bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence analysis and molecular characterization of the temperate lactococcal bacteriophage r1t
Authors: van Sinderen Douwe , Karsens Harma , Kok Jan , Terpstra Peter , Ruiters Marcel H. J. , Venema Gerard , Nauta Arjen
Published: October 2006

The temperate lactococcal bacteriophage r1t was isolated from its lysogenic host and its genome was subjected to nucleotide sequence analysis. The linear r1t genome is composed of 33 350 bp and was shown to possess 3′ staggered cohesive ends. Fifty open reading frames (ORFs) were identified which are, probably, organized in a life‐cycle‐specific manner. Nucleotide sequence comparisons, N‐terminal amino acid sequencing and functional analyses enabled the assignment of possible functions to a number of DNA sequences and ORFs. In this way, ORFs specifying regulatory proteins, proteins involved in DNA replication, structural proteins, a holin, a lysin, an integrase, and a dUTPase were putatively identified. One ORF seems to be contained within a self‐splicing group I intron. In addition, the bacteriophage att site required for site‐specific integration into the host chromosome was determined.
Atomic structure and specificity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes
Authors: Quiocho Florante A. , Ledvina Polly S.
Published: October 2006

Crystallographic structure refinement at very high resolutions of a dozen periplasmic receptors has revealed that, though they have different sizes (26 to 60kDa) and little sequence homology, they have high tertiary structure similarity. They consist of two distinct globular domains bisected by a cleft or groove wherein the ligand binds and is buried by a hinge‐bending motion between the two domains. Structural analysis also reveals how hydrogen‐bonding interactions can be tailored to a wide spectrum of specificity, ranging from the stringent specificity for phosphate and sulphate to the more loose specificity for peptides.
Cloning of a second gene encoding 6‐phosphofructo‐2‐kinase in yeast, and characterization of mutant strains without fructose‐2,6‐bisphosphate
Authors: Bolesm Eckhard , Göhlmann Hinrich W. H. , Zimmermann Friedrich K.
Published: October 2006

We have identified a new gene, PFK27, that encodes a second inducible 6‐phosphofructo‐2‐kinase in the yeast Saccharomyces cerevisiae. Sequencing shows an open reading frame of 397 amino acids and 45.3 kDa. Amino acid sequence comparisons with other bifunctional 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase isoenzymes of various organisms revealed similarities only to the kinase domains. Expression of PFK27 was induced severalfold by glucose and sucrose, but not by galactose or maltose, suggesting that sugar transport might be involved in triggering the induction signal. We have constructed a mutant strain devoid of any fructose‐2,6‐bisphosphate. The mutant strain grew well on several kinds and concentrations of carbon sources. The levels of hexose phosphates in the cells were increased, but flux rates for glucose utilization and ethanol production were similar to the wild‐type strain. However, after the transfer of the mutant cells from respiratory to fermentative growth conditions, growth, glucose consumption and ethanol production were delayed in a transition phase. Our results show that fructose‐2,6‐bisphosphate is an important effector in vivo of the 6‐phosphofructo‐1‐kinase/fructose‐1,6‐bisphospha‐tase enzyme pair, and is involved in the initiation of glycolysis during the transition to a fermentative mode of metabolism. Nevertheless, it can be effectively replaced by other effectors and regulatory mechanisms during growth on glucose.
The ATP‐binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3
Authors: Mahé Yannick , Parle‐McDermott Anne , Nourani Amine , Delahodde Agnès , Lamprecht Andrea , Kuchler Karl
Published: October 2006

Pleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP‐binding cassette (ABC) efflux pumps such as Pdr5 and Snq2. Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR. We show here that a pdr1–3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4‐nitroquinoline‐N‐oxide, a known substrate for Snq2 but not for Pdr5. Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10‐fold overexpressed in a pdr1–3 gain‐of‐function mutant strain, whereas Snq2 expression is severely reduced in a Δpdr1 deletion strain, and almost abolished in a Δpdr1Δpdr3 double disruptant when compared to the PDR1 strain. However, expression of the Ste6 a‐factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1–3 mutant cells and in strains carrying Δpdr1, Δpdr3, or Δpdr1Δpdr3 deletions. Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3. Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1–3 mutant strain results from overexpression of more than one ABC drug‐efflux pump.
Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of Listeria monocytogenes
Authors: Guzmán Carlos A. , Domann Eugen , Ronde Manfred , Bruder Dunja , Darji Ayub , Weiss Siegfried , Wehland Jürgen , Chakraborty Trinad , Timmis Kenneth N.
Published: October 2006

Infection of a murine‐spleen dendritic cell line by Listeria monocytogenes was found to induce cell death through apoptosis. To characterize the bacterial product(s) involved in induction of apoptosis, dendritic cells were infected with the L. monocytogenes EGD strain and several isogenic mutants deficient in the production of individual listerial virulence factors. The ability to induce cellular apoptosis was retained by all mutants tested, except the prfA and Δhly mutants, both of which are unable to produce listeriolysin. Apoptosis was also induced by purified listeriolysin suggesting that this protein directly induces apoptosis. Purified recombinant listeriolysins rendered either weakly haemolytic by a C‐484 to S mutation, or non‐haemolytic by a W‐491 to A mutation exhibited little or no capacity to induce apoptosis, indicating that both activities are associated within the same protein region. Treatment with purified listeriolysin or L. monocytogenes infection also triggers apoptosis in explanted bone‐marrow dendritic cells. Thus invasion of dendritic cells by L. monocytogenes, which results in cell death, may play an important role in the pathogenesis of listerial infections by impairing immune responses, hindering bacterial clearance and promoting spread of the infection.
Vibrio parahaemolyticus FlaJ, a homologue of FliS, is required for production of a flagellin
Authors: Stewart Bonnie J. , McCarter Linda L.
Published: October 2006

The flaA locus of Vibrio parahaemolyticus encodes one of the four polar flagellin genes, the flagellum‐capping protein HAP2, and three additional flagellar genes. Sequence analysis downstream of the gene encoding HAP2 revealed the region to be similar to the fliD (HAP2) locus of Pseudomonas aeruginosa. The deduced protein sequences for the newly identified genes suggest that one protein belongs to the family of transcriptional regulatory proteins known to interact with σ54, one may be a rod component of the flagellum, and one resembles the FMS protein. fliS is an essential flagellar gene in many bacteria; however its function is not clear. The V. parahaemolyticus polar flaC flagellin gene was poorly expressed in Escherichia coli. Production of FlaC was stimulated by provision of the flaA locus in trans. Dissection of this locus revealed that the fliS‐like gene, flaJ, was required for increased expression of flaC. Stimulation by FlaJ occurred in E. coli mutants defective in either the master flagellar‐controlling operon or the gene encoding the flagellar σ28. Therefore the effect of FlaJ was not mediated through flagellar proteins. Nor was it mediated through σ54, for enhanced FlaC production was observed in mutants with defects in the gene encoding σ54.
Tandem repeats of the tetramer 5′‐CAAT‐3’present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae
Authors: High Nicola J. , Jennings Michael P. , Moxon E. Richard
Published: October 2006

A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase‐variable LPS structure expressed by Haemo philus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neis seria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)n encoded by multiple tandem repeats of 5′‐CAAT‐3′. This observation suggested that (SINQ)n might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5′‐CAAT‐3’repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine‐SDS‐PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)n is not a prerequisite for the enzymatic function of Lic2A and that the 5′‐CAAT‐3’repeats in lic2A function solely as a mechan ism for generating phase variation. This observation suggested that wide variation in the number of 5’‐CAAT‐3’repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5′‐CAAT‐3’repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)n indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.
A gene product related to Tral is required for the mobilization of Bacteroides mobilizable transposons and plasmids
Authors: Smith C. Jeffrey , Parker Anita C.
Published: October 2006

The antibiotic‐resistance transposon Tn4555 from Bacteroides can be transferred between strains by conjugation. The transposon is not self‐transmissible and must be mobilized by resident chromosomal tetracy‐cline‐resistance elements. In the present report, the mechanism of transfer was examined at the genetic level by deletion analysis and nucleotide sequencing of clones that conferred a transmissible phenotype on a non‐mobilizable plasmid. The results suggested that the product of mobATn was required for mobilization and it worked in concert with a cis‐acting oriT‐like sequence. This mechanism was compared with the mobilization system of a cryptic Bacteroides plasmid, pBl143, and the two systems were found to share a common transfer strategy. The mobA gene products from both genetic elements were related and they had limited homology to the broad group of mobilization proteins (relaxases) typified by Tral of RP4. Phylogenetic analysis of MobA and several other mobilization proteins from commensal gastrointestinal tract organisms suggested that they formed a new subgroup of the Tral superfamily. The mobilization regions of both Tn4555 and pBl143 were located on discrete segments of DNA within the parent genetic element. These segments were delineated by regions of secondary structure, suggesting that they could be defined mobilization cassettes.
Ste50p sustains mating pheromone‐induced signal transduction in the yeast Saccharomyces cerevisiae
Authors: Xu Gang , Jansen Gregor , Thomas David Y. , Hollenberg Cornelis P. , Rad Massoud Ramezani
Published: October 2006

In the yeast Saccharomyces cerevisiae, the hetero‐trimeric G protein transduces the mating pheromone signal from a cell‐surface receptor. Free Gβγ then activates a mitogen‐activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal‐transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone‐induced signal which leads cells to hormone‐induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone‐induced signal transduction, and overexpression of STE50 intensifies the pheromone‐induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α‐pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two‐hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.
A single substitution in the putative helix‐turn‐helix motif of the pleiotropic activator PrfA attenuates Listeria monocytogenes virulence
Authors: Sheehan Brian , Klarsfeld André , Ebright Richard , Cossart Pascale
Published: October 2006

Prf A, the regulator of virulence‐gene expression in the pathogenic bacterium Listeria monocytogenes, displays sequence similarity to members of the CAP‐FNR family of transcriptional regulators. To test the functional significance of this similarity, we constructed and analysed substitutions of two amino acids of Prf A predicted to contact DNA, i.e. Ser‐184 and Ser‐183. Substitution of Ser‐184 by Ala reduced DNA binding and virulence‐gene activation, and attenuated the virulence in a mouse model of infection. In contrast, substitution of Ser‐183 by Ala had the opposite effect in these functional assays. A 17bp DNA sequence, which includes a putative PrfA site, was shown to be sufficient for target‐site recognition by PrfA and PrfA‐S183A. Our results strongly support the hypothesis that PrfA is a structural and functional homologue of CAP. In addition, they establish a clear correlation between DNA binding by PrfA, virulence‐gene activation, and virulence.
Interaction of Cody, a novel Bacillus subtillis DNA‐binding protein, with the dpp promoter region
Authors: Serror† Pascale , Sonenshein Abraham L.
Published: October 2006

The product of the codY gene is required for nutritional repression of the Bacillus subtilis dipeptide permease operon (dpp), an operon expressed at early stationary phase in nutrient‐rich medium. Though unrelated to any known DNA‐binding protein, Cody was shown to bind specifically to the dpp promoter region. DNase I footprinting experiments revealed that the Cody‐protected region encompasses the dpp transcription start site and overlaps with the region protected by another regulatory protein, AbrB. Cody and AbrB were found to compete, in vitro, for binding to the dpp promoter region. Binding of Cody was altered in mutants defective in nutritional regulation.
Characterization of an Escherichia coli mutant, feeA, displaying resistance to the calmodulin inhibitor 48/80 and reduced expression of the rare tRNA3Leu
Authors: Bouquin Nicolas , Chen Mao X. , Kim Sunghoon , Vannier Françoise , Bernard Sylvie , Holland I. Barry , Séror Simone J.
Published: October 2006

We previously described a mutation feeB1 conferring a temperature‐sensitive filamentation phenotype and resistance to the calmodulin inhibitor 48/80 in Escherichia coli, which constitutes a single base change in the acceptor stem of the rare tRNA3Leu recognizing CUA codons. We now describe a second mutant, feeA1, unlinked to feeB, but displaying a similar phenotype, 48/80 resistance and a reduced growth rate at the permissive temperature, 30°C, and temperature‐sensitive, forming short filaments at 42°C. In the feeA mutant, tRNA3Leu expression (but not that of tRNA1Leu) was reduced approximately fivefold relative to the wild type. We previously showed that the synthesis of β‐galactosidase, which unusually requires the translation of 6‐CUA codons, was substantially reduced, particularly at 42°C, in feeB mutants. The feeA mutant also shows drastically reduced synthesis of β‐galactosidase at the non‐permissive temperature and reduced levels even at the permissive temperature. We also show that increased copy numbers of the abundant tRNA1Leu, which can also read CUA codons at low efficiency, suppressed the effects of feeA1 under some conditions, providing further evidence that the mutant was deficient in CUA translation. This, and the previous study, demonstrates that mutations which either reduce the activity of tRNA3Leu or the cellular amount of tRNA3Leu confer resistance to the drug 48/80, with concomitant inhibition of cell division at 42°C.
From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli
Authors: Pratt Leslie A. , Hsing Weihong , Gibson Katherine E. , Silhavy Thomas J.
Published: October 2006

In Escherichia coli, levels of the two major outer membrane porin proteins, OmpF and OmpC, are regulated in response to a variety of environmental parameters, and numerous factors have been shown to influence porin synthesis. EnvZ and OmpR control porin‐gene transcription in response to osmolarity, and the anti‐sense RNA, MicF, influences ompF translation. In contrast to these characterized factors, some of the components reported to influence porin expression have only modest effects and/or act indirectly. For others, potential regulatory roles, although intriguing, remain elusive. Here we review many of the components that have been reported to influence porin expression, address the potential regulatory nature of these components, and discuss how they may contribute to a regulatory network controlling porin synthesis.
Assembly of human contact phase proteins and release of bradykinin at the surface of curli‐expressing Escherichia coli
Authors: Ben Nasr Abdelhakim , Olsén Arne , Sjöbring Ulf , Müller‐Esterl Werner , Björck Lars
Published: October 2006

Previous work has demonstrated that most strains of the human pathogen Streptococcus pyogenes bind kininogens through M protein, a fibrous surface protein and virulence determinant. Here we find that strains of several other pathogenic bacterial species, both Gram‐positive and Gram‐negative, isolated from patients with sepsis, also bind kininogens, especially H‐kininogen (HK). The most pronounced interaction was seen between HK and Escherichia coli. Among clinical isolates of E. coli, the majority of the entero‐haemorrhagic, enterotoxigenic, and sepsis strains, but none of the enteroinvasive and enteropathogenic strains, bound HK. Binding of HK to E. coli correlated with the expression of curli, another fibrous bacterial surface protein, and the binding of HK to purified curli was specific, saturable, and of high affinity; Ka = 9 107M‐1. Other contact phase proteins such as factor XI, factor XII, and prekallikrein bound to curliated E. coli, but not to an isogenic curli‐deficient mutant strain, suggesting that contact phase activation may occur at the surface of curliated bacteria. Kininogens are also precursor molecules of the vasoactive kinins. When incubated with human plasma, curli‐expressing bacteria absorbed HK. Addition of purified plasma kallikrein to the HK‐loaded bacteria resulted in a rapid and efficient release of bradykinin from surface‐bound HK. The assembly of contact phase factors at the surface of pathogenic bacteria and the release of the potent proinflammatory and vasoactive peptide bradykinin, should have a major impact on the host‐microbe relationship and may contribute to bacterial pathogencity and virulence.
Escherichia coli strains in which chromosome replication is controlled by a P1 or F replicon integrated into oriC
Authors: Eliasson Åsa , Nordström Kurt , Bernander Rolf
Published: October 2006

We report the construction of intP1 and intFs strains, in which the basic replicon from either plasmid P1 or plasmid F (oriS) has been integrated in both orientations into the origin of replication, oriC, of the Escherichia coli chromosome. In these strains, oriC is no longer functional and chromosome‐replication is instead controlled by the integrated plasmid replicon. The strains were viable, showing that the deviation from normal chromosome‐replication control was not large enough to prohibit cell survival. The strains showed a broader cell‐size distribution than a wild‐type strain and were more filamentous in rich than in minimal media, although cells of wild‐type size were also present. Cells which contained aberrantly shaped or aberrantly distributed nucleoids were also observed. Marker‐frequency analysis indicated that chromosome replication was predominantly bidirectional in both intFs strains. In the intP1 strains, the degree of bidirectionality depended upon the orientation of the integrated replicon.
Circadian expression of genes involved in the purine biosynthetic pathway of the cyanobacterium Synechococcus sp. strain PCC 7942
Authors: Liu Yi , Tsinoremas Nicholas F. , Golden Susan S. , Kondo Takao , Johnson Carl Hirschie
Published: October 2006

Extensive circadian (daily) control over gene expression in the cyanobacterium Synechococcus sp. strain PCC 7942 is programmed into at least two differentially phased groups. The transcriptional activity of the smaller group of genes is maximal at about dawn and minimal at about dusk. We identified one of the genes belonging to this latter group as purF, which encodes the key regulatory enzyme in the de novo purine synthetic pathway, glutamine PRPP amidotransferase (also known as amidophosphoribosyltransferase). Its expression pattern as a function of circadian time was confirmed by both luminescence from a purF:: luxAB reporter strain and the abundance of purF mRNA. By fusing sequences upstream of the purF coding region to promoterless luxAB genes, we identified a limited upstream region, which potentially regulates purF circadian expression patterns in vivo. We also identified the purL gene immediately upstream of purF. The purL gene encodes FGAM synthetase, the fourth enzyme in the purine nucleotide biosynthesis pathway. Although these genes are expressed as part of a larger operon in other bacteria, reporter gene fusions revealed that purF and purL are transcribed independently in Synechococcus and that they are expressed at different phases of the circadian cycle. This differential expression pattern may be related to the oxygen sensitivity of amidophosphoribosyltransferase.
RbfA, a 30S ribosomal binding factor, is a cold‐shock protein whose absence triggers the cold‐shock response
Authors: Jones Pamela G. , Inouye Masayori
Published: October 2006

The cold‐shock response, characterized by a specific pattern of gene expression, is induced upon a downshift in temperature and in the presence of inhibitors of ribosomal function. Here, we demonstrate that RbfA of Escherichia coli, considered to be involved in ribosomal maturation and/or initiation of translation, is a cold‐shock protein. Shifting the rbfA mutant to a lower temperature resulted in a constitutive induction of the cold‐shock response accompanied by slower growth at low temperatures, while shifting the rbfA mutant that overproduces wild‐type RbfA resulted in an increase in total protein synthesis accompanied by faster growth adaptation to the lower temperature. Furthermore, the cold‐shock response was also constitutively induced in a cold‐sensitive 16S rRNA mutant at low temperatures. Accompanying the transient induction of the cold‐shock response, we also report that shifting E. coli from 37°C to 15°C resulted in a temporary inhibition of initiation of translation, as evidenced by the transient decrease in polysomes accompanied by the transient increase in 70S monosomes. The accumulative data indicate that the inducing signal for the cold‐shock response is the increase in the level of cold‐unadapted non‐translatable ribo‐somes which are converted to cold‐adapted translatable ribosomes by the association of cold‐shock proteins such as RbfA. Therefore, the expression of the cold‐shock response, and thus cellular adaptation to low temperature, is regulated at the level of translation. The data also indicate that cold‐shock proteins can be translated by ribosomes under conditions that are not translatable for most mRNAs.
The vacuolating cytotoxin of Helicobacter pylori
Authors: Cover Timothy L.
Published: October 2006

Helicobacter pylori, the causative agent of chronic superficial gastritis and duodenal ulcer disease in humans, produces a unique cytotoxin (VacA) that induces cytoplasmic vacuolation in eukaryotic cells. The structural organization and processing of the vacuolating cytotoxin are characteristic of a family of proteins exemplified by Neisseria gonorrhoeae IgA protease. Although only 50% of H. pylori isolates produce detectable cytotoxin activity in vitro, vacA homologues are present in virtually all isolates. Several families of vacA alleles have been identified, and there is a strong correlation between presence of specific vacA genotypes, cytotoxin activity, and peptic ulceration. Experiments in a mouse model of H. pylori‐induced gastric damage indicate that the cytotoxin plays an important role in inducing gastric epithelial necrosis.
EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells
Authors: Kenny Brendan , Lai Li‐Ching , Finlay B. Brett , Donnenberg Michael S.
Published: October 2006

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhoea. EPEC mediates several effects on host epithelial cells, including activation of signal‐transduction pathways, cytoskeletal rearrangement along with pedestal and attachingleffacing lesion formation. It has been previously shown that the EPEC eaeB (espB) gene encodes a secreted protein required for signal transduction and adherence, while eaeA encodes intimin, an EPEC membrane protein that mediates intimate adherence and contributes to focusing of cytoskeletal proteins beneath bacteria. DNA‐sequence analysis of a region between eaeA and eaeB identified a predicted open reading frame (espA) that matched the amino‐terminal sequence of a 25 kDa EPEC secreted protein. A mutant with a non‐polar insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement. These phenotypes were complemented by a cloned espA gene. The espA mutant is also defective for invasion. It is concluded that espA encodes an EPEC secreted protein that is necessary for activating epithelial signal transduction, intimate contact, and formation of attaching and effacing lesions, processes which are central to pathogenesis.
A cluster of fourteen genes from enteropathogenic Escherichia coli is sufficient for the biogenesis of a type IV pilus
Authors: Stone Kelly D. , Zhang Hong‐Zhong , Carlson Lisa K. , Donnenberg Michael S.
Published: October 2006

Enteropathogenic Escherichia coli (EPEC) adhere to epithelial cells in microcolonies, a pattern termed localized adherence (LA). LA is dependent upon the presence of 50–70MDa plasmids, termed EPEC adherence factor (EAF) plasmids. Expression of an EAF plasm id‐encoded type IV fimbria, the bundle‐forming pilus (BFP), is associated with the LA phenotype. TnphoA insertions in bfpA, the gene encoding the major structural subunit of the BFP, abolish LA. While bfpA::TnphoA mutants cannot be complemented for LA by plasmids carrying the bfpA gene alone in trans, this work shows that they can be complemented by plasmids carrying the bfpA gene, as well as approximately 10kb of downstream sequence, suggesting that such mutations have polar effects on downstream genes. The identification and characterization of a cluster of 13 genes immediately downstream of bfpA are described. The introduction into a laboratory Escherichia coli strain of a plasmid containing these 14 bfp gene cluster genes, along with pJPN14, a plasmid containing another fragment derived from the EAF plasmid, confers LA ability and BFP biogenesis. However, when a mutation is introduced into the last gene of the bfp cluster, neither LA nor BFP biogenesis is conferred. This work also provides evidence to show that the fragment cloned in pJPN14 encodes a factor(s) which results in increased levels of the pilin protein. Finally, it is shown that expression of the 14 genes in the bfp cluster from an IPTG‐inducible promoter, in the absence of pJPN14, is sufficient to reconstitute BFP biogenesis in a laboratory E. cob strain, but is insufficient for LA. This is the first report demonstrating the reconstitution of a type IV pilus in a laboratory E. coli strain with a defined set of genes. The 8FP system should prove to be a useful model for studying the molecular mechanisms of type IV pilus biogenesis.
Bacillus subtilis operon under the dual control of the general stress transcription factor σB and the sporulation transcription factor σH
Authors: Varón Débora , Brody Margaret S. , Price Chester W.
Published: October 2006

The σB transcription factor of Bacillus subtilis is activated in response to a variety of environmental stresses, including those imposed by entry into the stationary‐growth phase, and by heat, salt or ethanol challenge to logarithmically growing cells. Although σB is thought to control a general stress regulon, the range of cellular functions it directs remains largely unknown. Our approach to understand the physiological role of σB is to characterize genes that require this factor for all or part of their expression, i.e. the csb genes. In this study, we report that the transposon insertion csb40::Tn917lac identifies an operon with three open reading frames, the second of which resembles plant proteins induced by desiccation stress. Primer‐extension and operon‐fusion experiments showed that the csb40 operon has a σB‐dependent promoter which is strongly induced by the addition of salt to logarithmically growing cells. The csb40 operon also has a second, σH‐dependent promoter that is unaffected by salt addition. These results provide support for the hypothesis that σB controls a general stress regulon, and indicate that the σB and σH regulons partly overlap. We suggest that in addition to its acknowledged role in the sporulation process, σH is also involved in controlling a subclass of genes that are broadly involved in a general stress response.
Differential regulation of the virulence genes of Listeria monocytogenes by the transcriptional activator PrfA
Authors: Bohne Jutta , Kestler Hubert , Uebele Corinna , Sokolovic Zeljka , Goebel Werner
Published: October 2006

The two Listeria monocytogenes strains EGD and NCTC 7973 display a different regulation pattern of their PrfA‐dependent genes. All PrfA‐dependent genes from L. monocytogenes NCTC 7973 are much more efficiently transcribed in brain‐heart infusion medium than those from strain EGD. Transcription of these genes in EGD is, however, induced after shift into Minimal Essential Medium (MEM) to a level that is comparable to that of strain NCTC 7973. Expression of the internalin gene (inIA) is also influenced by PrfA, but only one (P2) out of three mapped promoters is strictly dependent on PrfA. In contrast to the other PrfA‐regulated genes, transcription of inIA even from the P2 promoter is reduced in both strains after shift to MEM. The prfA deletion mutant SLCC 53 complemented with multiple copies of prfA synthesizes large amounts of monocistronic prfA transcript, but there is no concomitant increase in the transcripts of the PrfA‐dependent genes. However, upon a shift into MEM, transcription of the PrfA‐dependent genes (with the exception of the inIA gene) is highly induced even in the absence of de novo protein synthesis. The PrfA proteins of the two studied L. monocytogenes strains differ in their ability to activate PrfA‐dependent genes. This difference is probably the result of amino acid exchange(s) in the C‐terminal part of these proteins. Strain EGD supplemented with multiple copies of prfA‐7973 shows a similar regulation of the PrfA‐dependent genes as strain NCTC 7973, whereas multiple copies of prfA‐EGD introduced into strain EGD hardly change the rate of transcription of the PrfA‐dependent genes. Our data thus suggest that PrfA‐mediated gene expression depends not only on the amount of the PrfA protein and the ‘quality’ of the putative PrfA‐binding sites, but also on the ‘quality’ of the PrfA protein which seems to be influenced by its amino acid composition and by environmental parameters.
Localization of OccR‐activated and TraR‐activated promoters that express two ABC‐type permeases and the traR gene of Ti plasmid pTiR10
Authors: Fuqua Clay , Winans Stephen C.
Published: October 2006

Conjugation of Agrobacterium tumefaciens wide‐host‐range octopine‐type Ti plasmids is regulated by the LuxR‐type transcriptional activator TraR in conjunction with an acylated homoserine lactone designated AAI. Expression of traR in octopine‐type Ti plasmids is stimulated by OccR in response to octopine, an opine released from crown gall tumours, and is also positively autoregulated by TraR and AAI. Genetic and physical mapping of these promoters indicates that the OccR‐activated promoter lies 14.5 kb upstream of traR, while the TraR‐activated promoter lies 6kb upstream. The upstream portion of the 14.5 kb operon contains seven previously characterized genes that direct the uptake and catabolism of octopine. The TraR‐activated promoter lies just downstream from the octopine catabolic genes, and transcribes six genes in addition to traR, including five genes (ophABCDE) that show strong homology to oligopeptide permeases of Salmonella typhimurium and Bacillus subtilis. Several TraR‐regulated promoters overlap with 18bp inverted repeats called tra boxes. In contrast, the traR autoregulatory promoter is not associated with a consensus tra box.
Capsule phase variation in Neisseria meningitidis serogroup B by slipped‐strand mispairing in the polysialyltransferase gene (siaD): correlation with bacterial invasion and the outbreak of meningococcal disease
Authors: Hammerschmidt Sven , Müller Astrid , Sillmann Hanna , Miihlenhoff Martina , Borrow Raymond , Fox Andrew , van Putten Jos , Zollinger Wendell D. , Gerardy‐Schahn Rita , Frosch Matthias
Published: October 2006

A mechanism of capsular polysaccharide phase variation in Neisseria meningitidis is described. Meningococcal cells of an encapsulated serogroup B strain were used in invasion assays. Only unencapsulated variants were found to enter epithelial cells. Analysis of one group of capsule‐deficient variants indicated that the capsular polysaccharide was re‐expressed at a frequency of 10−3. Measurement of enzymatic activities involved in the biosynthesis of the α‐2,8 polysialic acid capsule showed that polysialyltransferase (PST) activity was absent in these capsule‐negative variants. Nucleotide sequence analysis of siaD revealed an insertion or a deletion of one cytidine residue within a run of (dC)7 residues at position 89, resulting in a frameshift and premature termination of translation. We analysed unencapsulated isolates from carriers and encapsulated case isolates collected during an outbreak of meningococcal disease. Further paired blood‐culture isolates and unencapsulated nasopharyngeal isolates from patients with meningococcal meningitis were examined. In all unencapsulated strains analysed we found an insertion or deletion within the oligo‐(dC) stretch within siaD, resulting in a frameshift and loss of capsule formation. All encapsulated isolates, however, had seven dC residues at this position, indicating a correlation between capsule phase variation and bacterial invasion and the out‐break of meningococcal disease.
FIS is a regulator of metabolism in Escherichia coli
Authors: González‐Gil Gardenia , Bringmann Peter , Kahmann Regine
Published: October 2006

The Escherichia coli DNA‐binding protein FIS (factor for inversion stimulation) stimulates site‐specific recombination reactions catalysed by DNA invertases and is an activator of stable RNA synthesis. To address the question of whether FIS is involved in other cellular processes we have identified and sequenced proteins whose expression pattern is affected by FIS. This has led to the identification of several E. coli genes whose expression in vivo is either enhanced or repressed by FIS. All of these genes encode enzymes or transport proteins involved in the catabolism of sugars or nucleic acids, and their expression is also dependent on the cAMP‐CRP complex. In most cases studied the regulation by FIS is indirect and occurs through effects on the synthesis of the respective repressor proteins. We conclude that FIS is a transcriptional modulator involved in the regulation of metabolism in E. coli.
Preferential processing of the S1 subunit of pertussis toxin that is bound to eukaryotic cells
Authors: Finck‐Barbançon Viviane , Barbieri Joseph T.
Published: October 2006

Labelled [125l]‐pertussis toxin was prepared and used to measure the association of pertussis toxin (PT) to eukaryotic cells. PT was radioiodinated by the lactoperoxidase method which preferentially radioiodinated the S1 subunit. PT was radioiodinated at a high specific activity and possessed the same cytotoxicity as native PT as demonstrated by the ability to cluster Chinese hamster ovary (CHO) cells. Cell association of [125l]‐PT was not inhibited by excess non‐radiolabelled PT, which indicated that the initial interaction between PT and CHO cells involved a large number of low‐affinity receptors. At 37° C, the S1 within cell‐associated PT was preferentially processed to an S1 with a lower apparent molecular weight (termed S1p). This processing was inhibited by the addition of unlabelled PT, indicating that the processing event was saturable and specific. S1 processing occurred in CHO, Madin‐Darby canine kidney (MDCK) cells, and pig kidney (LLC‐PK1) cells. A pulse‐chase experiment showed that, at 37° C but not at 22° C, essentially all of the cell‐associated S1 was processed within 3 h of a chase. Reagents that were previously shown to inhibit the ability of PT to ADP‐ribosylate G1 proteins in intact CHO cells also inhibited the preferential processing of S1 within cell‐associated PT, in the order of efficiency: 22°C chloroquine nocodazole brefeldin A. This indicates that S1 processing requires an early endosomal function.
Identification and characterization of AlgZ, an AlgT‐dependent DNA‐binding protein required for Pseudomonas aeruginosa algD transcription
Authors: Baynham Patricia J. , Wozniak Daniel J.
Published: October 2006

Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high‐level synthesis of the capsular polysaccharide alginate, an important P. aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease. In this study, electrophoretic mobility‐shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter. While AlgZ‐binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT. Electrophoretic mobility‐shift assays and copper‐phenanthroline foot‐printing localized AlgZ binding to a 36 bp algD region, which includes several helical repeats. A collection of alginate‐producing (mucoid) and non‐mucoid P. aeruginosa strains, derived from CF patients, was characterized for AlgZ‐binding activity. In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P. aeruginosa CF isolates were examined. However, this binding activity was not present when extracts from non‐mucoid P. aeruginosa CF isolates were tested. Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ‐binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ‐binding site was replaced with a nonspecific DNA sequence of identical size. When the algD38 mutation was cloned into an algD‐cat transcriptional fusion, this resulted in a 28‐fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription. These results support the hypothesis that activation of algD involves the formation of a high‐order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT. Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.
Functional in vivo studies of the Neurospora crassa cys‐14 gene upstream region: importance of CYS3‐binding sites for regulated expression
Authors: Li Qunhui , Zhou Liwei , Marzluf George A.
Published: October 2006

Sulphate transport in Neurospora crassa is achieved by two distinct sulphate permeases, I and II, encoded by the cys‐13 and cys‐14 genes, respectively. The synthesis of both sulphate permeases is subject to sulphur repression and requires the global positive‐acting regulatory protein CYS3. CYS3, a bZIP DNA‐binding protein, regulates cys‐14 expression at the transcriptional level and binds in vitro specifically to three DNA‐recognition sites, A, B, and C, in the cys‐14 upstream region. In vivo functional analysis of the cys‐14 promoter was carried out with 5’deletions and by deletions or mutations of CYS3 DNA‐binding sites. The most distal CYS3‐binding site, C, located 1.4kb upstream of the transcriptional start site, is necessary and sufficient to mediate strong transcriptional activation by CYS3; moreover, site C was able to function equally well when it was located at variable distances upstream of the cys‐14 gene. Site B, located 1 kb upstream, alone is able to support a moderate degree of cys‐14 expression. Site A is not required and does not appear to play any functional role in cys‐14 expression, even though it is in close proximity to the transcriptional start site. The presence of multiple copies of CYS3‐binding elements A or B in the cys‐14 promoter results in a parallel increase of regulated gene expression. When a transforming cys‐14 gene becomes integrated at ectopic locations in the host genome, it can be expressed in an unregulated fashion, presumably by coming under the control of other promoter elements. Our results also suggested that at least one enzyme in the sulphate catabolic pathway requires a functional CYS3 protein for expression.
Involvement of the sensor kinase EnvZ in the in vivo activation of the response‐regulator PhoB by acetyl phosphate
Authors: Kim Soo‐Ki , Wilmes‐Riesenberg Mary R. , Wanner Barry L.
Published: October 2006

Three signalling pathways lead to activation of the phosphate (Pho) regulon by phosphorylation of the response‐regulator PhoB in Escherichia coil One pathway responds to the extracellular inorganic phosphate (Pi) level and leads to activation by the Pi sensor kinase, PhoR. The other two pathways are Pi independent and are apparent in the absence of PhoR. One Pi‐independent pathway responds to the level of an unknown catabolite and leads to activation by the catabolite regulatory sensor kinase, CreC (originally called PhoM); the other Pi‐independent pathway responds to acetyl phosphate and leads to activation by a process requiring acetyl phosphate. Here we show that activation of PhoB by acetyl phosphate can require the sensor kinase EnvZ. Accordingly, we propose that the in vivo activation of PhoB by acetyl phosphate (and perhaps other two‐component response‐regulators as well) probably always requires a certain kinase that can vary depending upon the growth conditions.
Virulence and vaccine potential ofSalmonella typhlmuriummutants deficient in the expression of the RpoS (σs) regulon
Authors: Coynault Colette , Robbe‐Saule Véronique , Norel Françoise
Published: October 2006

The alternative sigma factor RpoS (σs) is required forSalmonellavirulence in mice. We report the immunizing capacity ofSalmonella typhimurlum rpoSandrpoS aroAmutants to protect susceptible BALB/c mice against subsequent oral challenge with virulent S.typhimurium.When administered orally or intraperitoneally,rpoSderivatives of the mouse‐virulent S.typhimuriumstrains, C52 and SL1344, were highly attenuated and were efficient single‐dose live vaccines.rpoS aroAmutants were more attenuated than corresponding singlearoAorrpoSmutants, as assessed after oral or intraperitoneal administration, but retained significant ability to protect mice against salmonellosis.Salmonella rpoSandrpoS aroAmutants therefore deserve serious consideration for rational vaccine design. Consistent with this,Salmonella typhiTy2, a ‘wild‐type’ strain used widely for the development of human live‐vaccine candidates against typhoid fever, was shown to be defective forrpoS.In addition, our results demonstrate thatrpoSnot only controls the growth and persistence of S.typhimuriumin deep lymphoid organs, but also plays a role during the initial stages of oral infection.
Measurement and Significance of 3‐Nitrotyrosine in Systemic Lupus Erythematosus
Authors: Khan F. , Siddiqui A. A. , Ali R.
Published: October 2006

Nitration of free and protein associated tyrosine represents, in vivo, a mechanism that can severely compromise the cell function. The detection of 3‐nitrotyrosine (3‐NT) in pathological tissues is suggestive of the occurrence of nitrating pathways and has been identified as a marker of inflammation and a stable end product of increased reactive nitrogen intermediate production. Protein nitration occurs in many disease conditions including systemic lupus erythematosus (SLE). In this study we show that the level of both free and protein bound 3‐NT, which is produced by reactive nitrogen species (RNS)‐dependent oxidative damage, is elevated in patients with SLE and that there is a possible role of RNS‐modified epitopes in the aetiology of the disease. Commercially available poly L‐tyrosine was exposed to nitrating species, inducing nitration in tyrosine residues. Immunoglobulin‐G (IgG) purified on Protein‐A‐Sepharose matrix from 24 SLE patients was studied for their recognition of native and nitrated poly L‐tyrosine by direct binding and competition enzyme‐linked immunosorbent assay (ELISA). The formation of immune complex between SLE IgG and nitrated poly L‐tyrosine was visualized by gel retardation assay. Free 3‐NT in patients’ sera was detected and quantitated by high performance liquid chromatography whereas protein‐bound 3‐NT was analysed by Western blotting and the concentration was calculated by sandwich ELISA. The concentration of free 3‐NT was found to be 1.4 ± 0.09 μm whereas the concentration of protein bound 3‐NT was 96.52 ± 21.12 μm nitrated bovine serum albumin equivalents/mg protein, which was significantly higher when compared with healthy controls. Elevated level of 3‐NT was observed in SLE patients using two different techniques, when compared with healthy subjects confirms the overproduction of RNS in the pathogenesis of human SLE.