does not function as a chaperone in PDR. No role for Ssb was found in PDR; overexpression did not affect PDR, nor was its presence required for Ssz1's or Zuo1's effect on PDR. As our results also indicate that Ssz1 and Zuo1 must be free of ribosomes to induce PDR, we propose that Ssz1's and Zuo1's function in PDR is distinct from their role as ribosome‐associated co‐chaperones and may be regulatory in nature.
Hfq is essential for Vibrio cholerae virulence and downregulates σE expression
Authors: Ding Yanpeng , Davis Brigid M. , Waldor Matthew K.
Published: December 2004

Hfq is an RNA‐binding protein that interacts with both small untranslated RNAs (sRNAs) and mRNAs to modulate gene expression post‐transcriptionally. In Escherichia coli and Salmonella typhimurium, Hfq is required for efficient expression of the stationary phase sigma factor σS, and consequently is critical for Salmonella virulence. We have found that Hfq is also essential for the virulence of Vibrio cholerae, as strains lacking hfq fail to colonize the suckling mouse intestine. Deletion of the V. cholerae hfq does not prevent production of σS, nor does it prevent expression of TCP, V. cholerae's primary colonization factor. The expression and activity of the alternative sigma factor σE are dramatically increased in a V. cholerae hfq mutant. Comparison of the transcriptome of an hfq mutant with that of an rseA mutant, which also overexpresses σE, revealed that σE controls approximately half the genes found to be upregulated in the hfq mutant. However, increased σE does not appear to account for this strain's reduced virulence. It is likely that sRNAs, in conjunction with Hfq, are critical regulators of V. cholerae pathogenicity.
Euroconference on the Biology of Type IV Secretion Processes: bacterial gates into the outer world
Authors: Llosa Matxalen , O'Callaghan David
Published: December 2004

Type IV secretion systems (T4SSs) mediate both protein and ssDNA secretion from a wide range of bacteria into virtually any cell type or into the milieu. It is this versatility that confers on them the ability to participate in many processes of bacterial life that imply communication with their environment. Type IV secretion systems are involved in horizontal DNA transfer to other bacteria and to plant cells, in DNA uptake from the milieu, in toxin secretion into the milieu, and in the injection of virulence factors into the eukaryotic host cell in a number of mammalian and plant pathogens. Recently, a EuroConference addressed the different aspects of the biology of these transmembrane multiprotein complexes, from the crystal structure of the individual components to the modification that the secreted substrates induce in the recipient cell. Significant progress has been made in the understanding of the molecular architecture and mechanism of secretion. The analysis of protein–protein interactions confirms the role of coupling proteins as substrate recruiters for the transporter. The VirB10 component of the complex has come up as a strong candidate for signal transducer. The wide range of effects on the recipient suggests that many effector proteins are secreted. New effector proteins are being identified for both plant and animal pathogens, as are their targets within the host cells. New T4SS members are being identified that perform novel roles, beyond DNA transfer and virulence, such as establishment of symbiotic processes. Our current knowledge of the Biology of Type IV Secretion Processes increases our ability to exploit them as biotechnological tools or to use them as new targets for inhibitors that could constitute a new generation of antimicrobials in the near future.
Advances in the detection of prion protein in peripheral tissues of variant Creutzfeldt‐Jakob disease patients using paraffin‐embedded tissue blotting
Authors: Ritchie D. L. , Head M. W. , Ironside J. W.
Published: December 2004

The accumulation of PrPSc, an abnormal and disease‐associated form of the normal prion protein (PrPc), within the central nervous system (CNS) is a key pathological feature of Creutzfeldt‐Jakob disease (CJD). Following limited proteolytic digestion of PrPSc, the detection of PrPres within lymphoid tissues is a unique characteristic of variant CJD in comparison with other human prion diseases, raising fears of an increased risk of iatrogenic spread. Because levels of PrPres in lymphoid tissues are lower than those found in CNS tissue, there is concern that other peripheral tissues may harbour infectivity at levels that current detection systems cannot demonstrate PrPres. We have modified the paraffin‐embedded tissue blot (PET blot), a technique combining immunohistochemistry (IHC), histoblot and Western blotting, for the detection of PrPres in paraffin sections in peripheral tissues in variant CJD. Five cases of variant CJD were examined, using a panel of anti‐PrP antibodies. In each of these five cases, spleen, tonsil, lymph nodes and dorsal root ganglia showed an increase in the sensitivity and specificity of labelling using the PET blot when compared with optimized PrPres IHC methods. Control cases showed no evidence of PrP accumulation in either peripheral or CNS tissues. Autopsy and biopsy brain material from sporadic CJD cases also showed an increased sensitivity of PrPres detection with the PET blot, confirming its value as an important diagnostic and research tool in human prion diseases.
Cytokine‐induced enhancement of autoimmune inflammation in the brain and spinal cord: implications for multiple sclerosis
Authors: Sun D. , Newman T. A. , Perry V. H. , Weller R. O.
Published: December 2004

Multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are autoimmune inflammatory diseases in which cytokines are intimately involved. Here we test the hypothesis that injection of pro‐inflammatory cytokines, tumour necrosis factor‐α (TNFα) and interferon gamma (IFNγ) into the brain of animals in the prodromal phase of EAE significantly enhances inflammation in the central nervous system (CNS). We were particularly interested to learn whether a local increase in cytokines influenced the pathology locally, or more extensively, within the CNS. EAE was induced in female adult Lewis rats. Eight days post‐inoculation, TNFα or INFγ was injected into one cerebral hemisphere. Days 11 and 13 post‐inoculation (3 and 5 days after the injection of cytokine) inflammation was quantified by the number of perivascular cuffs and the degree of major histocompatibility complex (MHC) class II expression by microglia. Normal animals injected with cytokines, and EAE animals with saline injection served as controls. Results: microglial activation was increased three‐ to fourfold in the brain and eightfold in the spinal cord (P ≤ 0.05); lymphocyte invasion was increased sixfold in the brain and three‐ to fourfold in the spinal cord (P ≤ 0.01) following injections of TNFα or INFγ in EAE animals compared with controls. Significant axonal damage was observed in white matter associated with the perivascular cuffs. Conclusion: local changes in the release of pro‐inflammatory cytokines within the brain in EAE results in the widespread enhancement of autoimmune inflammation within the brain and cord, and exacerbation of clinical symptoms.