bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel deoxynucleoside‐phosphorylating enzymes in mycoplasmas: evidence for efficient utilization of deoxynucleosides
Authors: Wang Liya , Westberg Joakim , Bölske Göran , Eriksson Staffan
Published: March 2003

Mycoplasmas are unable to synthesize purine and pyrimidine bases de novo. Therefore, salvage of existing nucleosides and bases is essential for their survival. Four mycoplasma species were studied with regard to their ability to phosphorylate deoxynucleosides. High levels of thymidine kinase (TK), deoxycytidine kinase (dCK), deoxyguanosine kinase (dGK) and deoxyadenosine kinase (dAK) activities were detected in extracts from Mycoplasma pneumoniae, Mycoplasma mycoides subsp. mycoides SC (M. mymySC), Acholeplasma laidlawii (A. laidlawii) and Mycoplasma arginini (M. arginini). Nucleoside phosphotransferase activities were found at high levels in A. laidlawii and low levels in M. arginini. Pyrophosphate‐dependent deoxynucleoside kinase activities were detected mainly in A. laidlawii and M. mymySC extracts. Two open reading frames were identified in the M. mymySC genome; one showed 25% sequence identity to human dGK and the other one had about 26% sequence identity to human TK1. The M. mymySC dGK‐like enzyme was cloned, expressed in Escherichia coli and affinity‐purified. This enzyme phosphorylated dAdo, dGuo and dCyd, and the highest catalytic rate was with dAdo as substrate. Therefore, we suggest that this enzyme should be named deoxyadenosine kinase. The physiological role of mycoplasma dAK and TK may be to support the unusually large dATP and dTTP pools required for replication of mycoplasma genomes.
Leaderless mRNAs in bacteria: surprises in ribosomal recruitment and translational control
Authors: Moll Isabella , Grill Sonja , Gualerzi Claudio O. , Bläsi Udo
Published: March 2003

It is commonly believed that the translational efficiency of prokaryotic mRNAs is intrinsically determined by both primary and secondary structures of their translational initiation regions. However, for leaderless mRNAs starting with the AUG initiating codon occurring in bacteria, archaea and eukaryotes, there is no evidence for ribosomal recruitment signals downstream of the 5′‐terminal AUG that seems to
be the only necessary and constant element. Studies in Escherichia coli have brought to light that the ratio of initiation factors IF2 and IF3 plays a decisive role in translation initiation of leaderless mRNA, indicating that the translational efficiency of this mRNA class can be modulated depending on the availability of components of the translational machinery. Recent data suggested that the start codon of bacterial leaderless mRNAs is recognized by a ribosome‐IF2‐fMet‐tRNA complex, an intermediate equivalent to that obligatorily formed during translation initiation in eukaryotes, which points to a conceptual similarity in all initiation pathways. In fact, the faithful translation of lead‐erless mRNAs in heterologous systems shows that the ability to translate leaderless mRNAs is an evo‐lutionarily conserved function of the translational apparatus.
Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli
Authors: Guzmán Elena C. , Caballero José Luis , Jiménez‐Sánchez Alfonso
Published: March 2003

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42°C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42°C of an Escherichia coli strain containing this allele allows DNA replication for about 40 min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, to‐gether with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run‐out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.
Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae
Authors: Merrell D. Scott , Hava David L. , Camilli Andrew
Published: March 2003

Despite over 100 years of study, the intestinal pathogen Vibrio cholerae
still causes epidemic disease in areas of the world where there is poor sanitation.
While cholera toxin and the toxin‐coregulated pilus (TCP) are known to be essential
for full virulence, the role that other factors play has remained ill‐defined. Herein,
we describe a large‐scale signature‐tagged mutagenesis (STM) screen utilizing 100
pools of 96 mutants each to identify factors involved in colonization of the infant
mouse small intestine. A total of 164 mutants representing transposition events into
95 different open reading frames were shown to be recovered at greatly reduced numbers
from the infant mouse model. Analysis of the sites of insertion revealed multiple
independent mutations within the rfb gene cluster, needed for synthesis of
lipopolysaccharide (LPS), and the tcp gene cluster, needed for synthesis of
the TCP. More importantly, in addition to these previously known colonization factors,
we identified many genes whose activity in colonization was not previously appreciated.
These can be divided into a number of functional groups, which include production
of factors involved in metabolic activities, regulation of cellular processes, transport,
adaptation to stress and unknown functions. In addition, we describe the reiterative
use of STM, whereby colonization‐defective mutants were assembled into virulence‐attenuated
pools (VAPs), which were used to begin to reveal roles that the identified virulence
factors play in the infection process. Nine new factors were shown to be crucial
for the V. cholerae acid tolerance response, which has previously been hypothesized
to be important for epidemic spread of cholera. Competition assays of these nine
acid tolerance response (ATR)‐defective mutants revealed that mutations in gshB,
hepA and recO result in a 1000‐fold reduction in colonization.
Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana
Authors: Boch Jens , Joardar Vinita , Gao Lisa , Robertson Tara L. , Lim Melisa , Kunkel Barbara N.
Published: March 2003

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology
(IVET) approach to identify genes from the plant pathogen Pseudomonas syringae
pv. tomato that are induced upon infection of Arabidopsis thaliana
and isolated over 500 in planta‐expressed (ipx) promoter fusions. Sequence
analysis of 79 fusions revealed several known and potential virulence genes, including
hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified
metabolic genes presumably important for adaptation to growth in plant tissue, as
well as several genes with unknown function that may encode novel virulence factors.
Many ipx fusions, including several corresponding to novel genes, are dependent
on HrpL, an alternative RNA polymerase sigma factor that regulates the expression
of virulence genes. Expression analysis indicated that several ipx fusions
are strongly induced upon inoculation into plant tissue. Disruption of one ipx
gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein
transglycosylase, resulted in decreased virulence of P. syringae. Our results
demonstrate that this screen can be used successfully to isolate genes that are induced
in planta, including many novel genes potentially involved in pathogenesis.
Primosome assembly requirement for replication restart in the Escherichia coli holDG10 replication mutant
Authors: Flores Maria Jose , Ehrlich S. Dusko , Michel Bénédicte
Published: March 2003

In this report, we study the role of pre‐primosome proteins in a strain in which the frequency of replication arrest is increased because of a mutation in a replication protein. The holDG10 mutant was used, in which replication restart
involves replication fork reversal. As expected, PriA primosome assembly function
is essential for growth of the holDG10 mutant. The priA300
mutation, which inactivates only the helicase function of PriA in vitro, and
priB inactivation strongly impair viability. In contrast, priC inactivation
has no effect. Therefore, PriB is more important than PriC for PriA‐dependent replication
fork restart in vivo. The gain of function mutation dnaC809 restores
the viability of holDG10 priA and holDG10 priB
mutants only to some extent. The dnaC809 820 double mutation restores full
viability to the holDG10 mutant lacking either PriA or PriB. Similarly
to the holDG10 single mutant, the holDG10 priA dnaC809
820 strain is depend‐ent on RecBC for viability, indicating that facilitating
primosome assembly using the dnaC809 820 mutation does not allow bypass of replication fork reversal.
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

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

A 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

Leaderless 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

The 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

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

Phospholipases 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

The 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

The 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

Deoxyadenosine 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

SsrA 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

Repeat‐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

Pyoverdines, 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

African 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

Folate 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.
Modulation of pPS10 host range by DnaA
Authors: Maestro Beatriz , Sanz Jesús M. , Faelen Michel , Couturier Martine , Díaz‐Orejas Ramón , Fernández‐Tresguerres Elena
Published: March 2003

Narrow‐host‐range plasmid pPS10, originally found inPseudomonas savastanoi, is unable to replicate in other strains such asEscherichia coli. Here, we report that the establishment of pPS10 inE. colican be achieved by a triple mutation in thednaAgene ofE. coli(dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As theE. colistrain used contained double amber suppressor mutations (supE,supF), the amber codon indnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon inE. coli.The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon inE. coliand provides support for the hypothesis that interactions between RepA and DnaA modulate the establish‐ment of pPS10 in that bacteria and probably in other species.
Acidic phospholipids inhibit the DNA‐binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli
Authors: Makise Masaki , Mima Shinji , Katsu Takashi , Tsuchiya Tomofusa , Mizushima Tohru
Published: March 2003

In order to initiate chromosomal DNA replication in Escherichia coli, the DnaA protein must bind to both ATP and the origin of replication (oriC). Acidic phospholipids are known to inhibit DnaA binding to ATP, and here we examine the effects of various phospholipids on DnaA binding to oriC. Among the phospholipids in E. coli membrane, cardiolipin showed the strongest inhibition of DnaA binding to oriC. Synthetic phosphatidylglycerol containing unsaturated fatty acids inhibited binding more potently than did synthetic phosphatidylglycerol containing saturated fatty acids, suggesting that membrane fluidity is important. Thus, acidic phospholipids seem to inhibit DnaA binding to both oriC and adenine nucleotides in the same manner. Adenine nucleotides bound to DnaA did not affect the inhibitory effect of cardiolipin on DnaA binding to oriC. A mobility‐shift assay re‐vealed that acidic phospholipids inhibited formation of a DnaA–oriC complex containing several DnaA molecules. DNase I footprinting of DnaA binding to oriC showed that two DnaA binding sites (R2 and R3) were more sensitive to cardiolipin than other DnaA binding sites. Based on these in vitro data, the physiological relevance of this inhibitory effect of acidic phospholipids on DnaA binding to oriC is discussed.
Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111
Authors: Huber Birgit , Riedel Kathrin , Köthe Manuela , Givskov Michael , Molin Søren , Eberl Leo
Published: March 2003

Burkholderia cepacia
and
Pseudomonas aeruginosa
often co‐exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini‐Tn
5
insertion mutants of
B. cepacia
H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild‐type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three‐dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i) genes encoding for surface proteins; (ii) genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii) genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of
N
‐octanoylhomoserine lactone (C8‐HSL). This compound serves as the major signal molecule of the
cep
quorum‐sensing system. As this density‐dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum‐sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the
cep
system, indicating that the quorum‐sensing system of
B. cepacia is a major checkpoint for biofilm formation.
The glycosylphosphatidylinositol (GPI) signal sequence of human placental alkaline phosphatase is not recognized by human Gpi8p in the context of the yeast GPI anchoring machinery
Authors: Meyer Urs , Fraering Patrick , Bosson Régine , Imhof Isabella , Benghezal Mohammed , Vionnet Christine , Conzelmann Andreas
Published: March 2003

Biosynthesis of glycosylphosphatidylinositol (GPI)‐anchored proteins involves the action of a GPI trans‐amidase, which replaces the C‐terminal GPI signal sequence (GPI‐SS) of the primary translation product with a preformed GPI lipid. The transamidation depends on a complex of four proteins, Gaa1p, Gpi8p, Gpi16p and Gpi17p. Although the GPI anchoring pathway is conserved throughout the eukaryotic kingdom, it has been reported recently that the GPI‐SS of human placental alkaline phosphatase (hPLAP) is not recognized by the yeast transamidase, but is recognized in yeast that contain the human Gpi8p homologue . This finding suggests that Gpi8p is intimately involved in the recognition of GPI precursor proteins and may also be responsible for the subtle taxon‐specific differences in transamidase specificity that sometimes prevent the efficient GPI anchoring of heterologously expressed GPI proteins. Here, we confirm that the GPI signal sequence of hPLAP is indeed not recognized by the yeast GPI‐anchoring machinery. However, in our hands, GPI attachment cannot be restored by the co‐expression of human Gpi8p in yeast cells under any circumstances.
The VirB/VirD4 type IV secretion system of Bartonella is essential for establishing intraerythrocytic infection
Authors: Schulein Ralf , Dehio Christoph
Published: March 2003

Bartonellae are pathogenic bacteria uniquely adapted to cause intraerythrocytic infection in their human or animal reservoir host(s). Experimental infection of rats by Bartonella tribocorum revealed the initial colonization of a yet unidentified niche outside of circulating blood. This primary niche periodically seeds bacteria into the bloodstream, resulting in the invasion and persistent intracellular colonisation of erythrocytes. Here, this animal model was used for a genetic analysis of the virB locus (virB2‐11) and the downstream located virD4 gene, which together encode a putative type IV secretion system (T4SS). A generic method for marker‐less gene replacement allowed the generation of non‐polar in‐frame deletions in either virB4 or virD4. Both mutants were unable to cause bacteraemia, whereas complementation with the full‐length genes in trans completely restored infectivity. Segregation analysis of the complementation plasmids further denoted that VirB4 and VirD4 are required at an early stage of the infection course before the onset of intraerythrocytic bacteraemia. This analysis of defined mutants in an in vivo model identified components of the VirB/VirD4 T4SS as the first bona fide pathogenicity factors in Bartonella.
On the mechanism of substrate specificity by resistance nodulation division (RND)‐type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition
Authors: Mao Weimin , Warren Mark S. , Black Deborah S. , Satou Takahumi , Murata Takeshi , Nishino Takeshi , Gotoh Naomasa , Lomovskaya Olga
Published: March 2003

Tripartite efflux systems of Gram‐negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD–OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non‐transported β‐lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD–OprJ‐mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD–OprJ susceptible to inhibition by a thiol‐reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu–Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.
ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon
Authors: McCaw Michelle L. , Lykken Guinevere L. , Singh Pradeep K. , Yahr Timothy L.
Published: March 2003

Expression of the Pseudomonas aeruginosa type III secretion system is induced by contact with eukaryotic cells, serum or low Ca2+ concentrations. We report that ExsD, a unique protein, is a negative regulator of the type III regulon. Localization studies indicate that ExsD is not secreted by P. aeruginosa. To determine the role of exsD, a non‐polar deletion was returned to the chromosome by allelic exchange. The ΔexsD mutant is competent for type III secretion and translocation of the ExoU cytotoxin to eukaryotic host cells. To examine the effect of ExsD on transcription, lacZ transcriptional reporter fusions were integrated into the chromosome. Promoters controlling transcription of genes encoding the type III secretory, regulatory and effector proteins demonstrated significant derepression in the ΔexsD background. Expression of ExsD from a multicopy plasmid completely repressed transcription of the regulon. Although a mutant in pscC, encoding a structural component of the type III translocase, is repressed for expression of the regulon, a ΔexsD, pscC::Ω double mutant is derepressed. Bacterial two‐hybrid data indicate that ExsD binds the transcriptional activator of the regulon, ExsA. We conclude that ExsD is a negative regulator and propose that ExsD functions as an ExsA antiactivator to regulate transcription of the regulon.
A novel apoptosis‐inducing protein from Helicobacter pylori
Authors: Shibayama Keigo , Kamachi Kazunari , Nagata Noriyo , Yagi Tetsuya , Nada Toshi , Doi Yohei , Shibata Naohiro , Yokoyama Keiko , Yamane Kunikazu , Kato Haru , Iinuma Yoshitsugu , Arakawa Yoshichika
Published: March 2003

Helicobacter pylori
infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis‐inducing protein that functions as a leading factor in
H. pylori
‐mediated apoptosis induction. We purified the protein from
H. pylori
by separating fractions that showed apoptosis‐inducing activity. This protein induced apoptosis of AGS cells in a dose‐dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N‐terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited γ‐glutamyl transpeptidase activity, the inhibition of which by 6‐diazo‐5‐oxo‐
l
‐norleucine resulted in a complete loss of apoptosis‐inducing activity. To the best of our knowledge, the apoptosis‐inducing function is a newly identified physiological role for bacterial γ‐glutamyl transpeptidase. The apoptosis‐inducing activity of the isogenic mutant γ‐glutamyl transpeptidase‐deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ‐glutamyl transpeptidase plays a significant role in
H. pylori
‐mediated apoptosis. Our findings provide new insights into
H. pylori pathogenicity and reveal a novel aspect of the bacterial γ‐glutamyl transpeptidase function.
Putrescine or a combination of methionine and arginine restores virulence gene expression in a tRNA modification‐deficient mutant of Shigella flexneri: a possible role in adaptation of virulence
Authors: Durand Jérôme M. B. , Björk Glenn R.
Published: March 2003

The wild‐type strain YSH6000 of Shigella flexneri growing in minimal medium contains the modified nucleoside epoxy‐Q (oQ) in a subset of tRNAs. This nucleoside is lacking in tRNA from a tgt mutant of this bacterium. When these bacteria are growing in minimal medium, the expression of virulence genes is 10‐fold lower in the tgt mutant than in the wild type, although only a twofold reduction in the expression of these virulence factors is observed in broth. Such a strong media‐dependant expression of virulence genes was not observed in the wild type. Accordingly, the level of the positive regulator of virulence, VirF, is much lower in the mutant than in the wild type. However, the transcription of the virF gene in minimal medium is the same in the wild type and in the tgt mutant. As the undermodification of tRNA is not affected by the quality of the growth medium, we conclude that such an environmental change in growth conditions partly restores virulence gene expression by counteracting poor translation of the virF mRNA mediated by an oQ‐deficient tRNA. Virulence gene expression is partly restored in the tgt mutant by the addition of a mixture of arginine and methionine. Addition of the polyamine putrescine, synthesis of which is metabolically related to that of arginine and methionine, has a comparable stimulatory effect on virulence gene expression. These results not only suggest a role for amino acids and polyamines in the environmental regulation of virulence gene expression in S. flexneri, but also demonstrate a strong and specific involvement of tRNA modifications, and especially oQ, in the adaptation of virulence gene expression to the nutritional quality of the growth medium.