bluuebunny/crossref_metadata_2025 huggingface dataset:
Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis
Authors: Hodgman T. C. , Griffiths H. , Summers D. K.
Published: March 2003

Dimers of plasmid ColE1 are converted to monomers by site‐specific recombination, a process that requires 240 bp of DNA (cer ) and four host‐encoded proteins (XerC, XerD, ArgR and PepA). Here, we propose structures for nucleoprotein complexes involved in cer–Xer recombination based upon existing knowledge of the structures of component proteins and computational analyses of protein structure and DNA curvature. We propose that, in the nucleoprotein complex at a single cer site, a PepA hexamer acts as an adaptor, connecting the heterodimeric recombinase (XerCD) to an ArgR hexamer. This provides a protein core around which the cer site wraps, its exact path being defined by strong sequence‐specific interactions with ArgR and XerCD, weak interactions with PepA and sequence‐dependent flexibility of cer. The initial association of single‐site complexes (pairing) is proposed to occur via an ArgR–PepA interaction. Pairing between sites in a plasmid dimer is stabilized by DNA supercoiling and is followed by a structural isomerization to form a recombination‐proficient synaptic complex. We propose that paired structures formed between sites in trans are too short‐lived to permit synaptic complex formation. There is thus an energetic barrier to inappropriate recombination reactions. Our proposals are consistent with a wide range of experimental observations.
Novel genes that upregulate the Proteus mirabilis flhDC master operon controlling flagellar biogenesis and swarming
Authors: Dufour Alain , Furness Richard B. , Hughes Colin
Published: March 2003

By screening for restoration of multicellular migration in a non‐swarming but motile Proteus mirabilis mutant lacking the FlgN facilitator of flagella assembly, we identified four distinct genes that, in trans and multicopy, increased flagella production and cell length. Each of the genes upregulated expression of the flhDC master operon that controls flagellar biogenesis, cell division and swarming, not only in the mutant but also in the wild type. The genes were named umoA, umoB, umoC and umoD. Disruption of each of the wild‐type chromosomal umo genes caused corresponding reductions in swarming and cell elongation, which correlated with decreased expression of the flhDC operon. The umoA, umoB, umoC and umoD genes are not closely linked, and only umoB is part of an operon. The sequences of the calculated gene products, UmoA (20.6 kDa), UmoB (78.0 kDa), UmoC (15.2 kDa) and UmoD (19.2 kDa), contain putative N‐terminal secretion signals and predict a location in the cell membranes or periplasm. UmoB and UmoD have sequence similarity to the Escherichia coli uncharacterized open reading frames YrfF and YcfJ respectively; UmoA and UmoC have no known homologues. The umoB and umoC gene transcripts were present at very low levels, but umoA and umoD expression was similar to that of flhDC and increased in parallel with flhDC expression during differentiation into elongated hyperflagellated swarm cells. Like flhDC, umoA and umoD expression was subject to negative feedback in a flagellar assembly mutant lacking the FlhA inner membrane component of the export machinery. Assays of umo gene expression and cross‐complementation indicated that the umo genes do not act in sequence within a pathway to upregulate flhDC, but revealed that umoA and umoD are reciprocally upregulated by FlhDC. Our findings strengthen the picture of the flhDC master operon as a major assimilatory checkpoint in Proteus mirabilis and other Gram‐negative bacteria and expand the view of a complex regulatory network coupled to flagellar biogenesis.
Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe
Authors: Arellano Manuel , Coll Pedro M. , Yang Wenli , Duran Angel , Tamanoi Fuyuhiko , Perez Pilar
Published: March 2003

The Schizosaccharomyces pombe cwg2+ gene encodes the β‐subunit of geranylgeranyl transferase I (GGTase I), which participates in the post‐translational C‐terminal modification of several small GTPases, allowing their targeting to the membrane. Using the two‐hybrid system, we have identified the cwp1+ gene that encodes the α‐subunit of the GGTase I. cwp1p interaction with cwg2p was mapped to amino acids 1–244 or 137–294 but was not restricted to amino acids 137–244. The genomic cwp1+ was isolated and sequenced. It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron. The predicted amino acid sequence shows significant similarity to GGTase I α‐subunits from different species. However, complementation of Saccharomyces cerevisiae ram2‐1 mutant by overexpressing the cwp1+ gene was not possible. Expression of both cwg2+ and cwp1+ in Escherichia coli allowed ‘in vitro’ reconstitution of the GGTase I activity. S. pombe cells expressing the mutant enzyme containing the cwg2‐1 mutation do not grow at 37°C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2‐1 mutant strain grown at 37°C showed an abnormal distribution of actin patches. The cwg2‐1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p. Deletion of the cwg2 gene is lethal; Δcwg2 spores can divide two or three times before losing viability. Most cells have aberrant morphology and septation defects. Overexpression of the rho1G15VC199R double‐mutant allele in S. pombe caused loss of polarity but was not lethal and did not render the (1–3)β‐D‐glucan synthase activity independent of GTP. Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase.
Pleiotropic effects of cAMP on germination, antibiotic biosynthesis and morphological development in Streptomyces coelicolor
Authors: Süsstrunk Urs , Pidoux Josette , Taubert Stefan , Ullmann Agnes , Thompson Charles J.
Published: March 2003

In wild‐type Streptomyces coelicolor MT1110 cultures, cyclic adenosine 3′,5′ monophosphate (cAMP) was synthesized throughout the developmental programme with peaks of accumulation both during germination and later when aerial mycelium and actinorhodin were being produced. Construction and characterization of an adenylate cyclase disruption mutant (BZ1) demonstrated that cAMP facilitated these developmental processes. Although pulse‐labelling experiments showed that a similar germination process was initiated in BZ1 and MT1110, germ‐tube emergence was severely delayed in BZ1 and never occurred in more than 85% of the spores. Studies of growth and development on solid glucose minimal medium (SMMS, buffered or unbuffered) showed that MT1110 and BZ1 produced acid during the first rapid growth phase, which generated substrate mycelium. Thereafter, on unbuffered SMMS, only MT1110 resumed growth and produced aerial mycelium by switching to an alternative metabolism that neutralized its medium, probably by reincorporating and metabolizing extracellular acids. BZ1 was not able to neutralize its medium or produce aerial mycelium on unbuffered SMMS; these defects were suppressed by high concentrations (>1 mM) of cAMP during early growth or on buffered medium. Other developmental mutants (bldA, bldB, bldC, bldD, bldG) also irreversibly acidified this medium. However, these bald mutants were not suppressed by exogenous cAMP or neutralizing buffer. BZ1 also differentiated when it was cultured in close proximity to MT1110, a property observed in cross‐feeding experiments between bald mutants and commonly thought to reflect diffusion of a discrete positively acting signalling molecule. In this case, MT1110 generated a more neutral pH environment that allowed BZ1 to reinitiate growth and form aerial mycelium. The fact that actinorhodin synthesis could be induced by concentrations of cAMP (< 20 μM) found in the medium of MT1110 cultures, suggested that it may serve as a diffusible signalling molecule to co‐ordinate antibiotic biosynthesis.
Cell division genes ftsQAZ in Escherichia coli require distant cis‐acting signals upstream of ddlB for full expression
Authors: Flärdh Klas , Palacios Pilar , Vicente Miguel
Published: March 2003

A transcriptional reporter fusion has been introduced into the chromosomal ftsZ locus in such a way that all transcription that normally reaches ftsZ can be monitored. The new Φ(ftsZ–lacZ ) fusion yields four times more β‐galactosidase activity than a ddlB–ftsQAZ–lacZ fusion on a lambda prophage vector. A strongly polar ddlB ::Ω insertion prevents contributions from signals upstream of the ftsQAZ promoters and decreases transcription of the chromosomal Φ(ftsZ–lacZ ) fusion by 66%, demonstrating that around two‐thirds of total ftsZ transcription require cis‐acting elements upstream of ddlB. We suggest that those elements are distant promoters, and thus that the cell division and cell wall synthesis genes in the dcw gene cluster are to a large extent co‐transcribed. The ddlB ::Ω insertion is lethal unless additional copies of ftsQA are provided or a compensatory decrease in FtsZ synthesis is made. This shows that ddlB is a dispensable gene, and reinforces the critical role of the FtsA/FtsZ ratio in septation. Using the new reporter fusion, it is demonstrated that ftsZ expression is not autoregulated.
Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii
Authors: Ruiz‐Dueñas Francisco Javier , Martínez María Jesús , Martínez Angel T.
Published: March 2003

A haem peroxidase different from other microbial, plant and animal peroxidases is described. The enzyme is secreted as two isoforms by dikaryotic Pleurotus eryngii in peptone‐containing liquid medium. The corresponding gene, which presents 15 introns and encodes a 361‐amino‐acid protein with a 30‐amino‐acid signal peptide, was isolated as two alleles corresponding to the two isoforms. The alleles differ in three amino acid residues and in a seven nucleotide deletion affecting a single metal response element in the promoter. When compared with Phanerochaete chrysosporium peroxidases, the new enzyme appears closer to lignin peroxidase (LiP) than to Mn‐dependent peroxidase (MnP) isoenzymes (58–60% and 55% identity respectively). The molecular model built using crystal structures of three fungal peroxidases as templates, also showed high structural affinity with LiP (Cα‐distance 1.2 Å). However, this peroxidase includes a Mn2+ binding site formed by three acidic residues (E36, E40 and D175) near the haem internal propionate, which accounts for the ability to oxidize Mn2+. Its capability to oxidize aromatic substrates could involve interactions with aromatic residues at the edge of the haem channel. Another possibility is long‐range electron transfer, e.g. from W164, which occupies the same position of LiP W171 recently reported as involved in the catalytic cycle of LiP.
Genomic rearrangements in the flagellin genes of Proteus mirabilis
Authors: Murphy Catherine A. , Belas Robert
Published: March 2003

Molecular analyses have revealed that Proteus mirabilis possesses two genes, flaA and flaB, that are homologous to each other and to flagellin genes of many other species. Both swimmer and swarmer cells transcribe flaA, but not flaB. FlaA− mutants are non‐motile and do not differentiate showing the essential role of flaA in swarmer cell differentiation and behaviour. At a low frequency, motile, differentiation‐proficient revertants have been found in FlaA− populations. These revertants produce an antigenically and biochemically distinct flagellin protein. The revertant flagellin is the result of a genetic fusion between highly homologous regions of flaA and flaB that places the active flaA promoter and the 5′ coding region of flaA adjacent to previously silent regions of flaB generating a hybrid flagellin protein. Analysis of the flaA–flaB region of two such revertants reveals that a portion of this locus has undergone a rearrangement and deletion event that is unique to each revertant. Using a polymerase chain reaction (PCR) to amplify the flaA–flaB locus from wild‐type swimmer cells, swarmer cells and cells obtained after urinary tract infection, we uncover at least six general classes of rearrangements between flaA and flaB. Each class of rearrangement occurs within one of nine domains of homology between flaA and flaB. Rearrangement of flaA and flaB results in a hybrid flagellin protein of nearly identical size and biochemical properties, suggesting a concerted mechanism may be involved in this process. The data also reveal that the frequency and distribution of flaAB rearrangements is predicated on environmental conditions. Thus, rearrangement between flaA and flaB may be a significant virulence component of P. mirabilis in urinary tract infections.
Listeriolysin O‐dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF‐κB and upregulation of adhesion molecules and chemokines
Authors: Kayal Samer , Lilienbaum Alain , Poyart Claire , Memet Sylvie , Israel Alain , Berche Patrick
Published: March 2003

The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system. Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis. By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L. monocytogenes, we found that wild‐type bacteria induced the expression of the adhesion molecules (ICAM‐1 and E‐selectin), chemokine secretion (IL‐8 and monocyte chemotactic protein‐1) and NF‐κB nuclear translocation. The activation of HUVEC required viable bacteria and was abolished in prfA‐deficient mutants of L. monocytogenes, suggesting that virulence genes are associated with endothelial cell activation. Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)‐deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes. Adhesion molecule expression, chemokine secretion and NF‐κB activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO. The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF‐κB‐responsive lacZ reporter gene. NF‐κB activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non‐haemolytic isogenic mutant. Direct evidence that LLO is involved in NF‐κB activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF‐κB in endothelial cells of blood capillaries. Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process.
Environmental regulation of Salmonella pathogenicity island 2 gene expression
Authors: Deiwick Jörg , Nikolaus Thomas , Erdogan Sezgin , Hensel Michael
Published: March 2003

The enteric pathogen Salmonella typhimurium co‐ordinates the expression of virulence determinants in response to environmental cues from the host organism. S. typhimurium possesses Salmonella pathogenicity island 2 (SPI2), a large virulence locus encoding a type III secretion system for virulence determinants required for systemic infections and accumulation inside host cells. We have generated transcriptional fusions to SPI2 genes to analyse expression and used antibodies against recombinant SPI2 proteins to monitor levels of SPI2 proteins under various conditions. Here, we demonstrate that SPI2 gene expression is induced by Mg2+ deprivation and phosphate starvation. These conditions are likely to represent the environmental cues encountered by S. typhimurium inside the phagosome of infected host cells. The induction of SPI2 gene expression is modulated by the global regulatory system PhoPQ and is dependent on SsrAB, a two‐component regulatory system encoded by SPI2.
The assembly and migration of SeqA–Gfp fusion in living cells of Escherichia coli
Authors: Onogi Toshinari , Niki Hironori , Yamazoe Mitsuyoshi , Hiraga Sota
Published: March 2003

SeqA protein, which binds to hemi‐methylated GATC sequences of DNA, is localized to discrete fluorescent foci in wild‐type Escherichia coli cells. In this work, we observed cellular localization of the SeqA–Gfp fusion in living cells. SeqA–Gfp was localized to a discrete focus or foci in wild‐type and seqA null mutant cells, but the fusion was dispersed in the whole cell in dam null mutant cells lacking Dam methyltransferase. These results were consistent with the previous description of the localization of SeqA by immunofluorescence microscopy. Time‐lapse experiments revealed that duplicated SeqA–Gfp foci migrated rapidly in opposite directions. Flow cytometry demonstrated that the fusion restored synchronous replication of chromosomal DNA from multiple origins in seqA null mutant cells, indicating that SeqA–Gfp is biologically active. Immunoprecipitation of the fusion from cell extracts using anti‐Gfp antibody indicated that the fusion was assembled with the wild‐type SeqA protein.
Escherichia coli nusG mutations that block transcription termination by coliphage HK022 Nun protein
Authors: Burova Elena , Hung Siu Chun , Chen John , Court Donald L. , Zhou Jian‐Guang , Mogilnitskiy Grigoriy , Gottesman Max E.
Published: March 2003

The Escherichia coli nusG gene product is required for transcription termination by phage HK022 Nun protein at the λnutR site in vivo. We show that it is also essential for Nun termination at λnutL. Three recessive missense nusG mutations have been isolated that inhibit termination by Nun at λnutR. The mutations are ineffective in a λpL nutL fusion, even when λnutR replaces λnutL. The mutant strains support λ growth, indicating that λ N antitermination activity is not impaired. Transcription arrest by Nun in vitro is stimulated by NusG protein at both λnutR and λnutL. Mutant NusG protein fails to enhance transcriptional arrest by Nun at either site. The mutant protein, like the wild‐type protein, suppresses transcriptional pausing by RNA polymerase and stimulates Rho‐dependent termination. These results imply that the role of NusG in Nun termination may be distinct from its roles in other transcription reactions.
FtsZ dimerization in vivo
Authors: Di Lallo G. , Anderluzzi D. , Ghelardini P. , Paolozzi L.
Published: March 2003

A hybrid assay, based on the properties of the λ repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N‐terminal end of the λcI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional λ repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N‐terminal of about 150 amino acids; the C‐terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ.
Dissection of a surface‐exposed portion of the cAMP–CRP complex that mediates transcription activation and repression
Authors: Meibom K. L. , Søgaard‐Andersen L. , Mironov A. S. , Valentin‐Hansen P.
Published: March 2003

The Escherichia coli cAMP receptor protein (CRP) is essential for the activation and repression of transcription initiation at promoters in the CytR regulon. CRP performs these activities by making direct protein–protein interactions to the α‐subunits of RNA polymerase and to the CytR regulator. Strikingly, it has been shown that amino acids of CRP that are critical for communication with the two partner proteins are located in close proximity on the surface of CRP. Here, we have dissected this surface in order to pinpoint the ‘repression region’ of CRP and to assess whether it overlaps with the characterized ‘activating region’. Our results established that residues 12, 13, 17, 105, 108 and 110 are essential for the interaction with CytR and confirmed that ‘activating region’ 2 of CRP is made up of residues 19, 21 and 101. In the crystallographic structure of the CRP–DNA complex, the two sets of determinants are located immediately adjacent to each other forming a consecutive surface‐exposed patch. The ‘repression region’ is chemically complementary to the characterized region on CytR that is essential for protein–protein communication to CRP. Moreover, the results provide insight into the mechanism by which CytR might prevent CRP‐mediated transcription.
Two AraC/XylS family members can independently counteract the effect of repressing sequences upstream of the hilA promoter
Authors: Schechter Lisa M. , Damrauer Scott M. , Lee Catherine A.
Published: March 2003

During infection of its hosts, Salmonella enterica serovar Typhimurium (S. typhimurium) enters the epithelial cells of the small intestine. This process requires a number of invasion genes encoded on Salmonella pathogenicity island 1 (SPI1), a 40 kb stretch of DNA located near minute 63 of the S. typhimurium chromosome. Expression of S. typhimurium SPI1 invasion genes is activated by the transcription factor HilA. hilA is tightly regulated in response to many environmental conditions, including oxygen, osmolarity and pH. Regulation of hilA expression may serve to limit invasion gene expression to the appropriate times during Salmonella infection. We have mapped the transcription start site of hilA and identified regions of the promoter that are required for the repression of hilA expression by conditions unfavourable for Salmonella invasion. We have also identified two SPI1‐encoded genes, hilC and hilD, that can independently derepress hilA expression. HilC and HilD are both members of the AraC/XylS family of transcriptional regulators. A mutation in hilD significantly reduces the ability of S. typhimurium to enter tissue culture cells, whereas a mutation in hilC only modestly affects Salmonella invasion. Based on these results, we have updated our model of Salmonella SPI1 invasion gene regulation. We also speculate on the possible significance of this model for Salmonella pathogenesis.
Genetic redundancy and gene fusion in the genome of the baker's yeast Saccharomyces cerevisiae : functional characterization of a three‐member gene family involved in the thiamine biosynthetic pathway
Authors: Llorente Bertrand , Fairhead Cécile , Dujon Bernard
Published: March 2003

Redundancy is a salient feature of all living organisms' genome. The yeast genome contains a large number of gene families of previously uncharacterized functions that can be used to explore the functional significance of structural redundancy in a systematic manner. In this work, we describe results on a three‐member gene family with moderately divergent sequences (YOL055c, YPL258c and YPR121w ). We demonstrate that two members are isofunctional and encode a hydroxymethylpyrimidine phosphate (HMP‐P) kinase (EC 2.7.4.7), an activity required for the final steps of thiamine biosynthesis, whose genes were not previously known in yeast. In addition, we show that the three genes are each composed of two distinct domains, each corresponding to individual genes in prokaryotes, suggesting gene fusion during evolution. The function of the carboxy‐terminal part of the proteins is not yet understood, but it is not required for HMP‐P kinase activity. Expression of all three genes is regulated in the same way. Several other examples of gene fusions exist in the same biosynthetic pathway when eukaryotic genes are compared with prokaryotic ones.
A novel mutation in the KH domain of polynucleotide phosphorylase affects autoregulation and mRNA decay in Escherichia coli
Authors: García‐Mena Jaime , Das Asis , Sánchez‐Trujillo Alejandra , Portier Claude , Montañez Cecilia
Published: March 2003

Polynucleotide phosphorylase (PNPase) is a key 3′–5′ exonuclease for mRNA decay in bacteria. Here, we report the isolation of a novel mutant of Escherichia coli PNPase that affects autogenous control and mRNA decay. We show that the inactivation of PNPase by a transposon insertion increases the half‐life of galactokinase mRNA encoded by a plasmid. When the bacteriophage lambda int gene retroregulator (sib/tI ) is placed between pgal and galK, it severely diminishes galactokinase expression because of transcription termination. The expression of galK from this construct is increased by a single base mutation, sib1, which causes a partial readthrough of transcription at tI. We have used this plasmid system with sib1 to select E. coli mutants that depress galK expression. Genetic and molecular analysis of one such mutant revealed that it contains a mutation in the pnp gene, which encodes the PNPase catalytic subunit α. The mutation responsible (pnp‐71 ) has substituted a highly conserved glycine residue in the KH domain of PNPase with aspartate. We show that this G‐570D substitution causes a higher accumulation of the α‐subunit as a result of defective autoregulation, thereby increasing the PNPase activity in the cell. The purified mutant α‐subunit shows the same electrophoretic mobility in denaturing gels as the wild‐type subunit, as expected. However, the mutant protein present in crude extracts displays an altered electrophoretic mobility in non‐denaturing gels that is indicative of a novel enzyme complex. We present a model for how the pnp‐71 mutation might affect autoregulation and mRNA decay based on the postulated role of the KH domain in RNA–protein and protein–protein interactions.
Identification and characterization of a Chlamydia trachomatis early operon encoding four novel inclusion membrane proteins
Authors: Scidmore‐Carlson Marci A. , Shaw Edward I. , Dooley Cheryl A. , Fischer Elizabeth R. , Hackstadt Ted
Published: March 2003

Chlamydia trachomatis is a bacterial obligate intracellular parasite that replicates within a vacuole, termed an inclusion, that does not fuse with lysosomes. Within 2 h after internalization, the C. trachomatis inclusion ceases to interact with the endocytic pathway and, instead, becomes fusogenic with exocytic vesicles containing exogenously synthesized NBD‐sphingomyelin. Both fusion of exocytic vesicles and long‐term avoidance of lysosomal fusion require early chlamydial gene expression. Modification of the chlamydial inclusion probably occurs through the expression and insertion of chlamydial protein(s) into the inclusion membrane. To identify candidate inclusion membrane proteins, antisera were raised against a total membrane fraction purified from C. trachomatis‐infected HeLa cells. By indirect immunofluorescence, this antisera recognized the inclusion membrane and, by immunoblot analysis, recognized three chlamydial‐specific antigens of approximate molecular weights 15, 18 and 21 kDa. IncG, encoding an 18 kDa and 21 kDa doublet chlamydial antigen, was identified by screening a C. trachomatis, serovar L2, genomic expression library. Three additional genes, incD, incE and incF, were co‐transcribed with incG. Monospecific antisera against each of the four genes of this operon demonstrated that the gene products were localized to the chlamydial inclusion membrane. Immediately downstream from the operon containing incD–G was the C. trachomatis homologue of incA. Like IncD, E, F and G, C. trachomatis IncA is also localized to the inclusion membrane. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis demonstrated that IncD–G, but not incA, are transcribed within the first 2 h after internalization, making them candidates for chlamydial factors required for the modification of the nascent chlamydial inclusion.
Quorum‐sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram‐negative bacteria
Authors: Holden Matthew T.G. , Ram Chhabra Siri , De Nys Rocky , Stead Paul , Bainton Nigel J. , Hill Philip J. , Manefield Mike , Kumar Naresh , Labatte Maurice , England Dacre , Rice Scott , Givskov Mike , Salmond George P.C. , Stewart Gordon S.A.B. , Bycroft Barrie W. , Kjelleberg Staffan , Williams Paul
Published: March 2003

In cell‐free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N‐acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(ΔAla‐l‐Val) and cyclo(l‐Pro‐l‐Tyr) respectively. These compounds were also found in cell‐free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(ΔAla‐l‐Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(l‐Phe‐l‐Pro). Dose–response curves using a LuxR‐based AHL biosensor indicated that cyclo(ΔAla‐l‐Val), cyclo(l‐Pro‐l‐Tyr) and cyclo(l‐Phe‐l‐Pro) activate the biosensor in a concentration‐dependent manner, albeit at much higher concentrations than the natural activator N‐(3‐oxohexanoyl)‐l‐homoserine lactone (3‐oxo‐C6‐HSL). Competition studies showed that cyclo(ΔAla‐l‐Val), cyclo(l‐Pro‐l‐Tyr) and cyclo(l‐Phe‐l‐Pro) antagonize the 3‐oxo‐C6‐HSL‐mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR‐binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR‐based quorum‐sensing systems, such as the N‐butanoylhomoserine lactone‐dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.
Novel Rhodobacter capsulatus genes required for the biogenesis of various c‐type cytochromes
Authors: Deshmukh Meenal , Brasseur Gael , Daldal Fevzi
Published: March 2003

Following chemical mutagenesis and screening for the inability to grow by photosynthesis and the absence of cyt cbb3 oxidase activity, two c‐type cytochrome (cyt)‐deficient mutants, 771 and K2, of Rhodobacter capsulatus were isolated. Both mutants were completely deficient in all known c‐type cyts, and could not be complemented by the previously known cyt c biogenesis genes of R. capsulatus. Complementation of 771 and K2 with a wild‐type chromosomal library led to the identification of two novel genes, cycJ and ccdA respectively. The cycJ is highly homologous to ccmE/cycJ, encountered in various Gram‐negative species. Unlike in other species, where cycJ is a part of an operon essential for cyt c biogenesis, in R. capsulatus, it is located immediately downstream from argC, involved in arginine biosynthesis. Mutation of its universally conserved histidine residue, which is critical for its proposed haem chaperoning role, to an alanine led to loss of its function. All R. capsulatus cycJ mutants studied so far excrete copious amounts of coproporphyrin and protoporphyrin when grown on enriched media, suggesting that its product is also a component of the haem delivery branch of cyt c biogenesis in this species. In contrast, the R. capsulatus ccdA was homologous to the cyt c biogenesis gene ccdA, found in the Gram‐positive bacterium Bacillus subtilis, and to the central region of dipZ, encoding a protein disulphide reductase required for cyt c biogenesis in Escherichia coli. Membrane topology of CcdA was established in R. capsulatus using ccdA ::phoA and ccdA ::lacZ gene fusions. The deduced topology revealed that the two conserved cysteine residues of CcdA are, as predicted, membrane embedded. Mutagenesis of these cysteines showed that both are required for the function of CcdA in cyt c biogenesis. This study demonstrated for the first time that CcdA homologues are also required for cyt c biogenesis in some Gram‐negative bacteria such as R. capsulatus.
Identification and characterization of mycobacteriophage L5 excisionase
Authors: Lewis John A. , Hatfull Graham F.
Published: March 2003

The well‐characterized mycobacteriophage L5 forms stable lysogens in Mycobacterium smegmatis. Establishment of lysogeny involves integration of the phage genome into the chromosome of its mycobacterial hosts through an integrase‐mediated site‐specific recombination event. As L5 lysogens spontaneously generate free phage particles, prophage excision must also occur, although an L5 excisionase gene had not been identified. We show here that L5 gene 36 encodes the phage excisionase and is a small, heat‐stable 56‐amino‐acid protein that strongly stimulates excisive recombination both in vivo and in vitro. The ability to manipulate the highly directional phage integration and excision reactions will provide powerful tools for the introduction, curing and recovery of foreign genes in recombinant mycobacterial strains.
Characterization of the Lactococcus lactis transcription factor FlpA and demonstration of an in vitro switch
Authors: Scott Colin , Guest John R. , Green Jeffrey
Published: March 2003

The commercially important bacterium Lactococcus lactis contains two FNR‐like proteins (FlpA and FlpB) which have a high degree of identity to each other and to the FLP of Lactobacillus casei. FlpA was isolated from a GST–FlpA fusion protein produced in Escherichia coli. Like FLP, isolated FlpA is a homodimeric protein containing both Zn and Cu. However, the properties of FlpA were more like those of the E. coli oxygen‐responsive transcription factor FNR than the FLP of L. casei. As prepared FlpA recognized an FNR site (TTGAT‐N4‐ATCAA) but not an FLP site (CCTGA‐N4‐TCAGG) in band‐shift assays. In contrast to FLP, DNA binding by FlpA did not require the formation of an intramolecular disulphide bond. However, despite containing only two cysteine residues per monomer, FlpA was able to acquire an FNR‐like, oxygen‐labile [4Fe 4S] cluster. But, whereas the incorporation of a [4Fe 4S] cluster into FNR enhances interaction with target DNA, it abolished DNA binding by FlpA. An FlpA variant (FlpA′) with an N‐terminal region designed to be more FLP‐like failed to incorporate an iron–sulphur cluster but could now form an intramolecular disulphide. This simple example of protein engineering, converting an oxygen‐labile [4Fe 4S] containing FNR‐like protein into a dithiol–disulphide FLP‐like redox sensor demonstrates the versatility of the basic CRP structure. Attempts to demonstrate an FlpA‐based aerobic–anaerobic switch in the heterologous host E. coli were unsuccessful. However, studies with a series of FNR‐dependent lac reporter fusions in strains of E. coli expressing flpA or flpB revealed that both homologues were able to activate expression of FNR‐dependent promoters in vivo but only when positioned 61 base pairs upstream of the transcription start.
Transcriptional and post‐transcriptional control of polynucleotide phosphorylase during cold acclimation in Escherichia coli
Authors: Zangrossi Sandro , Briani Federica , Ghisotti Daniela , Regonesi Maria Elena , Tortora Paolo , Dehò Gianni
Published: March 2003

Polynucleotide phosphorylase (PNPase, polyribonucleotide nucleotidyltransferase, EC 2.7.7.8) is one of the cold shock‐induced proteins in Escherichia coli and pnp, the gene encoding it, is essential for growth at low temperatures. We have analysed the expression of pnp upon cold shock and found a dramatic transient variation of pnp transcription profile: within the first hour after temperature downshift the amount of pnp transcripts detectable by Northern blotting increased more than 10‐fold and new mRNA species that cover pnp and the downstream region, including the cold shock gene deaD, appeared; 2 h after temperature downshift the transcription profile reverted to a preshift‐like pattern in a PNPase‐independent manner. The higher amount of pnp transcripts appeared to be mainly due to an increased stability of the RNAs. The abundance of pnp transcripts was not paralleled by comparable variation of the protein: PNPase steadily increased about twofold during the first 3 h at low temperature, as determined both by Western blotting and enzymatic activity assay, suggesting that PNPase, unlike other known cold shock proteins, is not efficiently translated in the acclimation phase. In experiments aimed at assessing the role of PNPase in autogenous control during cold shock, we detected a Rho‐dependent termination site within pnp. In the cold acclimation phase, termination at this site depended upon the presence of PNPase, suggesting that during cold shock pnp is autogenously regulated at the level of transcription elongation.
Resolution of Holliday junctions by RuvABC prevents dimer formation in rep mutants and UV‐irradiated cells
Authors: Michel Bénédicte , Recchia Gavin D. , Penel‐Colin Marion , Ehrlich S. Dusko , Sherratt David J.
Published: March 2003

In this work, we present evidence that indicates that RuvABC proteins resolve Holliday junctions in a way that prevents dimer formation in vivo. First, although arrested replication forks are rescued by recombinational repair in cells deficient for the Rep helicase, rep mutants do not require the XerCD proteins or the dif site for viability. This shows that the recombination events at arrested replication forks are generally not accompanied by the formation of chromosome dimers. Secondly, resolution of dimers into monomers is essential in the rep ruv strain because of an increased frequency of RecFOR recombination events in the chromosome of this mutant. This suggests that, in the absence of the Ruv proteins, chromosomal recombination leads to frequent dimerization. Thirdly, dif or xerC mutations increase the UV sensitivity of ruv‐deficient cells 100‐fold, whereas they do not confer UV sensitivity to ruv+ cells. This shows that recombinational repair of UV lesions is not accompanied by dimer formation provided that the RuvABC proteins are active. The requirement for dimer resolution in ruv strains is suppressed by the expression of the RusA Holliday junction resolvase; therefore, RusA also prevents dimer formation. We conclude that the inviability arising from a high frequency of dimer formation in rep or UV‐irradiated cells is only observed in the absence of known enzymes that resolve Holliday junctions.
Plasmid rolling‐circle replication: recent developments
Authors: Khan Saleem A.
Published: March 2003

It is now well established that a large majority of small, multicopy plasmids of Gram‐positive bacteria use the rolling‐circle (RC) mechanism for their replication. Furthermore, the host range of RC plasmids now includes Gram‐negative organisms as well as archaea. RC plasmids can be broadly classified into at least five families, individual members of which are spread among widely different bacteria. There is significant homology in the basic replicons of plasmids belonging to a particular family, and there is compelling evidence that such plasmids have evolved from common ancestors. Major advances have recently been made in our understanding of plasmid RC replication, including the characterization of the biochemical activities of the plasmid initiator proteins and their interaction with the double‐strand origin, the domain structure of the initiator proteins and the molecular basis for the function of single‐strand origins in plasmid lagging strand synthesis. Over the past several years, there has been a ‘renaissance’ in studies on RC replication as a result of the discovery that many plasmids replicate by this mechanism, and studies in the next few years are likely to reveal new and novel mechanisms used by RC plasmids for their regulated replication.
Regulation of growth inhibition at high temperature, autolysis, transformation and adherence in Streptococcus pneumoniae by ClpC
Authors: Charpentier Emmanuelle , Novak Rodger , Tuomanen Elaine
Published: March 2003

The ClpC ATPase is a subfamily of HSP100/Clp molecular chaperones–regulators of proteolysis. By screening a library of loss of function mutants for the ability to survive treatment with penicillin, we identified the gene clpC. The corresponding protein was identified as a ClpC ATPase, sharing strong peptide sequence identity with ClpC of Bacillus subtilis, Listeria monocytogenes and Lactococcus lactis. Northern blot experiments showed that expression of clpC was induced in response to high temperature (40–42°C) versus 37°C, suggesting that ClpC is a heat shock protein. Insertional duplication mutagenesis of clpC resulted in increased tolerance to high temperature; a result in contrast to other bacterial Clp proteases. The clpC‐deficient mutant formed long chains and failed to undergo lysis after treatment with penicillin or vancomycin. The effect of the clpC mutation extended to deficiency of adherence to the human type II alveolar cells. Finally, the clpC disruption resulted in decreased genetic transformation. Western blot analysis demonstrated that the mutant failed to express pneumolysin and the choline‐binding proteins LytA, CbpA, CbpE, CbpF, CbpJ. These results suggest that the heat shock protein ClpC plays an essential complex pleiotropic role in pneumococcal physiology, including cell growth under heat stress, cell division, autolysis, adherence and transformation.
Individual functions of the HAK and TRK potassium transporters of Schwanniomyces occidentalis
Authors: Bañuelos María A. , Madrid Ricardo , Rodríguez‐Navarro Alonso
Published: March 2003

We have cloned the gene encoding the TRK transporter of the soil yeast Schwanniomyces occidentalis and obtained the HAK1 trk1Δ and the hak1Δ TRK1 mutant strains. Analyses of the transport capacities of these mutants have shown that (i) the HAK1 and the TRK1 potassium transporters are the only transporters operating at low and medium K+ concentrations (< 1 mM); (ii) the HAK1 transporter is functional at low pH but fails at high pH; and (iii) the TRK1 transporter functions at neutral and high pH and fails at low pH. At neutral pH, both transporters are functional, but HAK1 is not expressed, except at very low K+ concentrations (< 50 µM) where HAK1 is very effective. TRK1 is also involved in the control of the membrane potential.
HrpB2 and HrpF from Xanthomonas are type III‐secreted proteins and essential for pathogenicity and recognition by the host plant
Authors: Rossier Ombeline , Van den Ackerveken Guido , Bonas Ulla
Published: March 2003

The interaction between the plant pathogen Xanthomonas campestris pv. vesicatoria and its host plants is controlled by hrp genes (hypersensitive reaction and pathogenicity), which encode a type III protein secretion system. Among type III‐secreted proteins are avirulence proteins, effectors involved in the induction of plant defence reactions. Using non‐polar mutants, we investigated the role of 12 hrp genes in the secretion of the avirulence protein AvrBs3 from X. c. pv. vesicatoria and a heterologous protein, YopE, from Yersinia pseudotuberculosis. Genes conserved among type III secretion systems (hrcQ, hrcR, hrcS and hrcT) as well as non‐conserved genes (hrpB1, hrpB2, hrpB4, hrpB5, hrpD5 and hrpD6) were shown to be required for secretion. Protein localization studies using specific antibodies showed that HrpB1 and HrpB4, as well as the putative ATPase HrcN, were mainly found in the soluble fraction of the bacterial cell. In contrast, HrpB2 and HrpF, which is related to NolX of Rhizobium fredii, are secreted into the culture medium in an hrp‐dependent manner. As HrpB2, but not HrpF, is essential for type III protein secretion, there might be a hierarchy in the secretion process. We propose that HrpF, which is dispensable for protein secretion but required for AvrBs3 recognition in planta, functions as a translocator of effector proteins into the host cell.
A two‐component system involving an HD‐GYP domain protein links cell–cell signalling to pathogenicity gene expression in Xanthomonas campestris
Authors: Slater Holly , Alvarez‐Morales Ariel , Barber Christine E. , Daniels Michael J. , Dow J. Maxwell
Published: March 2003

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris (Xcc) is regulated by a cluster of genes called rpf (for regulation of pathogenicity factors). Two of the genes, rpfF and rpfB, have previously been implicated in the synthesis of a diffusible regulatory molecule, DSF. Here, we describe a screen of transposon insertion mutants of Xcc that identified two DSF‐overproducing strains. In each mutant, the gene disrupted is rpfC, which encodes a hybrid two‐component regulatory protein in which the sensor and regulator domains are fused and which contains an additional C‐terminal phosphorelay (HPt) domain. We show that rpfC is in an operon with rpfH and rpfG. The predicted protein RpfG has a regulatory input domain attached to a specialized version of an HD domain, previously suggested to function in signal transduction. The predicted protein RpfH is structurally related to the sensory input domain of RpfC. We show that RpfC and RpfG act positively to regulate the synthesis of extracellular enzymes and EPS, but that RpfC acts negatively to regulate the synthesis of DSF. We propose that RpfGHC is a signal transduction system that couples the synthesis of pathogenicity factors to sensing of environmental signals that may include DSF itself.
The proteolytic control of restriction activity in Escherichia coli K‐12
Authors: Doronina Victoria A. , Murray Noreen E.
Published: March 2003

The endonuclease activity of EcoKI is regulated by the ClpXP‐dependent degradation of the subunit that is essential for restriction, but not modification. We monitored proteolysis in mutants blocked at different steps in the restriction pathway. Mutations that prevent DNA translocation render EcoKI refractory to proteolysis, whereas those that permit DNA translocation, but block endonuclease activity, do not. Although proteolysis alleviates restriction in a mutant that lacks modification activity, some restriction activity remains; our evidence indicates residual EcoKI associated with the membrane fraction. ClpXP protects the bacterial chromosome, but little effect was detected on unmodified foreign DNA within the cytoplasm of a restriction‐proficient cell. The molecular basis for the distinction between unmodified resident and foreign DNA remains to be determined.
A novel promoter architecture for microaerobic activation by the anaerobic transcription factor FNR
Authors: Marshall Fiona A. , Messenger Sarah L. , Wyborn Neil R. , Guest John R. , Wing Helen , Busby Stephen J. W. , Green Jeffrey
Published: March 2003

The yfiD gene of Escherichia coli has an unusual promoter architecture in which an FNR dimer located at −93.5 inhibits transcription activation mediated by another FNR dimer bound at the typical class II position (−40.5). In vitro transcription from the yfiD promoter indicated that FNR alone can downregulate yfiD expression. Analysis of yfiD::lac reporters showed that five turns of the DNA helix between FNR sites was optimal for downregulation. FNR heterodimers, in which one subunit carried a defective repression surface, revealed that the upstream subunit of the −40.5 dimer and the downstream subunit of the −93.5 dimer were most important for downregulating yfiD expression. Deletion of the C‐terminal domain of the α‐subunit of RNA polymerase (RNAP) did not affect FNR‐mediated repression, suggesting that repression is mediated through FNR–FNR and not FNR–RNAP interactions. Maximum yfiD::lac expression was observed in cultures exposed to 10 µM oxygen. More or less oxygen reduced expression dramatically. This pattern of response was dependent on the combination of a high‐affinity site at the activating class II position and a lower affinity site at the upstream position.