bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression in Mycoplasma pneumoniae of the recombinant gene encoding the cytadherence‐associated protein HMW1 and identification of HMW4 as a product
Authors: Hahn Tae‐Wook , Krebes Kathleen A. , Krause Duncan C.
Published: November 2003

Mycoplasma pneumoniae is a major cause of tracheobronchitis and pneumonia in older children and young adults. The lack of adequate tools for genetic analysis has hindered the elucidation of function and regulation of mycoplasma virulence determinants. We describe here the use of a transposon vector to deliver the cloned gene for the cytadherence‐associated protein HMW1 in M. pneumoniae. A 4.95 kbp BamHI fragment encoding all but the C‐terminal end of HMW1 was cloned into a modified Tn4001 and transformed into wild‐type M. pneumoniae and into a non‐cytadhering mutant lacking HMW1–HMW5. Southern blot hybridizations confirmed insertion of the transposon and the presence of both the resident and recombinant hmw1 alleles. Analysis by Western immunoblotting revealed a truncated HMW1 (HMW1′) in the transformants, the level of HMW1′ being dependent upon the orientation of the hmw1 gene in the transposon and the site of insertion. Similar expression patterns were noted in wild‐type and mutant backgrounds. However, expression of wild‐type levels of HMW1′ in the mutant did not restore adherence. Finally, HMW4 and HMW1 were shown to be products of the same gene, HMW4 being a heat‐modified derivative of HMW1.
A novel peptidoglycan‐linked lipoprotein (ComL) that functions in natural transformation competence of Neisseria gonorrhoeae
Authors: Fussenegger Martin , Facius Dirk , Meier Jürgen , Meyer Thomas F.
Published: November 2003

A novel peptidoglycan‐linked lipoprotein (ComL) has been identified which is required for efficient transformation of Neisseria gonorrhoeae by species‐related DNA. Although most mutations in comL appear to be lethal, transposon shuttle mutagenesis was successful in generating a single viable comL mutant of N. gonorrhoeae strain MS11. This mutant, N457, exhibits a cratered and crinkled colony morphology and grows slower than wild‐type MS11. However, as indicated by electron microscopy, this retardation is due to a small bacterial size rather than to a decreased generation time of the mutant bacteria. Complementation of N457 with an intact comL gene via the Hermes shuttle system fully reconstitutes bacterial size, colony morphology, and transformation competence of the wild‐type strain. comL is a single‐copy gene and maps downstream of the previously described comA gene It is transcribed in the opposite direction, probably using the same transcriptional terminator. ComL has a predicted size of 29 kDa and is synthesized in Escherichia coli under the control of its native promoter, which is highly conserved with the E. coli promoter consensus sequence. The 5′ end of the coding sequence reveals a lipoprotein secretion signal shown to be functional by gene fusion with alkaline phosphatase (phoA′ ). In E. coli, cloned ComL can be labelled with [3H]‐palmitic acid, thus demonstrating its lipoproteinaceous nature. Palmitoylated ComL appears to be covalently bound to the murein sacculus of E. coli and N. gonorrhoeae since it resists boiling in 4% sodium dodecyl sulphate and is released only by lysozyme treatment. Homologous counterparts of the comL gene are found in Neisseriameningitidis as well as in several non‐pathogenic Neisseria species.
Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis
Authors: Van Der Ley Peter , Kramer Marco , Steeghs Liana , Kuipers Betsy , Andersen Svein R. , Jennings Michael P. , Moxon E. Richard , Poolman Jan T.
Published: November 2003

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin‐resistance marker (ermC), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin‐resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti‐lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3‐negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin‐resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)2–(Hep)2. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild‐type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N‐acetylglucosamine to heptose.
Translational initiation competence, ‘leaky scanning’ and translational reinitiation in areA mRNA of Aspergillus nidulans
Authors: Arst, Jr Herbert N. , Sheerins Alan
Published: November 2003

(1) AUG codons that either permit or prevent ‘leaky scanning’ of mRNA encoding AREA, the transcriptional activator mediating nitrogen metabolite repression in Aspergillus nidulans, have been identified. The consensus context for a strong initiation codon (i.e. one preventing ‘leaky scanning’) derived from this work is GXX AUG C/UCX. However, AUG codons which do not conform to this consensus are nevertheless able to initiate translation. (2) Translational reinitiation can occur within areA mRNA, although there is a limitation determined by the distance between the chain termination and reinitiation codons and/or the strength of the reinitiation codon. (3) The minimum size of the region of areA mRNA that is competent for initiation of translation has been determined to be 329 nucleotides (nt), and might be at least 975 nt if a 709 bp deletion does not alter it.
Exotoxin A production in Pseudomonas aeruginosa requires the iron‐regulated pvdS gene encoding an alternative sigma factor
Authors: Ochsner Urs A. , Johnson Zaiga , Lamont Iain L. , Cunliffe Heather E. , Vasil Michael L.
Published: November 2003

Exotoxin A (ETA) is secreted by Pseudomonas aeruginosa under iron‐limiting growth conditions. The ETA structural gene, toxA, is regulated at the transcriptional level by the gene products of the regAB operon. The expression of both toxA and regAB is repressed under iron‐replete conditions, suggesting a role for the ferric uptake regulator (Fur) in regulation of ETA synthesis; however, the Fur protein does not interact directly with the toxA or the regAB promoters. Evidence is presented that the iron control of ETA synthesis is mediated by a Fur‐regulated alternative sigma factor, PvdS, which had initially been identified as a positive activator for the production of the siderophore pyoverdin. In a ΔpvdS deletion mutant, ETA was produced at low levels of less than 5% compared to wild type, but still in response to iron starvation, and introduction of a functional pvdS gene on a plasmid fully restored wild‐type levels and normal iron regulation of ETA synthesis. Therefore, a functional pvdS locus is essential for ETA production. Neither toxA nor regAB mRNA was detectable in a ΔpvdS mutant. Overexpression of pvdS from the tac promoter on a plasmid resulted in a high‐level and iron‐independent production of ETA in wild‐type PAO1, in the ΔpvdS strain, but not in a ΔregA strain as a host. These findings suggest that PvdS is required for the activation of the regAB promoters. The transcription of regAB and toxA after induction of the Ptac–pvdS gene was monitored in cells grown in high‐iron medium. While both regAB and toxA were highly expressed during all growth phases under microaerobic conditions, toxA transcripts were detected only during the exponential but not the early stationary phase of growth under aerobic conditions. These results suggest that a second regulatory mechanism besides the Fur–PvdS system is involved in iron regulation of ETA production.
Transcriptional activation of the dnaA gene encoding the initiator for oriC replication by IciA protein, an inhibitor of in vitro oriC replication in Escherichia coli
Authors: Lee Yong Sun , Kim Hakjung , Hwang Deog Su
Published: November 2003

Transcription of the Escherichia coli dnaA gene, encoding DnaA protein required for initiation of chromosomal DNA replication at oriC in E. coli, starts from two promoters, 1P and 2P. Gel‐shift and DNase I‐protection assays revealed that IciA protein, an inhibitor of initiation of in vitro E. coli chromosomal DNA replication at oriC, bound to two sites in the dnaA promoter region. One site is located upstream of promoter 1P, and the second is located downstream of promoter 2P. Whereas IciA protein did not affect transcription from the promoter 2P, transcription from the promoter 1P was specifically enhanced by IciA protein in vivo and in vitro. DnaA protein bound to the DnaA box between the two promoters 1P and 2P, acts as an transcriptional repressor. Under this condition, IciA protein counteracted the repressive effect of DnaA protein on the promoter 1P. These findings suggest that IciA protein may regulate the initiation of chromosomal DNA replication at oriC by controlling expression of the dnaA gene, as well as by inhibiting the initiation of chromosomal DNA replication at oriC
Role of the periplasmic domain of the Escherichia coli NarX sensor‐transmitter protein in nitrate‐dependent signal transduction and gene regulation
Authors: Cavicchioli Ricardo , Chiang Robin C. , Kalman Lisa V. , Gunsalus Robert P.
Published: November 2003

The narX, narQ and narL genes of Escherichia coli encode a nitrate‐responsive two‐component regulatory system that controls the expression of many anaerobic electron‐transport‐ and fermentation‐related genes. When nitrate is present, the NarX and NarQ sensor‐transmitter proteins function to activate the response‐regulator protein, NarL, which in turn binds to its DNA‐recognition sites to modulate gene expression. The sensor‐transmitter proteins are anchored in the cytoplasmic membrane by two transmembrane domains that are separated by a periplasmic region of ≈115 amino acids. In this study we report the isolation and characterization of narX* (star) mutants that constitutively activate nitrate reductase (narGHJI) gene expression and repress fumarate reductase (frdABCD) gene expression when no nitrate is provided for the cell. An additional narX mutant was identified that has lost its ability to respond to environmental signals. Each narX defect was caused by a single amino acid substitution within a conserved 17 amino acid sequence, called the ‘P‐box’, in the periplasmic exposed region of the NarX protein. As a result, DNA binding is then ‘locked‐on’ or ‘locked‐off’ to give the observed pattern of gene expression. Diploid analysis of these narX mutants showed that a NarX P‐box mutant which confered a ‘locked‐on’ phenotype was trans dominant over wild‐type NarX. Both were also trans dominant over the NarX P‐box mutant which conferred a ‘locked‐off’ phenotype. Certain narX P‐box mutations, when combined with a narX‘linker’ region mutation, were recessive to the NarX linker mutation. Finally, a truncated form of the NarX protein that lacked the periplasmic and membrane regions also showed a ‘locked‐on’ phenotype in vivo. Thus, the periplasmic and membrane domains are essential for signal transduction to NarL. From these findings, we propose that nitrate is detected in the periplasmic space of the cell, and that a signal‐transduction event through the cytoplasmic membrane into the interior of the cell modulates the NarX‐dependent phosphorylation/dephosphorylation of NarL.
Phosphorylation‐dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis
Authors: Karimova Gouzel , Bellalou Jacques , Ullmann Agnes
Published: November 2003

In Bordetella pertussis,transcription of virulence‐associated genes is regulated by the BvgS and BvgA proteins, members of the bacterial two‐component signal‐transduction family. BvgS is the transmembrane sensor and BvgA, in its phosphorylated form, is believed to be the key transcriptional activator in B. pertussis. However, the BvgA recognition sites in most virulence promoters have not yet been identified. To investigate the interaction of BvgA with the upstream region of cyaA, the gene encoding adenylate cyclase haemolysin, we have produced large amounts of BvgA in Escherichia coli. The protein was purified from inclusion bodies and then phosphorylated by acetyl phosphate. Using electrophoretic mobility‐shift and footprinting assays, we provide evidence that BvgA cannot bind to the cyaA promoter unless it is phosphorylated. The phosphorylated form of BvgA (BvgA‐P) is able to bind specifically to the upstream region of cyaA. Analysis of this region revealed that an unexpectedly large sequence, from −137 to −51, appears to be the target for BvgA‐P binding, and probably contains multiple binding sites.
The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane
Authors: Håkansson Sebastian , Galyov Edouard E. , Rosqvist Roland , Wolf‐Watz Hans
Published: November 2003

Multiple yop mutant strains of Yersinia pseudotuberculosis not expressing several virulence effector Yop proteins (YopH, M, E, K and YpkA) were engineered. When high‐copy‐number plasmids carrying the ypkA or the yopE gene with their endogenous promoters were introduced into the engineered strains, the corresponding Yop protein was secreted at high levels in vitro. These multiple yop mutant strains, when harbouring the yopE gene in trans, behaved as the wild‐type strain with respect to YopB‐dependent translocation of YopE through the HeLa cell plasma membrane. Using these multiple yop mutant strains, it was demonstrated that the YpkA Ser/Thr protein kinase mediates morphological alterations of infected cultured HeLa cells different from those mediated by YopE and YopH. Furthermore, YpkA is shown to be translocated by a YopB‐dependent translocation mechanism from surface‐located bacteria and subsequently targeted to the inner surface of the target‐cell plasma membrane. The pattern of YpkA localization after infection suggests that this Yop effector is involved in interference with signal transduction.
Cloning and characterization of bfpTVW, genes required for the transcriptional activation of bfpA in enteropathogenic Escherichia coli
Authors: Tobe Toru , Schoolnik Gary K. , Sohel Indira , Bustamante Victor H. , Puente Jose Luis
Published: November 2003

Expression of the bundle‐forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) is regulated at the transcriptional level by growth phase, temperature, calcium and ammonium. Genes required for the transcriptional activation of bfpA were localized to a 1.8 kb fragment of the enteroadherent factor (EAF) plasmid of EPEC that is separated from the bfp operon by 6 kb. Within this fragment three identically oriented and closely spaced open reading frames (ORFs) were identified and designated bfpT, bfpV and bfpW. bfpT is predicted to encode a 31.8 kDa protein that shares homology with the AraC family of transcriptional regulators, including the presence of a conserved C‐terminal DNA‐binding helix‐turn‐helix motif. Insertional inactivation of bfpT led to the loss of bfpA transcription, BfpA protein production and the localized adherence (LA) phenotype; this mutant phenotype could be complemented by introduction of bfpTVW and, on separate plasmids, bfpT + bfpW. However, introduction of bfpT + bfpV, bfpV alone, bfpW alone, or bfpV + bfpW did not enable recovery of the wild‐type phenotype. Maximal efficiency of bfpA transcription required all three genes, but bfpV and bfpW each enhanced transcription providing bfpT was also present. A series of deletions of the bfpA upstream promoter region was prepared; with respect to the bfpA transcription start site, sequence between nucleotides −94 and −55 was found to bind bfpT. BfpT also bound a DNA fragment containing the eaeA promoter region on the EPEC chromosome. From these results we conclude that bfpTVW causes transcriptional activation of bfpA, and possibly eaeA, by a trans‐acting mechanism that may co‐ordinately regulate the expression of EPEC virulence determinants.
A flexible partnership: the CytR anti‐activator and the cAMP–CRP activator protein, comrades in transcription control
Authors: Valentin‐Hansen Poul , Søgaard‐Andersen Lotte , Pedersen Henrik
Published: November 2003

A vital point in gene regulation is control at the level of transcription initiation. Recent research has established that this regulation can involve sophisticated networks of interacting proteins that modulate the activity of the transcription machinery by DNA looping, direct protein–protein interactions or changing DNA topology in the promoter region. This MicroReview focuses on our investigations of a relatively simple prokaryotic gene regulatory system, the Escherichia coli CytR regulon, which exhibits a number of these features. This work has opened the door to the molecular understanding of how a prokaryotic repressor can be correctly positioned at specific DNA sequences with the help of a global activator, and how the repressor subsequently inhibits factor‐dependent transcription initiation.
The centromere‐like parC locus of plasmid R1
Authors: Breüner Anne , Jensen Rasmus Bugge , Dam Mette , Pedersen Susanne , Gerdes Kenn
Published: November 2003

The parA partitioning system of plasmid R1 consists of three components: the cis‐acting centromere‐like parC locus, and two proteins, ParM and ParR. The parC locus contains two sets of five direct repeats (iterons) to which the ParR protein binds. The parA promoter is located in the core region between the two sets of iterons. Mini‐R1 replicons carrying parC are stabilized by the simultaneous presence of ParM and ParR. The parC locus present on a co‐resident plasmid leads to instability of the mini‐R1 replicon (incompatibility). Here we present a genetic analysis of the stability and incompatibility phenotypes associated with parC. We show that all 10 iterons are required for maximum stabilization and incompatibility. Replacement of the core promoter region between the repeats by a foreign promoter region did not reduce stabilization. Thus, the only structural components in parC seem to be the two sets of iterons. The parA promoter, PparA, is repressed by ParR. We show that all 10 iterons are required for full repression of the promoter. The activity of the promoter was influenced by sequences located outside the core region. An A‐rich region located upstream of the −35 element of PparA was found to increase promoter activity. The region encoding the parA mRNA leader region also strongly influenced the expression level of PparA–lacZ fusions. We show that this high expression (hex) element is a transcriptional antiterminator that prevents Rho‐dependent termination.
Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea
Authors: Sayavedra‐Soto Luis A. , Hommes Norman G. , Russell Sterling A. , Arp Daniel J.
Published: November 2003

In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2−). A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins). A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO). Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+). To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+. The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion. In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation. HAO mRNA also increased in the NH4+‐treated cells. Other proteins and RNAs induced by NH4+ were detected in 14CO2‐labelling experiments. The AMO and HAO mRNAs were preferentially synthesized during energy‐limiting conditions.
Characterization of a Penicillium chrysogenum gene encoding a PacC transcription factor and its binding sites in the divergent pcbAB–pcbC promoter of the penicillin biosynthetic cluster
Authors: Suárez Teresa , Peñalva Miguel Angel
Published: November 2003

Previous work established that pH regulation of gene expression in Aspergillus nidulans, a major determinant of penicillin biosynthesis, is mediated by the zinc‐finger transcription factor PacC, an activator of transcription of the isopenicillin N synthase gene. We characterize here the pacC gene from the efficient penicillin producer Penicillium chrysogenum, which functionally complements an A. nidulans pacC null mutation. It encodes a 641‐residue polypeptide showing 64% identity to A. nidulans PacC and containing three putative zinc fingers specifically recognizing a 5′‐GCCARG‐3′ hexanucleotide. Penicillium pacC transcript levels are higher under alkaline than under acidic growth conditions and elevate at late stages of growth. The gene contains three PacC‐binding sites in its 5′‐upstream region. Transcript levels of pcbC (encoding P. chrysogenum isopenicillin N synthase) are low on a repressing carbon source and elevated on a derepressing carbon source. With either carbon source, alkaline pH elevates pcbC transcript levels, correlating with the presence of seven PacC‐binding sites in the 1.1 kb pcbAB–pcbC intergenic region and strongly suggesting that pcbC is under direct pacC control. However, in contrast to the situation in A. nidulans, alkaline pH does not override the negative effects of a repressing carbon source, revealing differences in the regulation of the penicillin pathway between Penicillium and Aspergillus
Essential roles of core starvation‐stress response loci in carbon‐starvation‐inducible cross‐resistance and hydrogen peroxide‐inducible adaptive resistance to oxidative challenge in Salmonella typhimurium
Authors: Seymour Robert L. , Mishra Prashant V. , Khan M. Akbar , Spector Michael P.
Published: November 2003

The starvation‐stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g. C‐source. A subset of SSR genes, known as core SSR genes, are required for the long‐term starvation survival of the bacteria. Four core SSR loci have been identified in S. typhimuriumrpoSstiAstiB, and stiC. Here we report that in S. typhimurium C‐starvation induced a greater and more sustainable cross‐resistance to oxidative challenge (15 mM hydrogen peroxide (H2O2) for 40 min) than either N‐ or P‐starvation. Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C‐starvation‐inducible cross‐resistance to H2O2 challenge. Interestingly, (unadapted) log‐phase S. typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge. Based on this, we determined if these core SSR loci were important for H2O2 resistance developed during a 60 min adaptive exposure to 60 μM H2O2 (adapted cells). Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H2O2 challenge. In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H2O2 challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild‐type strain. The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C‐starvation‐inducible cross‐resistance to oxidative challenge, and (ii) the rpoSstiA, and, in a delayed effect, stiB loci are needed for H2O2‐inducible adaptive resistance to oxidative challenge. Moreover, we found that both stiA and stiB are induced by a 60 μM H2O2 exposure, but only stiA was regulated (repressed) by (reduced form) OxyR.
Identification and characterization of FrzZ, a novel response regulator necessary for swarming and fruiting‐body formation in Myxococcus xanthus
Authors: Trudeau Katherine G. , Ward Mandy J. , Zusman David R.
Published: November 2003

The frz genes of Myxococcus xanthus constitute a signal‐transduction pathway that processes chemotactic information in a manner analogous to that found in enteric bacteria. Ultimately, these genes regulate the frequency of individual cell reversal. We report here the identification of a novel component of this signal‐transduction pathway, designated frzZ, which was discovered as an open reading frame located 5′ to the frz operon but transcribed in the opposite orientation. The translational start site of frzZ  is 170 base pairs from that of frzAfrzZ  utilizes a promoter similar to the σ70 promoters of Escherichia coli, and encodes a 290‐amino‐acid soluble protein, FrzZ (Mr 30 500). FrzZ contains two domains, both of which show strong homology to CheY and other members of the response‐regulator family. Linking these domains is a 39‐amino‐acid region that is very rich in alanine and proline (38% Ala and 33% Pro). A frzZ null mutant showed abnormally low reversal rates when compared to the wild‐type control and was unable to form fruiting bodies on starvation medium, but it did form ‘frizzy’ aggregates. In addition, the frzZ mutant was defective in swarming, particularly on soft agar (0.3% w/v). However, unlike most frz mutants, the frzZ mutant was able to respond to attractants and repellents in the spatial chemotaxis assay. The discovery of FrzZ demonstrates that the M. xanthusfrz signal‐transduction pathway utilizes multiple response‐regulator (CheY‐like) proteins.
New components of protein folding in extracytoplasmic compartments of Escherichia coli SurA, FkpA and Skp/OmpH
Authors: Missiakas Dominique , Betton Jean‐Michel , Raina Satish
Published: November 2003

A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the σE regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the σE‐dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM (rfaD) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM (rfaD) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn10 insertions were isolated, which led to an increased activity of the σE regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA, fkpA and ompH/skpgenes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.
Activation of stable DNA replication in rapidly growing Escherichia coli at the time of entry to stationary phase
Authors: Hong Xiankang , Cadwell Gregory W. , Kogoma Tokio
Published: November 2003

The conditions are described in which DNA replication can occur, in the absence of protein synthesis, in wild‐type Escherichiacoli cells. Chromosome replication, which is normally inhibited by addition of chloramphenicol, becomes resistant to this drug after nutritional shiftup, e.g. from minimal medium to Luria broth. This replication activity appears transiently when nutritionally upshifted cells enter stationary phase. The activity strictly requires recA+, but it is independent of recB + and dnaA+. It can occur in the absence of concomitant transcription. Activation of the replication does not result from induction of the SOS response. As the characteristics of this DNA replication resemble those of the previously characterized stable DNA replication, it is termed nutritional shiftup‐activatable stable DNA replication, nSDR. Possible mechanisms of the activation of nSDR in rapidly growing cells at the time of entry to stationary phase are discussed.
Purification and activities of the Rhodobacter capsulatus RpoN (σN) protein
Authors: Cannon Wendy , Missailidis Sotiris , Austin Sara , Moore Madeleine , Drake Alex , Buck Martin
Published: November 2003

The rpoN‐encoded sigma factors (σN) are a distinct class of bacterial sigma factors, with no obvious homology to the major σ70 class. The σN‐containing RNA polymerase holoenzyme functions in enhancer‐dependent transcription to allow expression of positively controlled genes. We have purified the Rhodobacter capsulatusσN protein, which is distinctive in lacking an acidic region implicated in the melting of promoter DNA by the Escherichia coliσN holoenzyme, and may represent a minor subclass of σN proteins. Assays of promoter recognition and holoenzyme formation and function showed that the purified R. capsulatusσN protein is distinct in activity compared to the enteric proteins, but retains the broad functions described for these proteins. As first described for the Kleb‐siella pneumoniae protein, promoter recognition in the absence of core RNA polymerase was detected, but contact of certain promoter bases by the R. capsulatusσN protein and its response to core RNA polymerase was clearly different from that determined for the K. pneumoniae and E. coli proteins. Results are discussed in the context of a requirement to modulate the activity of the DNA‐binding surfaces of σN to regulate σN function. Circular dichroism was used to evaluate the structure of the R. capsulatus protein and revealed differences in the tertiary signals as compared to the K. pneumoniae protein, some of which are attributable to the DNA‐binding domain of σN
A set of ordered cosmids and a detailed genetic and physical map for the 8 Mb Streptomyces coelicolor A3(2) chromosome
Authors: Redenbach M. , Kieser H. M. , Denapaite D. , Eichner A. , Cullum J. , Kinashi H. , Hopwood D. A.
Published: November 2003

A Supercos‐1 library carrying chromosomal DNA of a plasmid‐free derivative of Streptomyces coelicolor A3(2) was organized into an ordered encyclopaedia of overlapping clones by hybridization. The minimum set of overlapping clones representing the entire chromosome (with three short gaps) consists of 319 cosmids. The average insert size is 37.5 kb and the set of clones therefore divides the chromosome into 637 alternating unique and overlapping segments which have an average length of approx. 12.5 kb. More than 170 genes, gene clusters and other genetic markers were mapped to their specific segment by hybridization to the encyclopaedia. Genes could be cloned by direct transformation and complementation of S. coelicolor mutants with cosmids isolated from Escherichia coli, selecting for insertion into the chromosome by homologous recombination. As in other streptomycetes, the ends of the chromosome have long terminal inverted repeats.
Fate of peptides in peptidase mutants of Lactococcus lactis
Authors: Kunji Edmund R. S. , Mierau Igor , Poolman Bert , Konings Wil N. , Venema Gerard , Kok Jan
Published: November 2003

The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepNpepCpepOpepX and pepT were deleted. Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu–Gly–Gly, Gly–Phe–Leu, Leu–Gly–Pro, Ala–Pro–Leu and Gly–Leu–Gly–Leu, respectively, indicating that no other peptidases are present to release the essential amino acid Leu. In these mutants, peptides accumulate intracellularly, demonstrating that peptides are translocated as whole entities prior to degradation. The mutant lacking all five peptidases could still grow on Gly–Leu and Tyr–Gly–Gly–Phe–Leu, which confirmed the presence of a dipeptidase and led to the identification of an unknown PepO‐like endopeptidase. These studies have also shown that the general aminopeptidases PepN, PepC and PepT have overlapping but not identical specificities and differ in their overall activity towards individual peptides. In contrast, PepX has an unique specificity, because it is the only enzyme which can efficiently degrade Ala–Pro–Leu. The concerted action of peptidases in the breakdown of particular peptides revealed how these substrates are utilized as sources of nitrogen.
Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production
Authors: Hamood Abdul N. , Colmer Jane A. , Ochsner Urs A. , Vasil Michael L.
Published: November 2003

Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes. In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P. aeruginosa. In an iron‐deficient medium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 resulted in a four‐ to fivefold increase in exotoxin A synthesis. No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease. Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb KpnI–BglII fragment. Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system. The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino‐terminus region of PtxR contains a putative helix‐turn‐helix DNA‐binding motif. Specific ptxR‐deletion mutants in P. aeruginosa strains PAO1 and PA103 were constructed. In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity. However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1. Transcriptional studies, using both toxA–lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription. These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.
An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli
Authors: Van Heeswijk Wally C. , Hoving Sjouke , Molenaar Douwe , Stegeman Brenda , Kahn Daniel , Westerhoff Hans V.
Published: November 2003

The PII protein has been considered pivotal to the dual cascade regulating ammonia assimilation through glutamine synthetase activity. Here we show that PII, encoded by the glnB gene, is not always essential; for instance upon ammonia deprivation of a glnB deletion strain, glutamine synthetase can be deadenylylated as effectively as in the wild‐type strain. We describe a new operon, glnK amtB, which encodes a homologue of PII and a putative ammonia transporter. We cloned and overexpressed glnK and found that the expressed protein had almost the same molecular weight as PII, reacted with polyclonal PII antibody, and was 67% identical in terms of amino acid sequence with Escherichia coli PII. Like PII, purified GlnK can activate the adenylylation of glutamine synthetase in vitro, and, in vivo, the GlnK protein is uridylylated in a glnD‐dependent fashion. Unlike PII, however, the expression of glnK depends on the presence of UTase, nitrogen regulator I (NRI), and absence of ammonia. Because of a NRI and a σN (σ54) RNA polymerase‐binding consensus sequence upstream from the glnK gene, this suggests that glnK is regulated through the NRI/NRII two‐component regulatory system. Indeed, in cells grown in the presence of ammonia, glutamine synthetase deadenylylation upon ammonia depletion depended on PII. Possible regulatory implications of this conditional redundancy of PII are discussed.
Rhizobium leguminosarum bv. viciae contains a second fnr/fixK‐like gene and an unusual fixL homologue
Authors: Patschkowski Thomas , Schlüter Andreas , Priefer Ursula B.
Published: November 2003

Genes of Rhizobium leguminosarum bv. viciae VF39 coding for the regulatory elements NifA, FixL and FixK were isolated, sequenced and genetically analysed. The fixK–fixL region is located upstream of the fixNOQP operon on the non‐nodulation plasmid pRleVF39c. The deduced amino acid sequence of FixL revealed an unusual structure in that it contains a receiver module (homologous to the N‐terminal domain of response regulators) fused to its transmitter domain. An oxygen‐sensing haem‐binding domain, found in other FixL proteins, is conserved in R. leguminosarum bv. viciae FixL. R. leguminosarum bv. viciae possesses a second fnr‐like gene, designated fixK, whose encoded gene product is very similar to Rhizobium meliloti and Azorhizobium caulinodans FixK. Individual R. leguminosarum bv. viciae fixK and fixL insertion mutants displayed a Fix+ phenotype. A reduced nitrogen‐fixation activity was found for a R. leguminosarum bv. viciae fnrN‐deletion mutant, whereas no nitrogen‐fixation activity was detectable for a fixK/fnrN double mutant. The R. leguminosarum bv. viciae nifA gene is expressed independently of FixL and FixK under aerobic and microaerobic conditions, whereas fixL gene expression is induced under microaerobiosis. Another orf was identified downstream of fixK–fixL and encodes a product which has homology to pseudoazurins from different species. Mutation of this azu gene showed that it is dispensable for nitrogen fixation.
A stringently controlled expression system for analysing lateral gene transfer between bacteria
Authors: Jaenecke Susanne , De Lorenzo Víctor , Timmis Kenneth N. , Díaz Eduardo
Published: November 2003

The lateral transfer of genetic information among microorganisms is a major force driving the outstanding adaptability of microbial communities to environmental changes. Until now little information has been obtained on gene transfer in natural ecosystems. We present here a genetic circuit for detecting and quantifying horizontal gene transfer from a defined donor microorganism to recipient organisms in the absence of selection for a recipient‐specific phenotype. The system consists of an engineered lacZ (encoding β‐galactosidase) reporter gene whose expression is controlled by a synthetic regulatory element based on a fusion between the Pr promoter‐operator from lambda bacteriophage and the 5′ non‐coding leader region of the tnp gene encoding the IS10 transposase function. Expression of this reporter cassette in the recombinant microorganism is completely shut down by two chromosomally encoded trans‐acting repressors working at the level of transcription (the CI‐EK117 protein from the lambda phage), and at the level of translation (the antisense RNA‐OUT of the IS10 element). When the reporter element is transferred to a different host by any mechanism, it escapes repression and becomes expressed. The system was validated with Pseudo‐monas putida, and conjugational transfer frequencies of the reporter element as low as 10−6 were detected. The modular design and broad host range of the genetic circuit, in combination with biomarkers which permit real‐time in situ detection, will facilitate the monitor‐ing of gene flow in a non‐disruptive manner within the environment.
Fucosylation and arabinosylation of Nod factors in Azorhizobium caulinodans: involvement of nolKnodZ as well as noeC and/or downstream genes
Authors: Mergaert Peter , D'Haeze Wim , Fernández‐López Manuel , Geelen Danny , Goethals Koen , Promé Jean‐Claude , Van Montagu Marc , Holsters Marcelle
Published: November 2003

The DNA region downstream of the nodABCSUIJ operon of Azorhizobium caulinodans was further characterized and two new genes, nodZ and noeC were identified in the same operon. The A. caulinodans wild‐type strain produces a population of Nod factors that, at the reducing end, are either unmodified or carry a D‐arabinosyl and/or an L‐fucosyl branch. Nod factors produced by Tn5‐insertion mutants in nodZnoeC, and the separate nolK locus, were analysed by thin‐layer chromatography and mass spectrometry. Fucosylation of Nod factors depended on both nodZ and nolK. Arabinosylation depended on noeC and/or downstream genes. Protein extracts of A. caulinodans contained an enzymatic activity for fucose transfer from GDP‐fucose to chitooligosaccharides and to Nod factors. By mutant analysis and expression of nodZ in Escherichia coli, the fucosyltransferase activity was ascribed to the protein encoded by nodZ. In addition, a Nod factor fucosyltransferase activity, independent of nodZ or other known nod genes, was detected in A. caulinodans. Finally, on the basis of sequence similarity of the nolK gene product, and mass spectrometric analysis of Nod factors produced by a nolK mutant, we propose that this gene is involved in the synthesis of GDP‐fucose.
Common cis‐acting elements critical for the expression of several genes of Toxoplasma gondii
Authors: Mercier Corinne , Lefebvre‐Van Hende Sophie , Garber Gary E. , Lecordier Laurence , Capron André , Cesbron‐Delauw Marie‐France
Published: November 2003

Transient transformation of Toxoplasma using the CAT (chloramphenicol acetyl transferase) reporter gene has been used to map promoter elements of four genes encoding dense granule proteins (GRA1, GRA2, GRA5 and GRA6). Intense CAT activities (GRA1 > GRA5 > GRA2 > GRA6) are detected for constructs containing 379 bp, 276 bp, 209 bp and 265 bp upstream of the transcription start site of the GRA1GRA2GRA5 and GRA6 genes, respectively. Deletion analysis shows that optimal promoter activity of each gene is contained in the proximal region of the transcription start site: −129 to −47 for GRA1, −87 to −37 for GRA2, −156 to −30 for GRA5 and −146 to −27 for GRA6. Quantitative CAT assay and mutation analysis show that repeated motifs (A/TGAGACG) found in either orientation with respect to transcription are critical elements of these defined promoter regions. We have found such sequence elements in the upstream region of other Toxoplasma genes such as Tub1 and within the stretch of 27 bp repeats of the SAG1 promoter.
Expression, secretion and antigenic variation of bacterial S‐layer proteins
Authors: Boot Hein J. , Pouwels Peter H.
Published: November 2003

The function of the S‐layer, a regularly arranged structure on the outside of numerous bacteria, appears to be different for bacteria living in different environments. Almost no similarity exists between the primary sequences of S‐proteins, although their amino acid composition is comparable. S‐protein production is directed by single or multiple promoters in front of the S‐protein gene, yielding stable mRNAs. Most bacteria secrete S‐proteins via the general secretory pathway (GSP). Translocation of S‐protein across the outer membrane of Gram‐negative bacteria sometimes occurs by S‐protein‐specific branches of the GSP. O‐polysaccharide side‐chains of the lipopolysaccharide component of the cell wall of Gram‐negative bacteria appear to function as receptors for attachment of the S‐layer. Silent S‐protein genes have been found in Campylobacter fetus and Lactobacillus acidophilus. These silent genes are placed in the expression site in a fraction of the bacterial population via inversion of a chromosomal segment.
Cloning of the ASN1 and ASN2 genes encoding asparagine synthetases in Saccharomyces cerevisiae: differential regulation by the CCAAT‐box‐binding factor
Authors: Dang Van‐Dinh , Valens Michèle , Bolotin‐Fukuhara Monique , Daignan‐Fornier Bertrand
Published: November 2003

Two new yeast genes, named ASN1 and ASN2, were isolated by complementation of the growth defect of an asparagine auxotrophic mutant. Genetical analysis indicates that these two genes are allelic to the asnA and asnB loci described previously. Simultaneous disruption of both genes leads to a total asparagine auxotrophy, while disruption of asn1 or asn2 alone has no effect on growth under tested conditions. Nucleotide sequences of ASN1 and ASN2 revealed striking similarities with genes encoding asparagine synthetase (AS) from other organisms. Regulation of ASN1 and ASN2 expression was studied using lacZ fusions and both genes were found to be several times less expressed in the absence of the transcription activator Gcn4p. The HAP complex, another transcription factor that binds to CCAAT‐box sequences, was shown to specifically affect ASN1 expression. Hap2p and Hap3p subunits of the HAP complex are required for optimal expression of ASN1, while the Hap4p regulatory subunit, which is required for regulation by the carbon source, plays a minor role in this process. Consistent with the weak effect of Hap4p, the carbon source does not significantly affect expression of ASN1. Our results show that the role of the HAP complex is not limited to activation of genes required for respiratory metabolism.
Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression
Authors: Wylie John L. , Berry Jody D. , McClarty Grant
Published: November 2003

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase–polymerase chain reaction (RT–PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid‐ and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP‐2‐keto‐3‐deoxyoctanoic acid synthetase (CMP‐KDO synthetase) as part of a polycistronic mRNA. The co‐expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP‐KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP‐KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co‐operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the Km of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.