bluuebunny/crossref_metadata_2025 huggingface dataset:
Riboregulation by DsrA RNA: trans‐actions for global economy
Authors: Lease Richard A. , Belfort Marlene
Published: March 2003

DsrA is an 87 nucleotide Escherichia coli RNA with extraordinary regulatory properties. The profound impact of its actions stems from DsrA regulating translation of two global transcription regulators, H‐NS and RpoS (σs), by sequence‐specific RNA–RNA interactions. H‐NS is a major nucleoid‐structuring and global repressor protein, and RpoS is the stationary phase and stress response sigma factor of RNA polymerase. DsrA changes its conformation to bind to these two different mRNA targets and thereby inhibits H‐NS translation, while stimulating that of RpoS in a mechanistically distinct fashion. DsrA apparently binds both the start and the stop codons of hns mRNA and sharply decreases the mRNA half‐life. DsrA also binds sequences in the 5′‐untranslated leader region of rpoS mRNA, enhancing rpoS mRNA stability and RpoS translation. A cohort of genes, governed by H‐NS repression and RpoS activation, are thus regulated. Low temperatures increase the levels of DsrA, with differential effects on H‐NS and RpoS. Additionally, the RNA chaperone protein Hfq is involved with DsrA regulation, as well as with other small RNAs that also act on RpoS to co‐ordinate stress responses. We address the possible functions of this genetic regulatory mechanism, as well as the advantages of using small RNAs as global regulators to orchestrate gene expression.
P fimbriae enhance the early establishment of Escherichia coli in the human urinary tract
Authors: Wullt Björn , Bergsten Göran , Connell Hugh , Röllano Piotr , Gebretsadik Negash , Hull Richard , Svanborg Catharina
Published: March 2003

This study examined the role of P fimbriae in the establishment of bacteriuria. Patients (n = 17) were subjected to intravesical inoculation with an asymptomatic bacteriuria strain, Escherichia coli 83972, or its P‐fimbriated (pap+/prs+) transformants. As shown by groupwise analysis, the pap+/prs+ transformants established bacteriuria more rapidly than E. coli 83972 (P = 0.021) and required a lower number of inoculations to reach 105 cfu ml−1 (P = 0.018). Intraindividual analysis showed that the pap+/prs+ transformants established bacteriuria more rapidly than E. coli 83972 in the patients who subsequently became carriers of both strains. Finally, bacterial establishment was shown to vary with the in vivo expression of P fimbriae. Bacterial counts were higher when P‐fimbrial expression was detected than when the pap+/prs+ strain showed a negative phenotype. The results suggested that P fimbriae enhance the establishment of bacteriuria and fulfil the molecular Koch postulates as a colonization factor in the human urinary tract.
Four genes are required for the system II cytochrome c biogenesis pathway in Bordetella pertussis, a unique bacterial model
Authors: Beckett Caroline S. , Loughman Jennifer A. , Karberg Katherine A. , Donato Gina M. , Goldman William E. , Kranz Robert G.
Published: March 2003

Unlike other cytochromes, c‐type cytochromes have two covalent bonds formed between the two vinyl groups of haem and two cysteines of the protein. This haem ligation requires specific assembly proteins in prokaryotes or eukaryotic mitochondria and chloroplasts. Here, it is shown that Bordetella pertussis is an excellent bacterial model for the widespread system II cytochrome c synthesis pathway. Mutations in four different genes (ccsA, ccsB, ccsX and dipZ) result in B. pertussis strains unable to synthesize any of at least seven c‐type cytochromes. Using a cytochrome c4:alkaline phosphatase fusion protein as a bifunctional reporter, it was demonstrated that the B. pertussis wild‐type and mutant strains secrete an active alkaline phosphatase fusion protein. However, unlike the wild type, all four mutants are unable to attach haem covalently, resulting in a degraded N‐terminal apocytochrome c4 component. Thus, apocytochrome c secretion is normal in each of the four mutants, but all are defective in a periplasmic assembly step (or export of haem). CcsX is related to thioredoxins, which possess a conserved CysXxxXxxCys motif. Using phoA gene fusions as reporters, CcsX was proven to be a periplasmic thioredoxin‐like protein. Both the B. pertussis dipZ (i.e. dsbD) and ccsX mutants are corrected for their assembly defects by the thiol‐reducing compounds, dithiothreitol and 2‐mercaptoethanesulphonic acid. These results indicate that DipZ and CcsX are required for the periplasmic reduction of the cysteines of apocytochromes c before ligation. In contrast, the ccsA and ccsB mutants are not corrected by exogenous reducing agents, suggesting that CcsA and CcsB are required for the haem ligation step itself in the periplasm (or export of haem to the periplasm). Related to this suggestion, the topology of CcsB was determined experimentally, demonstrating that CcsB has four transmembrane domains and a large 435‐amino‐acid periplasmic region.
Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth
Authors: Coers Jörn , Kagan Jonathan C. , Matthews Miguelina , Nagai Hiroki , Zuckman Deborah M. , Roy Craig R.
Published: March 2003

Legionella pneumophila is a bacterial pathogen that can enter the human lung and grow inside alveolar macrophages. To grow within phagocytic host cells, the bacteria must create a specialized organelle that restricts fusion with lysosomes. Biogenesis of this replicative organelle is controlled by 24 dot and icm genes, which encode a type IV‐related transport apparatus. To understand how this transporter functions, isogenic L. pneumophila dot and icm mutants were characterized, and three distinct phenotypic categories were identified. Our data show that, in addition to genes that encode the core Dot/Icm transport apparatus, subsets of genes are required for pore formation and modulation of phagosome trafficking. To understand activities required for virulence at a molecular level, we investigated protein–protein interactions. Specific interactions between different Icm proteins were detected by yeast two‐hybrid and gel overlay analysis. These data support a model in which the IcmQ–IcmR complex regulates the formation of a translocation channel that delivers proteins into host cells, and the IcmS–IcmW complex is required for export of virulence determinants that modulate phagosome trafficking.
Cross‐regulation of competence pheromone production and export in the early control of transformation in Streptococcus pneumoniae
Authors: Martin Bernard , Prudhomme Marc , Alloing Geneviève , Granadel Chantal , Claverys Jean‐Pierre
Published: March 2003

Two operons, comAB and comCDE, play a key role in the co‐ordination of spontaneous competence development in cultures of Streptococcus pneumoniae. ComAB is required for export of the comC‐encoded competence‐stimulating peptide (CSP). Upon CSP binding, the histidine kinase ComD activates ComE, its cognate response regulator, required for autoinduction of comCDE and for induction of the late competence genes. To understand better the early control of competence development, mutants upregulating comCDE (ComCDEUP) were isolated using a comC–lacZ transcriptional fusion. Mutants were generated by polymerase chain reaction mutagenesis of the comCDE region and by in vitro transposon mutagenesis of the chromosome. Both types of ComCDEUP mutants exhibited similar phenotypes. They differed from wild type in displaying trypsin‐resistant transformation, competence under acid growth conditions and expression of comCDE under microaerobiosis; increased production of CSP in the mutants could account for the various phenotypes. The ComCDEUP transposon mutations included four independent insertions in the ciaR gene, which encodes the response regulator of a two‐component system previously found to affect competence, and two immediately upstream of the comAB operon. The latter two resulted in comAB overexpression, indicating that CSP export is rate limiting. Among comDE point mutations, a single amino acid change in ComD (T233I) conferred constitutive, CSP‐independent competence and resulted in comAB overexpression, providing support for the hypothesis that ComE regulates comAB; a ComE mutant (R120S) exhibited altered kinetics of competence shut‐off. Collectively, these data indicate that pheromone autoinduction, cross‐regulation of the comAB and comCDE operons and, possibly, competence shut‐off contribute to the early control of competence development in S. pneumoniae. They argue for a metabolic control of competence, mediated directly or indirectly by CiaR, and they suggest that both comAB and comCDE are potential targets for regulation.
Group II introns in the bacterial world
Authors: Martínez‐Abarca Francisco , Toro Nicolás
Published: March 2003

Group II introns are large catalytic RNA molecules that act as mobile genetic elements. They were initially identified in the organelle genomes of lower eukaryotes and plants, and it has been suggested that they are the progenitors of nuclear spliceosomal introns. Group II self‐splicing introns were shown to be present in bacteria in 1993, since when the various bacterial genome sequencing projects have led to a significant increase in the number of group II intron sequences present in databases. However, few of these introns have been characterized, and most were identified on the basis of their intron‐encoded protein (IEP), with little data available concerning their ribozyme/RNA structure. Their frequency in prokaryotes is also unknown. We attempt here to provide a first comprehensive review of bacterial group II introns based on recent genome sequencing data and mechanistic studies.
Cold‐temperature induction of Escherichia coli polynucleotide phosphorylase occurs by reversal of its autoregulation	†‡
Authors: Beran Rudolf K. , Simons Robert W.
Published: March 2003

When Escherichia coli cells are shifted to low temperatures (e.g. 15°C), growth halts while the ‘cold shock response’ (CSR) genes are induced, after which growth resumes. One CSR gene, pnp, encodes polynucleotide phosphorylase (PNPase), a 3′‐exoribonuclease and component of the RNA degradosome. At 37°C, ribonuclease III (RNase III, encoded by rnc) cleaves the pnp untranslated leader, whereupon PNPase represses its own translation by an unknown mechanism. Here, we show that PNPase cold‐temperature induction involves several post‐transcriptional events, all of which require the intact pnp mRNA leader. The bulk of induction results from reversal of autoregulation at a step subsequent to RNase III cleavage of the pnp leader. We also found that pnp translation occurs throughout cold‐temperature adaptation, whereas lacZ+ translation was delayed. This difference is striking, as both mRNAs are greatly stabilized upon the shift to 15°C. However, unlike the lacZ+ mRNA, which remains stable during adaptation, pnp mRNA decay accelerates. Together with other evidence, these results suggest that mRNA is generally stabilized upon a shift to cold temperatures, but that a CSR mRNA‐specific decay process is initiated during adaptation.
Molecular and physiological characterization of the NAD‐dependent glycerol 3‐phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans
Authors: Fillinger Sabine , Ruijter George , Tamás Markus J. , Visser Jaap , Thevelein Johan M. , D'enfert Christophe
Published: March 2003

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3‐phosphate dehydrogenase (G3PDH)‐encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3‐phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin‐interacting agent calcofluor and the membrane‐destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.
The variable P5 proteins of typeable and non‐typeable Haemophilus influenzae target human CEACAM1
Authors: Hill Darryl J. , Toleman Mark A. , Evans Debbie J. , Villullas Silvia , Van Alphen Loek , Virji Mumtaz
Published: March 2003

Haemophilus influenzae, a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We have recently shown that numerous strains of capsulate (typeable) and acapsulate (non‐typeable) H. influenzae target the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CEACAMs). Moreover, the ligands appeared to be antigenically variable and, when using viable typeable bacteria, their adhesive functions were inhibited by the presence of capsule. In this report, we show that the antigenically variable outer membrane protein, P5, expressed by typeable and non‐typeable H. influenzae targets human CEACAM1. Variants and mutants lacking the expression of P5 of all strains tested were unable to target purified soluble receptors. A non‐typeable strain that did not interact with CEACAM1 was made adherent to both the soluble receptors and CEACAM1‐transfected Chinese hamster ovary cells by transformation with the P5 gene derived from the adherent typeable strain Rd. However, several H. influenzae mutants lacking P5 expression continued to bind the cell‐bound CEACAM1 receptors. These observations suggest that (i) CEACAM1 alone can support P5 interactions and (ii) some strains contain additional ligands with the property to target CEACAM1 but require the receptor in the cellular context. The identification of a common ligand in diverse strains of H. influenzae and the presence of multiple ligands for the same receptor suggests that targeting of members of the CEACAM family of receptors may be of primary significance in colonization and pathogenesis of H. influenzae strains.
Characterization of the bacteriophage φ29‐encoded protein p16.7: a membrane protein involved in phage DNA replication
Authors: Meijer Wilfried J. J. , Serna‐Rico Alejandro , Salas Margarita
Published: March 2003

An early expressed operon, located at the right end of the linear bacteriophage φ29 genome, contains open reading frame (ORF)16.7, whose deduced protein sequence of 130 amino acids is conserved in φ29‐related phages. Here, we show that this ORF actually encodes a protein, p16.7, which is abundantly and early expressed after infection. p16.7 is a membrane protein, and the N‐terminally located transmembrane‐spanning domain is required for its membrane localization. The variant p16.7A, in which the N‐terminal membrane anchor was replaced by a histidine‐tag, was purified and characterized. Purified p16.7A was shown to form dimers in solution. To study the in vivo role of p16.7, a φ29 mutant containing a suppressible mutation in gene 16.7 was constructed. In vivo phage DNA replication was affected in the absence of p16.7, especially at early infection times. Based on the results, the putative role of p16.7 in in vivoφ29 DNA replication is discussed.
Dispensable nature of phosphatidylglycerol in Escherichia coli: dual roles of anionic phospholipids
Authors: Matsumoto Kouji
Published: March 2003

The major anionic phospholipids of Escherichia coli, phosphatidylglycerol (PG) and cardiolipin (CL), have been considered to be indispensable for essential cellular functions, such as the initiation of DNA replication and translocation of proteins across the cytoplasmic membrane. However, we successfully constructed a null pgsA mutant of E. coli that had undetectable levels of PG and CL if the major outer membrane lipoprotein was deficient, clearly indicating that these anionic phospholipids are not indispensable. In the null mutant, we observed the accumulation of phosphatidic acid, an acidic biosynthetic precursor. This suggests a functionally substitutable nature of these anionic phospholipids and allows us to formulate a dual role model for the physiological roles of the anionic phospholipids in E. coli. The anionic phospholipids may play dual roles in E. coli as (i) substrates for head group‐specific enzyme reactions, albeit the viability of null PG mutants indicates that the products of head group‐specific reactions are not essential; and (ii) those that are replaceable, partly or entirely, by other phospholipids bearing net negative charges, because of their rather loose head group specificity. These two aspects of the physiological roles of anionic phospholipids are discussed with special reference to the phospholipids of other bacteria and eukaryotic organelles.
Subcellular localization and characterization of chorismate synthase in the apicomplexan Plasmodium falciparum
Authors: Fitzpatrick Teresa , Ricken Sigrid , Lanzer Michael , Amrhein Nikolaus , Macheroux Peter , Kappes Barbara
Published: March 2003

The resurgence of drug‐resistant apicomplexa, in particular Plasmodium falciparum, the most fatal human malarial parasite, has focused attention on the recent discovery of the shikimate pathway in these organisms, as it may provide the urgently required, novel drug targets resulting from the absence of this pathway in mammals. The direction of a parasiticidal drug design programme obviously requires knowledge of the subcellular localization and indeed full characterization of the possible enzyme targets. Here, we report the cloning and characterization of chorismate synthase from P. falciparum and present the first biochemical and immunological studies of an enzyme of the shikimate pathway from an apicomplexan parasite. We show that this chorismate synthase does not possess an intrinsic flavin reductase activity and is therefore monofunctional like the plant and bacterial chorismate synthases. Highest immunological cross‐reactivity was found with a plant chorismate synthase. However, in contrast to the plant enzyme, which is located to the plastid, P. falciparum chorismate synthase is found in the parasite cytosol, akin to the fungal enzymes that possess an intrinsic flavin reductase activity (i.e. are bifunctional). Thus, P. falciparum chorismate synthase has a combination of properties that distinguishes it from other described chorismate synthases.
The Saccharomyces cerevisiae Sko1p transcription factor mediates HOG pathway‐dependent osmotic regulation of a set of genes encoding enzymes implicated in protection from oxidative damage
Authors: Rep Martijn , Proft Markus , Remize Fabienne , Tamás Markus , Serrano Ramón , Thevelein Johan M. , Hohmann Stefan
Published: March 2003

A major part of the transcriptional response of yeast cells to osmotic shock is controlled by the HOG pathway and several downstream transcription factors. Sko1p is a repressor that mediates HOG pathway‐dependent regulation by binding to CRE sites in target promoters. Here, we report five target genes of Hog1p–Sko1p: GRE2, AHP1, SFA1, GLR1 and YML131w. The two CREs in the GRE2 promoter function as activating sequences and, hence, bind (an) activator protein(s). However, the two other yeast CRE‐binding proteins, Aca1p and Aca2p, are not involved in regulation of the GRE2 promoter under osmotic stress. In the absence of the co‐repressor complex Tup1p–Ssn6p/Cyc8p, which is recruited by Sko1p, stimulation by osmotic stress is still observed. These data indicate that Sko1p is not only required for repression, but also involved in induction upon osmotic shock. All five Sko1p targets encode oxidoreductases with demonstrated or predicted roles in repair of oxidative damage. Altered basal expression levels of these genes in hog1Δ and sko1Δ mutants may explain the oxidative stress phenotypes of these mutants. All five Sko1p target genes are induced by oxidative stress, and induction involves Yap1p. Although Sko1p and Yap1p appear to mediate osmotic and oxidative stress responses independently, Sko1p may affect Yap1p promoter access or activity. The five Sko1p target genes described here are suitable models for studying the interplay between osmotic and oxidative responses at the molecular and physiological levels.
Formation of disulphide bonds during secretion of proteins through the periplasmic‐independent type I pathway
Authors: Fernández Luis A. , De Lorenzo Víctor
Published: March 2003

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single‐chain Fv (scFv) fragment secreted by theEscherichia colihaemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein‐folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen‐binding activity). We show that an scFv–HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane‐bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv–HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly‐dependent secretion of disulphide‐containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide‐containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.
Acetylation of the response regulator, CheY, is involved in bacterial chemotaxis
Authors: Barak Rina , Eisenbach Michael
Published: March 2003

It is well established that the response regulator of the chemotaxis system of Escherichia coli, CheY, can undergo acetylation at lysine residues 92 and 109 via a reaction mediated by acetyl‐CoA synthetase (Acs). The outcome is activation of CheY, which results in increased clockwise rotation. Nevertheless, it has not been known whether CheY acetylation is involved in chemotaxis. To address this question, we examined the chemotactic behaviour of two mutants, one lacking the acetylating enzyme Acs, and the other having an arginine‐for‐lysine substitution at residue 92 of CheY – one of the acetylation sites. The Δacs mutant exhibited much reduced sensitivity to chemotactic stimuli (both attractants and repellents) in tethering assays and greatly reduced responses in ring‐forming, plug and capillary assays. Likewise, the cheY(92KR) mutant had reduced sensitivity to repellents in tethering assays and a reduced response in capillary assays. However, its response to the addition or removal of attractants was normal. These observations suggest that Acs‐mediated acetylation of CheY is involved in chemotaxis and that the acetylation site Lys‐92 is only involved in the response to repellents. The observation that, in the cheY(92KR) mutant, the addition of a repellent was not chemotactically equivalent to the removal of an attractant also suggests that there are different signalling pathways for attractants and repellents in E. coli.
RNA polymerase II and TBP occupy the repressed CYC1 promoter
Authors: Martens Craig , Krett Brian , Laybourn Paul J.
Published: March 2003

Saccharomyces cerevisiae CYC1 gene expression has been studied in great detail with regard to the response to oxygen availability and carbon source. In the absence of oxygen and the presence of glucose, the CYC1 gene is completely repressed. Chromatin structure is thought to play an important role in CYC1 gene regulation, as nucleosome depletion results in 94‐fold derepression. In addition, the CYC1 core promoter has been used extensively in hybrid constructs to study activation by heterologous transcription factors. Therefore, we set out to map the chromatin structure of the CYC1 promoter and determine its role in CYC1 gene regulation. We report here that the repressed CYC1 promoter contains no positioned nucleosomes over the core promoter. However, we did find TFIID and RNA polymerase II bound in a complex on the repressed promoter. These results indicate that recruitment of TFIID and RNA polymerase II are not rate‐limiting steps in CYC1 activation.
α‐Enolase of Streptococcus pneumoniae is a plasmin(ogen)‐binding protein displayed on the bacterial cell surface
Authors: Bergmann Simone , Rohde Manfred , Chhatwal Gursharan S. , Hammerschmidt Sven
Published: March 2003

Binding of human plasminogen to Streptococcus pneumoniae and its subsequent activation promotes penetration of bacteria through reconstituted basement membranes. In this study, we have characterized a novel pneumococcal surface protein with a molecular mass of 47 kDa, designated Eno, which specifically binds human plasmin(ogen), exhibits α‐enolase activity and is necessary for viability. Using enzyme assays, we have confirmed the α‐enolase activity of both pneumococcal surface‐displayed Eno and purified recombinant Eno protein. Immunoelectron microscopy indicated the presence of Eno in the cytoplasm as well as on the surface of encapsulated and unencapsulated pneumococci. Plasminogen‐binding activity was demonstrated with whole pneumococcal cells and purified Eno protein. Binding of activated plasminogen was also shown for Eno; however, the affinity for plasmin is significantly reduced compared with plasminogen. Results from competitive inhibition assays indicate that binding is mediated through the lysine binding sites in plasmin(ogen). Carboxypeptidase B treatment and amino acid substitutions of the C‐terminal lysyl residues of Eno indicated that the C‐terminal lysine is pivotal for plasmin(ogen)‐binding activity. Eno is ubiquitously distributed among pneumococcal serotypes, and binding experiments suggested the reassociation of secreted Eno to the bacterial cell surface. The reassociation was also confirmed by immunoelectron microscopy. The results suggest a mechanism of plasminogen activation for human pathogens that might contribute to their virulence potential in invasive infectious processes.
Carbon catabolite repression in Aspergillus nidulans involves deubiquitination
Authors: Lockington Robin A. , Kelly Joan M.
Published: March 2003

The best studied role of ubiquitination is to mark proteins for destruction by the proteasome but, in addition, it has recently been shown to promote macromolecular assembly and function, and alter protein function, thus playing a regulatory role distinct from protein degradation. Deubiquinating enzymes, the ubiquitin‐processing proteases (ubps) and the ubiquitin carboxy‐terminal hydrolases (uchs), remove ubiquitin from ubiquitinated substrates. We show here that the creB gene involved in carbon catabolite repression in Aspergillus nidulans encodes a functional member of the novel subfamily of the ubp family defined by the human homologue UBH1, thus implicating ubiquitination in the process of carbon catabolite repression. Members of the novel subfamily of ubps that include CreB are widespread amongst eukaryotes, with homologues present in mammals, nematodes, Drosophila and Arabidopsis, but mutations in the genes have only been identified in A. nidulans. From phenotypes of the A. nidulans mutants it is probable that this subfamily is involved in complex regulatory pathways. Mutations in the gene encoding the WD40 repeat protein CreC result in an identical phenotype, implicating both genes in this pathway.
Alkaline shock induces the Bacillus subtilisσW regulon
Authors: Wiegert Thomas , Homuth Georg , Versteeg Saskia , Schumann Wolfgang
Published: March 2003

When confronted with a stress factor, bacteria react with a specific stress response, a genetically encoded programme resulting in the transiently enhanced expression of a subset of genes. One of these stress factors is a sudden increase in the external pH. As a first step to understand the response of Bacillus subtilis cells towards an alkali shock at the transcriptional level, we attempted to identify alkali‐inducible genes using the DNA macroarray technique. To define the appropriate challenging conditions, we used the ydjF gene, the orthologue of the Escherichia coli pspA, as a model gene for an alkali‐inducible gene. Hybridization of 33P‐labelled cDNA to a DNA macroarray revealed induction of more than 80 genes by a sudden increase in the external pH value from 6.3 to 8.9. It was discovered that a large subset of these genes belong to the recently described σW regulon, which was confirmed by the analysis of a sigW knockout. A comparison of B. subtilis wild type with the congenic sigW knockout also led to the discovery of new members of the σW regulon. In addition, we found several genes clearly not belonging to that regulon. This analysis represents the first report of an extracellular stimulus inducing the σW regulon.
Transcription of glycolytic genes and operons in Bacillus subtilis: evidence for the presence of multiple levels of control of the gapA operon
Authors: Ludwig Holger , Homuth Georg , Schmalisch Matthias , Dyka Frank M. , Hecker Michael , Stülke Jörg
Published: March 2003

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Although the biochemical activity of glycolytic enzymes has been studied in detail, no information about the expression of glycolytic genes has so far been available in this organism. Therefore, transcriptional analysis of all glycolytic genes was performed. The genes cggR, gapA, pgk, tpi, pgm and eno, encoding the enzymes required for the interconversion of triose phosphates, are transcribed as a hexacistronic operon as demonstrated by Northern analysis. This gapA operon is repressed by the regulator CggR. The presence of sugars and amino acids synergistically results in the induction of the gapA operon. The transcriptional start site upstream of cggR was mapped by primer extension. Transcripts originating upstream of cggR are processed near the 3′ end of cggR. This endonucleolytic cleavage leads to differential stability of the resulting processing products: the monocistronic cggR message is very rapidly degraded, whereas the mRNA species encoding glycolytic enzymes exhibit much higher stability. An additional internal constitutive promoter was identified upstream of pgk. Thus, gapA is the most strongly regulated gene of this operon. The pfk pyk operon encoding phosphofructokinase and pyruvate kinase is weakly induced by glucose. In contrast, the genes pgi and fbaA, coding for phosphoglucoisomerase and fructose‐1,6‐bisphosphate aldolase, are constitutively expressed.
De novo formation of focal complex‐like structures in host cells by invading Streptococci
Authors: Ozeri Vered , Rosenshine Ilan , Ben‐Ze'Ev Avri , Bokoch Gary M. , Jou Tzuu‐Shu , Hanski Emanuel
Published: March 2003

Group A streptococcus (GAS) induces its own entry into eukaryotic cells in vitro and in vivo. Fibronectin (Fn) bound to protein F1, a GAS surface protein, acts as a bridge connecting the bacterium to host cell integrins. This triggers clustering of integrins, which acquire a polar pattern of distribution similar to that of protein F1 on the GAS surface. A unique and transient adhesion complex is formed at the site of GAS entry, which does not contain α‐actinin. Vinculin is recruited to the site of GAS entry but is not required for uptake. The invading GAS recruits focal adhesion kinase (FAK), which is required for uptake and is tyrosine phosphorylated. The Src kinases, Src, Yes and Fyn, enhance the efficiency of GAS uptake but are not absolutely required for GAS entry. In addition, Rac and Cdc42, but not Rho, are required for the entry process. We suggest a model in which integrin engagement by Fn‐occupied protein F1 triggers two independent signalling pathways. One is initiated by FAK recruitment and tyrosine phosphorylation, whereas the other is initiated by the recruitment and activation of Rac. The two pathways subsequently converge to trigger actin rearrangement leading to bacterial uptake.
Type III secretion in plant growth‐promoting Pseudomonas fluorescens SBW25
Authors: Preston Gail M. , Bertrand Nicolas , Rainey Paul B.
Published: March 2003

In vivo expression technology (IVET) analysis of rhizosphere‐induced genes in the plant growth‐promoting rhizobacterium (PGPR) Pseudomonas fluorescens SBW25 identified a homologue of the type III secretion system (TTSS) gene hrcC. The hrcC homologue resides within a 20‐kb gene cluster that resembles the type III (Hrp) gene cluster of Pseudomonas syringae. The type III (Rsp) gene cluster in P. fluorescens SBW25 is flanked by a homologue of the P. syringae TTSS‐secreted protein AvrE. P. fluorescens SBW25 is non‐pathogenic and does not elicit the hypersensitive response (HR) in any host plant tested. However, strains constitutively expressing the rsp‐specific sigma factor RspL elicit an AvrB‐dependent HR in Arabidopsis thaliana ecotype Col‐0, and a host‐specific HR in Nicotiana clevelandii. The inability of wild‐type P. fluorescens SBW25 to elicit a visible HR is therefore partly attributable to low expression of rsp genes in the leaf apoplast. DNA hybridization analysis indicates that rsp genes are present in many plant‐colonizing Pseudomonas and PGPR, suggesting that TTSSs may have a significant role in the biology of PGPR. However, rsp and rsc mutants retain the ability to reach high population levels in the rhizosphere. While functionality of the TTSS has been demonstrated, the ecological significance of the rhizosphere‐expressed TTSS of P. fluorescens SBW25 remains unclear.
Nuclease activity is essential for RecBCD recombination in Escherichia coli
Authors: Jockovich M. E. , Myers R. S.
Published: March 2003

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double‐stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of χ recombination hot‐spots. These paradoxical roles are accommodated by χ‐dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that χ converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5′→ 3′ exonuclease activity is essential for recombination by RecBCD at χ recombination hot‐spots and at dsDNA ends in recD mutants. In the absence of RecB‐dependent nuclease function, recombination becomes entirely dependent on the 5′ → 3′ single‐stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).
Discovery and distribution of super‐integrons among Pseudomonads
Authors: Vaisvila Romualdas , Morgan Richard D. , Posfai Janos , Raleigh Elisabeth A.
Published: March 2003

Until recently, integrons (systems for acquisition and expression of new genetic materials) have been associated generally with antibiotic resistance gene cassettes. The discovery of ‘super‐integrons’ in Vibrionaceae suggests a greater impact of this gene acquisition mechanism on bacterial genome evolution than initially believed. Super‐integrons may contain more than 100 gene cassettes and may encode other determinants, including biochemical functions or virulence factors. Here, we report the genetic organization of a super‐integron from Pseudomonas alcaligenes ATCC 55044. This is the first evidence of a super‐integron in a non‐pathogenic bacterium, one which is widely distributed in a great number of ecological niches such as soil and aquatic habitats. Here, the sequence composition, open reading frame (ORF) content and organization of In55044 are described and found to have features intermediate between the multidrug‐resistant integrons and the Vibrio cholerae super‐integron. Similar structures are inferred to be present in several Pseudomonas species, based on polymerase chain reaction (PCR) experiments.
Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response
Authors: Turgay Kürsad , Persuh Marjan , Hahn Jeanette , Dubnau David
Published: March 2003

MecA targets the competence transcription factor ComK to ClpC. As a consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C‐terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C‐terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation of the N‐terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. In vivo, this mutation had no effect on thermal resistance, but had a clpC null phenotype with respect to competence. Mutation of the C‐terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this mutant protein displayed no response to 0.2 mM ATP, unlike the wild‐type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10‐fold but was not eliminated. In vivo, the K551Q mutation showed a partial defect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C‐terminal domain. Although the evidence suggests that the functions of the two ATP‐binding domains overlap, it appears that the N‐terminal nucleotide‐binding domain of ClpC is particularly concerned with MecA‐related functions, whereas the C‐terminal domain plays a more general role in the activities of ClpC.
Regulation of the HpyII restriction–modification system of Helicobacter pylori by gene deletion and horizontal reconstitution
Authors: Aras Rahul A. , Takata Tohru , Ando Takafumi , Van Der Ende Arie , Blaser Martin J.
Published: March 2003

Helicobacter pylori, Gram‐negative, curved bacteria colonizing the human stomach, possess strain‐specific complements of functional restriction–modification (R–M) systems. Restriction–modification systems have been identified in most bacterial species studied and are believed to have evolved to protect the host genome from invasion by foreign DNA. The large number of R–Ms homologous to those in other bacterial species and their strain‐specificity suggest that H. pylori may have horizontally acquired these genes. A type IIs restriction–modification system, hpyIIRM, was active in two out of the six H. pylori strains studied. We demonstrate now that in most strains lacking M.HpyII function, there is complete absence of the R–M system. Direct DNA repeats of 80 bp flanking the hpyIIRM system allow its deletion, resulting in an ‘empty‐site’ genotype. We show that strains possessing this empty‐site genotype and strains with a full but inactive hpyIIRM can reacquire the hpyIIRM cassette and functional activity through natural transformation by DNA from the parental R–M+ strain. Identical isolates divergent for the presence of an active HpyII R–M pose different restriction barriers to transformation by foreign DNA. That H. pylori can lose HpyII R–M function through deletion or mutation, and can horizontally reacquire the hpyIIRM cassette, is, in composite, a novel mechanism for R–M regulation, supporting the general hypothesis that H. pylori populations use mutation and transformation to regulate gene function.
IpaC from Shigella and SipC from Salmonella possess similar biochemical properties but are functionally distinct
Authors: Osiecki John C. , Barker Jeff , Picking Wendy L. , Serfis Alexa Barnoski , Berring Erin , Shah Shreya , Harrington Amanda , Picking William D.
Published: March 2003

Invasion plasmid antigen C (IpaC) is secreted via the type III secretion system (TTSS) of Shigella flexneri and serves as an essential effector molecule for epithelial cell invasion. The only homologue of IpaC identified thus far is Salmonella invasion protein C (SipC/SspC), which is essential for enterocyte invasion by Salmonella typhimurium. To explore the biochemical and functional relatedness of IpaC and SipC, recombinant derivatives of both proteins were purified so that their in vitro biochemical properties could be compared. Both proteins were found to: (i) enhance the entry of wild‐type S. flexneri and S. typhimurium into cultured cells; (ii) interact with phospholipid membranes; and (iii) oligomerize in solution; however, IpaC appeared to be more efficient in carrying out several of the biochemical properties examined. Overall, the data indicate that purified IpaC and SipC are biochemically similar, although not identical with respect to their in vitro activities. To extend these observations, complementation analyses were conducted using S. flexneri SF621 and S. typhimurium SB220, neither of which is capable of invading epithelial cells because of non‐polar null mutations in ipaC and sipC respectively. Interestingly, both ipaC and sipC restored invasiveness to SB220 whereas only ipaC restored invasiveness to SF621, suggesting that SipC lacks an activity possessed by IpaC. This functional difference is not at the level of secretion because IpaC and SipC are both secreted by SF621 and it does not appear to be because of SipC dependency on this native chaperone as coexpression of sipC and sicA in SF621 still failed to restore detectable invasiveness. Taken together, the data suggest that IpaC and SipC differ in either their ability to be translocated into host cells or in their function as effectors of host cell invasion. Because IpaB shares significant sequence homology with the YopB translocator of Yersinia species, the ability for IpaC and SipC to associate with this protein was explored as a potential indicator of translocation function. Both proteins were found to bind to purified IpaB with an apparent dissociation constant in the nanomolar range, suggesting that they may differ with respect to effector function. Interestingly, whereas SB220 expressing sipC behaved like wild‐type Salmonella, in that it remained within its membrane‐bound vacuole following entry into host cells, SB220 expressing ipaC was found in the cytoplasm of host cells. This observation indicates that IpaC and SipC are responsible for a major difference in the invasion strategies of Shigella and Salmonella, that is, they escape into the host cell cytoplasm. The implications of the role of each protein's biochemistry relative to its in vivo function is discussed.
The basis of asymmetry in IS2 transposition
Authors: Lewis Leslie A. , Gadura Nidhi , Greene Marsha , Saby Robert , Grindley Nigel D. F.
Published: March 2003

In the first step of IS2 transposition, the formation of an IS2 minicircle, the roles of the two IS ends differ. Terminal cleavage initiates exclusively at the right inverted repeat (IRR) – the donor end – whereas IRL is always the target. At the resulting minicircle junction, the two abutted ends are separated by a spacer of 1 or 2 basepairs. In this study, we have identified the determinants of donor and target function. The inability of IRL to act as a donor results largely from two sequence differences between IRL and IRR – an extra basepair between the conserved transposase binding sequences and the end of the element, and a change of the terminal dinucleotide from CA‐3′ to TA‐3′. These two changes also impose a characteristic size on the minicircle junction spacer. The only sequences required for the efficient target function of IRL appear to be contained within the segment from position 11–42. Although IRR can function as a target, its shorter length and additional contacts with transposase (positions 1–7) result in minicircles with longer, and inappropriate, spacers. We propose a model for the synaptic complex in which the terminus of IRL makes different contacts with the transposase for the initial and final strand transfer steps. The sequence differences between IRR and IRL, and the behavioural characteristics of IRL that result from them, have probably been selected because they optimize expression of transposase from the minicircle junction promoter, Pjunc.
Sensing nitrogen limitation inCorynebacterium glutamicum: the role ofglnKandglnD†
Authors: Nolden Lars , Ngouoto‐Nkili Carole‐Estelle , Bendt Anne Kathrin , Krämer Reinhard , Burkovski Andreas
Published: March 2003

A novel nitrogen control system regulating the transcription of genes expressed in response to nitrogen starvation inCorynebacterium glutamicumwas identified by us recently. In this communication, we also show that the nitrogen regulation cascade inC. glutamicumfunctions by a new mechanism, although components highly similar to sensor and signal transmitter proteins ofEscherichia coliare used, namely uridylyltransferase and a PII‐type GlnK protein. The genes encoding these key components of the nitrogen regulation cascade,glnDandglnK, are organized in an operon together withamtB, which codes for an ammonium permease. Using a combination of site‐directed mutagenesis, RNA hybridization experiments, reporter gene assays, transport measurements and non‐denaturing gel electrophoresis followed by immunodetection, we showed that GlnK is essential for nitrogen control and that signal transduction is transmitted by uridylylation of this protein. As a consequence of the latter, aglnDdeletion strain lacking uridylyltransferase is impaired in its response to nitrogen shortage. TheglnDmutant revealed a decreased growth rate in the presence of limiting amounts of ammonium or urea; additionally, changes in its protein profile were observed, as shown byin vivolabelling and two‐dimensional PAGE. In contrast toE. coli, expression ofglnDis upregulated upon nitrogen limitation inC. glutamicum. This indicates that theglnDgene product is probably not the primary sensor of nitrogen status inC. glutamicumas shown for enterobacteria. In accordance with this hypothesis, we found a deregulated nitrogen control as a result of the overexpression ofglnD. Furthermore, quantification of cytoplasmic amino acid pools excluded the possibility that a fall in glutamine concentration is perceived as the signal for nitrogen starvation byC. glutamicum, as is found in enterobacteria. Direct measurements of the intracellular ammonium pool indicated that the concentration of this compound might indicate the cellular nitrogen status. Deduced fromglnKandglnDexpression patterns and the genetic organization of these genes, this regulatory mechanism is also present inCorynebacterium diphtheriae, the causative agent of diphtheria.