bluuebunny/crossref_metadata_2025_split huggingface dataset:
Competitive interaction of the OxyR DNA‐binding protein and the Dam methylase at the antigen 43 gene regulatory region in Escherichia coli
Authors: Waldron Denise E. , Owen Peter , Dorman Charles J. 
Published: March 2003

SummaryThe antigen 43 surface protein of Escherichia coli is expressed in a phase‐variable manner by a mechanism involving alternative activation and repression of transcription of the agn43 gene. The repressor is the OxyR DNA‐binding protein, and its binding site was found to be located downstream of the agn43 transcription start site in a region of DNA that encompasses three 5′‐GATC‐3′ sequences that are subject to Dam‐mediated DNA methylation. It has been suggested previously that the phase‐variable expression of antigen 43 results from a competition between Dam methylase and the OxyR repressor for these sites. The 5′‐GATC‐3′ sequences were inactivated for methylation by site‐directed mutagenesis, and all possible combinations of inactive and active sites were assessed for effects on phase‐variable expression of the agn43 gene. Inactivation of any 5′‐GATC‐3′ site individually had no effect; at least two sites had to be inactivated to disrupt the normal pattern of expression. Studies of OxyR interaction with agn43 DNA showed that methylation of any two 5′‐GATC‐3′ sites was necessary and sufficient to block binding of the repressor. It was also found that the adenines of the second and third 5′‐GATC‐3′ sites are required for OxyR binding, demonstrating that the sites for Dam methylation and for repressor binding are intimately associated. This is consistent with a competition model in which Dam and OxyR share a preference for specific DNA sequences in the regulatory region of the agn43 gene.
Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli
Authors: Wagner Patrick L. , Livny Jonathan , Neely Melody N. , Acheson David W. K. , Friedman David I. , Waldor Matthew K. 
Published: March 2003

SummaryThe stx genes of many Shiga toxin‐encoding Escherichia coli (STEC) strains are encoded by prophages of the λ bacteriophage family. In the genome of the Stx1‐encoding phage H‐19B, the stx1AB genes are found ≈ 1 kb downstream of the late phage promoter, pR′, but are known to be regulated by the associated iron‐regulated promoter, pStx1. Growth of H‐19B lysogens in low iron concentrations or in conditions that induce the prophage results in increased Stx1 production. Although the mechanism by which low iron concentration induces Stx1 production is well understood, the mechanisms by which phage induction enhances toxin production have not been extensively characterized. The studies reported here identify the factors that contribute to Stx1 production after induction of the H‐19B prophage. We found that replication of the phage genome, with the associated increase in stx1AB copy number, is the most quantitatively important mechanism by which H‐19B induction increases Stx1 production. Three promoters are shown to be involved in stx1AB transcription after phage induction, the iron‐regulated pStx1 and the phage‐regulated pR and pR′ promoters, the relative importance of which varies with environmental conditions. Late phage transcription initiating at the pR′ promoter, contrary to previous findings in the related Stx2‐encoding phage φ361, was found to be unnecessary for high‐level Stx1 production after phage induction. Finally, we present evidence that phage‐mediated lysis regulates the quantity of Stx1 produced by determining the duration of Stx1 accumulation and provides a mechanism for Stx1 release. By amplifying stx1AB copy number, regulating stx1AB transcription and allowing for Stx1 release, the biology of the Stx‐encoding phages contributes greatly to the production of Stx, the principal virulence factor of STEC.
Specific activation of the Bacillus quorum‐sensing systems by isoprenylated pheromone variants
Authors: Ansaldi Mireille , Marolt Darja , Stebe Tina , Mandic‐Mulec Ines , Dubnau David 
Published: March 2003

SummaryNatural genetic competence in Bacillus subtilis is controlled by quorum‐sensing
(QS). The ComP– ComA two‐component system detects the signalling molecule
ComX, and this signal is transduced by a conserved phosphotransfer mechanism. ComX
is synthesized as an inactive precursor and is then cleaved and modified by ComQ
before export to the extracellular environment. The comQXP′ loci
of a set of natural Bacillus isolates have been sequenced and shown to possess a striking polymorphism that determines specific patterns of both activation and inhibition of the quorum‐sensing response. We have developed a simple purification method for the modified peptide signalling pheromones allowing the characterization of four distinct ComX molecules representing different pherotypes. Genetic and biochemical evidence demonstrate that all the ComX variants are isoprenylated by the post‐translational modification of a conserved tryptophan residue and that the modifications on the ComX peptide backbones vary in mass among the various pherotypes. These results give new insights into peptidemediated quorum‐sensing signalling in Gram‐positive bacteria and emphasize the role of isoprenylation in bacterial signal transduction.
Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface
Authors: Claessen Dennis , Wösten Han A. B. , Keulen Geertje van , Faber Onno G. , Alves Alexandra M. C. R. , Meijer Wim G. , Dijkhuizen Lubbert 
Published: March 2003

SummaryThe filamentous bacteria Streptomyces coelicolor and Streptomyces lividans exhibit a complex life cycle. After a branched submerged mycelium has been established, aerial hyphae are formed that may septate to form chains of spores. The aerial structures possess several surface layers of unknown nature that make them hydrophobic, one of which is the rodlet layer. We have identified two homologous proteins, RdlA and RdlB, that are involved in the formation of the rodlet layer in both streptomycetes. The rdl genes are expressed in growing aerial hyphae but not in spores. Immunolocalization showed that RdlA and RdlB are present at surfaces of aerial structures, where they form a highly insoluble layer. Disruption of both rdlA and rdlB in S. coelicolor and S. lividans (ΔrdlAB strains) did not affect the formation and differentiation of aerial hyphae. However, the characteristic rodlet layer was absent. Genes rdlA and rdlB were also expressed in submerged hyphae that were in contact with a hydrophobic solid. Attachment to this substratum was greatly reduced in the ΔrdlAB strains. Sequences homologous to rdlA and rdlB occur in a number of streptomycetes representing the phylogenetic diversity of this group of bacteria, indicating a general role for these proteins in rodlet formation and attachment.
Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis
Authors: Kadoya Ryosuke , Hassan Anwarul K. M. , Kasahara Yasuhiro , Ogasawara Naotake , Moriya Shigeki 
Published: March 2003

SummaryCurrent views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic‐like apparatus. Furthermore, little is known about cis‐acting elements for chromosome segregation in bacteria. In this report, we show that two separate DNA regions, a 3′ coding region of dnaA and the AT‐rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis. When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC‐deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation. However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1%. In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences. These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B. subtilis.
A bZIP protein from halophilic archaea: structural features and dimer formation of cGvpE from Halobacterium salinarum
Authors: Plößer Petra , Pfeifer Felicitas 
Published: March 2003

SummaryThe cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c‐gvpA gene encoding the major gas vesicle structural protein cGvpA. Molecular modelling of the carboxy‐terminal region of cGvpE suggests that this protein resembles a basic leucine‐zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants. Mutations in the key residues of the putative leucine‐zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c‐gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function. Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE. A cGvpE protein containing a his‐tag at the C‐terminus was still able to activate the expression of c‐gvpA in vivo. The cGvpE his‐purified from Hf. volcanii formed a dimer in Blue‐native polyacrylamide gels that could be resolved into monomers by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Dimers of cGvpE were already seen using SDS–PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper. These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.
Pseudomonas aeruginosa RoxR, a response regulator related to Rhodobacter sphaeroides PrrA, activates expression of the cyanide‐insensitive terminal oxidase
Authors: Comolli James C. , Donohue Timothy J. 
Published: March 2003

SummaryThe facultative anaerobe Pseudomonas aeruginosa has multiple aerobic electron transport pathways, one of which is terminated by a cyanide‐insensitive oxidase (CIO). This study characterizes a P. aeruginosa two‐component system that regulates CIO production. The response regulator of this system (RoxR) has significant amino acid sequence similarity to PrrA of Rhodobacter sphaeroides and related proteins in other α‐proteobacteria. In heterologous complementation analysis, R. sphaeroides PrrA rescued the growth defect of a P. aeruginosa mutant lacking RoxR, and RoxR enabled photosynthetic growth of an R. sphaeroides PrrA mutant. Also, RoxR could substitute for PrrA in activating transcription in vitro, demonstrating that these proteins are functional homologues. P. aeruginosa strains lacking RoxR or the sensor kinase (RoxS) were more sensitive than wild type to the respiratory inhibitors cyanide and azide. The phenotypes of these mutant strains correlated with reduced cyanide‐insensitive O2 utilization and less cyanide‐dependent expression of the locus encoding the CIO (cioAB). The ability of purified RoxR to bind to the cioAB promoter region also suggests that this protein acts directly to regulate cioAB transcription. Therefore, RoxR appears to play a role in regulating the transcription of loci for P. aeruginosa energy‐generating enzymes similar to that of its homologues in α‐proteobacteria.
Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake
Authors: Ireland Marcia M. E. , Karty Jonathan A. , Quardokus Ellen M. , Reilly James P. , Brun Yves V. 
Published: March 2003

Summary Caulobacter crescentus
, a Gram‐negative
α
‐purple proteobacterium, is an oligotroph that lives in aquatic environments dilute in nutrients. This bacterium divides asymmetrically. Part of this asymmetric cell division involves the formation of a prosthecum at one pole, referred to as the stalk, which replaces the flagellum of the motile swarmer cell. Little is known about the synthesis or function of the stalk. The stalk is an extension of the cell membranes and peptidoglycan layer, and stalk elongation is stimulated by phosphate starvation. In this study, we have taken advantage of two‐dimensional gel (2D gel) electro‐phoresis as well as the fully sequenced genome of
Caulobacter
to study the proteome of the stalk. We modified a stalk‐shedding mutant strain of
Caulobacter crescentus
to increase the yield of stalk material shed and performed 2D gel electrophoresis of purified stalks and cellular fractions. Comparison of the stalk 2D gel with the 2D gels of cell membrane and soluble fractions showed that the stalk is mostly free of cytoplasmic proteins and has a profile very similar to that of the cell membrane. Of the 172 proteins on a stalk 2D gel, we report the identification of 64 spots, corresponding to 39 different proteins present in the stalk of
Caulobacter. The identifications include several TonB‐dependent receptors, two OmpA family proteins, a dipeptidase, GlpQ, two alkaline phosphatases, 3‐phytase, a putative TolC protein and 11 proteins of unknown function. These identifications are consistent with the hypothesis that the stalk plays a role in nutrient uptake.
Identification of a second Listeria secA gene associated with protein secretion and the rough phenotype
Authors: Lenz Laurel L. , Portnoy Daniel A. 
Published: March 2003

SummaryWe describe the identification and characterization of a second secA gene in Listeria monocytogenes. This gene, termed secA2, is involved in smooth–rough phenotypic variation and secA2 expression contributes to bacterial virulence. Spontaneous rough (R‐) variants of L. monocytogenes grow in chains and form rough colonies on solid media. A subset of R‐variants, classified here as type I, also shows reduced secretion of an autolysin, p60. We find that disruptions and in frame deletions in secA2 confer phenotypes identical to those of spontaneous type I R‐variants. Additionally, the secA2 genes from two spontaneous type I R‐variants encoded truncated SecA2 proteins. Mutations were not found in the secA2 genes from the remaining five independent R‐variants, four of which showed a distinct (type II) rough morphology and secreted wild‐type levels of p60. Expression of an epitope‐tagged SecA2 in the ΔsecA2 strain and a spontaneous R‐variant restored normal cell septation and smooth colony morphology. These data suggest that mutations in both secA2 and other genes contribute to smooth–rough phase variation in L. monocytogenes. Expression of the full‐length SecA2 also promotes secretion of p60 and a set of additional L. monocytogenes proteins. We hypothesize that SecA2‐dependent protein secretion plays a role in the colonization of environmental and host surfaces.
GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes
Authors: Ochsner Urs A. , Wilderman Paula J. , Vasil Adriana I. , Vasil Michael L. 
Published: March 2003

SummaryUpon iron restriction, the opportunistic pathogen Pseudomonas aeruginosa produces various virulence factors, including siderophores, exotoxin, proteases and haemolysin. The ferric uptake regulator (Fur) plays a central role in this response and also controls other regulatory genes, such as pvdS, which encodes an alternative sigma factor. This circuit leads to a hierarchical cascade of direct and indirect iron regulation. We used the GeneChip® to analyse the global gene expression profiles in response to iron. In iron‐starved cells, the expression of 118 genes was increased at least fivefold compared with that in iron‐replete cells, whereas the expression of 87 genes was decreased at least fivefold. The GeneChip® data correlated well with results obtained using individual lacZ gene fusions. Strong iron regulation was observed for previously identified genes involved in biosynthesis or uptake of the siderophores pyoverdine and pyochelin, utilization of heterologous siderophores and haem and ferrous iron transport. A low‐iron milieu led to increased expression of the genes encoding TonB, alkaline protease, PrpL protease, exotoxin A, as well as fumarase C, Mn‐dependent superoxide dismutase SodA, a ferredoxin and ferredoxin reductase and several oxidoreductases and dehydrogenases. Iron‐controlled regulatory genes included seven alternative sigma factors and five other transcriptional regulators. Roughly 20% of the iron‐regulated genes encoded proteins of unknown function and lacked any conclusive homologies. Under low‐iron conditions, expression of 26 genes or operons was reduced in a ΔpvdS mutant compared with wild type, including numerous novel pyoverdine biosynthetic genes. The GeneChip® proved to be a very useful tool for rapid gene expression analysis and identification of novel genes controlled by Fur or PvdS.
A malaria scavenger receptor‐like protein essential for parasite development
Authors: Claudianos Charles , Dessens Johannes T. , Trueman Holly E. , Arai Meiji , Mendoza Jacqui , Butcher Geoff A. , Crompton Tessa , Sinden Robert E. 
Published: March 2003

SummaryMalaria parasites suffer severe losses in the mosquito as they cross the midgut, haemolymph and salivary gland tissues, in part caused by immune responses of the insect. The parasite compensates for these losses by multiplying during the oocyst stage to form the infectious sporozoites. Upon human infection, malaria parasites are again attenuated by sustained immune attack. Here, we report a single copy gene that is highly conserved amongst Plasmodium species that encodes a secreted protein named PxSR. The predicted protein is composed of a unique combination of metazoan protein domains that have been previously associated with immune recognition/activation and lipid/protein adhesion interactions at the cell surface, namely: (i) scavenger receptor cysteine rich (SRCR); (ii) pentraxin (PTX); (iii) polycystine‐1, lipoxygenase, alpha toxin (LH2/PLAT); (iv) Limulus clotting factor C, Coch‐5b2 and Lgl1 (LCCL). In our assessment the PxSR molecule is completely novel in biology and is only found in Apicomplexa parasites. We show that PxSR is expressed in sporozoites of both human and rodent malaria species. Disruption of the PbSR gene in the rodent malaria parasite P. berghei results in parasites that form normal numbers of oocysts, but fail to produce any sporozoites. We suggest that, in addition to a role in sporogonic development, PxSR may have a multiplicity of functions.
Differential binding to and biofilm formation on, HEp‐2 cells by Salmonella enterica Serovar Typhimurium is dependent upon allelic variation in the fimH gene of the fim gene cluster
Authors: Boddicker Jennifer D. , Ledeboer Nathan A. , Jagnow Jennifer , Jones Bradley D. , Clegg Steven 
Published: March 2003

SummaryType 1 fimbria‐mediated adherence to HEp‐2 cells by two strains of Salmonella enterica serovar Typhimurium was found to be different. Although both strains exhibited a strong mannose‐sensitive haemagglutination reaction with guinea pig erythrocytes, characteristic of the expression of type 1 fimbriae, only one of the strains adhered in large numbers to HEp‐2 cells. Characterization of the fimH genes, encoding the fimbrial adhesins, indicated two allelic variants. Using fimH mutants of the two strains it was possible to demonstrate that binding to HEp‐2 cells was associated with the presence of one of the alleles regardless of the host strain. Therefore, this differential binding was not a function of the type I fimbrial shaft or the presence of other types of fimbriae produced by one strain but not the other. These observations may explain the differences in HEp‐2 binding by type 1 fimbriate strains of Salmonella previously reported by several groups. Also, our studies demonstrate that the FimH adhesin can influence the efficiency of biofilm formation on HEp‐2 cells using once‐flow‐through continuous culture conditions. The formation of biofilms on eukaryotic cells using this procedure is more likely to represent those conditions found in the intestinal tract than conditions using batch culture techniques to investigate adherence and biofilm formation. Indeed, the increased efficiency of biofilm formation in the murine intestinal tract confirmed the role of one of the fimH alleles in this process.
Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier
Authors: Purdy Des , O'Keeffe Triona A. T. , Elmore Michael , Herbert Mike , McLeod Anne , Bokori‐Brown Monika , Ostrowski Anna , Minton Nigel P. 
Published: March 2003

SummaryProgress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT‐based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M. CdiCD6II, with equivalent specificities to Sau96I/M. Sau96I (5′‐GGNMCC‐3′) and MboI/M. MboI (5′‐GMATC‐3′) respectively. A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5′‐CATCG‐3′ between the fourth and fifth nucleotide to give a blunt‐ended fragment. This is the first time that an enzyme with this specificity has been reported. The sequential addition of this site to vectors showed that each site caused between a five‐ and 16‐fold reduction in transfer efficiency. The transfer efficiencies achieved with both strains equated to between 1.0 × 10−6 and 5.5 × 10−5 transconjugants per donor.
Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL‐37
Authors: Schmidtchen Artur , Frick Inga‐Maria , Andersson Emma , Tapper Hans , Björck Lars 
Published: March 2003

SummaryEffectors of the innate immune system, the anti‐bacterial peptides, have pivotal roles in preventing infection at epithelial surfaces. Here we show that proteinases of the significant human pathogens Pseudomonas aeruginosa, Enterococcus faecalis, Proteus mirabilis and Streptococcus pyogenes, degrade the antibacterial peptide LL‐37. Analysis by mass spectrometry of fragments generated by P. aeruginosa elastase in vitro revealed that the initial cleavages occurred at Asn‐Leu and Asp‐Phe, followed by two breaks at Arg‐Ile, thus inactivating the peptide. Proteinases of the other pathogens also degraded LL‐37 as determined by SDS‐PAGE. Ex vivo, P. aeruginosa elastase induced LL‐37 degradation in human wound fluid, leading to enhanced bacterial survival. The degradation was blocked by the metalloproteinase inhibitors GM6001 and 1, 10‐phenantroline (both of which inhibited P. aeruginosa elastase, P. mirabilis proteinase, and E. faecalis gelatinase), or the inhibitor E64 (which inhibited S. pyogenes cysteine proteinase). Additional experiments demonstrated that dermatan sulphate and disaccharides of the structure [ΔUA(2S)‐GalNAc(4,6S)], or sucroseoctasulphate, in‐hibited the degradation of LL‐37. The results indicate that proteolytic degradation of LL‐37 is a common virulence mechanism and that molecules which block this degradation could have therapeutic potential.