bluuebunny/crossref_metadata_2025 huggingface dataset:
FNR‐dependent activation of the class II dmsA and narG promoters of Escherichia coli requires FNR‐activating regions 1 and 3
Authors: Lamberg Karin E. , Kiley Patricia J.
Published: March 2003

In Escherichia coli, the anaerobic expression of genes encoding the nitrate (narGHJI) and dimethyl sulphoxide (dmsABC) terminal reductases is stimulated by the global anaerobic regulator FNR. The ability of FNR to activate transcription initiation has been proposed to be dependent on protein–protein interactions between RNA polymerase and two activating regions (AR) of FNR, FNR‐AR1 and FNR‐AR3. To further our understanding of the role of FNR‐AR1 and FNR‐AR3 in transcription activation, we measured the effects of FNR‐AR mutants on expression of the narG and dmsA promoters, PnarG and PdmsA. All the FNR‐AR1 (FNR‐S73F, FNR‐T118A, FNR‐S187P), FNR‐AR3 (FNR‐G85A) and FNR‐AR1‐AR3 (FNR‐G85A‐S187P) mutants that were tested decreased expression from PnarG and PdmsAin vivo. Transcription assays of PdmsA also showed that the FNR‐AR mutant proteins impaired transcription activation in vitro. Furthermore, DNase I footprinting analysis confirmed that this transcription defect was not a result of altered DNA‐binding properties. The function of FNR‐S187P and FNR‐G85A was also measured in strains containing σ70 mutants (σ70‐K593A, σ70‐R596A and σ70‐K597A) known to be impaired in FNR‐dependent transcription activation. Of all of the combinations analysed, only FNR‐G85 and σ70‐K597 showed a genetic interaction, supporting the notion that FNR‐AR3 and σ70 interact functionally in the process of transcription activation. Lastly, the transcription activation defect of the FNR‐AR1 and FNR‐AR3 mutants was greatly reduced when expression of PnarG was assayed in the presence of nitrate. As these growth conditions promote maximal activity of PnarG as a result of the combined function of NarL, IHF and FNR, these results suggest that the requirements for FNR‐AR1 and FNR‐AR3 are altered in the presence of additional activators.
Evidence for the secretion of Chlamydia trachomatis CopN by a type III secretion mechanism
Authors: Fields K. A. , Hackstadt T.
Published: March 2003

The medically significant, obligate intracellular pathogen Chlamydia trachomatis replicates within vacuoles termed inclusions. A developmental cycle is initiated after entry into a host cell and is manifested by the transformation of infectious elementary bodies (EBs) to larger, non‐infectious reticulate bodies (RBs). Analysis of the C. trachomatis genome has revealed that chlamydiae possess genes that may encode a type III secretion apparatus. In other Gram‐negative pathogens, the type III secretion mechanism is used to target virulence factors directly to the host cell cytoplasm and is essential for full virulence. To evaluate the possibility of a functional type III secretion mechanism in C. trachomatis, we initially focused on a locus containing genes encoding products with similarity to chaperones (Scc1), secretion pore components (Cds1 and Cds2) and secreted proteins (CopN) from other type III systems. Gene expression was tested by reverse transcriptase–polymerase chain reaction (RT–PCR) of total RNA extracted from infected HeLa cell monolayers at 2, 6, 12 and 20 h after infection and normalized for the number of C. trachomatis genomes present. Message was detected for Scc1 at all times, whereas message for all other tested genes was detected in significant amounts at 12 h and 20 h. Immunoblot analysis with Scc1‐ and CopN‐specific antibodies revealed that CopN and Scc1 were present in EBs, RBs and whole‐culture extracts harvested 20 h after infection. CopN is homologous to the secreted protein YopN of Yersinia sp., and analysis of monolayers 20 h after infection via indirect immunofluorescence showed specific labelling of inclusion membranes when probed with CopN‐specific antibodies but not with Scc1‐specific antibodies. His‐tagged CopN and a chlamydial cytoplasmic control protein (NrdB) were expressed in Yersinia enterocolitica containing or lacking the virulence plasmid pYV. CopN, but not NrdB, was secreted by Y. enterocolitica in a Ca2+‐ and pYV‐dependent fashion. These data indicate that components of the putative type III apparatus of C. trachomatis are expressed and that at least one of these products is secreted by chlamydiae to the inclusion membrane. The observation that CopN is also secreted by the Yersinia type III apparatus provides support for the notion that chlamydiae secrete proteins via a type III mechanism.
Structure and composition of the Shigella flexneri‘needle complex’, a part of its type III secreton
Authors: Blocker Ariel , Jouihri Noureddine , Larquet Eric , Gounon Pierre , Ebel Frank , Parsot Claude , Sansonetti Philippe , Allaoui Abdelmounaaïm
Published: March 2003

Type III secretion systems (TTSSs or secretons), essential virulence determinants of many Gram‐negative bacteria, serve to translocate proteins directly from the bacteria into the host cytoplasm. Electron microscopy (EM) indicates that the TTSSs of Shigella flexneri are composed of: (1) an external needle; (2) a transmembrane domain; and (3) a cytoplasmic bulb. EM analysis of purified and negatively stained parts 1, 2 and a portion of 3 of the TTSS, together termed the ‘needle complex’ (NC), produced an average image at 17 Å resolution in which a base, an outer ring and a needle, inserted through the ring into the base, could be discerned. This analysis and cryoEM images of NCs indicated that the needle and base contain a central 2–3 nm canal. Five major NC components, MxiD, MxiG, MxiJ, MxiH and MxiI, were identified by N‐terminal sequencing. MxiG and MxiJ are predicted to be inner membrane proteins and presumably form the base. MxiD is predicted to be an outer membrane protein and to form the outer ring. MxiH and MxiI are small hydrophilic proteins. Mutants lacking either of these proteins formed needleless secretons and were unable to secrete Ipa proteins. As MxiH was present in NCs in large molar excess, we propose that it is the major needle component. MxiI may cap at the external needle tip.
Low external pH induces HOG1‐dependent changes in the organization of the Saccharomyces cerevisiae cell wall
Authors: Kapteyn J. C. , Ter Riet B. , Vink E. , Blad S. , De Nobel H. , Van Den Ende H. , Klis F. M.
Published: March 2003

Low environmental pH strongly affected the organization of the Saccharomyces cerevisiae cell wall, resulting in rapidly induced resistance to β1,3‐glucanase. At a molecular level, we found that a considerable amount of Cwp1p became anchored through a novel type of linkage for glycosylphosphatidylinositol (GPI)‐dependent cell wall proteins, namely an alkali‐labile linkage to β1,3‐glucan. This novel type of modification for Cwp1p did not require the presence of a GPI‐derived structure connecting the protein with β1,6‐glucan. In addition, we found high levels of Cwp1p, which was double‐anchored through both the novel alkali‐sensitive bond to β1,3‐glucan and the alkali‐resistant GPI‐derived linkage to β1,6‐glucan. Further cell wall analyses demonstrated that Pir2p/Hsp150 and possibly other Pir cell wall proteins, which were already known to be linked to the β1,3‐glucan framework by an alkali‐sensitive linkage, were also more efficiently retained in the cell wall at pH 3.5 than at pH 5.5. Consequently, the alkali‐sensitive type of linkage of cell wall proteins to β1,3‐glucan was induced by low pH. The low pH‐induced alterations in yeast cell wall architecture were demonstrated to be dependent on a functional HOG1 gene, but not on the Slt2p‐mediated MAP kinase pathway. Consistent with this observation, DNA microarray studies revealed transcriptional induction of many known high‐osmolarity glycerol (HOG) pathway‐dependent genes, including four cell wall‐related genes, namely CWP1, HOR7, SPI1 and YGP1.
MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR‐1
Authors: Beliaev Alex S. , Saffarini Daad A. , McLaughlin Jennifer L. , Hunnicutt David
Published: March 2003

Shewanella putrefaciens is a facultative anaerobe that can use metal oxides as terminal electron acceptors during anaerobic respiration. Two proteins, MtrB and Cct, have been identified that are specifically involved in metal reduction. Analysis of S. putrefaciens mutants deficient in metal reduction led to the identification of two additional proteins that are involved in this process. MtrA is a periplasmic decahaem c‐type cytochrome that appears to be part of the electron transport chain, which leads to Fe(III) and Mn(IV) reduction. MtrC is an outer membrane decahaem c‐type cytochrome that appears to be required for the activity of the terminal Fe(III) reductase. Membrane fractions of mutants deficient in MtrC exhibited a decreased level of Fe(III) reduction compared with the wild type. We suggest that MtrC may be a component of the terminal reductase or may be required for its assembly.
Actin is ADP‐ribosylated by the Salmonella enterica virulence‐associated protein SpvB
Authors: Tezcan‐Merdol Dilek , Nyman Tomas , Lindberg Uno , Haag Friedrich , Koch‐Nolte Friedrich , Rhen Mikael
Published: March 2003

The Salmonella enterica virulence‐associated protein SpvB was recently shown to contain a carboxy‐terminal mono(ADP‐ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full‐length SpvB modifies a 43 kDa protein from macrophage‐like J774‐A.1 and epithelial MDCK cells as shown by label transfer from [32P]‐nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two‐dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325‐infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]‐NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB‐mediated modification, most probably ADP‐ribosylation, and that the modification of G actin interferes with actin polymerization.
The Nudix hydrolases of Deinococcus radiodurans
Authors: Xu WenLian , Shen JianYing , Dunn Christopher A. , Desai Seema , Bessman Maurice J.
Published: March 2003

All 21 of the Nudix hydrolase genes from the radiation‐resistant organism Deinococcus radiodurans have been cloned into vectors under the control of T7 promoters and expressed as soluble proteins in Escherichia coli. Their sizes range from 9.8 kDa (91 amino acids) to 59 kDa (548 amino acids). Two novel proteins were identified, each with two Nudix boxes in its primary structure, unique among all other known Nudix hydrolases. Extracts of each of the expressed proteins were assayed by a generalized procedure that measures the hydrolysis of nucleoside diphosphate derivatives, and several enzymatic activities were tentatively identified. In addition to representatives of known Nudix hydrolase subfamilies active on ADP‐ribose, NADH, dinucleoside polyphosphates or (deoxy)nucleoside triphosphates, two new enzymes, a UDP‐glucose pyrophosphatase and a CoA pyrophosphatase, were identified.
Yersinia YopE is targeted for type III secretion by N‐terminal, not mRNA, signals
Authors: . Lloyd Scott A , Norman Mari , Rosqvist Roland , Wolf‐Watz Hans
Published: March 2003

Pathogenic Yersinia species inject virulence proteins, known as Yops, into the cytosol of eukaryotic cells. The injection of Yops is mediated via a type III secretion system. Previous studies have suggested that YopE is targeted for secretion by two signals. One is mediated by its cognate chaperone YerA, whereas the other consists of either the 5′ end of yopE mRNA or the N‐terminus of YopE. In order to characterize the YopE N‐terminal/5′ mRNA secretion signal, the first 11 codons of yopE were systematically mutagenized. Frameshift mutations, which completely alter the amino acid sequence of residues 2–11 but leave the mRNA sequence essentially intact, drastically reduce the secretion of YopE in a yerA mutant. In contrast, a mutation that alters the yopE mRNA sequence, while leaving the amino acid sequence of YopE unchanged, does not impair the secretion of YopE. Therefore, the N‐terminus of YopE, and not the 5′ end of yopE mRNA, serves as a targeting signal for type III secretion. In addition, the chaperone YerA can target YopE for type III secretion in the absence of a functional N‐terminal signal. Mutational analysis of the YopE N‐terminus revealed that a synthetic amphipathic sequence of eight residues is sufficient to serve as a targeting signal. YopE is also secreted rapidly upon a shift to secretion‐permissive conditions. This ‘rapid secretion’ of YopE does not require de novo protein synthesis and is dependent upon YerA. Furthermore, this burst of YopE secretion can induce a cytotoxic response in infected HeLa cells.
The two TonB systems of Vibrio cholerae: redundant and specific functions
Authors: Seliger Stefan S. , Mey Alexandra R. , Valle Ana‐Maria , Payne Shelley M.
Published: March 2003

The two TonB systems in Vibrio cholerae were found to have unique as well as common functions. Both systems can mediate transport of haemin and the siderophores vibriobactin and ferrichrome. However, TonB1 specifically mediates utilization of the siderophore schizokinen, whereas TonB2 is required for utilization of enterobactin by V. cholerae. Although either TonB system was sufficient for the use of haemin as an iron source, in vitro competition between TonB1 and TonB2 system mutants indicates a preferential role for TonB1 in haemin utilization. This was most pronounced in conditions of high osmolarity, in which TonB1 system mutants were unable to grow with haemin as the sole iron source. Sequence analysis predicted that the two TonB proteins differ in both amino acid sequence and protein size. An internal deletion in TonB1 was constructed in order to generate a protein of approximately the same size as TonB2. A strain expressing the TonB1 deletion protein, and no other TonB, used haemin as the iron source in low‐osmolarity medium, but could not use haemin in high osmolarity. This is the same phenotype as a strain expressing only TonB2 and suggests that TonB1, but not TonB2, can span the increased periplasmic space in high osmolarity and thus mediate haemin transport. Mouse colonization assays indicated a role for both TonB systems, and mutations in either system resulted in reduced ability to compete with the wild type in vivo.
Two‐partner secretion in Gram‐negative bacteria: a thrifty, specific pathway for large virulence proteins
Authors: Jacob‐Dubuisson Françoise , Locht Camille , Antoine Rudy
Published: March 2003

A collection of large virulence exoproteins, including Ca2+‐independent cytolysins, an iron acquisition protein and several adhesins, are secreted by the two‐partner secretion (TPS) pathway in various Gram‐negative bacteria. The hallmarks of the TPS pathway are the presence of an N‐proximal module called the ‘secretion domain’ in the exoproteins that we have named the TpsA family, and the channel‐forming β‐barrel transporter proteins we refer to as the TpsB family. The genes for cognate exoprotein and transporter protein are usually organized in an operon. Specific secretion signals are present in a highly conserved region of the secretion domain of TpsAs. TpsBs probably serve as specific receptors of the TpsA secretion signals and as channels for the translocation of the exoproteins across the outer membrane. A subfamily of transporters also mediates activation of their cognate cytolysins upon secretion. The exoproteins are synthesized as precursors with an N‐terminal cleavable signal peptide, and a subset of them carries an extended signal peptide of unknown function. According to our current model, the exoproteins are probably translocated across the cytoplasmic membrane in a Sec‐dependent fashion, and their signal peptide is probably processed by a LepB‐type signal peptidase. The N‐proximal secretion domain directs the exoproteins towards their transporters early, so that translocation across both membranes is coupled. The exoproteins transit through the periplasm in an extended conformation and fold progressively at the cell surface before eventually being released into the extracellular milieu. Several adhesins also undergo extensive proteolytic processing upon secretion. The genes of many new TpsAs and TpsBs are found in recently sequenced genomes, suggesting that the TPS pathway is widespread.
A functional genomic analysis of type 3 Streptococcus pneumoniae virulence
Authors: Lau Gee W. , Haataja Sauli , Lonetto Michael , Kensit Sarah E. , Marra Andrea , Bryant Alexander P. , McDevitt Damien , Morrison Donald A. , Holden David W.
Published: March 2003

Streptococcus pneumoniae remains a serious cause of morbidity and mortality in humans, but relatively little is known about the molecular basis of its pathogenesis. We used signature‐tagged mutagenesis together with an analysis of S. pneumoniae genome sequence to identify and characterize genes required for pathogenesis. A library of signature‐tagged mutants was created by insertion–duplication mutagenesis, and 1786 strains were analysed for their inability to survive and replicate in murine models of pneumonia and bacteraemia. One hundred and eighty‐six mutant strains were identified as attenuated, and 56 were selected for further genetic characterization based on their ability to excise the integrated plasmid spontaneously. The genomic DNA inserts of the plasmids were cloned in Escherichia coli and sequenced. These sequences were subjected to database searches, including the S. pneumoniae genome sequence, which allowed us to examine the chromosomal regions flanking these genes. Most of the insertions were in probable operons, but no pathogenicity islands were found. Forty‐two novel virulence loci were identified. Five strains mutated in genes involved in gene regulation, cation transport or stress tolerance were shown to be highly attenuated when tested individually in a murine respiratory tract infection model. Additional experiments also suggest that induction of competence for genetic transformation has a role in virulence.
The Salmonella spvB virulence gene encodes an enzyme that ADP‐ribosylates actin and destabilizes the cytoskeleton of eukaryotic cells
Authors: Lesnick Marc L. , Reiner Neil E. , Fierer Joshua , Guiney Donald G.
Published: March 2003

ADP‐ribosylating enzymes, such as cholera and diphtheria toxins, are key virulence factors for a variety of extracellular bacterial pathogens but have not been implicated previously during intracellular pathogenesis. Salmonella strains are capable of invading epithelial cells and localizing in macrophages during infection. The spvB virulence gene of Salmonella is required for human macrophage cytotoxicity in vitro and for enhancing intracellular bacterial proliferation during infection. Here, we present evidence that spvB encodes an ADP‐ribosylating enzyme that uses actin as a substrate and depolymerizes actin filaments when expressed in CHO cells. Furthermore, site‐directed mutagenesis demonstrates that the ADP‐ribosylating activity of SpvB is essential for Salmonella virulence in mice. As spvB is expressed by Salmonella strains after invasion of epithelial cells or phagocytosis by macrophages, these results suggest that SpvB functions as an intracellular ADP‐ribosylating toxin critical for the pathogenesis of Salmonella infections.
Genetic dissection of the roles of chaperones and proteases in protein folding and degradation in the Escherichia coli cytosol
Authors: Tomoyasu Toshifumi , Mogk Axel , Langen Hanno , Goloubinoff Pierre , Bukau Bernd
Published: March 2003

We investigated the roles of chaperones and proteases in quality control of proteins in the Escherichia coli cytosol. In ΔrpoH mutants, which lack the heat shock transcription factor and therefore have low levels of all major cytosolic proteases and chaperones except GroEL and trigger factor, 5–10% and 20–30% of total protein aggregated at 30°C and 42°C respectively. The aggregates contained 350–400 protein species, of which 93 were identified by mass spectrometry. The aggregated protein species were similar at both temperatures, indicating that thermolabile proteins require folding assistance by chaperones already at 30°C, and showed strong overlap with previously identified DnaK substrates. Overproduction of the DnaK system, or low‐level production of the DnaK system and ClpB, prevented aggregation and provided thermotolerance to ΔrpoH mutants, indicating key roles for these chaperones in protein quality control and stress survival. In rpoH+ cells, DnaK depletion did not lead to protein aggregation at 30°C, which is probably the result of high levels of proteases and thus suggests that DnaK is not a prerequisite for proteolysis of misfolded proteins. Lon was the most efficient protease in degrading misfolded proteins in DnaK‐depleted cells. At 42°C, ClpXP and Lon became essential for viability of cells with low DnaK levels, indicating synergistic action of proteases and the DnaK system, which is essential for cell growth at 42°C.
The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome
Authors: Weigel Christoph , Messer Walter , Preiss Susanne , Welzeck Michaela ,   Morigen, Boye Erik
Published: March 2003

We have developed a simple three‐step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild‐type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild‐type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild‐type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.
A phase‐variable capsule is involved in virulence of Campylobacter jejuni 81‐176
Authors: Bacon David J. , Szymanski Christine M. , Burr Don H. , Silver Richard P. , Alm Richard A. , Guerry Patricia
Published: March 2003

Campylobacter jejuni strain 81‐176 (HS36, 23) synthesizes two distinct glycan structures, as visualized by immunoblotting of proteinase K‐digested whole‐cell preparations. A site‐specific insertional mutant in the kpsM gene results in loss of expression of a high‐molecular‐weight (HMW) glycan (apparent Mr 26 kDa to > 85 kDa) and increased resolution of a second ladder‐like glycan (apparent Mr 26–50 kDa). The kpsM mutant of 81‐176 is no longer typeable in either HS23 or HS36 antisera, indicating that the HMW glycan structure is the serodeterminant of HS23 and HS36. Both the kpsM‐dependent HMW glycan and the kpsM‐independent ladder‐like structure appear to be capsular in nature, as both are attached to phospholipid rather than lipid A. Additionally, the 81‐176 kpsM gene can complement a deletion in Escherichia coli kpsM, allowing the expression of an α2,8 polysialic acid capsule in E. coli. Loss of the HMW glycan in 81‐176 kpsM also increases the surface hydrophobicity and serum sensitivity of the bacterium. The kpsM mutant is also significantly reduced in invasion of INT407 cells and reduced in virulence in a ferret diarrhoeal disease model. The expression of the kpsM‐dependent capsule undergoes phase variation at a high frequency.
Systematic mutagenesis of the DNA binding sites for SoxS in the Escherichia coli zwf and fpr promoters: identifying nucleotides required for DNA binding and transcription activation
Authors: Griffith Kevin L. , Wolf Richard E.
Published: March 2003

SoxS is the direct transcriptional activator of at least 15 genes of the Escherichia coli superoxide regulon. SoxS is small (107 amino acids), binds DNA as a monomer and recognizes a highly degenerate DNA binding site, termed ‘soxbox’. Like other members of the AraC/XylS family, SoxS has two putative helix–turn–helix (HTH) DNA‐binding motifs, and it has been proposed that each HTH motif recognizes a highly conserved recognition element of the soxbox. To determine which nucleotides are important for SoxS binding, we conducted a systematic mutagenesis of the DNA binding sites for SoxS in the zwf and fpr promoters and determined the effect of the soxbox mutations on SoxS DNA binding and transcription activation in vivo by measuring β‐galactosidase activity in strains with fusions to lacZ. We found that the sequences GCAC and CAAA, termed recognition elements 1 and 2 (RE 1 and RE 2), respectively, are critical for SoxS binding, as mutations within these elements severely hinder or eliminate SoxS‐dependent transcription activation; substitutions within RE 2 (CAAA), however, are tolerated better than changes within RE 1 (GCAC). Although substitutions at the seven positions separating the two REs had only a modest effect on SoxS binding, AT basepairs were favoured within this ‘spacer’ region, presumably because, by facilitating DNA bending, they help bring the two recognition elements into proper juxtaposition. We also found that the ‘invariant A’ present at position 1 of 14/15 functional soxboxes identified thus far is important for SoxS binding, as a change to any other nucleotide at this position reduced SoxS‐dependent transcription by ≈ 50%. In addition, positions surrounding the REs seem to show a context effect, in that certain substitutions there have little or no effect when the RE has the optimal binding sequence, but produce a pronounced effect when the RE has a suboptimal sequence. We propose that these nucleotides play an important role in effecting differential expression from the various promoters. Lastly, we used gel retardation assays to show that alterations in transcription activation in vivo are caused by effects on DNA binding. Based on this exhaustive mutagenesis, we propose the following optimal sequence for SoxS binding: AnVGCACWWWnKRHCAAAHn (n = A, C, G, T; V = A, C, G; W = A, T; K = G, T; R = A, G; H = A, C, T).
The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides
Authors: Martin Angela C. , Wadhams George H. , Armitage Judith P.
Published: March 2003

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in‐frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non‐chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl‐accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.
A novel ligand from Plasmodium falciparum that binds to a sialic acid‐containing receptor on the surface of human erythrocytes
Authors: Thompson Jennifer K. , Triglia Tony , Reed Michael B. , Cowman Alan F.
Published: March 2003

Invasion of the merozoite form of Plasmodium falciparum into human erythrocytes involves multiple receptor–ligand interactions. The EBA175 protein of P. falciparum has been shown to be the ligand that binds to a sialic acid‐dependent site on glycophorin A. We have identified a novel P. falciparum ligand, termed erythrocyte‐binding antigen 140 (EBA140), that shares structural features and homology with EBA175. Subcellular localization of EBA140 suggests that it is located in the micronemes, the same localization as EBA175. EBA140 binds to a sialic acid‐dependent receptor on the surface of human erythrocytes. Binding of EBA140 to this erythrocyte receptor is sensitive to neuraminidase and resistant to trypsin, proteinase K and pronase. The protease‐resistant properties of the erythrocyte receptor suggests that it is not glycophorin A or C. Additionally, analysis of mutant erythrocytes from humans has shown that EBA140 does not bind glycophorin B. Interestingly, we have identified a parasite line that lacks the eba140 gene, suggesting that this protein is not essential for in vitro invasion. These results suggest that EBA140 may be involved in merozoite invasion using a sialic acid‐dependent receptor on human erythrocytes.
The operator‐early promoter regions of Shiga‐toxin bearing phage H‐19B
Authors: Shi Ting , Friedman David I.
Published: March 2003

Genes (stx) encoding Shiga toxins (Stx), major virulence factors in some pathogenic strains of Escherichia coli (STEC), are located in prophages of the λ family. Agents that induce prophages lead to high levels of Stx, suggesting a role for the prophage in stx expression. Activation of the phage regulatory cascade has been shown to contribute to Stx production and release. Therefore, repressor–operator interactions that maintain prophage repression appear important in regulating expression of a major bacterial virulence factor. To determine if the operators of an stx‐bearing phage have distinctive features, we characterized the operator regions of H‐19B, a lambdoid phage carrying stx1 genes. H‐19B mutants that grow in the presence of repressor (classically called virulent mutants) were selected and the mutations definitively identified the operators. The H‐19B operators, as those in other lambdoid phages, comprise variations of an inverted repeat. Four repeats were identified in OR rather than the three found in each of the operators of other lambdoid phages. Primer extensions identified the transcription start sites of PR and PRM, the two promoters in OR regulated by repressor.
Valency conversion in the type 1 fimbrial adhesin of Escherichia coli
Authors: Sokurenko Evgeni V. , Schembri Mark A. , Trintchina Elena , Kjærgaard Kristian , Hasty David L. , Klemm Per
Published: March 2003

FimH protein is a lectin‐like adhesive subunit of type 1, or mannose‐sensitive, fimbriae that are found on the surface of most Escherichia coli strains. All naturally occurring FimH variants demonstrate a conserved mannotriose‐specific (i.e. multivalent) binding. Here, we demonstrate that replacement of residues 185–279 within the FimH pilin domain with a corresponding segment of the type 1C fimbrial adhesin FocH leads to a loss of the multivalent mannotriose‐specific binding property accompanied by the acquisition of a distinct monomannose‐specific (i.e. monovalent) binding capability. Bacteria expressing the monovalent hybrid adhesins were capable of binding strongly to uroepithelial tissue culture cells and guinea pig erythrocytes. They could not, however, agglutinate yeast or bind human buccal cells – functions readily accomplished by the E. coli‐ expressing mannotriose‐specific FimH variants. Based on the relative potency of inhibiting compounds of different structures, the receptor binding site within monovalent FimH–FocH adhesin has an extended structure with an overall configuration similar to that within the multivalent FimH of natural origin. The monomannose‐only specific phenotype could also be invoked by a single point mutation, E89K, located within the lectin domain of FimH, but distant from the receptor binding site. The structural alterations influence the receptor‐binding valency of the FimH adhesin via distal effects on the combining pocket, obviously by affecting the FimH quaternary structure.
Deficiency of essential GTP‐binding protein ObgE in Escherichia coli inhibits chromosome partition
Authors: Kobayashi Gengo , Moriya Shigeki , Wada Chieko
Published: March 2003

GTP‐binding proteins are involved in cell proliferation, development, signal transduction, protein elongation, etc. and construct the GTPase superfamily, whose structures and sequence motifs (G‐1 to G‐5) are highly conserved from prokaryote to eukaryote. Obg of Bacillus subtilis and Obg homologues of other bacteria belong to the GTPase superfamily and have been suggested as being essential for cell growth, development and monitoring of intracellular levels of GTP. We identified the Obg homologue in Escherichia coli, a protein previously known as YhbZ, which we have renamed ObgE. Double cross‐over experiments showed that the obgE gene is essential for growth in E. coli. From characterization of the obgE temperature‐sensitive mutant, we found that DNA replication was not inhibited, that the nucleoids did not partition and instead remained in the middle of cell, and that the cells elongated. Overproduction of ObgE also resulted in aberrant chromosome segregation. These data suggested that ObgE is involved directly or indirectly in E. coli chromosome partitioning. Characterization studies showed that ObgE is abundant in normal cells, partially associated with the membrane and does not associate with ribosomes such as in Obg of B. subtilis. We purified ObgE protein from a cell extract of E. coli, and the purified ObgE had GTPase activity and DNA‐binding ability.
Quantitative analysis of expression of two circadian clock‐controlled gene clusters coding for the bidirectional hydrogenase in the cyanobacterium Synechococcus sp. PCC7942
Authors: Schmitz Oliver , Boison Gudrun , Bothe Hermann
Published: March 2003

Hydrogen metabolism is of central interest in cyanobacterial research because of its potential applications. The gene expression and physiological role of the cyanobacterial bidirectional NAD(P)+‐reducing hydrogenase are poorly understood. Transcription rates of hoxEF and hoxUYH encoding this enzyme have been studied in Synechococcus sp. PCC7942. PhoxU activity was about three times higher than that of PhoxE. Circadian phasing of both promoters was found to be synchronous and influenced expression levels by at least one order of magnitude. This is the first demonstration of circadian control of gene expression for any hydrogenase. For the majority of PhoxU‐driven messages, transcription presumably terminates between hoxU and hoxH. Being part of a polycistronic hoxUYHW… operon, hoxW, encoding a protease involved in C‐terminal processing of the hydrogenase large‐subunit HoxH, is mainly expressed by its own promoter, PhoxW. The complex transcript formation may be a key feature for controlling bidirectional hydrogenase expression in vivo.
The att locus of Rhodococcus fascians strain D188 is essential for full virulence on tobacco through the production of an autoregulatory compound
Authors: Maes Tania , Vereecke Danny , Ritsema Tita , Cornelis Karen , Thu Hang Ngo Thi , Van Montagu Marc , Holsters Marcelle , Goethals Koen
Published: March 2003

The ability of Rhodococcus fascians strain D188 to provoke leafy gall formation on a variety of plant species is correlated with the linear plasmid pFiD188, on which different pathogenicity loci were identified. The att locus affects the severity of symptom development on tobacco, whereas the fas locus is essential for virulence. To gain insight into the function of the att locus, sequence and expression analyses were performed. The att locus contains nine open reading frames homologous to arginine and β‐lactam biosynthetic genes. att gene expression is transcriptionally induced by leafy gall extracts, but not by extracts of uninfected plants, and depends on the attR gene that encodes a LysR‐type transcriptional regulator. The att locus proves to be essential for the formation of inducing factors (IFs) that are present in gall extracts. Because the induction of the fas locus also requires the presence of IFs in gall extracts, the att locus is proposed to play an important role in regulating the expression of the virulence loci of R. fascians.
The RepA protein of plasmid pSC101 controls Escherichia coli cell division through the SOS response
Authors: Ingmer Hanne , Miller Christine , Cohen Stanley N.
Published: March 2003

Although plasmid copy number varies widely among different plasmid species, normally copy number is maintained within a narrow range for any given plasmid. Such copy number control has been shown to occur by regulation of the rate of plasmid DNA replication. Here we report a novel mechanism by which the pSC101 plasmid also can detect an imbalance between the cellular level of its replication protein, RepA, and plasmid‐borne RepA binding sites to inhibit bacterial DNA replication and delay host cell division when RepA is in relative excess. We show that delayed cell division occurs by RepA‐mediated induction of the SOS response and can be reversed by over‐expression of the host DNA primase, DnaG. The effects of RepA excess are prevented by introducing a surfeit of RepA binding sites. The mechanism reported here may help to limit variation in plasmid copy number and allow repopulation of cells with plasmids when copy number falls – potentially pre‐empting plasmid loss in cultures of dividing cells.
Receptor‐mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor
Authors: Harvey H. A. , Jennings M. P. , Campbell C. A. , Williams R. , Apicella M. A.
Published: March 2003

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP‐R) and the terminal lactosamine of lacto‐N‐neotetraose‐expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP‐R traffics to the cell surface after gonococcal challenge. Co‐localization of ASGP‐R with gonococci was observed. As ASGP‐R‐mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co‐localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin‐coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(β1–4)GlcNac(β1–3)Gal(β1–4)Glc (lacto‐N‐neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP‐R ligand, significantly reduced invasion. A dose–response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH‐deacylated 1291 LOS. These studies indicated that an interaction between lacto‐N‐neotetraose‐terminal LOS and ASGP‐R allows gonococcal entry into PHUECs.
The LuxS‐dependent autoinducer AI‐2 controls the expression of an ABC transporter that functions in AI‐2 uptake in Salmonella typhimurium
Authors: Taga Michiko E. , Semmelhack Julia L. , Bassler Bonnie L.
Published: March 2003

In a process called quorum sensing, bacteria communicate with one another using secreted chemical signalling molecules termed autoinducers. A novel autoinducer called AI‐2, originally discovered in the quorum‐sensing bacterium Vibrio harveyi, is made by many species of Gram‐negative and Gram‐positive bacteria. In every case, production of AI‐2 is dependent on the LuxS autoinducer synthase. The genes regulated by AI‐2 in most of these luxS‐containing species of bacteria are not known. Here, we describe the identification and characterization of AI‐2‐regulated genes in Salmonella typhimurium. We find that LuxS and AI‐2 regulate the expression of a previously unidentified operon encoding an ATP binding cassette (ABC)‐type transporter. We have named this operon the lsr (luxS regulated) operon. The Lsr transporter has homology to the ribose transporter of Escherichia coli and S. typhimurium. A gene encoding a DNA‐binding protein that is located adjacent to the Lsr transporter structural operon is required to link AI‐2 detection to operon expression. This gene, which we have named lsrR, encodes a protein that represses lsr operon expression in the absence of AI‐2. Mutations in the lsr operon render S. typhimurium unable to eliminate AI‐2 from the extracellular environment, suggesting that the role of the Lsr apparatus is to transport AI‐2 into the cells. It is intriguing that an operon regulated by AI‐2 encodes functions resembling the ribose transporter, given recent findings that AI‐2 is derived from the ribosyl moiety of S‐ribosylhomocysteine.
Autoregulation allows Escherichia coli RNase E to adjust continuously its synthesis to that of its substrates
Authors: Sousa Sandra , Marchand Isabelle , Dreyfus Marc
Published: March 2003

The Escherichia coli endonuclease RNase E plays a key role in rRNA maturation and mRNA decay. In particular, it controls the decay of its own mRNA by cleaving it within the 5′‐untranslated region (UTR), thereby autoregulating its synthesis. Here, we report that, when the synthesis of an RNase E substrate is artificially induced to high levels in vivo, both the rne mRNA concentration and RNase E synthesis increase abruptly and then decrease to a steady‐state level that remains higher than in the absence of induction. Using rne–lacZ fusions that retain or lack the rne 5′UTR, we show that these variations reflect a transient mRNA stabilization mediated by the rne 5′UTR. Finally, by putting RNase E synthesis under the control of an IPTG‐controlled promoter, we show that a similar, rne 5′UTR‐mediated mRNA stabilization can result from a shortage of RNase E. We conclude that the burst in substrate synthesis has titrated RNase E, stabilizing the rne mRNA by protecting its 5′UTR. However, this stabilization is self‐correcting, because it allows the RNase E pool to expand until its mRNA is destabilized again. Thus, autoregulation allows RNase E to adjust its synthesis to that of its substrates, a behaviour that may be common among autoregulated proteins. Incidentally, this adjustment cannot occur when translation is blocked, and we argue that the global mRNA stabilization observed under these conditions originates in part from this defect.
The Helicobacter pylori UreI protein: role in adaptation to acidity and identification of residues essential for its activity and for acid activation
Authors: Bury‐Moné Stéphanie , Skouloubris Stéphane , Labigne Agnès , De Reuse Hilde
Published: March 2003

Helicobacter pylori is a human gastric pathogen that survives the strong acidity of the stomach by virtue of its urease activity. This activity produces ammonia, which neutralizes the bacterial microenvironment. UreI, an inner membrane protein, is essential for resistance to low pH and for the gastric colonization of mice by H. pylori. In the heterologous Xenopus oocytes expression system, UreI behaves like an H+‐gated urea channel, and His‐123 was found to be important for low pH activation. We investigated the role of UreI directly in H. pylori and showed that, in the presence of urea, strains expressing wild‐type UreI displayed very rapid stimulation of extracellular ammonia production upon exposure to pH ≤ 5. This response was not observed when acetamide was used as a source of ammonia; therefore, it is specific for urea hydrolysis. To identify residues critical for UreI activity or activation, we constructed H. pylori strains carrying individual chromosomal mutations of UreI (i) in the four conserved histidine residues (H71, H123, H131, H193) and (ii) in a conserved region of the third intracellular loop (L165, G166, K167, F168). The distal H193 (and not H123) was found to be crucial for stimulating the production of ammonia at low pH; a single mutation in this residue uncoupled the UreI activity from its acid activation. The third intracellular loop of UreI was shown to be important for UreI activity. Thus, in H. pylori, UreI is necessary for the adaptation of urease activity to the extracellular pH. UreI behaves like a novel type of urea transporter, and the identification of residues essential for its function in H. pylori provides new insight into the unusual molecular mechanism of low pH activation.
The universal stress protein paralogues of Escherichia coli are co‐ordinately regulated and co‐operate in the defence against DNA damage
Authors: Gustavsson N. , Diez . A. , Nyström T.
Published: March 2003

We have cloned, characterized and inactivated genes encoding putative UspA paralogues in Escherichia coli. The yecG (uspC), yiiT (uspD)
and ydaA (uspE) genes were demonstrated to encode protein pro‐ducts
and these were mapped to spots in the E. coli proteomic database. Expression
analysis using chromosomal transcriptional lacZ fusions and two‐dimensional
gels revealed that all usp genes analysed are regulated in a similar fashion.
Thus, uspC, D and E are all induced in stationary phase and
by a variety of stresses causing growth arrest of cells. Induction is independent
of rpoS but is abolished in a ΔrelAΔspoT
(ppGpp0) background and rescued by suppressor mutations rendering the
β‐subunit of RNA polymerase to behave like a stringent polymerase. Ectopic
elevation of ppGpp levels in growing cells, by overproducing the RelA protein, triggered
the induction of all usp genes. The expression of all usp genes was
also elevated by a mutation in the ftsK cell division gene, and this super‐induction
could be suppressed by inactivating recA indicating that the usp paralogues
are involved in the management of DNA. Indeed, uspC, uspD and uspE
deletion mutants were all found to be sensitive to UV exposure. Overexpression of
UspD could compensate for the lack of a chromosomal uspD gene but not a uspA
gene. Similarly, UspA overproduction could only compensate for the lack of chromosomal
uspA. Moreover, combination of usp mutations had no additive effect on UV sensitivity indicating that they are all co‐operating and required in the same pathway, which could explain the co‐ordinated regulation of the genes.