bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential role of Helicobacter pyloriγ‐glutamyltranspeptidase for the colonization of the gastric mucosa of mice
Authors: Chevalier Catherine , Thiberge Jean‐Michel , Ferrero Richard L. , Labigne Agnès
Published: March 2003

Constitutive expression of γ‐glutamyltranspeptidase (GGT) activity is common to all Helicobacter pylori strains, and is used as a marker for identifying H. pylori isolates. Helicobacter pylori GGT was purified from sonicated extracts of H. pylori strain 85P by anion exchange chromatography. The N‐terminal amino acid sequences of two of the generated endoproteolysed peptides were determined, allowing the cloning and sequencing of the corresponding gene from a genomic H. pylori library. The H. pylori ggt gene consists of a 1681 basepair (bp) open reading frame encoding a protein with a signal sequence and a calculated molecular mass of 61 kDa. Escherichia coli clones harbouring the H. pylori ggt gene exhibited GGT activity at 37°C, in contrast to E. coli host cells (MC1061, HB101), which were GGT negative at 37°C. GGT activity was found to be constitutively expressed by similar genes in Helicobacter felis, Helicobacter canis, Helicobacter bilis, Helicobacter hepaticus and Helicobacter mustelae. Western immunoblots using rabbit antibodies raised against a His‐tagged‐GGT recombinant protein demonstrated that H. pylori GGT is synthesized in both H. pylori and E. coli as a pro‐GGT that is processed into a large and a small subunit. Deletion of a 700 bp fragment within the GGT‐encoding gene of a mouse‐adapted H. pylori strain (SS1) resulted in mutants that were GGT negative yet grew normally in vitro. These mutants, however, were unable to colonize the gastric mucosa of mice when orally administered alone or together (co‐infection) with the parental strain. These results demonstrate that H. pylori GGT activity has an essential role for the establishment of the infection in the mouse model, demonstrating for the first time a physiological role for a bacterial GGT enzyme.
Amino acid residue Ala‐62 in the FimH fimbrial adhesin is critical for the adhesiveness of meningitis‐associated Escherichia coli to collagens
Authors: Pouttu Riitta , Puustinen Terhi , Virkola Ritva , Hacker Jörg , Klemm Per , Korhonen Timo K.
Published: March 2003

Adhesion of meningitis‐associated Escherichia coli O18acK1H7 to collagens was characterized. The E. coli strain IHE 3034 adhered to type IV and type I collagens but not to type III collagen immobilized on glass. Collagens lack terminal mannosyl units, yet the bacterial adhesion was completely abolished in the presence of α‐methyl‐D‐mannoside. A cat cassette was introduced into the fimA gene of IHE 3034, and the resulting mutant strain IHE 3034‐2 failed to adhere to collagens. In contrast, insertion of a Gm cassette into the sfaA gene of IHE 3034, encoding the S‐fimbrillin, had no significant effect on the adhesiveness. The fim cluster from IHE 3034 was cloned and expressed in trans in the fimA::cat mutant strain IHE 3034‐2. The complemented strain IHE 3034‐2(pRPO‐1) exhibited adhesiveness to type IV and type I collagens, confirming the function of the type 1 fimbria in the adhesion. We have previously shown that the type 1 fimbria from E. coli K‐12 strain PC31 does not confer bacterial adhesiveness to collagens. The fimH genes from E. coli IHE 3034 as well as from PC31 were expressed in the fimH‐null strain MS4. The FimH from IHE 3034 potentiated collagen adherence, whereas the FimH from PC31 was inactive. Sequence comparison of fimH from IHE 3034 and PC31 revealed five amino‐acid differences in the predicted mature FimH proteins: at residues 27, 62, 70, 78 and 201. Each of these residues in the IHE 3034‐FimH were individually substituted to the corresponding amino acid in the PC31‐FimH. The substitution S62→A completely abolished collagen adhesiveness. The reverse substitution A62→S in the PC31‐FimH as well as in the FimH from another E. coli strain induced collagen adhesiveness to the level seen with IHE 3034‐FimH. Out of nine fimH genes analysed from isolates of E. coli, collagen adhesiveness as well as alanine at position 62 in FimH were found only in two O18acK1H7 isolates with the isoenzyme profile ET type 1. Our results demonstrate that the amino‐acid residue Ala‐62 in the FimH lectin is critical for the adhesion to collagens by a highly virulent clonal group of E. coli.
PcaR‐mediated activation and repression of pca genes from Pseudomonas putida are propagated by its binding to both the −35 and the −10 promoter elements
Authors: Guo Zhu , Houghton John E.
Published: March 2003

Degradation of protocatechuate in Pseudomonas putida is accomplished by the products of the pca genes (pcaH,G, pcaBDC, pcaI, J and pcaF ). In P. putida, all these genes (with the exception of pcaH,G ) are activated by the regulatory protein PcaR, in association with the pathway intermediate β‐ketoadipate. Having previously cloned and characterized the pcaR locus, we have overexpressed and purified the PcaR protein to homogeneity. The purified PcaR protein was shown to form a homodimer in solution and to bind specifically to its own promoter, as well as to the promoter regions of pcaI, J and pcaF. Subsequent footprint analyses demonstrated that the binding of PcaR to its own promoter occurs within a footprint that extends from the −20 to the +4 position. In contrast, PcaR appeared to interact with the inducible pcaI, J promoter as a dimer of dimers; binding in tandem within a dual footprint encompassing both the ‘−35′ and the ‘−10’ regions of the promoter sequence. The similarities and differences between the two binding patterns correlate well with the very different effects that PcaR has upon transcription at each of the promoter sequences. The interactions at the pcaI, J promoter are reminiscent of those exhibited by the MerR family of regulatory proteins. However, as PcaR bears very little primary sequence homology to any of the regulatory proteins within this family, the results presented here reveal the possible existence of a new series of functionally related transcriptional inducers.
Two evolutionarily closely related ABC transporters mediate the uptake of choline for synthesis of the osmoprotectant glycine betaine in Bacillus subtilis
Authors: Kappes Rainer M. , Kempf Bettina , Kneip Susanne , Boch Jens , Gade Jutta , Meier‐Wagner Jana , Bremer Erhard
Published: March 2003

Biosynthesis of the compatible solute glycine betaine in Bacillus subtilis confers a considerable degree of osmotic tolerance and proceeds via a two‐step oxidation process of choline, with glycine betaine aldehyde as the intermediate. We have exploited the sensitivity of B. subtilis strains defective in glycine betaine production against glycine betaine aldehyde to select for mutants resistant to this toxic intermediate. These strains were also defective in choline uptake, and genetic analysis proved that two mutations affecting different genetic loci (opuB and opuC ) were required for these phenotypes. Molecular analysis allowed us to demonstrate that the opuB and opuC operons each encode a binding protein‐dependent ABC transport system that consists of four components. The presumed binding proteins of both ABC transporters were shown to be lipoproteins. Kinetic analysis of [14C]‐choline uptake via OpuB (Km = 1 μM; Vmax = 21 nmol min−1 mg−1 protein) and OpuC (Km = 38 μM; Vmax = 75 nmol min−1 mg−1 protein) revealed that each of these ABC transporters exhibits high affinity and substantial transport capacity. Western blotting experiments with a polyclonal antiserum cross‐reacting with the presumed substrate‐binding proteins from both the OpuB and OpuC transporter suggested that the expression of the opuB and opuC operons is regulated in response to increasing osmolality of the growth medium. Primer extension analysis confirmed the osmotic control of opuB and allowed the identification of the promoter of this operon. The opuB and opuC operons are located close to each other on the B. subtilis chromosome, and their high sequence identity strongly suggests that these systems have evolved from a duplication event of a primordial gene cluster. Despite the close relatedness of OpuB and OpuC, these systems exhibit a striking difference in substrate specificity for osmoprotectants that would not have been predicted readily for such closely related ABC transporters.
Pore‐forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth
Authors: Zuckman Deborah M. , Hung Jerry B. , Roy Craig R.
Published: March 2003

Bacterial pathogens often subvert eukaryotic cellular processes in order to establish a replicative niche and evade host immunity. Inhibition of phagosome lysosome fusion is a strategy used by several intracellular bacteria that grow within mammalian cells. It was shown recently that Legionella pneumophila possesses a cytolytic activity that results from the insertion of pores in the macrophage membrane upon contact, and that this activity requires the dot/icm gene products, which are necessary for intracellular growth and phagosome trafficking. Other bacteria that inhibit phagosome lysosome fusion, such as Mycobacterium tuberculosis, demonstrate similar cytolytic activities, which suggests that formation of pores in the phagosome membrane may account for the defects observed in phagosome trafficking. In this study, we identify a new class of L. pneumophila mutant that retains the pore‐forming activity found in virulent bacteria, but is defective in phagosome lysosome fusion inhibition and intracellular growth. These data indicate that cytolytic activity is not sufficient for L. pneumophila‐induced alterations in phagosome trafficking. Rather, the pore may be a vehicle that facilitates delivery of bacterial‐derived effector molecules to the host cell cytoplasm.
P fimbriae‐dependent, lipopolysaccharide‐independent activation of epithelial cytokine responses
Authors: Hedlund M. , Wachtler C. , Johansson E. , Hang L. , Somerville J. E. , Darveau R. P. , Svanborg C.
Published: March 2003

Cells in the mucosal barrier are equipped to sense and respond to microbes in the lumen and translate this molecular information into signals that can reach local or distant sites. The interaction of P‐fimbriated Escherichia coli with human uroepithelial cells is a model to study the molecular mechanism of epithelial cell activation by mucosal pathogens. Here, we examine the role of lipopolysaccharide (LPS) as a co‐stimulatory molecule in epithelial cell activation by P‐fimbriated E. coli. P‐fimbriated clinical isolates or recombinant strains were shown to trigger a fimbriae‐dependent epithelial cell cytokine response. Mutational inactivation of the msbB sequences that control lipid A myristoylation drastically impaired monocyte stimulation but not epithelial responses to P‐fimbriated bacteria. Polymyxin B or bactericidal/permeability increasing factor (BPI) neutralized the effects of lipid A in the monocyte assay, but did not reduce epithelial responses. Finally, isolated LPS of the smooth, rough and deep rough chemotypes were poor epithelial cell activators. The cells were shown to lack surface CD14 or CD14 mRNA as well as the CD14 co‐receptor function and were also very poor LPS responders in the presence of human serum. These results demonstrate that epithelial cell responses to P‐fimbriated E. coli are CD14 and LPS independent, and suggest that attaching pathogens can overcome the LPS unresponsiveness of epithelial cells by fimbriae‐dependent activation mechanisms.
Ambient pH signal transduction in Aspergillus: completion of gene characterization
Authors: Negrete‐Urtasun Susana , Reiter Wolfgang , Diez Eliecer , Denison Steven H. , Tilburn Joan , Espeso Eduardo A. , Peñalva Miguel A. , Arst, Jr Herbert N.
Published: March 2003

Completing the molecular analysis of the six pal genes of the ambient pH signal transduction pathway in Aspergillus nidulans, we report the characterization of palC and palH. The derived translation product of palH contains 760 amino acids with prediction of seven transmembrane domains in its N‐terminal moiety. Remarkably, a palH frameshift mutant lacking just over half the PalH protein, including almost all of the long hydrophilic region C‐terminal to the transmembrane domains, retains some PalH function. The palC‐derived translation product contains 507 amino acids, and the null phenotype of a frameshift mutation indicates that at least one of the C‐terminal 142 residues is essential for function. Uniquely among the A. nidulans pH‐signalling pal genes, palC appears to have no Saccharomyces cerevisiae homologue, although it does have a Neurospora crassa expressed sequence tag homologue. In agreement with findings for the palA, palB and palI genes of this signalling pathway, levels of the palC and palH mRNAs do not appear to be pH regulated.
The β‐barrel domain of FhuAΔ5‐160 is sufficient for TonB‐dependent FhuA activities of Escherichia coli
Authors: Braun Michael , Killmann Helmut , Braun Volkmar
Published: March 2003

FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and φ80. The previously determined crystal structure reveals that residues 160–714 of the mature protein form a β‐barrel that is closed from the periplasmic side by the globular N‐proximal fragment, residues 1–159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuAΔ5‐160, that was incorporated in the outer membrane. Cells that synthesized FhuAΔ5‐160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuAΔ5‐160. These results demonstrate non‐specific diffusion of compounds across the outer membrane of cells that synthesize FhuAΔ5‐160. However, growth of a FhuAΔ5‐160 tonB wild‐type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild‐type rate despite the lack of ferrichrome binding sites provided by the cork. FhuAΔ5‐160 conferred sensitivity to the phages and colicin M at levels similar to that of wild‐type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuAΔ5‐160 depended on TonB, although the mutant lacks the TonB box (residues 7–11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB‐dependent transport of ferrichrome through FhuAΔ5‐160. FhuAΔ5‐160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB‐mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the β‐barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuAΔ5‐160, interaction of TonB with the β‐barrel is sufficient to release ferrichrome from the residual binding sites at the β‐barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB‐dependent phages T1 and φ80.
Developmental gene regulation in Giardia lamblia: first evidence for an encystation‐specific promoter and differential 5′ mRNA processing
Authors: Knodler Leigh A. , Svärd Staffan G. , Silberman Jeffrey D. , Davids Barbara J. , Gillin Frances D.
Published: March 2003

Giardia lamblia must encyst to survive in the environment and subsequently infect new hosts. We investigated the expression of glucosamine‐6‐phosphate isomerase (Gln6PI), the first enzyme required for biosynthesis of N‐acetylgalactosamine, for the major cyst wall polysaccharide. We isolated two Gln6PI genes that encode proteins with large areas of identity, but distinctive central and terminal regions. Both recombinant enzymes have comparable kinetics. Interestingly, these genes have distinct patterns of expression. Gln6PI‐A has a conventional, short 5′ untranslated region (UTR), and is expressed at a low level during vegetative growth and encystation. The Gln6PI‐B gene has two transcripts — one is expressed constitutively and the second species is highly upregulated during encystation. The non‐regulated Gln6PI‐B transcript has the longest 5′‐UTR known for Giardia and is 5′ capped or blocked. In contrast, the Gln6PI‐B upregulated transcript has a short, non‐capped 5′‐UTR. A small promoter region (< 56 bp upstream from the start codon) is sufficient for the regulated expression of Gln6PI‐B. Gln6PI‐B also has an antisense overlapping transcript that is expressed constitutively. A shorter antisense transcript is detected during encystation. This is the first report of a developmentally regulated promoter in Giardia, as well as evidence for a potential role of 5′ RNA processing and antisense RNA in differential gene regulation.
Isolation of the topB gene encoding DNA topoisomerase III as a multicopy suppressor of topA null mutations in Escherichia coli
Authors: Broccoli Sonia , Phoenix Pauline , Drolet Marc
Published: March 2003

One major function of DNA topoisomerase I in Escherichia coli is to repress R‐loop formation during transcription elongation, which may otherwise inhibit cell growth. We have previously shown that the growth problems of topA mutants can be corrected by overproducing RNase H, an enzyme that degrades the RNA moiety of an R‐loop. The goal of the present study was to identify other potential regulators of R‐loop formation. To this end, we have screened for multicopy suppressors of topA null mutations. As expected using this procedure, we cloned the rnhA gene encoding RNase H. In addition, we also identified the topB gene encoding DNA topoisomerase III as an efficient suppressor of topA null mutations and, hence, of R‐loop formation. We show that DNA topoisomerase III is able to relax transcription‐induced negative supercoiling both in vitro and in vivo. An R‐loop is also shown to be a hot‐spot for relaxation by DNA topoisomerase III, and we found that R‐loop‐dependent hypernegative supercoiling can be prevented by the activity of this topoisomerase in vivo. It is also shown that the topB gene can act synergistically with the rnhA gene to correct the growth defect of topA null mutants efficiently. This synergistic effect can be explained by the fact that some R‐loops must not be degraded in order for the RNA to be available for protein synthesis. Topoisomerase III can presumably repress the formation of such R‐loops or cause their destabilization to prevent RNA degradation. This is supported by the fact that overproduction of this topoisomerase corrects the negative effect of overexpressing RNase H activity on the growth of topA null mutants at low temperatures. Moreover, the fact that DNA topoisomerase III does not relax global supercoiling supports our previous conclusion that R‐loop formation, and therefore the essential function of DNA topoisomerase I, involves local, rather than global, supercoiling.
UvrD‐dependent replication of rolling‐circle plasmids in Escherichia coli
Authors: Bruand Claude , Ehrlich S. Dusko
Published: March 2003

The Escherichia coli UvrD helicase (or helicase II) is known for its involvement in DNA repair. We report that UvrD is required for DNA replication of several different rolling‐circle plasmids in E. coli, whereas its homologue, the Rep helicase, is not. Lack of UvrD helicase does not impair the first step of plasmid replication, nicking of the double‐stranded origin by the plasmid initiator protein. However, replication proceeds no further without UvrD. Indeed, the nicked plasmid molecules accumulate to a high level in uvrD mutants. We conclude that UvrD is the replicative helicase of various rolling‐circle plasmids. This is the first description of a direct implication of UvrD in DNA replication in vivo.
The Brucella abortus Lon functions as a generalized stress response protease and is required for wild‐type virulence in BALB/c mice
Authors: Robertson Gregory T. , Kovach Michael E. , Allen Chris A. , Ficht Thomas A. , Roop R. Martin
Published: March 2003

The gene encoding a Lon protease homologue has been cloned from Brucella abortus. The putative Brucella abortus Lon shares > 60% amino acid identity with its Escherichia coli counterpart and the recombinant form of this protein restores the capacity of an Escherichia coli lon mutant to resist killing by ultraviolet irradiation and regulate the expression of a cpsB::lacZ fusion to wild‐type levels. A σ32 type promoter was identified upstream of the predicted lon coding region and Northern analysis revealed that transcription of the native Brucella abortus lon increases in response to heat shock and other environmental stresses. ATP‐dependent proteolytic activity was also demonstrated for purified recombinant Lon. To evaluate the capacity of the Brucella abortus Lon homologue to function as a stress response protease, the majority of the lon coding region was removed from virulent strain Brucella abortus 2308 via allelic exchange. In contrast to the parent strain, the Brucella abortus lon mutant, designated GR106, was impaired in its capacity to form isolated colonies on solid medium at 41°C and displayed an increased sensitivity to killing by puromycin and H2O2. GR106 also displayed reduced survival in cultured murine macrophages and significant attenuation in BALB/c mice at 1 week post infection compared with the virulent parental strain. Beginning at 2 weeks and continuing for 6 weeks post infection, however, GR106 and 2308 displayed equivalent spleen and liver colonization levels in mice. These findings suggest that the Brucella abortus Lon homologue functions as a stress response protease that is required for wild‐type virulence during the initial stages of infection in the mouse model, but is not essential for the establishment and maintenance of chronic infection in this host.
Promoter discrimination by the related transcriptional activators MarA and SoxS: differential regulation by differential binding
Authors: Martin R. G. , Gillette W. K. , Rosner J. L.
Published: March 2003

MarA and SoxS are closely related proteins (≈45% identical) that transcriptionally activate a common set of unlinked genes, resulting in multiple antibiotic and superoxide resistance in Escherichia coli. Both proteins bind as monomers to a 20 bp degenerate asymmetric recognition sequence, the ‘marbox’, located upstream of the promoter. However, the proteins differ widely in the extents to which they activate particular promoters, with the consequence that overexpression of SoxS leads to greater superoxide resistance than does overexpression of MarA. This ‘discrimination’ between activators by promoters was demonstrated in vivo, using promoters fused to lacZ, and in vitro, using purified RNA polymerase, promoter DNA and MarA or SoxS. The marbox was found to be a critical element in discrimination by in vivo and in vitro assays of hybrid promoters containing the marbox from one gene and the core promoter from another. Furthermore, by sequential mutation of its marbox, a promoter that discriminated 35‐fold in favour of SoxS was converted into one that did not discriminate. The relative activation of a promoter by MarA or SoxS was paralleled by the relative binding of the two activators to the promoter's marbox as assayed by band shift experiments. Thus, differential recognition of closely related marbox sequences by the closely related activators is the primary basis for promoter discrimination. Discrimination enables the cell to customize its response to the stresses that trigger synthesis of the activators.
Outer‐membrane phospholipase A: known structure, unknown biological function
Authors: Dekker Niek
Published: March 2003

Outer‐membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Gram‐negative bacteria. The enzymatic activity of OMPLA is strictly regulated to prevent uncontrolled breakdown of the surrounding phospholipids. The activity of OMPLA can be induced by membrane perturbation and concurs with dimerization of the enzyme. The recently elucidated crystal structures of the inactive, monomeric and an inhibited dimeric form of the enzyme provide detailed structural insight into the functional properties of the enzyme. OMPLA is a serine hydrolase with a unique Asn‐156–His‐142–Ser‐144 catalytic triad. Only in the dimeric state, complete substrate binding pockets and functional oxyanion holes are formed. A model is proposed for the activation of OMPLA in which membrane perturbation causes the formation of non‐bilayer structures, resulting in the presentation of phospholipids to the active site of OMPLA and leading to the formation of the active dimeric species. Possible roles for OMPLA in maintaining the cell envelope integrity and in pathogenicity are discussed.
Lewis X structures in the O antigen side‐chain promote adhesion of Helicobacter pylori to the gastric epithelium
Authors: Edwards Nicola J. , Monteiro Mario A. , Faller Gerhard , Walsh Evelyn J. , Moran Anthony P. , Roberts Ian S. , High Nicola J.
Published: March 2003

Helicobacter pylori NCTC11637 expresses a lipopolysaccharide (LPS) that comprises an O antigen side‐chain with structural homology to the human blood group antigen Lewis X (Lex). The role of this molecule in adhesion of H. pylori to gastric epithelial cells was investigated. Mutants expressing truncated LPS structures were generated through insertional mutagenesis of rfbM and galE; genes encode GDP mannose pyrophosphorylase and galactose epimerase respectively. Compositional and structural analysis revealed that the galE mutant expressed a rough LPS that lacked an O antigen side‐chain. In contrast, an O antigen side‐chain was still synthesized by the rfbM mutant, but it lacked fucose and no longer reacted with anti‐Lex monoclonal antibodies (Mabs). The ability of these mutants to bind to paraffin‐embedded sections from the antrum region of a human stomach was assessed. Adhesion of the wild type was characterized by tropic binding to the apical surface of mucosal epithelial cells and cells lining gastric pits. In contrast, both the rfbM and galE mutants failed to demonstrate tropic binding and adhered to the tissue surface in a haphazard manner. These results indicate that LPS and, more specifically, LeX structures in the O antigen side‐chain play an important role in targeting H. pylori to specific cell lineages within the gastric mucosa. The role of LeX in this interaction was confirmed by the tropic binding of synthetic Lex, conjugated to latex beads, to gastric tissue. The observed pattern of adhesion was indistinguishable from that of wild‐type H. pylori.
Multidrug resistance mechanisms: drug efflux across two membranes
Authors: Zgurskaya Helen I. , Nikaido Hiroshi
Published: March 2003

A set of multidrug efflux systems enables Gram‐negative bacteria to survive in a hostile environment. This review focuses on the structural features and the mechanism of major efflux pumps of Gram‐negative bacteria, which expel from the cells a remarkably broad range of antimicrobial compounds and produce the characteristic intrinsic resistance of these bacteria to antibiotics, detergents, dyes and organic solvents. Each efflux pump consists of three components: the inner membrane transporter, the outer membrane channel and the periplasmic lipoprotein. Similar to the multidrug transporters from eukaryotic cells and Gram‐positive bacteria, the inner membrane transporters from Gram‐negative bacteria recognize and expel their substrates often from within the phospholipid bilayer. This efflux occurs without drug accumulation in the periplasm, implying that substrates are pumped out across the two membranes directly into the medium. Recent data suggest that the molecular mechanism of the drug extrusion across a two‐membrane envelope of Gram‐negative bacteria may involve the formation of the membrane adhesion sites between the inner and the outer membranes. The periplasmic components of these pumps are proposed to cause a close membrane apposition as the complexes are assembled for the transport.
Intracellular localization and processing of Pseudomonas aeruginosa ExoS in eukaryotic cells
Authors: Pederson Kristin J. , Pal Sangita , Vallis Amy J. , Frank Dara W. , Barbieri Joseph T.
Published: March 2003

ExoS is a type III cytotoxin of Pseudomonas aeruginosa, which modulates two eukaryotic signalling pathways. The N‐terminus (residues 1–234) is a GTPase activating protein (GAP) for RhoGTPases, while the C‐terminus (residues 232–453) encodes an ADP‐ribosyltransferase. Utilizing a series of N‐terminal deletion peptides of ExoS and an epitope‐tagged full‐length ExoS, two independent domains have been identified within the N‐terminus of ExoS that are involved in intracellular localization and expression of GAP activity. N‐terminal peptides of ExoS localized to the perinuclear region of CHO cells, and a membrane localization domain was localized between residues 36 and 78 of ExoS. The capacity to elicit CHO cell rounding and express GAP activity resided within residues 90–234 of ExoS, which showed that membrane localization was not required to elicit actin reorganization. ExoS was present in CHO cells as a full‐length form, which fractionated with membranes, and as an N‐terminally processed fragment, which localized to the cytosol. Thus, ExoS localizes in eukaryotic cells to the perinuclear region and is processed to a soluble fragment, which possesses both the GAP and ADP‐ribosyltransferase activities.
The antisense RNA of the par locus of pAD1 regulates the expression of a 33‐amino‐acid toxic peptide by an unusual mechanism
Authors: Greenfield Tony J. , Ehli Erik , Kirshenmann Todd , Franch Thomas , Gerdes Kenn , Weaver Keith E.
Published: March 2003

The par stability determinant of the Enterococcus faecalis plasmid pAD1 is the first antisense RNA‐regulated post‐segregational killing system (PSK) identified in a Gram‐positive organism. Par encodes two small, convergently transcribed RNAs, designated RNA I and RNA II, which are the toxin and antidote of the par PSK system respectively. RNA I encodes an open reading frame of 33 codons designated fst. The results presented here demonstrate that the peptide encoded by fst is the par toxin. The fst sequence was shown to be sufficient for cell killing, and removal of the final codon inactivated the toxin. In vitro translation reactions of purified RNA I transcript produced a product of the expected size for the fst‐encoded peptide. This product was not produced when purified RNA II transcript was added to the translation reaction. Toeprint analysis demonstrated that purified RNA II was able to inhibit ribosome binding to RNA I. These data suggest that fst expression is regulated by RNA II via an antisense RNA mechanism. In vitro translation studies and toeprint analyses also indicated that fst expression is internally regulated by a stem–loop structure at the 5′ end of RNA I. Removal of this structure resulted in better ribosome binding to RNA I and a 300‐fold increase in production of the fst‐encoded peptide. Finally, RNA II was shown to be less stable than RNA I in vivo, providing a basis for the selective expression of fst in plasmid‐free cells.
Interaction between the RP4 coupling protein TraG and the pBHR1 mobilization protein Mob
Authors: Szpirer Cédric Y. , Faelen Michel , Couturier Martine
Published: March 2003

It is currently believed that interaction between the relaxosome of a mobilizable plasmid and the transfer machinery of the helper conjugative plasmid is mediated by a TraG family coupling protein. The coupling proteins appear as an essential determinant of mobilization specificity and efficiency. Using a two‐hybrid system, we demonstrated for the first time the direct in vivo interaction between the coupling protein of a conjugative plasmid (the TraG protein of RP4) and the relaxase of a mobilizable plasmid (the Mob protein of pBHR1, a derivative of the broad host range plasmid pBBR1). This interaction was confirmed in vitro by an overlay assay and was shown to occur even in the absence of the transfer origin of pBHR1. We showed that, among 11 conjugative plasmids tested, pBHR1 is efficiently mobilized only by plasmids encoding an IncP‐type transfer system. We also showed that the RP4 TraG coupling protein is essential for mobilization of a pBBR1 derivative and is the element that allows its mobilization by R388 plasmid (IncW) at a detectable frequency.
Transcriptional regulation and immunity in mycobacteriophage Bxb1
Authors: Jain Shruti , Hatfull Graham F.
Published: March 2003

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis that shares a similar genome organization to mycobacteriophage L5, although the two phages are heteroimmune. We have investigated the regulatory circuitry of Bxb1 and found that it encodes a repressor, gp69, which regulates at least two promoters, an early lytic promoter, Pleft, and the divergent promoter, Pright. Bxb1 gp69 is 41% identical to the L5 repressor (gp71) and binds to repressor binding sites that conform to a similar, but distinct, 13 bp asymmetric consensus sequence to that for the L5 gp71 binding sites. The two phage repressors have a strong preference for their cognate binding sites, thus accounting for their immunity phenotypes. The Bxb1 genome contains 34 putative repressor binding sites located throughout the genome, but situated within short intergenic spaces and orientated in only one direction relative to the direction of transcription. Comparison with the locations of repressor binding sites within the L5 genome provides insights into how these unusual regulatory systems evolve.
The HspR regulon of Streptomyces coelicolor: a role for the DnaK chaperone as a transcriptional co‐repressor†
Authors: Bucca Giselda , Brassington Anna M. E. , Schönfeld Hans‐Joachim , Smith Colin P.
Published: March 2003

The dnaK operon of Streptomyces coelicolor encodes the DnaK chaperone machine and HspR, the transcriptional repressor of the operon; HspR confers repression by binding to several inverted repeat sequences in the promoter region, dnaKp. Here, we demonstrate that HspR specifically requires the presence of DnaK protein to retard a dnaKp fragment in gel‐shift assays. This requirement is independent of the co‐chaperones, DnaJ and GrpE, and it is ATP independent. Furthermore the retarded protein–DNA complex can be ‘supershifted’ by anti‐DnaK monoclonal antibody, demonstrating that DnaK forms an integral component of the complex. It was shown in DNase I footprinting experiments that refolding and specific binding of HspR to its DNA target does not require DnaK. We conclude that the formation of the stable DnaK–HspR–DNA ternary complex does not depend on the chaperoning activity of DnaK. In affinity chromatography experiments using whole‐cell extracts, DnaK was shown to co‐purify with HspR, providing additional evidence that the two proteins interact in vivo; it was not possible to purify HspR away from DnaK in any experiments unless a powerful denaturant was used. The level of heat shock induction of chromosomal DnaK could be partially suppressed by expressing dnaK extrachromosomally from a heterologous promoter. In addition, it is shown that DnaK confers enhanced HspR‐mediated repression of transcription in vitro. Taken together, these results suggest that DnaK functions as a transcriptional co‐repressor by binding to HspR at its operator sites. In this model, the DnaK–HspR system would represent a novel example of feedback regulation of gene expression by a molecular chaperone, in which DnaK directly activates a repressor, rather than inactivates an activator (as is the case in the DnaK–σ32 and Hsp70–HSF systems of other organisms).
Identification and characterization of BipA, a Bordetella Bvg‐intermediate phase protein
Authors: Stockbauer Kathryn E. , Fuchslocher Bryna , Miller Jeff F. , Cotter Peggy A.
Published: March 2003

The Bordetella BvgAS sensory transduction system has traditionally been viewed as controlling a transition between two distinct phenotypic phases: the Bvg+ or virulent phase and the Bvg− or avirulent phase. Recently, we identified a phenotypic phase of Bordetella bronchiseptica that displays reduced virulence in a rat model of respiratory infection concomitant with increased ability to survive nutrient deprivation. Characterization of this phase, designated Bvg‐intermediate (Bvgi), indicated the presence of antigens that are maximally, if not exclusively, expressed in this phase and therefore suggested the existence of a previously unidentified class of Bvg‐regulated genes. We now report the identification and characterization of a Bvgi phase protein, BipA (Bvg‐intermediate phase protein A), and its structural gene, bipA. Reverse transcriptase–polymerase chain reaction (RT–PCR) analysis indicates that bipA is expressed maximally under Bvgi phase conditions and thus represents the first identified Bvgi phase gene. bipA encodes a 1578‐amino‐acid protein that shares amino acid sequence similarity at its N‐terminus with the proposed outer membrane localization domains of intimin (Int) of enteropathogenic and enterohaemorrhagic Escherichia coli and invasin (Inv) of Yersinia spp. Although not apparent at the amino acid level, BipA is also similar to Int and Inv in that the proposed membrane‐spanning domain is followed by several 90‐amino‐acid repeats and a distinct C‐terminal domain. Localization studies using an antibody directed against the C‐terminus of BipA indicated that its C‐terminus is exposed on the bacterial cell surface. Western blot analysis with this same antibody indicated that BipA homologues are expressed in Bvgi phase Bordetella pertussis and Bordetella parapertussis. Comparison of a ΔbipA strain with wild‐type B. bronchiseptica indicated that BipA is not required for Bvgi phase‐specific aggregative adherence to rat lung epithelial cells in vitro or for persistent colonization of the rabbit respiratory tract in vivo. However, our data are consistent with the hypothesis that BipA, and the Bvgi phase in general, play an important role in the Bordetella infectious cycle, perhaps by contributing to aerosol transmission.
Concerted inhibition of the transcriptional activation functions of the enhancer‐binding protein NIFA by the anti‐activator NIFL
Authors: Barrett Jason , Ray Pampa , Sobczyk André , Little Richard , Dixon Ray
Published: March 2003

The Azotobacter vinelandii NIFL regulatory flavoprotein responds to the redox, energy and nitrogen status of the cell to inhibit transcriptional activation by the σN‐dependent enhancer binding protein, NIFA, via the formation of a NIFL–NIFA protein complex. The NIFA protein contains three domains: an N‐terminal domain of unknown function; a central catalytic domain required to couple nucleotide hydrolysis to activation of the σN‐RNA polymerase holoenzyme; and a C‐terminal DNA‐binding domain. We report that truncated NIFA proteins that either lack the amino‐terminal domain or contain only the isolated central domain remain responsive to inhibition by NIFL but, in contrast to native NIFA, continue to hydrolyse nucleotides when NIFL is present. We also report that NIFL is competent to inhibit the DNA‐binding function of NIFA. Taken together, these results suggest that NIFL inhibits NIFA via a concerted mechanism in which DNA binding, catalytic activity and, potentially, interaction with the polymerase are controlled by NIFL in order to prevent transcriptional activation under detrimental environmental conditions.
Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi
Authors: Stewart Philip E. , Thalken Rebecca , Bono James L. , Rosa Patricia
Published: March 2003

Borrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A non‐infectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.
The cold‐shock stress response in Mycobacterium smegmatis induces the expression of a histone‐like protein
Authors: Shires K. , Steyn L.
Published: March 2003

The response of Mycobacterium smegmatis to a cold shock was investigated by monitoring changes in both growth and cellular protein composition of the organism. The nature of the cellular response was influenced by the magnitude of the temperature reduction, with the shock from 37°C to 10°C having the most widespread effect on growth, metabolism and protein composition. This 27°C temperature reduction was associated with a lag period of 21–24 h before increases were seen in all the measured cellular activities. The response to cold shock was adaptive, with growth resuming after this period, albeit at a 50‐fold slower rate. The synthesis of at least 15 proteins was induced during the lag period. Two distinct patterns of cold‐induced synthesis were apparent, namely transient and continuous, indicating the production of both cold‐induced and cold‐acclimation proteins. One of these cold‐shock proteins, CipMa, was identified as the histone‐like protein, Hlp, of M. smegmatis, which is also induced during anaerobic‐induced dormancy. The corresponding gene demonstrated transient, cold‐inducible expression with a five‐ to sevenfold increase in mRNA occurring 9–12 h after temperature shift. Although bacterial survival was unaffected, CipMa/Hlp knock‐out mutants were unable to adapt metabolically to the cold shock and resume growth, thus indicating a key role for CipMa in the cold‐shock response.
How does oxygen inhibit central metabolism in the obligate anaerobe Bacteroides thetaiotaomicron
Authors: Pan Ning , Imlay James A.
Published: March 2003

The molecular basis of obligate anaerobiosis is not well established. Bacteroides thetaiotaomicron is an opportunistic pathogen that cannot grow in fully aerobic habitats. Because microbial niches reflect features of energy‐producing strategies, we suspected that aeration would interfere with its central metabolism. In anaerobic medium, this bacterium fermented carbohydrates to a mixture of succinate, propionate and acetate. When cultures were exposed to air, the formation of succinate and propionate ceased abruptly. In vitro analysis demonstrated that the fumarase of the succinate–propionate pathway contains an iron–sulphur cluster that is sensitive to superoxide. In vivo, fumarase activity fell to < 5% when cells were aerated; virtually all activity was recovered after extracts were chemically treated to rebuild iron–sulphur clusters. Aeration minimally affected the remainder of this pathway. However, aeration reduced pyruvate:ferredoxin oxidoreductase (PFOR), the first enzyme in the acetate fermentation branch, to 3% of its anaerobic activity. This cluster‐containing enzyme was damaged in vitro by molecular oxygen but not by superoxide. Thus, aerobic growth is precluded by the vulnerability of these iron–sulphur cluster enzymes to oxidation. Importantly, both enzymes were maintained in a stable, inactive form for long periods in aerobic cells; they were then rapidly repaired when the bacterium was returned to anaerobic medium. This result explains how this pathogen can easily recover from occasional exposure to oxygen.
The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation
Authors: Ferreira Paulo C. , Ness Frédérique , Edwards Susan R. , Cox Brian S. , Tuite Mick F.
Published: March 2003

In the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion‐like, self‐propagating conformation giving rise to the non‐Mendelian [PSI+] determinant. The replication of [PSI+] prion seeds can be readily blocked by growth in the presence of low concentrations of guanidine hydrochloride (GdnHCl), leading to the generation of prion‐free [psi–] cells. Here, we provide evidence that GdnHCl blocks seed replication in vivo by inactivation of the molecular chaperone Hsp104. Although growth in the presence of GdnHCl causes a modest increase in HSP104 expression (20–90%), this is not sufficient to explain prion curing. Rather, we show that GdnHCl inhibits two different Hsp104‐dependent cellular processes, namely the acquisition of thermotolerance and the refolding of thermally denatured luciferase. The inhibitory effects of GdnHCl protein refolding are partially suppressed by elevating the endogenous cellular levels of Hsp104 using a constitutive promoter. The kinetics of GdnHCl‐induced [PSI+] curing could be mimicked by co‐expression of an ATPase‐negative dominant HSP104 mutant in an otherwise wild‐type [PSI+] strain. We suggest that GdnHCl inactivates the ATPase activity of Hsp104, leading to a block in the replication of [PSI+] seeds.
Solute‐binding protein‐dependent ABC transporters are responsible for solute efflux in addition to solute uptake
Authors: Hosie A. H. F. , Allaway D. , Jones M. A. , Walshaw D. L. , Johnston A. W. B. , Poole P. S.
Published: March 2003

The ATP‐binding cassette (ABC) transporter superfamily is one of the most widespread of all gene families and currently has in excess of 1100 members in organisms ranging from the Archaea to man. The movement of the diverse solutes of ABC transporters has been accepted as being strictly unidirectional, with recent models indicating that they are irreversible. However, contrary to this paradigm, we show that three solute‐binding protein‐dependent (SBP) ABC transporters of amino acids, i.e. the general amino acid permease (Aap) and the branched‐chain amino acid permease (Bra) of Rhizobium leguminosarum and the histidine permease (His) of Salmonella typhimurium, are bidirectional, being responsible for efflux in addition to the uptake of solutes. The net solute movement measured for an ABC transporter depends on the rates of uptake and efflux, which are independent; a plateau is reached when both are saturated. SBP ABC transporters promote active uptake because, although the Vmax values for uptake and efflux are not significantly different, there is a 103−104 higher affinity for uptake of solute compared with efflux. Therefore, the SBP ABC transporters are able to support a substantial concentration gradient and provide a net uptake of solutes into bacterial cells.