bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evidence for a partial redundancy of the fibronectin‐binding proteins for the transfer of mycoloyl residues onto the cell wall arabinogalactan termini of Mycobacterium tuberculosis
Authors: Puech Virginie , Guilhot Christophe , Perez Esther , Tropis Marielle , Armitige Lisa Y. , Gicquel Brigitte , Daffé Mamadou 
Published: March 2003

Summary Mycobacterium tuberculosis produces a series of major secreted proteins, the fibronectin‐binding proteins (Fbps), also known as the antigen 85 complex, that are believed to play an essential role in the pathogenesis of tuberculosis through their mycoloyltransferase activity required for maintaining the integrity of the bacterial cell envelope. Four different fbp genes are found in the genome of M. tuberculosis, but the reason for the existence of these Fbps sharing the same substrate specificity in vitro in mycobacteria is unknown. We have shown previously that, in the heterologous host, Corynebacterium glutamicum, FbpA, FbpB and FbpC can all add mycoloyl residues to the cell wall arabinogalactan and that, in M. tuberculosis, the cell wall mycoloylation decreases by 40% when fbpC is knocked out. To investigate whether the remaining 60% mycoloylation came from the activity of FbpA and/or FbpB, fbpA‐ and fbpB‐inactivated mutant strains were biochemically characterized and compared with the previously studied fbpC‐disrupted mutant. Unexpectedly, both mutants produced normally mycoloylated cell walls. Overproduction of FbpA, FbpB or FbpC, but not FbpD, in the fbpC‐inactivated mutant strain of M. tuberculosis restored both the cell wall‐linked mycolate defect and the outer cell envelope permeability barrier property. These results are consistent with all three enzymes being involved in cell wall mycoloylation and FbpC playing a more critical role than the others or, alternatively, FbpC is able to compensate for FbpA and FbpB in ways that these enzymes cannot compensate for FbpC, pointing to a partial redundancy of Fbps. In sharp contrast, FbpD does not appear to be an active mycoloyltransferase enzyme, as it cannot complement the fbpC‐inactivated mutant. Most importantly, application of Smith degradation to the cell walls of transformants demonstrated that the multiple Fbp enzymes are redundant rather than specific for the various arabinogalactan mycoloylation regions. Neither FbpA nor FbpB attaches mycoloyl residues to specific sites but, like FbpC, each enzyme transfers mycoloyl residues onto the four sites present in the arabinogalactan non‐reducing end hexaarabinosides.
Topology of the Salmonella invasion protein SipB in a model bilayer
Authors: McGhie Emma J. , Hume Peter J. , Hayward Richard D. , Torres Jaume , Koronakis Vassilis 
Published: March 2003

SummaryA critical early event in Salmonella infection is entry into intestinal
epithelial cells. The Salmonellainvasion protein SipB
is required for the delivery of bacterial effector proteins into target eukaryotic
cells, which subvert signal transduction pathways and cytoskeletal dynamics. SipB
inserts into the host plasma membrane during infection, and the purified protein
has membrane affinity and heterotypic membrane fusion activity in vitro. We
used complementary biochemical and biophysical techniques to inves‐tigate the topology
of purified SipB in a model membrane. We show that the 593 residue SipB is predominantly
α‐helical in aqueous solution, and that no significant change in secondary
structural content accompanies lipid interaction. SipB contains two α‐helical transmembrane domains (residues 320–353 and 409–427), which insert deeply into the bilayer. Their integration allowed the hydrophilic region between the hydrophobic domains (354–408) to cross the bilayer. SipB membrane integration required both the hydrophobic domains and an additional helical C‐terminal region (428–593). Further spectroscopic analysis of these domains in isolation showed that the hydrophobic regions insert obliquely into the bilayer, whereas the C‐terminal domain associates with the bilayer surface, tilted parallel to the membrane. The combined data suggest a topological model for membrane‐inserted SipB.
Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD‐box helicase on translation of leaderless and canonical mRNAs in Escherichia coli
Authors: Moll Isabella , Grill Sonja , Gründling Angelika , Bläsi Udo 
Published: March 2003

SummaryLeaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless λcI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal
protein S2. Here, we have studied this phenomenon at the molecular level by making
use of an E. coli rpsBts mutant. The analysis of the ribosomes
isolated under the non‐permissive conditions revealed that in addition to ribosomal
protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for
binding of S1 to the 30S ribosomal subunit. In vitro translation assays and
the selective translation of a leaderless mRNA in vivo at the non‐permissive
temperature corroborate and extend previous in vitro ribosome binding studies
in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA
gene, encoding the ‘DeaD/CsdA’ DEAD‐box helicase, has been isolated
as a multicopy suppressor of rpsBts mutations. Here, we show that
expression of a plasmid borne deaD/csdA gene restores both S1 and S2 on the
ribosome at the non‐permissive temperature in the rpsBts strain, which in turn leads to suppression of the translational defect affecting canonical mRNAs. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.
Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea‡
Authors: Cortés Jesús , Velasco Javier , Foster Graham , Blackaby Andrew P. , Rudd Brian A. M. , Wilkinson Barrie 
Published: March 2003

SummaryThe soluble, diffusible red‐brown pigment produced by a Saccharopolyspora erythraea‘red variant’ has been shown to contain glycosylated and polymerized
derivatives of 2,5,7‐trihydroxy‐1,4‐naphthoquinone (flaviolin). Flaviolin is a spontaneous
oxidation product of 1,3,6,8‐tetrahydroxynaphthalene (THN), which is biosynthesized
in bacteria by a chalcone synthase‐like (CS‐like) type III polyketide synthase (PKS).
A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8‐7
was amplified by polymerase chain reaction (PCR) using degenerate primers based on
conserved regions of known plant CS and bacterial CS‐like genes. From the isolated
fragment, a suicide vector was prepared, which was subsequently used to disrupt the
red‐brown pigment‐producing (rpp) locus in S. erythraea, generating
a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant
was subsequently used in a vector‐recapture protocol to isolate a plasmid that contained
an insert spanning the entire rpp locus. Sequencing of the insert revealed
that the disrupted open reading frame (ORF) encodes a CS‐like protein displaying
69% sequence identity to the rppA gene of Streptomyces griseus.
The S. griseus rppA gene encodes RppA, the first characterized bacterial CS‐like
protein, which is sufficient in vitro for the synthesis of THN from malonyl‐CoA.
The rppA disruption mutant and rppA sequence provided a means by which
to address the mechanism of diffusible pigment biosynthesis, as well as to investigate
any link between this and the modulation of erythromycin A titre, which has been
observed for S. erythraea variants.
StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor
Authors: Lathem Wyndham W. , Grys Thomas E. , Witowski Sarah E. , Torres Alfredo G. , Kaper James B. , Tarr Phillip I. , Welch Rodney A. 
Published: March 2003

Summary Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1‐INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1‐INH to produce (unique) ≈ 60–65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage‐like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1‐INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE‐encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1‐INH by StcE could plausibly cause localized pro‐inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.
Phospholipases C are involved in the virulence of Mycobacterium tuberculosis
Authors: Raynaud Catherine , Guilhot Christophe , Rauzier Jean , Bordat Yann , Pelicic Vladimir , Manganelli Riccardo , Smith Issar , Gicquel Brigitte , Jackson Mary 
Published: March 2003

SummaryPhospholipases C play a role in the pathogenesis of several bacteria. Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses four genes encoding putative phospholipases C, plcA, plcB, plcC and plcD. However, the contribution of these genes to virulence is unknown. We constructed four single mutants of M. tuberculosis each inactivated in one of the plc genes, a triple plcABC mutant and a quadruple plcABCD mutant. The mutants all exhibited a lower phospholipase C activity than the wild‐type parent strain, demonstrating that the four plc genes encode a functional phospholipase C in M. tuberculosis. Functional complementation of the ΔplcABC triple mutant with the individual plcA, plcB and plcC genes restored in each case about 20% of the total Plc activity detected in the parental strain, suggesting that the three enzymes contribute equally to the overall Plc activity of M. tuberculosis. RT‐PCR analysis of the plc genes transcripts showed that the expression of these genes is strongly upregulated during the first 24 h of macrophage infection. Moreover, the growth kinetics of the triple and quadruple mutants in a mouse model of infection revealed that both mutants are attenuated in the late phase of the infection emphasizing the importance of phospholipases C in the virulence of the tubercle bacillus.
A protein kinase specifically associated with proliferative forms of Trypanosoma brucei is functionally related to a yeast kinase involved in the co‐ordination of cell shape and division
Authors: García‐Salcedo José A. , Nolan Derek P. , Gijón Purificación , Gómez‐Rodriguez Julio , Pays Etienne 
Published: March 2003

SummaryThe life cycle of African trypanosomes is characterized by the alternation of proliferative and quiescent stages but the molecular details of this process remain unknown. Here, we describe a new cytoplasmic protein kinase from Trypanosoma brucei, termed TBPK50, that belongs to a family of protein kinases involved in the regulation of the cell cycle, cell shape and proliferation. TBPK50 is expressed only in proliferative forms but is totally absent in quiescent cells despite the fact that the gene is constitutively transcribed at the same level throughout the life cycle. It is probable that TBPK50 has very specific substrate requirements as it was unable to transphosphorylate a range of classical phosphoacceptor substrates in vitro, although an autophosphorylation activity was readily detectable in the same assays. Complementation studies using a fission yeast mutant demonstrated that TBPK50 is a functional homologue of Orb6, a protein kinase involved in the regulation of cellular morphology and cell cycle progression in yeast. These results link the expression of TBPK50 and the growth status of trypanosomes and support the view that this protein kinase is likely to be involved in the control of life cycle progression and cell division of these parasites.
Cellular responses to environmental salinity in the halophilic black yeast Hortaea werneckii
Authors: Petrovicˇ Urosˇ , Gunde‐Cimerman Nina , Plemenitasˇ Ana 
Published: March 2003

SummaryThe development of crop plants with increased salt tolerance necessitates the study of naturally salt‐tolerant eukaryotic species. We studied the bio‐synthesis of glycerol as a compatible solute in the halophilic eukaryotic microorganism, black yeast Hortaea werneckii. A restriction fragment–differential display technique was used to investigate the transcriptome of the organism. Eight differentially expressed genes were identified in response to growth at different salinities. Although the putative functions of their products, P‐type ATPase, ubiquinone reductase, aconitase, RNA helicase, Asn‐tRNA ligase, isoamyl alcohol oxidase, and phosphatidylinositol‐3‐kinase, are not intimately related within the cellular machinery, the results presented here are sufficient to propose a model which describes how H. werneckii adapts to extremely high salinities. Some of these mechanisms of adaptation to raised environmental salinity are similar to those in other salt‐sensitive species, e.g. glycerol accumulation, there also appear to be novel mechanisms present such as the use of different energy production mechanisms and post‐transcriptional regulation of gene expression. Our results have also provided new data on two genes from two other fungal species, the Neurospora crassa B1D1.130 gene and the Aspergillus ustus amdS‐A gene.
Genome‐wide analysis of deoxyadenosine methyltransferase‐mediated control of gene expression in Escherichia coli
Authors: Oshima Taku , Wada Chieko , Kawagoe Yuya , Ara Takeshi , Maeda Maki , Masuda Yasushi , Hiraga Sota , Mori Hirotada 
Published: March 2003

SummaryDeoxyadenosine methyltransferase (Dam) methylates the deoxyadenine residues in 5′‐GATC‐3′ sequences and is important in many cellular processes in Escherichia coli. We performed a computational analysis of the entire E. coli genome and confirmed that GATC sequences are distributed unevenly in regulatory regions, which suggests that Dam might regulate gene transcription. To test this, a high‐density DNA microarray of 4097 E. coli genes was constructed and used to assess the gene expression profiles of the wild type and the dam‐16::kam mutant strain grown under four different conditions. We also used two‐dimensional electrophoretic analysis of the proteome to assess the protein profiles. The expression of a large number of genes was affected by the dam deficiency. Genes involved in aerobic respiration, stress and SOS responses, amino acid meta‐bolism and nucleotide metabolism were expressed at higher levels in the mutant cells, especially in aerobic conditions. In contrast, transcription of genes partici‐pating in anaerobic respiration, flagella biosynthesis, chemotaxis and motility was decreased in the dam mutant strain under both aerobic and low aerobic conditions. Thus, Dam‐controlled genes are involved in adjusting the metabolic and respiratory pathways and bacterial motility to suit particular environmental conditions. The promoters of most of these Dam‐controlled genes were also found to contain GATC sequences that overlap with recognition sites for two global regulators, fumarate nitrate reduction (Fnr) and catabolite activator protein (CRP). We propose that Dam‐mediated methylation plays an important role in the global regulation of genes, particularly those with Fnr and CRP binding sites.
Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli
Authors: Collier Justine , Binet Emmanuelle , Bouloc Philippe 
Published: March 2003

SummarySsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA‐tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C‐terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.
Repeat‐induced point mutation (RIP) in Magnaporthe grisea: implications for its sexual cycle in the natural field context
Authors: Ikeda Ken‐ichi , Nakayashiki Hitoshi , Kataoka Tomomori , Tamba Hiroki , Hashimoto Yoko , Tosa Yukio , Mayama Shigeyuki 
Published: March 2003

SummaryRepeat‐induced point mutation (RIP) is a process that detects DNA duplications and peppers their sequences with C:G to T:A transitions in the sexual phase of the life cycle. So far, this unique mechanism has been identified as a currently active process in only two fungal species, Neurospora crassa and Podospora anserina. To determine whether a RIP‐like process operates in the plant pathogenic fungus Magnaporthe grisea, the retrotransposon MAGGY and the hygromycin B phosphotransferase gene were introduced into the fungus as multiple transgenes and examined for sequence alterations after a cross. Frequent C:G to T:A transitions in the transgenes were found in the descendants, preferentially in (A/Tp)Cp(A/T) contexts, suggesting that a process similar to RIP functions in M. grisea. We also examined the sequence of another retrotransposon Pyret in six field isolates of M. grisea. Even though no perfect stage has been known in M. grisea under field conditions to date, RIP‐like transitions were found in all the field isolates tested. Interestingly, the frequency of the transitions mostly correlated with the fertility of the isolates examined under laboratory conditions. These results imply that the sexual cycle of this fungus exists or existed in the natural field context.
Identification of new, conserved, non‐ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine
Authors: Mossialos Dimitris , Ochsner Urs , Baysse Christine , Chablain Patrice , Pirnay Jean‐Paul , Koedam Nico , Budzikiewicz Herbert , Fernández Diana Uría , Schäfer Mathias , Ravel Jacques , Cornelis Pierre 
Published: March 2003

SummaryPyoverdines, the main siderophores of fluorescent pseudomonads, contain a peptide moiety, different for each pyoverdine, and an identical chromophore. While it has been shown that non‐ribosomal peptide synthetases (NRPSs) are involved in the biosynthesis of the peptide chain of pyoverdines, this was not demonstrated for the biosynthesis of the chromo‐phore part. We found that PvsA, from Pseudomonas fluorescens ATCC 17400, and PvdL (PA2424), from Pseudomonas aeruginosa are similar NRPSs and functional homologues, necessary for the production of pyoverdine. Transcriptional lacZ fusions showed that pvdL is co‐transcribed with the upstream PA2425 gene, encoding a putative thioesterase, and is iron‐regulated via PvdS. Similarly, RT‐PCR analysis revealed that expression of pvsA is repressed by iron. Analysis of the adenylation domains of PvsA, PvdL and their homologues, revealed that their N‐terminus starts with an acyl‐CoA ligase module, followed by three amino acid activation domains. Computer modelling of these domains suggests that PvsA in P. fluorescens and PvdL in P. aeruginosa are orthologues involved in the biosynthesis of the pyoverdine chromophore.
Two pathways of homologous recombination in Trypanosoma brucei
Authors: Conway Colin , Proudfoot Chris , Burton Peter , Barry J. David , McCulloch Richard 
Published: March 2003

SummaryAfrican trypanosomes are unicellular parasites that use DNA recombination to evade the mammalian immune response. They do this in a process called antigenic variation, in which the parasites periodically switch the expression of VSG genes that encode distinct Variant Surface Glycoprotein coats. Recombination is used to move new VSG genes into specialised bloodstream VSG transcription sites. Genetic and molecular evidence has suggested that antigenic variation uses homologous recombination, but the detailed reaction pathways are not understood. In this study, we examine the recombination pathways used by trypanosomes to integrate transformed DNA into their genome, and show that they possess at least two pathways of homologous recombination. The primary mechanism is dependent upon RAD51, but a subsidiary pathway exists that is RAD51‐independent. Both pathways contribute to antigenic variation. We show that the RAD51‐independent pathway is capable of recombining DNA substrates with very short lengths of sequence homology and in some cases aberrant recombination reactions can be detected using such microhomologies.
Transcriptional analysis of genes encoding enzymes of the folate pathway in the human malaria parasite Plasmodium falciparum
Authors: Nirmalan Niroshini , Wang Ping , Sims Paul F. G. , Hyde John E. 
Published: March 2003

SummaryFolate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one‐carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway‐specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy‐drofolate reductase–thymidylate synthase, dhfr‐ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.