bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear DNA analyses in genetic studies of populations: practice, problems and prospects
Authors: Zhang De‐Xing , Hewitt Godfrey M.
Published: March 2003

Population‐genetic studies have been remarkably productive and successful in the last decade following the invention of PCR technology and the introduction of mitochondrial and microsatellite DNA markers. While mitochondrial DNA has proven powerful for genealogical and evolutionary studies of animal populations, and microsatellite sequences are the most revealing DNA markers available so far for inferring population structure and dynamics, they both have important and unavoidable limitations. To obtain a fuller picture of the history and evolutionary potential of populations, genealogical data from nuclear loci are essential, and the inclusion of other nuclear markers, i.e. single copy nuclear polymorphic (scnp) sequences, is clearly needed. Four major uncertainties for nuclear DNA analyses of populations have been facing us, i.e. the availability of scnp markers for carrying out such analysis, technical laboratory hurdles for resolving haplotypes, difficulty in data analysis because of recombination, low divergence levels and intraspecific multifurcation evolution, and the utility of scnp markers for addressing population‐genetic questions. In this review, we discuss the availability of highly polymorphic single copy DNA in the nuclear genome, describe patterns and rate of evolution of nuclear sequences, summarize past empirical and theoretical efforts to recover and analyse data from scnp markers, and examine the difficulties, challenges and opportunities faced in such studies. We show that although challenges still exist, the above‐mentioned obstacles are now being removed. Recent advances in technology and increases in statistical power provide the prospect of nuclear DNA analyses becoming routine practice, allowing allele‐discriminating characterization of scnp loci and microsatellite loci. This certainly will increase our ability to address more complex questions, and thereby the sophistication of genetic analyses of populations.
Bacteriophage T7 mRNA is polyadenylated
Authors: Johnson M. D. , Popowski J. , Cao G.‐J. , Shen P. , Sarkar N.
Published: March 2003

To determine whether the RNA of bacterial viruses is polyadenylated like bacterial mRNAs, pulse‐labelled as well as the steady‐state population of bacteriophage T7‐specific transcripts were examined for the presence of poly(A) tracts by binding to oligo(dT) cellulose followed by hybridization with specific gene probes. Representatives of all classes of bacteriophage‐specific mRNA — early, middle and late — were found to be polyadenylated. This conclusion was confirmed by screening the products of oligo(dT)‐dependent cDNA synthesis. A cDNA library was prepared from RNA synthesized after bacteriophage T7 infection and the sequence of bacteriophage‐specific clones was determined to define the sites of polyadenylation. About half of the clones were polyadenylated near the end of a protein‐coding region, one of them at the site of post‐transcriptional processing by RNase III. Other clones were polyadenylated within protein‐coding regions. These observations suggest that polyadenylation occurs after the nucleolytic processing of primary transcripts and in some cases also after mRNA degradation has already begun.
Cell cycle arrest in Era GTPase mutants: a potential growth rate‐regulated checkpoint in Escherichia coli
Authors: Britton Robert A. , Powell Bradford S. , Dasgupta Santanu , Sun Qin , Margolin William , Lupski James R. , Court Donald L.
Published: March 2003

Era is a low‐molecular‐weight GTPase essential for Escherichia coli viability. The gene encoding Era is found in the rnc operon, and the synthesis of both RNase III and Era increases with growth rate. Mutants that are partially defective in Era GTPase activity or that are reduced in the synthesis of wild‐type Era become arrested in the cell cycle at the predivisional two‐cell stage. The partially defective Era GTPase mutation (era1) suppresses several temperature‐sensitive lethal alleles that affect chromosome replication and chromosome partitioning but not cell division. Our results suggest that Era plays an important role in cell cycle progression at a specific point in the cycle, after chromosome partitioning but before cytokinesis. Possible functions for Era in cell cycle progression and the initiation of cell division are discussed.
A second prepilin peptidase gene in Escherichia coli K‐12
Authors: Francetić Olivera , Lory Stephen , Pugsley Anthony P.
Published: March 2003

Escherichia coli K‐12 strains grown at 37°C or 42°C, but not at 30°C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase‐dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position −1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non‐specific proteases. These data suggested that E. coli K‐12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E. coli K‐12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E. coli when expressed from high‐copy‐number plasmids but not when present in a single chromosomal copy. The analysis of pppA–lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E. coli isolates.
Identification of a regulatory protein required for pressure‐responsive gene expression in the deep‐sea bacterium Photobacterium species strain SS9
Authors: Welch Timothy J. , Bartlett Douglas H.
Published: March 2003

Here, we report the characterization of a gene necessary for hydrostatic pressure regulation of gene expression in the deep‐sea bacterium Photobacterium species strain SS9. The deduced amino acid sequence of the gene product shares extensive similarity to ToxR, a transmembrane DNA‐binding protein first discovered as a virulence determinant in the pathogenic bacterium Vibrio cholerae. Changes in hydrostatic pressure induce changes in both the abundance and the activity of the SS9 ToxR protein (or the activity of a ToxR‐regulated protein). As with other high‐pressure‐inducible phenomena observed in higher organisms, anaesthetics antagonize high‐pressure signalling mediated by ToxR. It is suggested that SS9 ToxR has evolved the ability to respond to pressure‐mediated alterations in membrane structure. V. cholerae and SS9 also share similarity in a ToxR‐regulated protein, indicating that part of the ToxR regulon is conserved in diverse members of the family Vibrionaceae. The SS9 ToxR system represents a useful model for studies of signal transduction and environmental adaptation in the largest portion of the biosphere, the deep sea.
A novel protein kinase that controls carbon catabolite repression in bacteria
Authors: Reizer Jonathan , Hoischen Christian , Titgemeyer Friedrich , Rivolta Carlo , Rabus Ralf , Stülke Jörg , Karamata Dimitri , Saier Jr Milton H. , Hillen Wolfgang
Published: March 2003

HPr(Ser) kinase is the sensor in a multicomponent phosphorelay system that controls catabolite repression, sugar transport and carbon metabolism in Gram‐positive bacteria. Unlike most other protein kinases, it recognizes the tertiary structure in its target protein, HPr, a phosphocarrier protein of the bacterial phosphotransferase system and a transcriptional cofactor controlling the phenomenon of catabolite repression. We have identified the gene (ptsK) encoding this serine/threonine protein kinase and characterized the purified protein product. Orthologues of PtsK have been identified only in bacteria. These proteins constitute a novel family unrelated to other previously characterized protein phosphorylating enzymes. The Bacillus subtilis kinase is shown to be allosterically activated by metabolites such as fructose 1,6‐bisphosphate and inhibited by inorganic phosphate. In contrast to wild‐type B. subtilis, the ptsK mutant is insensitive to transcriptional regulation by catabolite repression. The reported results advance our understanding of phosphorylation‐dependent carbon control mechanisms in Gram‐positive bacteria.
Circularization of Tn916 is required for expression of the transposon‐encoded transfer functions: characterization of long tetracycline‐inducible transcripts reading through the attachment site
Authors: Celli Jean , Trieu‐Cuot Patrick
Published: March 2003

A detailed transcriptional analysis of the conjugative transposon Tn916 was carried out, which revealed that transcription of the transfer functions requires excision of the element and dramatically increases in the presence of tetracycline. The key components of this regulatory system are two contiguous transposon‐borne genes, orf7 and orf8, located downstream from and having the same polarity of transcription as the tetracycline resistance determinant tetM. The gene orf7 encodes a 140‐amino‐acid (aa) protein exhibiting limited homology with σF of Bacillus subtilis, whereas orf8 encodes a 76‐aa peptide that does not share any sequence homology with any cognate proteins. In the presence of tetracycline, an attenuation mechanism enables the transcription of orf7 and orf8 from the tetM promoter. The resulting increased synthesis of ORF7 and ORF8 activates the promoter Porf7 located upstream from orf7, which then directs the expression of the transfer functions in the transposon circular intermediate through long transcripts encompassing the attachment site. The apparently non‐regulated promoter Pxis located upstream of the excisionase encoding gene xis could also participate in the expression of the tra genes. We also demonstrate that Tn916 carries another regulated promoter, Porf9, which directs transcription of a single gene, orf9, located downstream from and transcribed counterclockwise to tetM. This gene encodes a 117‐aa putative transcriptional repressor, but the exact role of this protein in the mobility of Tn916, as well as the regulation of its expression, remains to be elucidated. Our results constitute the molecular basis for the observation that tetracycline increased the transfer frequency of this type of element.
Structure of the chromosomal insertion site for pSAM2: functional analysis in Escherichia coli
Authors: Raynal Alain , Tuphile Karine , Gerbaud Claude , Luther Tatjana , Guérineau Michel , Pernodet Jean‐Luc
Published: March 2003

The element pSAM2 from Streptomyces ambofaciens integrates into the chromosome through site‐specific recombination between the element (att P) and the chromosomal (att B) sites. These regions share an identity segment of 58 bp extending from the anti‐codon loop through the 3′ end of a tRNAPro gene. To facilitate the study of the att B site, the int and xis genes, expressed from an inducible promoter, and att P from pSAM2 were cloned on plasmids in Escherichia coli. Compatible plasmids carrying the different att B regions to be tested were introduced in these E. coli strains. Under these conditions, Int alone could promote site‐specific integration; Int and Xis were both required for site‐specific excision. This experimental system was used to study the sequences required in att B for efficient site‐specific recombination. A 26 bp sequence, centred on the anti‐codon loop region and not completely included in the identity segment, retained all the functionality of att B; shorter sequences allowed integration with lower efficiencies. By comparing the 26‐bp‐long att B with att P, according to the Lambda model, we propose that B and B′, C and C′ core‐type Int binding sites consist of 9 bp imperfect inverted repeats separated by a 5 bp overlap region.
Sequence, exon–intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans
Authors: Cazelle Béatrice , Pokorska Anna , Hull Elizabeth , Green Peter M. , Stanway Glyn , Scazzocchio Claudio
Published: March 2003

The prnA gene codes for a transcriptional activator that mediates proline induction of four other genes involved in proline utilization as a nitrogen and/or carbon source in Aspergillus nidulans. In this paper, we present the genomic and cDNA sequence and the transcript map of prnA. The PrnA protein belongs to the Zn binuclear cluster family of transcriptional activators. The gene shows a striking intron–exon organization, with the putative nuclear localization sequence and the Zn cluster domain in discrete exons. Although the protein sequence presents some interesting similarities with the isofunctional protein of Saccharomyces cerevisiæ Put3p, a higher degree of similarity is found with a functionally unrelated protein Thi1 of Schizosaccharomyces pombe. A number of mutations mapping in the prnA gene were sequenced. This comprises a deletion that results in an almost complete loss of the prnA‐specific mRNA, a mutation in the putative nuclear localization signal, a proline to leucine mutation in the second loop of the zinc cluster and a cold‐sensitive mutation in the so‐called ‘central region’. Other complete or partial loss of function mutations map in regions of unknown function. We establish that the transcription of the gene is neither self‐regulated nor significantly affected by carbon and/or nitrogen metabolite repression.
Target specificity of insertion element IS30
Authors: Olasz Ferenc , Kiss János , König Peter , Buzás Zsuzsa , Stalder Rolf , Arber Werner
Published: March 2003

The Escherichia coli resident mobile element IS30 has pronounced target specificity. Upon transposition, the element frequently inserts exactly into the same position of a preferred target sequence. Insertion sites in phages, plasmids and in the genome of E. coli are characterized by an exceptionally long palindromic consensus sequence that provides strong specificity for IS30 insertions, despite a relatively high level of degeneracy. This 24‐bp‐long region alone determines the attractiveness of the target DNA and the exact position of IS30 insertion. The divergence of a target site from the consensus and the occurrence of ‘non‐permitted’ bases in certain positions influence the target activity. Differences in attractiveness are emphasized if two targets are present in the same replicon, as was demonstrated by quantitative analysis. In a system of competitive targets, the oligonucleotide sequence representing the consensus of genomic IS30 insertion sites proved to be the most efficient target. Having compared the known insertion sites, we suppose that IS30‐like target specificity, which may represent an alternative strategy in target selection among mobile elements, is characteristic of the insertion sequences IS3, IS6 and IS21, too.
Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake
Authors: Roy Craig R. , Berger Karen H. , Isberg Ralph R.
Published: March 2003

Numerous intracellular bacterial pathogens modulate the nature of the membrane‐bound compartment in which they reside, although little is known about the molecular basis for this control. Legionella pneumophila is a bacterial pathogen able to grow within human alveolar macrophages and residing in a phagosome that does not fuse with lysosomes. This study demonstrates that the dotA product is required to regulate trafficking of the L. pneumophila phagosome. Phagosomes containing L. pneumophila dotA+ bacteria exhibited differential trafficking profiles when compared with isogenic dotA mutants. Phagosomes containing dotA mutants showed rapid accumulation of the lysosomal glycoprotein LAMP‐1 as early as 5 min after uptake, whereas the majority of wild‐type L. pneumophila phagosomes did not acquire LAMP‐1. The association of LAMP‐1 with phagosomes containing dotA mutant bacteria was concomitant with the appearance of the small GTP‐binding protein Rab7 on the vacuolar membrane. These data demonstrate that phagosomes containing replication‐competent L. pneumophila evade early endocytic fusion events. In contrast, the kinetics of LAMP‐1 and Rab7 association indicate that the dotA mutants are routed along a well‐characterized endocytic pathway leading to fusion with lysosomes. Genetic studies show that L. pneumophila requires DotA expression before macrophage uptake in order to establish an intracellular site for replication. However, the bacteria do not appear to require continuous expression of the DotA protein to maintain a replicative phagosome. These data indicate that DotA is one factor that plays a fundamental role in regulating initial phagosome trafficking decisions either upon or immediately after macrophage uptake.
The rpfA gene of Xanthomonas campestris pathovar campestris, which is involved in the regulation of pathogenicity factor production, encodes an aconitase
Authors: Wilson T. J. G. , Bertrand N. , Tang J.‐L. , Feng J.‐X. , Pan M.‐Q. , Barber C. E. , Dow J. M. , Daniels M. J.
Published: March 2003

Xanthomonas campestris pv campestris (Xcc) is a plant pathogenic bacterium that controls the production of pathogenicity factors in part by a cluster of genes designated rpf (regulation of pathogenicity factors). Sequence analysis of one of these genes (rpfA) revealed an open reading frame with amino acid sequence similarity to aconitases from other bacteria. Aconitase activity was lower in cellular extracts of an rpfA::Tn5 mutant than in those from the wild type. A zymogram of aconitase activity after native gel electrophoresis showed the presence of two distinct aconitases in Xcc; the major aconitase was absent in the rpfA::Tn5 mutant. This mutant also had reduced levels of extracellular enzymes and extracellular polysaccharide (EPS). Supplying rpfA in trans to the rpfA::Tn5 mutant restored both the major aconitase activity and the synthesis of these pathogenicity factors. The transcription of the genes for two extracellular enzymes (prtA, encoding a serine protease, and engXCA, encoding endoglucanase) was reduced in the rpfA mutant background. Because some eukaryotic aconitases are also involved in iron regulation, we explored a possible connection between rpfA and iron metabolism. Intracellular iron levels in the mutants were lower than in the wild type as assessed by sensitivity to the iron‐activated antibiotic, streptonigrin. Wild‐type bacteria grown in iron‐deficient conditions had a similar sensitivity to streptonigrin as the aconitase mutant. Overall, these results suggest that a prokaryotic aconitase can also act as a regulator of gene expression and that the regulation is possibly related to changes in intracellular iron levels.
Three rhamnosyltransferases responsible for assembly of the A‐band D‐rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A‐band and B‐band lipopolysaccharide synthesis
Authors: Rocchetta Heather L. , Burrows Lori L. , Pacan Jennifer C. , Lam Joseph S.
Published: March 2003

The Pseudomonas aeruginosa A‐band lipopolysaccharide (LPS) molecule has an O‐polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D‐rhamnose (Rha). The A‐band polysaccharide is assembled by the α‐D‐rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A‐band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A‐band polymer. The genes coding for these transferases were identified at the 3′ end of the A‐band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co‐enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A‐band LPS, while B‐band synthesis is unaffected. WbpL, a transferase encoded within the B‐band biosynthetic cluster, was previously proposed to initiate B‐band biosynthesis through the addition of Fuc2NAc (2‐acetamido‐2,6‐dideoxy‐D‐galactose) to undecaprenol phosphate (Und‐P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross‐complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B‐band synthesis with a Fuc2NAc residue and A‐band synthesis with either a GlcNAc (N‐acetylglucosamine) or GalNAc (N‐acetylgalactosamine) residue. These data indicate that A‐band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A‐band and B‐band LPS synthesis.
Thermoregulation of Escherichia coli pap transcription: H‐NS is a temperature‐dependent DNA methylation blocking factor
Authors: White‐Ziegler Christine A. , Angus Hill Melinda L. , Braaten Bruce A. , Van Der Woude Marjan W. , Low David A.
Published: March 2003

The expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26°C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23°C. The histone‐like nucleoid structuring protein H‐NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H‐NS or a factor regulated by H‐NS bound within the pap regulatory region at 23°C but not at 37°C, as evidenced by H‐NS‐dependent inhibition of methylation of the pap GATC sites designated GATC‐I and GATC‐II. These GATC sites lie upstream of the papBAp promoter and have been shown previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H‐NS bound specifically to a pap DNA fragment containing the GATC‐I and GATC‐II sites. Moreover, H‐NS blocked methylation of these pap GATC sites in vitro : H‐NS blocked pap GATC methylation at 1.4 μM but was unable to do so at higher concentrations at which non‐specific binding occurred. Thus, non‐specific binding of H‐NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H‐NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H‐NS with pap regulatory DNA. We hypothesize that a function of H‐NS such as oligomerization was altered at 23°C, which enabled H‐NS to repress pap gene expression through the formation of a specific nucleoprotein complex.
The ABC maltose transporter
Authors: Ehrmann Michael , Ehrle Rainer , Hofmann Eckhard , Boos Winfried , Schlösser Andreas
Published: March 2003

Bacterial ATP‐binding cassette (ABC) transporters and their homologues in eukaryotic cells form one of the largest superfamilies known today. They function as primary pumps that couple substrate translocation across the cytoplasmic membrane to ATP hydrolysis. Although ABC transporters have been studied for more than three decades, the structure of these multicomponent systems is unknown, and the mechanism of transport is not understood. This article reviews one of the most widely studied ABC systems, the maltose transporter of Escherichia coli. A first structural model of the transport channel allows discussion of possible mechanisms of transport. In addition, recent experimental evidence suggests that regulation of gene expression and transport activity is far more complex than expected.
PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling‐circle replication
Authors: Petit Marie‐Agnès , Dervyn Etienne , Rose Matthias , Entian Karl‐Dieter , McGovern Steven , Ehrlich S. Dusko , Bruand Claude
Published: March 2003

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus. This gene is located at 55° on the chromosome and belongs to a putative operon together with a ligase gene (lig ) and two unknown genes named pcrB and yerH. As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling‐circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading‐strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli. PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep− phenotype on E. coli. Altogether, these results show that PcrA is an helicase used for plasmid rolling‐circle replication and suggest that it is also involved in UV repair.
Osmoregulation of the opuE proline transport gene from Bacillus subtilis: contributions of the sigma A‐ and sigma B‐dependent stress‐responsive promoters
Authors: Spiegelhalter Frank , Bremer Erhard
Published: March 2003

The opuE gene from Bacillus subtilis encodes a transport system (OpuE) for osmoprotective proline uptake and is expressed from two osmoregulated promoters: opuE P‐1 recognized by the vegetative sigma factor A (σA) and opuE P‐2 dependent on the stress‐induced transcription factor sigma B (σB). The contributions of these two promoters to osmoregulation of opuE were analysed. Genetic studies using chromosomal opuE–treA operon fusions revealed that opuE transcription is rapidly induced after an osmotic upshock. The strength of opuE expression is proportionally linked to the osmolarity of the growth medium. Deletion analysis of the opuE regulatory region identified a 330 bp DNA segment carrying all sequences required in cis for full and osmoregulated transcription. The proper rotational orientation of the upstream region present within this fragment was essential for the functioning of both opuE promoters. Mutant opuE–treA fusions with defects in either the σA‐ or the σB‐dependent promoters revealed different contributions of these sequences to the overall osmoregulation of opuE. opuE P‐2 (σB) activity increased transiently after an osmotic upshock and did not significantly contribute to the level of opuE expression in cells subjected to long‐term osmotic stress. In contrast, transcription initiating from opuE P‐1 (σA) rose in proportion to the external osmolarity and was maintained at high levels. Moreover, both promoters exhibited a different response to the osmoprotectant glycine betaine in the medium. Our results suggest that at least two different signal transduction pathways operate in B. subtilis to communicate osmotic changes in the environment to the transcription apparatus of the cell.
An Escherichia coli gene (yaeO ) suppresses temperature‐sensitive mutations in essential genes by modulating Rho‐dependent transcription termination
Authors: Pichoff Sébastien , Alibaud Laetitia , Guédant Agnès , Castanié Marie‐Pierre , Bouché Jean‐Pierre
Published: March 2003

An extragenic multicopy suppressor of the cell division inhibition caused by a MalE–MinE fusion protein in Escherichia coli has been mapped and identified as yaeO, one of the two short open reading frames (ORFs) of an operon located at 4.6 min. Overexpressed yaeO also suppressed some temperature‐sensitive mutations in division genes ftsA and ftsQ, in chaperone gene groEL and in co‐chaperone gene grpE. Gene yaeO, whose expression is regulated by growth rate, codes for a 9 kDa acidic protein with no obvious resemblance to other proteins. Transcription termination protein Rho co‐purified with a histidine‐tagged derivative of YaeO protein on Ni2+‐NTA agarose columns in a manner that suggested direct YaeO–Rho interaction. In vivo, yaeO expression reduced termination at rho‐dependent bacteriophage terminator tL1 and at the terminator of autogenously regulated gene rho. The suppression of temperature‐sensitive phenotypes was a consequence of anti‐termination, as it could be mimicked by a Prho::Tn10 mutation that reduces the expression and activity of gene rho. Our data indicate that the suppression is not caused by overexpression of the mutated genes, but presumably by indirect stabilization of the mutated proteins.
Sequence and analysis of the 60 kb conjugative, bacteriocin‐producing plasmid pMRC01 from Lactococcus lactis DPC3147
Authors: Dougherty Brian A. , Hill Colin , Weidman Janice F. , Richardson Delwood R. , Venter J. Craig , Ross R. Paul
Published: March 2003

The complete sequence of pMRC01, a large conjugative plasmid from Lactococcus lactis ssp. lactis DPC3147, has been determined. Using a shotgun sequencing approach, the 60 232 bp plasmid sequence was obtained by the assembly of 1056 underlying sequences (sevenfold average redundancy). Sixty‐four open reading frames (ORFs) were identified. Analysis of the gene organization of pMRC01 suggests that the plasmid can be divided into three functional domains, with each approximately 20 kb region separated by insertion sequence (IS) elements. The three regions are (i) the conjugative transfer region, including a 16‐gene Tra (transfer) operon; (ii) the bacteriocin production region, including an operon responsible for the synthesis of the novel bacteriocin lacticin 3147; and (iii) the phage resistance and plasmid replication region of the plasmid. The complete sequence of pMRC01 provides important information about these industrially relevant phenotypes and gives insight into the structure, function and evolution of large Gram‐positive conjugative plasmids in general. The completely sequenced pMRC01 plasmid should also provide a useful framework for the design of novel plasmids to be incorporated into starter strain improvement programmes for the dairy industry.
Synthesis of the A‐band polysaccharide sugar D‐rhamnose requires Rmd and WbpW: identification of multiple AlgA homologues, WbpW and ORF488, in Pseudomonas aeruginosa
Authors: Rocchetta Heather L. , Pacan Jennifer C. , Lam Joseph S.
Published: March 2003

Pseudomonas aeruginosa is capable of producing various cell‐surface polysaccharides including alginate, A‐band and B‐band lipopolysaccharides (LPS). The D‐mannuronic acid residues of alginate and the D‐rhamnose (D‐Rha) residues of A‐band polysaccharide are both derived from the common sugar nucleotide precursor GDP‐D‐mannose (D‐Man). Three genes, rmd, gmd and wbpW, which encode proteins involved in the synthesis of GDP‐D‐Rha, have been localized to the 5′ end of the A‐band gene cluster. In this study, WbpW was found to be homologous to phosphomannose isomerases (PMIs) and GDP‐mannose pyrophosphorylases (GMPs) involved in GDP‐D‐Man biosynthesis. To confirm the enzymatic activity of WbpW, Escherichia coli PMI and GMP mutants deficient in the K30 capsule were complemented with wbpW, and restoration of K30 capsule production was observed. This indicates that WbpW, like AlgA, is a bifunctional enzyme that possesses both PMI and GMP activities for the synthesis of GDP‐D‐Man. No gene encoding a phosphomannose mutase (PMM) enzyme could be identified within the A‐band gene cluster. This suggests that the PMM activity of AlgC may be essential for synthesis of the precursor pool of GDP‐D‐Man, which is converted to GDP‐D‐Rha for A‐band synthesis. Gmd, a previously reported A‐band enzyme, and Rmd are predicted to perform the two‐step conversion of GDP‐D‐Man to GDP‐D‐Rha. Chromosomal mutants were generated in both rmd and wbpW. The Rmd mutants do not produce A‐band LPS, while the WbpW mutants synthesize very low amounts of A band after 18 h of growth. The latter observation was thought to result from the presence of the functional homologue AlgA, which may compensate for the WbpW deficiency in these mutants. Thus, WbpW AlgA double mutants were constructed. These mutants also produced low levels of A‐band LPS. A search of the PAO1 genome sequence identified a second AlgA homologue, designated ORF488, which may be responsible for the synthesis of GDP‐D‐Man in the absence of WbpW and AlgA. Polymerase chain reaction (PCR) amplification and sequence analysis of this region reveals three open reading frames (ORFs), orf477, orf488 and orf303, arranged as an operon. ORF477 is homologous to initiating enzymes that transfer glucose 1‐phosphate onto undecaprenol phosphate (Und‐P), while ORF303 is homologous to L‐rhamnosyltransferases involved in polysaccharide assembly. Chromosomal mapping using pulsed field gel electrophoresis (PFGE) and Southern hybridization places orf477, orf488 and orf303 between 0.3 and 0.9 min on the 75 min map of PAO1, giving it a map location distinct from that of previously described polysaccharide genes. This region may represent a unique locus within P. aeruginosa responsible for the synthesis of another polysaccharide molecule.
An essential GTP‐binding protein functions as a regulator for differentiation in Streptomyces coelicolor
Authors: Okamoto Susumu , Ochi Kozo
Published: March 2003

The Streptomyces coelicolor obg gene, which encodes a putative GTP‐binding protein of the Obg/Gtp1 family, was characterized. The obg gene was essential for viability. Introduction of multiple copies of obg into wild‐type S. coelicolor suppressed aerial mycelium formation. A single amino acid substitution at any of six positions was introduced into the GTP binding site of Obg, and the mutated proteins were expressed in wild‐type cells. ObgP168 → V exerted a more accentuated suppressive effect on aerial mycelium formation than did the wild‐type Obg protein. In contrast, ObgG171 → A accelerated the development of aerial mycelium. These results show that Obg protein functions as a pivotal regulator for the onset of cell differentiation through its ability to bind GTP. Western analysis revealed that expression of obg is regulated in a growth phase‐dependent manner, indicating a sharp decrease just after onset of aerial mycelium development or at the end of vegetative growth. Obg was a membrane‐bound protein as determined by immunoelectron microscopy.
Functional analysis of the Helicobacter pylori principal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription
Authors: Beier Dagmar , Spohn Gunther , Rappuoli Rino , Scarlato Vincenzo
Published: March 2003

We have cloned the rpoD gene encoding the principal sigma (σ) factor of Helicobacter pylori. The deduced amino acid sequence reveals a predicted polypeptide of 676 residues that has amino acid homology with the principal σ factors of a number of divergent prokaryotes. We have designated this factor σ80. Amino acid sequence analysis suggests that region 1.1 is missing in σ80 and that a region with homology to a regulatory protein from Bacillus subtilis phage SPO1 is present. Genetic studies have indicated that σ80 is not compatible with the transcriptional machinery of Escherichia coli. However, in vitroσ80 could be assembled into the E. coli RNA polymerase and could bind to E. coli and H. pylori promoters, suggesting that the σ80‐containing RNA polymerase has the same stoichiometry as the native complex. By exchanging protein domains between E. coli and H. pyloriσ factors, we demonstrate that the σ80 domain inhibiting transcription from E. coli promoters is confined within the non‐conserved spacer region, implying that the spacer region of prokaryotic primary σ factors plays an important role in the process of transcription. Consistent with its restricted niche and with the availability of a very restricted number of transcriptional regulators, H. pylori may have evolved a spacer region of the σ factor to modulate total transcription and to quickly respond to microenvironmental changes.
Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of IncQ plasmid RSF1010
Authors: Segal Gil , Shuman Howard A.
Published: March 2003

Previously we have reported that Legionella pneumophila can mediate plasmid DNA transfer at a frequency of about 10−3 transconjugants per donor and that this process is dependent on several icm genes. Here we characterize the icm‐dependent conjugal ability of L. pneumophila and study its relationship to intracellular multiplication and host cell killing. We found that three icm genes and the RSF1010 mobA gene are completely required and that three icm genes and the RSF1010 mobC gene are partially required for conjugation. Conjugation occurred during lag phase and stopped when the cell number increased. Inhibition of transcription or translation in the donor had only a minor effect on conjugation frequency. These results suggest that stationary‐phase bacteria contain a functional icm complex that can mediate conjugal DNA transfer and probably can initiate infection of human macrophages as well. We also found that a functional RSF1010 mobilization system inhibits intracellular multiplication and killing of human macrophages by L. pneumophila. The strongest inhibition was observed in icm insertion mutants complemented with wild‐type icm genes on an RSF1010‐derived plasmid. These results suggest that the conjugation substrate probably competes with the natural substrate of the L. pneumophila icm system for transfer outside the bacterial cell. We propose that the function of the L. pneumophila icm system is to transfer effector molecules to the host cell. These effector molecules may interact with components of the host cell that are involved in phagosome formation and fate.
Molecular basis for structural diversity in the core regions of the lipopolysaccharides of Escherichia coli and Salmonella enterica
Authors: Heinrichs David E. , Yethon Jeremy A. , Whitfield Chris
Published: March 2003

Bacterial lipopolysaccharides (LPS) are unique and complex glycolipids that provide characteristic components of the outer membranes of Gram‐negative bacteria. In LPS of the Enterobacteriaceae, the core oligosaccharide links a highly conserved lipid A to the antigenic O‐polysaccharide. Structural diversity in the core oligosaccharide is limited by the constraints imposed by its essential role in outer membrane stability and provides a contrast to the hypervariable O‐antigen. The genetics of core oligosaccharide biosynthesis in Salmonella and Escherichia coli K‐12 have served as prototypes for studies on the LPS and lipo‐oligosaccharides from a growing range of bacteria. However, despite the wealth of knowledge, there remains a number of unanswered questions, and direct experimental data are not yet available to define the precise mechanism of action of many gene products. Here we present a comparative analysis of the recently completed sequences of the major core oligosaccharide biosynthesis gene clusters from the five known core types in E. coli and the Ra core type of Salmonella enterica serovar Typhimurium and discuss advances in the understanding of the related biosynthetic pathways. Differences in these clusters reflect important structural variations in the outer core oligosaccharides and provide a basis for ascribing functions to the genes in these model clusters, whereas highly conserved regions within these clusters suggest a critical and unalterable function for the inner region of the core.
Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli
Authors: Tomoyasu Toshifumi , Ogura Teru , Tatsuta Takashi , Bukau Bernd
Published: March 2003

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the σ32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes σ32, and the FtsH protease, which is largely responsible for σ32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose‐controlled promoters. Small increases in levels of DnaK and the DnaJ co‐chaperone (< 1.5‐fold of wild type) resulted in decreased level and activity of σ32 at intermediate temperature and faster shut‐off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat‐denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut‐off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the σ32 levels, but the accumulated σ32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress‐sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.
Requirement of the VanY and VanX D,D‐peptidases for glycopeptide resistance in enterococci
Authors: Arthur Michel , Depardieu Florence , Cabanié Lucien , Reynolds Peter , Courvalin Patrice
Published: March 2003

Transposon Tn1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D‐peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D‐alanyl‐D‐lactate (D‐Ala‐D‐Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N‐terminal membrane anchor of the protein. The enzyme was a Zn2+‐dependent D,D‐carboxypeptidase that cleaved the C‐terminal residue of peptidoglycan precursors ending in R‐D‐Ala‐D‐Ala or R‐D‐Ala‐D‐Lac but not the dipeptide D‐Ala‐D‐Ala. The specificity constants kcat/Km were 17‐ to 67‐fold higher for substrates ending in the R‐D‐Ala‐D‐Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP‐MurNAc‐tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high‐level glycopeptide resistance in a medium supplemented with D‐Ala. The enzyme could not replace the VanX D,D‐dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D‐Ala:D‐Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D‐carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D‐Ala‐containing medium. Thus, VanX and VanY had non‐overlapping functions involving the hydrolysis of D‐Ala‐D‐Ala and the removal of D‐Ala from membrane‐bound lipid intermediates respectively.
Identification of a 47 kDa fibronectin‐binding protein expressed by Borrelia burgdorferi isolate B31
Authors: Probert William S. , Johnson Barbara J. B.
Published: March 2003

The attachment of pathogenic microorganisms to host cells and tissues is often mediated through the expression of surface receptors recognizing components of the extracellular matrix (ECM). Here, we investigate the ability of Borrelia spirochaetes to bind the ECM constituent, fibronectin. Borrelia lysates were separated by SDS–PAGE, transferred to nitrocellulose and probed with alkaline phosphatase‐labelled fibronectin (fibronectin‐AP). Five of six Borrelia species and four of eight B. burgdorferi sensu lato isolates expressed one or more fibronectin‐binding proteins. Borrelia burgdorferi isolate B31 expressed a 47 kDa (P47) fibronectin‐binding protein that was localized to the outer envelope based on susceptibility to proteinase K. The interaction of P47 with fibronectin was specific, and the region of fibronectin bound by P47 mapped to the gelatin/collagen binding domain. P47 was purified by affinity chromatography, digested with endoproteinase Lys‐C, and the peptide fragments analysed by liquid chromatography/tandem mass spectroscopy. A search of protein databases disclosed that the P47 peptide mass profile matched that predicted for the bbk32 gene product of B. burgdorferi isolate B31. The bbk32 gene was cloned into Escherichia coli, and the ability of recombinant BBK32 to bind fibronectin and inhibit the attachment of B. burgdorferi was demonstrated. The identification of BBK32 as a receptor for fibronectin binding may enhance our understanding of the pathogenesis and chronic nature of Lyme disease.
The long‐term cytoskeletal rearrangement induced by rabbit enteropathogenic Escherichia coli is Esp dependent but intimin independent
Authors: Nougayrède Jean‐Philippe , Marchès Olivier , Boury Michèle , Mainil Jacques , Charlier Gérard , Pohl Pierre , De Rycke Jean , Milon Alain , Oswald Eric
Published: March 2003

Attaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres. In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE. In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69. Non‐polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed. The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model. The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE. Each espA, ‐B, ‐D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE‐negative E2348/69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype. In conclusion, the CPE is the first example of an Esp‐dependent but Eae (intimin)‐independent alteration of the host cell cytoskeleton by certain EPEC strains.