bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutual control of the PecS/PecM couple, two proteins regulating virulence‐factor synthesis in Erwinia chrysanthemi
Authors: Praillet Thierry , Reverchon Sylvie , Nasser William
Published: November 2003

The Erwinia chrysanthemi pecS mutant displays constitutive production of virulence factors, such as pectinases or cellulases. Complementation of the pecS mutation can be obtained in the presence of the pecS wild‐type gene on a low‐copy‐number plasmid. Moreover, the resulting plasmid decreases the expression of a pecS::uidA chromosomal fusion, indicating the existence of an autoregulation mechanism. This negative autoregulation was confirmed and quantified by analysis of the pecS transcripts using primer‐extension experiments. Band‐shift assays and DNase I footprinting experiments demonstrated that the PecS protein could bind to the intergenic regulatory region, located between the pecS and pecM genes, with a relatively high affinity (apparent dissociation constant (K′d) close to 4 nM). These PecS‐binding sites overlap the pecS and pecM promoters. The comparison of these new PecS‐binding sites with those previously characterized on the target genes confirms the absence of a consensus. This observation was in accordance with the results of the missing‐contact experiments performed on the pecS–pecM intergenic regulatory region and the celZ operator. Concurrently, we demonstrated that the PecS protein negatively controls the expression of the divergently transcribed pecM gene located 400 bp upstream from the pecS gene. By following the efficiency of pecS autoregulation in a double E. chrysanthemi pecM–pecS mutant, we established that the PecM protein potentiates PecS activity in vivo.
In vivo mobility of a group I twintron in nuclear ribosomal DNA of the myxomycete Didymium iridis
Authors: Johansen Steinar , Elde Morten , Vader Anna , Haugen Peik , Haugli Kari , Haugli Finn
Published: November 2003

DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin‐ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron‐containing and intron‐less amoebae are mated. The mobility process is fast, being completed in 5–10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron‐encoded protein was expressed in Escherichia coli and found to be an endonuclease, I‐Dir I, that cleaves an intron‐less ribosomal DNA allele at the intron‐insertion site, and is probably involved in intron homing. The endonuclease I‐Dir I seems to be a rare example of a protein that is expressed from a ribozyme‐processed RNA polymerase I transcript in vivo.
The σS level in starving Escherichia coli cells increases solely as a result of its increased stability, despite decreased synthesis
Authors: Zgurskaya H. I. , Keyhan M. , Matin A.
Published: November 2003

The σS level in starving (stationary phase) Escherichia coli cells increases four‐ to sixfold following growth in a defined or a complex medium. Chemostat‐grown cells, subjected to increasing carbon starvation, also become progressively richer in σS content. These increases occur despite reduced transcription of the σS‐encoding gene, rpoS, and translation of rpoS mRNA, and result solely from a large increase in the stability of the sigma protein. Previous results, based on rpoS ::lacZ transcriptional and translational fusions, and on methionine incorporation in σS, had suggested increased synthesis of σS in starving cells. Alternative explanations for these results consistent with the conclusions of this paper are discussed.
Contribution of individual promoters in the ddlB–ftsZ region to the transcription of the essential cell‐division gene ftsZ in Escherichia coli
Authors: Flärdh Klas , Garrido Teresa , Vicente Miguel
Published: November 2003

The essential cell‐division gene ftsZ is transcribed in Escherichia coli from at least six promoters found within the coding regions of the upstream ddlB, ftsQand ftsA genes. The contribution of each one to the final yield of ftsZ transcription has been estimated using transcriptional lacZ fusions. The most proximal promoter, ftsZ2p, contributes less than 5% of the total transcription from the region that reaches ftsZ. The ftsZ4p and ftsZ3p promoters, both located inside ftsA, produce almost 37% of the transcription. An ftsAp promoter within the ftsQ gene yields nearly 12% of total transcription from the region. A large proportion of transcription (≈ 46%) derives from promoters ftsQ2p and ftsQ1p, which are located inside the upstream ddlB gene. Thus, the ftsQAZ genes are to a large extent transcribed as a polycistronic mRNA. However, we find that the ftsZ proximal region is necessary for full expression, which is in agreement with a recent report that mRNA cleavage by RNase E at the end of the ftsA cistron has a significant role in the contol of ftsZ expression.
Mutations in the terminase genes of bacteriophage λ that bypass the necessity for FI
Authors: Murialdo Helios , Tzamtzis Dimitra , Berrú Maribel , Fife Wendy L. , Becker Andrew
Published: November 2003

DNA maturation in bacteriophage λ is the process by which the concatemeric precursor DNA is cleaved at sites called cos to generate mature λ DNA molecules. These DNA molecules are then packaged into procapsids, the empty capsid precursors. The enzyme that catalyses these events is λ DNA terminase. It is composed of two subunits, made of 181 and 641 amino acids, the products of genes Nu1 and A, respectively. The product of the FI gene (gpFI ) stimulates the formation of an intermediate in capsid assembly called complex II, which contains a procapsid, terminase and DNA. The mechanism of stimulation remains unknown. It has been suggested that gpFI may also stimulate terminase‐mediated cos cleavage, in the absence of procapsids, by increasing enzyme turnover. Mutants in FI fail to mature and package DNA but, in comparison with other capsid gene mutants, FI mutants are leaky. Second site mutants of FI phages, called ‘fin’ (for FI independence), bypass the necessity for gpFI. These mutants were originally localized to the region of Nu1 and A and are of two classes: finA includes those that induce the synthesis of fourfold more gene A product (gpA ) than wild‐type phages, and finB includes those that produce normal amounts of gpA. Whereas all finA mutants analysed map to Nu1, finB mutants have been found both in E and in Nu1. The existence of E mutants able to bypass the necessity for gpFI in vivo shows that gpE and gpFI interact, directly or indirectly.

Here we have analysed and sequenced two finA mutants and one finB mutant. All of these map in Nu1. Of the two finA mutants, one corresponds to an Ala163Ser change and the other is a silent mutation. It is likely that the finA mutations alter mRNA conformation in a manner that results in an increase in the efficiency of A mRNA translation. The fourfold increase in gpA synthesis translates into a 10‐fold increase in terminase activity. These results show that terminase overproduction is sufficient to bypass the necessity for gpFI and that such an overproduction can be achieved by changes in the efficiency of translation of A due to subtle changes in the sequence upstream of the gene.

The finBcs103 mutation is a His‐87→Tyr change in Nu1. Therefore, an alternative way in which to bypass the requirement for gpFI involves an alteration in the structure of gpNu1. It is likely that the altered gpNu1 would increase cleavage and packaging efficiency directly or indirectly.

We have determined that DNA cleavage in vivo does not occur in the absence of gpFI. Therefore it seems that gpFI somehow facilitates an otherwise latent capacity of terminase to autoactivate its nucleolytic activity.
DNA‐binding activity of the A‐factor receptor protein and its recognition DNA sequences
Authors: Onaka Hiroyasu , Horinouchi Sueharu
Published: November 2003

The A‐factor receptor protein (ArpA) containing an α‐helix‐turn‐α‐helix DNA‐binding consensus sequence at its N‐terminal portion plays a key role in the regulation of secondary metabolism and cell differentiation in Streptomyces griseus. A binding site forming a palindrome 24 bp in length was initially recovered from a pool of random‐sequence oligonucleotides by rounds of a binding/immunoprecipitation/amplification procedure with histidine‐tagged ArpA and anti‐ArpA antibody. By means of further binding/gel retardation/amplification experiments on the basis of the recovered sequence, a 22 bp palindromic binding site with the sequence 5′‐GG(T/C)CGGT(A/T)(T/C)‐G(T/G)‐3′ as one half of the palindrome was deduced as a consensus sequence recognized and bound by ArpA. ArpA did not bind to the binding site in the presence of its ligand, A‐factor. In addition, exogenous addition of A‐factor to the ArpA–DNA complex induced immediate release of ArpA from the DNA. All of these data are consistent with the idea, obtained from previous genetic studies, that ArpA acts as a repressor‐type regulator for secondary metabolism and cellular differentiation by preventing the expression of a certain key gene(s) during the early growth phase. A‐factor, produced in a growth‐dependent manner, releases ArpA from the DNA, thus switching on the expression of the key gene(s), leading to the onset of secondary metabolism and aerial mycelium formation.
‘Locked‐on’ and ‘locked‐off’ signal transduction mutations in the periplasmic domain of the Escherichia coli NarQ and NarX sensors affect nitrate‐ and nitrite‐dependent regulation by NarL and NarP
Authors: Chiang Robin C. , Cavicchioli Ricardo , Gunsalus Robert P.
Published: November 2003

The Escherichia coli NarX, NarQ, NarL and NarP proteins comprise a two‐component regulatory system that controls the expression of many anaerobic electron‐transport and fermentation‐related genes in response to nitrate and nitrite. Either of the two sensor‐transmitter proteins, NarX and NarQ, can activate the response‐regulator proteins, NarL and NarP, which in turn are able to bind at their respective DNA regulatory sites to modulate gene expression. NarX contains a conserved 17 amino acid sequence, designated the ‘P‐box’ element, that is essential for nitrate sensing. In this study we characterize narQ mutants that also confer altered nitrate control of NarL‐dependent nitrate reductase (narGHJI ) and fumarate reductase (frdABCD) gene expression. While some narQ mutations cause the constitutive activation or repression of reporter‐gene expression even when the cells are grown in the absence of the nitrate signal (i.e. a ‘locked‐on’ phenotype), other mutations abolish nitrate‐dependent control (i.e. a ‘locked‐off’ phenotype). Interestingly the narQ (A42→T) and narQ (R50→Q) mutations along with the analogous narX18 (A46→T) and narX902 (R54→E) mutations also confer a ‘locked‐on’ or a ‘locked‐off’ phenotype in response to nitrite, the second environmental signal detected by NarQ and NarX. Furthermore, these narQ and narX mutations also affect NarP‐dependent gene regulation of nitrite reductase (nrfABCDEFG) and aeg‐46.5 gene expression in response to nitrite. We therefore propose that the NarQ sensor‐transmitter protein also detects nitrate and nitrite in the periplasmic space via its periplasmic domain. A signal transduction model, which we previously proposed for NarX, is now extended to NarQ, in which a nitrate‐ or nitrite‐detection event in the periplasmic region of the cell is followed by a signal transduction event through the inner membrane to the cytoplasmic domain of NarQ and NarX proteins to modulate their protein kinase/phosphatase activities.
Role of arginine‐43 and arginine‐69 of the Hin recombinase catalytic domain in the binding of Hin to the hix DNA recombination sites
Authors: Adams Curtis W. , Nanassy Oliver , Johnson Reid C. , Hughes Kelly T.
Published: November 2003

The Hin recombinase mediates the site‐specific inversion of a segment of the Salmonella chromosome between two flanking 26 bp hix DNA recombination sites. Mutations in two amino acid residues, R43 and R69 of the catalytic domain of the Hin recombinase, were identified that can compensate for loss of binding resulting from elimination of certain major and minor groove contacts within the hix recombination sites. With one exception, the R43 and R69 mutants were also able to bind a hix sequence with an additional 4 bp added to the centre of the site, unlike wild‐type Hin. Purified Hin mutants R43H and R69C had both partial cleavage and inversion activities in vitro while mutants R43L, R43C, R69S, and R69P had no detectable cleavage and inversion activities. These data support a model in which the catalytic domain plays a role in DNA‐binding specificity, and suggest that the arginine residues at positions 43 and 69 function to position the Hin recombinase on the DNA for a step in the recombination reaction which occurs either at and/or prior to DNA cleavage.
Induction of the Bacillus subtilis ptsGHI operon by glucose is controlled by a novel antiterminator, GlcT
Authors: Stülke Jörg , Martin‐Verstraete Isabelle , Zagorec Monique , Rose Matthias , Klier André , Rapoport Georges
Published: November 2003

Glucose is the preferred carbon and energy source of Bacillus subtilis. It is transported into the cell by the glucose‐specific phosphoenolpyruvatesugar phosphotransferase system (PTS) encoded by the ptsGHI locus. We show here that these three genes (ptsG, ptsH, and ptsI ) form an operon, the expression of which is inducible by glucose. In addition, ptsH and ptsI form a constitutive ptsHI operon. The promoter of the ptsGHI operon was mapped and expression from this promoter was found to be constitutive. Deletion mapping of the promoter region revealed the presence of a transcriptional terminator as a regulatory element between the promoter and coding region of the ptsG gene. Mutations within the ptsG gene were characterized and their consequences on the expression of ptsG studied. The results suggest that expression of the ptsGHI operon is subject to negative autoregulation by the glucose permease, which is the ptsG gene product. A regulatory gene located upstream of the ptsGHI operon, termed glcT, was also identified. The GlcT protein is a novel member of the BglG family of transcriptional antiterminators and is essential for the expression of the ptsGHI operon. A deletion of the terminator alleviates the need for GlcT. The activity of GlcT is negatively regulated by the glucose permease.
The Rhizobium meliloti exoK gene and prsD/prsE/exsH genes are components of independent degradative pathways which contribute to production of low‐molecular‐weight succinoglycan
Authors: York Gregory M. , Walker Graham C.
Published: November 2003

When grown on medium supplemented with the succinoglycan‐binding dye, Calcofluor, and visualized under UV light, colonies of Rhizobiummeliloti (Sinorhizobium meliloti ) exoK mutants produce a fluorescent halo with a delayed onset relative to wild‐type colonies. By conducting transposon mutagenesis of exoK mutants of R. meliloti and screening for colonies with even more severe delays in production of these fluorescent halos, we identified three genes, designated prsD, prsE, and exsH, which are required for the eventual production of fluorescent halos by exoK colonies. Nucleotide sequence indicates that the prsD and prsE genes encode homologues of ABC transporters and membrane fusion proteins of Type I secretion systems, respectively, whereas exsH encodes a homologue of endo‐1,3‐1,4‐β‐glycanases with glycine‐rich nonameric repeats typical of proteins secreted by Type I secretion systems. The exoK gene and the prsD/prsE/exsH genes were shown to be components of independent pathways for production of extracellular succinoglycan degrading activities and for production of low‐molecular‐weight succinoglycan by R. meliloti. Based on these results, we propose that ExsH is a succinoglycan depolymerase secreted by a Type I secretion system composed of PrsD and PrsE, and that the ExsH and ExoK glycanases contribute to production of low‐molecular‐weight succinoglycan.
beta, a novel repetitive DNA element associated with tRNA genes in the pathogenic yeast Candida albicans
Authors: Perreau Victoria M. , Santos Manuel A. S. , Tuite Mick F.
Published: November 2003

We have identified a novel 399 bp repetitive DNA element (which we designate beta ) 9 bp upstream of a seryl‐tRNACAG gene in the genome of Candida albicans. There are two copies of the seryl‐tRNACAG gene, one on each homologue of chromosome VI, and the beta element is found upstream of one copy of the gene in C. albicans strain 2005E. The beta element is not present upstream of either copy of the seryl‐tRNACAG gene in eight other laboratory strains of C. albicans tested, but was detected in this location in several fresh clinical isolates. Southern blot analysis indicated that there are approximately eight copies of the beta element per diploid C. albicans genome and that it is a mobile element, being present on at least two different chromosomes. Three unique genomic DNA clones containing the beta element were isolated from strain 2005E; in each case, a different tRNA gene was found immediately adjacent to the beta element. Three new tRNA genes from C. albicans have thus been identified: tRNAAsp, tRNAAla and tRNAIle. The beta element shows no significant sequence homology to other known prokaryotic or eukaryotic repetitive elements, although an 8 bp repeat at the 3′ end of the element is identical to that of the Ty3 retrotransposable element of Saccharomyces cerevisiae. We propose that the beta element is a solo long terminal repeat (LTR) sequence of a Ty3/gypsy‐like transposable element in C. albicans that is closely associated with tRNA genes.
The ssb‐113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli
Authors: Quiñones Ariel , Neumann Susanne
Published: November 2003

Mutations in the dnaQ gene, which encodes the proofreading ε‐subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication. In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication. We found that the ssb‐113 mutation suppresses the mutator phenotype of dnaQ49. The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions. S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter. This seems to be the consequence of an increased DNA supercoiling in the ssb‐113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling. These results support the hypothesis that the expression of the ε‐subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis.
The propeptide of Clostridium septicum alpha toxin functions as an intramolecular chaperone and is a potent inhibitor of alpha toxin‐dependent cytolysis
Authors: Sellman Bret R. , Tweten Rodney K.
Published: November 2003

Clostridium septicum alpha toxin is activated by a proteolytic cleavage at Arg‐398 in its carboxy terminus, which yields a 41.3‐kDa cytolytically active toxin and a 5.1‐kDa propeptide. Studies were performed to determine when the propeptide dissociated from the toxin after proteolytic activation of the protoxin (ATpro) and to demonstrate the chaperone activity of the propeptide. The propeptide was found to remain associated with the toxin after activation with trypsin (ATact) when analysed by gel filtration or affinity chromatography of a polyhistidine‐tagged derivative that contained the polyhistidine tag on the propeptide. The affinity of the propeptide for the toxin was decreased significantly when a mutation was introduced in which Val‐400 was converted to a cysteine residue. This mutation destabilized the interaction of the propeptide with the toxin and the propeptide was found to dissociate from the toxin under the same gel‐filtration conditions used for the wild‐type toxin. The separation of the propeptide in the V400C mutant did not affect the cytolytic activity of the toxin and therefore the propeptide was not necessary for cytolytic activity. These data suggested that the propeptide did not dissociate from the main body of the toxin after proteolysis. Further analysis demonstrated that purified propeptide was a potent inhibitor of alpha toxin activity, which inhibited the oligomerization of alpha toxin into a functional pore. These data suggest that the propeptide does not participate in the final oligomerized complex and that oligomerization appears to displace the propeptide from ATact. The importance of the propeptide to the solution stability of alpha toxin was also demonstrated. When ATpro was activated in solution with trypsin a significant level (approximately 50%) of inactive aggregate formed. This aggregate, which could be removed by centrifugation at 14 000 × g, was made up of both SDS‐sensitive and ‐resistant aggregates, suggesting that a variety of inactive aggregates formed when the monomers interacted in solution. Significantly higher levels of haemolytic activity (approximately 16‐fold) were observed when alpha toxin was proteolytically activated after membrane binding instead of in solution. These results support the role of the propeptide as an intramolecular chaperone that stabilizes the monomeric ATpro and shuttles it to the membrane where it is activated by protease, oligomerizes into a pre‐pore complex and forms a pore. The data suggest that oligomerization of the toxin displaces the propeptide from the monomer form of alpha toxin and that the propeptide does not participate in, and is not necessary to, the final cytolytic complex.
Functional analysis of chimerical plasma membrane H+‐ATPases from Saccharomyces cerevisiae and Schizosaccharomyces pombe
Authors: Kerchove d’Exaerde Alban de , Morsomme Pierre , Sempoux‐Thinès Denise , Supply Philip , Goffeau André , Ghislain Michel
Published: November 2003

The plasma membrane H+‐ATPase from the fission yeast Schizosaccharomyces pombe does not support growth of H+‐ATPase‐depleted cells of the budding yeast Saccharomyces cerevisiae, even after deletion of the enzyme’s carboxy terminus. Functional chimerical H+‐ATPase proteins in which appropriate regions of the S. pombe enzyme were replaced with their S. cerevisiae counterparts were generated by in vivo gene recombination. Site‐directed mutagenesis of the H+‐ATPase chimeras showed that a single amino acid replacement, tyrosine residue 596 by alanine, resulted in functional expression of the S. pombe H+‐ATPase. The reverse Ala‐598 →Tyr substitution was introduced into the S. cerevisiae enzyme to better understand the role of this alanine residue. However, no obvious effect on ATPase activity could be detected. The S. cerevisiae cells expressing the S. pombe H+‐ATPase substituted with alanine were enlarged and grew more slowly than wild‐type cells. ATPase activity showed a more alkaline pH optimum, lower Km values for MgATP and decreased Vmax compared with wild‐type S. cerevisiae activity. None of these kinetic parameters was found to be modified in glucose‐starved cells, indicating that the S. pombe H+‐ATPase remained fully active. Interestingly, regulation of ATPase activity by glucose was restored to a chimera in which the S. cerevisiae sequence spans most of the catalytic site.
FtsN, a late recruit to the septum in Escherichia coli
Authors: Addinall Stephen G. , Cao Chune , Lutkenhaus Joe
Published: November 2003

The localization of FtsN in Escherichia coli was investigated by immunofluorescence microscopy. FtsN is an essential cell division protein with a simple bitopic topology, a short N‐terminal cytoplasmic segment fused to a large carboxy periplasmic domain through a single transmembrane domain. FtsN was found to localize to the septum in a ring pattern similar to that observed for FtsZ and FtsA, although the frequency of cells with rings was less. A MalG–FtsN fusion was also localized to the septum, indicating that the information for FtsN localization is supplied by its periplasmic domain. FtsN localization was dependent upon the prior localization of FtsZ and FtsA and required the function of FtsI and FtsQ. Consistent with FtsN functioning after FtsZ, Z rings were observed in a mutant depleted of FtsN.
Oriented DNA binding by one‐armed λ repressor heterodimers and contacts between repressor and RNA polymerase at PRM
Authors: Kim Yong‐In , Hu James C.
Published: November 2003

Bacteriophage λ repressor activates transcription from PRM by contacting the σ subunit of E. coli RNA polymerase. Although mutations in repressors that are defective in activation affect exposed residues in the repressor–operator co‐crystal, the subunit in repressor dimers that is responsible for activation has not been determined experimentally. Here, we describe an oriented heterodimer approach using one‐armed repressor–leucine zipper fusion proteins to resolve this question. Protection against methylation of operator DNA in vitro by one‐armed and two‐armed repressor–leucine zipper heterodimers shows that the subunit containing the arm in a one‐armed repressor heterodimer binds preferentially in the consensus half‐site of λ operators. The patterns of activation of the PRM promoter in vivo by one‐armed heterodimers containing the pc2 mutation in only one subunit show that RNA polymerase at PRM contacts the N‐terminal domain of the promoter‐proximal subunit of the repressor dimer.
Identification of novel chromosomal loci affecting Yersinia enterocolitica pathogenesis
Authors: Young Glenn M. , Miller Virginia L.
Published: November 2003

Pathogenic species of the genus Yersinia have a marked tropism for lymphoid tissue during the early stages of infection. Bacterial survival at this site determines whether the disease is localized or progresses systemically, leading to a high rate of mortality. Several plasmid‐encoded virulence genes are known to be required for survival and pathogenesis, but the contribution of chromosomal genes has been largely unexplored. This study represents the first intensive effort to characterize and determine the function of Yersinia chromosomal genes expressed in lymphoid tissue after intragastric infection. Strains harbouring cat fusions expressed in the host were isolated from Peyer’s patch tissue of mice intragastrically infected and treated with chloramphenicol (Cm); genes identified in this manner were designated hre for host responsive element. The hre::cat strains that were Cm resistant in vivo (in mouse tissue) and Cm sensitive in vitro (on laboratory media at 26°C) were identified and shown to consist of 61 different allelic groups. The hre::cat fusions from 48 of the allelic groups were cloned and characterized by DNA sequence analysis. The results identified genes necessary for iron acquisition, protection from environmental stresses, biosynthesis of cell envelope components and other diverse metabolic activities. However, the DNA sequence of many clones had no homology to other known genes. Insertion mutations were constructed for four hre genes and the resulting Y. enterocolitica mutants were tested in the mouse model for effects on pathogenesis. All of the mutant strains were affected for virulence when assayed for survival in host tissues and LD50 analysis.
The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene
Authors: Tilly Kit , Casjens Sherwood , Stevenson Brian , Bono James L. , Samuels D. Scott , Hogan Daniel , Rosa Patricia
Published: November 2003

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non‐infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.
TraM of plasmid R1 controls transfer gene expression as an integrated control element in a complex regulatory network
Authors: Pölzleitner Elisabeth , Zechner Ellen L. , Renner Wilfried , Fratte Rainer , Jauk Bettina , Högenauer Gregor , Koraimann Günther
Published: November 2003

Site‐directed mutagenesis was used to investigate the functions of the traM gene in plasmid R1‐mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1‐16 plasmid, a transfer‐derepressed (finO −) variant of plasmid R1. The frequency of conjugative transfer of the traM null mutant derivative of R1‐16 was 107‐fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this gene in conjugative transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM gene of plasmid R1 has at least two functions in conjugation: (i) positive control of transfer gene expression; and (ii) a function in a process distinct from gene expression. Since expression of the negatively autoregulated traM gene is itself affected positively by the expression of the transfer operon genes, this gene constitutes a decisive element within a regulatory circuit that co‐ordinates expression of the genes necessary for horizontal DNA transfer. Based on our studies, we present a novel model for the regulation of the transfer genes of plasmid R1 that might also be applicable to other IncF plasmids.
Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation
Authors: Cotter Peggy A. , Melville Stephen B. , Albrecht Jeffrey A. , Gunsalus Robert P.
Published: November 2003

The cydAB operon of Escherichia coli encodes the cytochrome d oxidase complex, one of two aerobic terminal oxidases that catalyses the oxidation of ubiquinol‐8 and the reduction of oxygen to water. This enzyme has a higher affinity for oxygen than the cytochrome o oxidase complex and accumulates as oxygen becomes limiting. Expression of the cydAB operon is microaerobically controlled by the ArcA/ArcB two‐component regulatory system and by Fnr. To understand how ArcA and Fnr contribute to this control, a set of cyd–lacZ reporter fusions were constructed and analysed in vivo. Two cydAB promoters, designated P1 and P2, were identified by primer extension analysis and are located 288 and 173 bp upstream of the start of cydA translation respectively. Transcription from promoter P1 was shown to be regulated by both Fnr and ArcA in response to anaerobiosis. DNaseI footprint experiments revealed the locations of two Fnr binding sites at the P1 promoter: one is centred at the start of cyd transcription, while the other is positioned 53.5 bp upstream. A single ArcA‐phosphate binding site of 49 bp, centred 93 bp upstream of promoter P1, was identified to be sufficient for the activation of cydAB expression. Based on the results of the in vitro and in vivo studies, a working model for ArcA activation and Fnr repression of cydAB transcription is proposed.
Availability of the fibre subunit CsgA and the nucleator protein CsgB during assembly of fibronectin‐binding curli is limited by the intracellular concentration of the novel lipoprotein CsgG
Authors: Loferer Hannes , Hammar Mårten , Normark Staffan
Published: November 2003

Curli, an adhesive surface fibre produced by Escherichia coli and salmonellae, was proposed on the basis of genetic evidence to follow a distinct assembly pathway involving an extracellular intermediate of the fibre subunit CsgA, the polymerization of which can be induced at the cell surface by a ‘nucleator’ protein (CsgB). Here we show biochemically that CsgA is actively secreted to the extracellular milieu and that CsgB is surface located. We demonstrate that the putative curli assembly factor CsgG is an outer membrane‐located lipoprotein. CsgG is highly resistant to protease digestion both in vivo and in vitro. During curli assembly, CsgG is required to maintain the stability of CsgA and CsgB. In line with this, it is possible to modulate the steady‐state levels of CsgA and CsgB by varying intracellular levels of CsgG. This suggests that, in the absence of CsgG, CsgA and CsgB are proteolytically degraded. Moreover, curli production and steady‐state levels of CsgA and CsgB can be increased above wild‐type levels by overexpression of CsgG, meaning that the quantity of assembled curli fibres can be controlled by this lipoprotein.
Non‐palindromic attI sites of integrons are capable of site‐specific recombination with one another and with secondary targets
Authors: Hansson Karin , Sköld Ola , Sundström Lars
Published: November 2003

Genes borne on cassettes are mobile owing to site‐specific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R‐plasmids. In these processes, the palindromic site, attC (59‐base element), at cassette junctions has been proposed as being essential. Excised and circularized cassettes have been found to integrate with preference for an attI site at one end of the conserved sequence in integrons. In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attI sites. Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross‐overs between attI and secondary sites. To characterize recombination excluding the attC sites, we have used naturally occurring attI variants and constructed mutants. The cross‐over point was identified between a guanine and a thymine in attI using point mutations. Progressive deletions showed the extent of attI and identified two important regions in the conserved sequence 5′ of the cross‐over point. A region 27–36 bp 5′ of attI influenced recombination with attC sites only, whereas a sequence 9–14 bp 5′ of the cross‐over point in attI was important for recombination with both attI and attC. Recombination between attI and secondary sites could allow fusion of the conserved sequence encoding the integron site‐specific recombinase to new sequences.
Interspecies sequence differences in the Mip protein from the genus Legionella: implications for function and evolutionary relatedness
Authors: Ratcliff Rodney M. , Donnellan Stephen C. , Lanser Janice A. , Manning Paul A. , Heuzenroeder Michael W.
Published: November 2003

The nucleotide sequence of the mip genes and their inferred amino acid sequences were determined from 35 Legionella species and compared with the published sequences for L. pneumophila, L. micdadei and L. longbeachae. The sequences were 69–97% conserved at the nucleotide level and 82–99% at the amino acid level, with total conservation of amino acids determined to be associated with sites known to be involved in peptidyl prolyl cis–trans isomerase activity. No apparent difference could be determined in the arrangement of amino acids that would predict a functional difference in Mip from species associated with disease and Mip from species isolated only from the environment. Additionally, a phylogenetic comparison of the sequences with published 16S RNA sequences, using both genetic distance and maximum parsimony methods, was performed. Few relationships were apparent that were well supported by both data sets, the most robust being a clade comprising {[(cincinnatiensis, longbeachae, sainthelensi, santicrucis) gratiana] (moravica, quateirensis, shakespearei, worsleiensis) anisa, bozemanii, cherrii, dumoffii, gormanii, jordanis, parisiensis, pneumophila, steigerwaltii, tucsonensis, and wadsworthii }.
Control of the ToxR virulence regulon in Vibrio cholerae by environmental stimuli
Authors: Skorupski Karen , Taylor Ronald K.
Published: November 2003

Many bacterial pathogens regulate the expression of virulence genes in a co‐ordinate manner in response to changes in the environment. For example, the human pathogen, Vibrio cholerae, possesses a virulence regulon composed of over 20 genes involved in colonization, toxin production and bacterial survival within the host, which are co‐ordinately regulated by external stimuli, such as temperature, pH and osmolarity. Although the expression of the regulon is dependent upon the transcriptional activator ToxR, most of these genes are controlled by a second transcriptional activator, ToxT, which is itself positively regulated by ToxR. The mechanisms by which environmental stimuli influence the ToxR regulon are not yet understood, but ToxR‐mediated control over the expression of toxT clearly plays a role. The recent finding that the global regulator cAMP‐CRP also influences the expression of the ToxR regulon under various environmental conditions raises new issues regarding the pathways and mechanisms by which this regulation is achieved and indicates that multiple overlapping systems are involved.
A new Escherichia coli gene, dsbG, encodes a periplasmic protein involved in disulphide bond formation, required for recycling DsbA/DsbB and DsbC redox proteins
Authors: Andersen Catherine L. , Matthey‐Dupraz Anne , Missiakas Dominique , Raina Satish
Published: November 2003

We have identified and functionally characterized a new Escherichia coli gene, dsbG, whose product is involved in disulphide bond formation in the periplasm. The dsbG gene was cloned from a multicopy plasmid library lacking the dsbB redox protein‐encoding gene. Multicopy dsbG‐carrying clones were selected, since they allowed E. coli to grow at lethal concentrations of dithiothreitol. In a complementary genetic approach, point mutations were independently obtained and mapped to the dsbG gene. Such mutations led simultaneously to a dithiothreitol‐sensitive phenotype and an increased σE‐dependent heat shock response, which reflects the presence of misfolded proteins in the extracytoplasm. In agreement with these observations, dsbG mutants were shown to accumulate reduced forms of a variety of disulphide bond‐containing proteins in the periplasm. This DsbG defect could be rescued by addition to the growth medium of either oxidized dithiothreitol or cystine, or by overexpression of the dsbA or dsbB genes. DsbG is synthesized as a precursor form of 27.5 kDa and processed to a 25.7 kDa mature species located in the periplasm. DsbG was overproduced, purified to homogeneity and shown to have redox properties of thiol–disulphide oxidoreductases in vitro. Replacement of the first Cys residue of the predicted active site, Phe–(Xaa)4–Cys–Pro–Tyr–Cys by Ala, completely inactivated DsbG protein function. Taken together, all our results demonstrate that DsbG acts in vivo as an efficient thiol–disulphide oxidase. In addition, dsbG is the first member of the dsb family for which null mutations are conditionally lethal and can be propagated only if supplemented with oxidants in the growth medium. We propose that the main role of DsbG is to maintain the proper redox balance between the DsbA/DsbB and DsbC systems.
Differences in the control of the temperature‐dependent expression of four genes for desaturases in Synechocystis sp. PCC 6803
Authors: Los Dmitry A. , Ray Malay K. , Murata Norio
Published: November 2003

Cyanobacteria are capable of desaturating the fatty acids in their membrane lipids in response to decreases in temperature. The cyanobacterium, Synechocystis sp. PCC 6803, contains four desaturases, which specifically catalyse desaturation at the Δ6, Δ9, Δ12 and ω3 positions of fatty acids. The levels of the mRNAs transcribed from the genes that encode the Δ6, Δ12 and ω3 desaturases increased about 10‐fold, but at different rates, upon a decrease in temperature from 34°C to 22°C, whereas the level of the mRNA for the Δ9 desaturase remained constant. The increases in the levels of mRNAs were caused both by the enhanced transcription and by the increased stability of the mRNAs at the low temperature. Western blotting analysis demonstrated that levels of the Δ6, Δ12 and ω3 desaturases increased at different rates at the low temperature, while that of the Δ9 desaturase remained constant. These observations indicate that the expression of the genes for the four desaturases is regulated by temperature in different ways.
Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii
Authors: Dobrowolski Janice M. , Carruthers Vern B. , Sibley L. David
Published: November 2003

Toxoplasma gondii is an obligate intracellular parasite that actively invades mammalian cells using a unique form of gliding motility that critically depends on actin filaments in the parasite. To determine if parasite motility is driven by a myosin motor, we examined the distribution of myosin and tested the effects of specific inhibitors on gliding and host cell invasion. A single 90 kDa isoform of myosin was detected in parasite lysates using an antisera that recognizes a highly conserved myosin peptide. Myosin was localized in T. gondii beneath the plasma membrane in a circumferential pattern that overlapped with the distribution of actin. The myosin ATPase inhibitor, butanedione monoxime (BDM), reversibly inhibited gliding motility across serum‐coated slides. The myosin light‐chain kinase inhibitor, KT5926, also blocked parasite motility and greatly reduced host cell attachment; however, these effects were primarily caused by its ability to block the secretion of microneme proteins, which are involved in cell attachment. In contrast, while BDM partially reduced cell attachment, it prevented invasion even under conditions in which microneme secretion was not affected, indicating a potential role for myosin in cell entry. Collectively, these results indicate that myosin(s) probably participate(s) in powering gliding motility, a process that is essential for cell invasion by T. gondii.
Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol
Authors: De Bernard Marina , Arico' Beatrice , Papini Emanuele , Rizzuto Rosario , Grandi Guido , Rappuoli Rino , Montecucco Cesare
Published: November 2003

Cells exposed to Helicobacter pylori toxin VacA develop large vacuoles that originate from massive swelling of membranous compartments of late stages of the endocytic pathway. To determine if the toxin is active from the cell cytosol, cells were either microinjected with toxin or transfected with plasmids encoding VacA. Both procedures cause formation of intracellular vacuoles. Cytosolic localization of the toxin was assessed by indirect immunofluorescence with specific antibodies and by expression of an active green fluorescence protein (GFP)–VacA chimera. Vacuoles induced by internally produced VacA are morphologically and functionally identical to those induced by externally added toxin. It is concluded that VacA is a toxin acting intracellularly by altering a cytosol‐exposed target, possibly involved in the control of membrane trafficking.
The non‐standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation?
Authors: Santos Manuel A. S. , Ueda Takuya , Watanabe Kimitsuna , Tuite Mick F.
Published: November 2003

A number of yeasts of the genus Candida translate the standard leucine‐CUG codon as serine. This unique genetic code change is the only known alteration to the universal genetic code in cytoplasmic mRNAs, of either eukaryotes or prokaryotes, which involves reassignment of a sense codon. Translation of CUG as serine in these species is mediated by a novel serine‐tRNA (ser‐tRNACAG), which uniquely has a guanosine at position 33, 5′ to the anticodon, a position that is almost invariably occupied by a pyrimidine (uridine in general) in all other tRNAs. We propose that G‐33 has two important functions: lowering the decoding efficiency of the ser‐tRNACAG and preventing binding of the leucyl‐tRNA synthetase. This implicates this nucleotide as a key player in the evolutionary reassignment of the CUG codon. In addition, the novel ser‐tRNACAG has 1‐methylguanosine (m1G‐37) at position 37, 3′ to the anticodon, which is characteristic of leucine, but not serine tRNAs. Remarkably, m1G‐37 causes leucylation of the ser‐tRNACAG both in vitro and in vivo, making the CUG codon an ambiguous codon: the polysemous codon. This indicates that some Candida species tolerate ambiguous decoding and suggests either that (i) the genetic code change has not yet been fully established and is evolving at different rates in different Candida species; or (ii) CUG ambiguity is advantageous and represents the final stage of the reassignment. We propose that such dual specificity indicates that reassignment of the CUG codon evolved through a mechanism that required codon ambiguity and that ambiguous decoding evolved to generate genetic diversity and allow for rapid adaptation to environmental challenges.
Instability of artificially circularized chromosomes of Streptomyces lividans
Authors: Lin Yi‐Shing , Chen Carton W.
Published: November 2003

The chromosomes of Streptomyces species are linear molecules, containing long terminal inverted repeats and covalently bound terminal proteins. These chromosomes undergo spontaneous deletions of the terminal sequences at high frequencies and become circularized in several cases examined. Artificial circularization of the Streptomyces lividans chromosome was also achieved by targeted recombination in vivo, in which the terminal inverted repeats of the chromosome were connected by a kanamycin resistance gene (aphII ). Under kanamycin selection, the circularized chromosomes harboured tandem amplifications of a 20.2 kb sequence that included the aphII gene flanked by direct repeats and deletions nearby. On release from kanamycin selection, the aphII amplifications and the neighbouring sequences were deleted from the chromosomes, rendering all the cultures kanamycin sensitive. The chloramphenicol resistance gene, which was prone to deletion in wild‐type S. lividans, became much more stable in the kanamycin‐sensitive derivatives. These results indicate that the telomeres and/or certain terminal sequences may be involved in the structural instability of Streptomyces chromosomes.