bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemi‐methylated oriC DNA binding activity found in non‐specific acid phosphatase
Authors: Reshetnyak Elena , D'Alençon Emmanuelle , Kern Renée , Taghbalout Aziz , Guillaud Philippe , Kohiyama Masamichi
Published: March 2003

The lacZ–hobH fusion clone, containing an Escherichia coli DNA segment located at 92 min on the chromosomal map, was screened as a producer of E. coli oriC hemi‐methylated binding activity. We have purified the protein encoded by this locus to near homogeneity. The protein corresponds to the monomeric form of a non‐specific acid phosphatase (NAP) whose gene has been designated aphA. oriC DNA footprinting experiments showed protection of hemi‐methylated probe by partially purified NAP, but not by purified preparations. Yet, gel retardation experiments with an oriC oligonucleotide demonstrated DNA binding activity of purified NAP in the presence of Mg2+. This experiment also showed an increased affinity of the protein for the hemi‐methylated probe compared with the fully or unmethylated form. Indirect immunofluorescence microscopy revealed the existence of discrete NAP foci at mid‐cell in cells with two nucleoids, but at cell poles in those with one nucleoid.
The sodium ion translocating glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans: cloning and function of the genes forming a second operon
Authors: Braune Annett , Bendrat Klaus , Rospert Sabine , Buckel Wolfgang
Published: March 2003

Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans (clostridal cluster IX), a strict anaerobic inhabitant of animal intestines, uses the free energy of decarboxylation (ΔG°′ ≈ −30 kJ mol−1) in order to translocate Na+ from the inside through the cytoplasmic membrane. The proton, which is required for decarboxylation, most probably comes from the outside. The enzyme consists of four different subunits. The largest subunit, α or GcdA (65 kDa), catalyses the transfer of CO2 from glutaconyl‐CoA to biotin covalently attached to the γ‐subunit, GcdC. The β‐subunit, GcdB, is responsible for the decarboxylation of carboxybiotin, which drives the Na+ translocation (approximate Km for Na+ 1 mM), whereas the function of the smallest subunit, δ or GcdD, is unclear. The gene gcdA is part of the ‘hydroxyglutarate operon’, which does not contain genes coding for the other three subunits. This paper describes that the genes, gcdDCB, are transcribed in this order from a distinct operon. The δ‐subunit (GcdD, 12 kDa), with one potential transmembrane helix, probably serves as an anchor for GcdA. The biotin carrier (GcdC, 14 kDa) contains a flexible stretch of 50 amino acid residues (A26–A75), which consists of 34 alanines, 14 prolines, one valine and one lysine. The β‐subunit (GcdB, 39 kDa) comprising 11 putative transmembrane helices shares high amino acid sequence identities with corresponding deduced gene products from Veillonella parvula (80%, clostridial cluster IX), Archaeoglobus fulgidus (61%, Euryarchaeota), Propionigenium modestum (60%, clostridial cluster XIX), Salmonella typhimurium (51%, enterobacteria) and Klebsiella pneumoniae (50%, enterobacteria). Directly upstream of the promoter region of the gcdDCB operon, the 3′ end of gctM was detected. It encodes a protein fragment with 73% sequence identity to the C‐terminus of the α‐subunit of methylmalonyl‐CoA decarboxylase from V. parvula (MmdA). Hence, it appears that A. fermentans should be able to synthesize this enzyme by expression of gctM together with gdcDCB, but methylmalonyl‐CoA decarboxylase activity could not be detected in cell‐free extracts. Earlier observations of a second, lower affinity binding site for Na+ of glutaconyl‐CoA decarboxylase (apparent Km 30 mM) were confirmed by identification of the cysteine residue 243 of GcdB between the putative helices VII and VIII, which could be specifically protected from alkylation by Na+. The α‐subunit was purified from an overproducing Escherichia coli strain and was characterized as a putative homotrimer able to catalyse the carboxylation of free biotin.
The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon
Authors: Grandvalet Cosette , De Crécy‐Lagard Valérie , Mazodier Philippe
Published: March 2003

The clpB gene of Streptomyces albus was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides. Transcriptional analysis showed that the clpB gene was heat induced. Primer extension identified a transcription start site preceded by typical vegetative −10 and −35 hexamer sequences. The Streptomyces HspR repressor is known to bind to three inverted repeat motifs (IR1, IR2, IR3) upstream from the S. coelicolor dnaK operon. We identified an inverted repeat motif identical to IR3 upstream from the S. albus clpB gene. DNA‐binding experiments showed that HspR regulates clpB transcription by interacting directly with this motif. Streptomyces albus is the first Gram‐positive organism for which the co‐regulation of DnaK and ClpB has been described. Such co‐regulation suggests that there is a physiological relationship between these two proteins in this bacterium. Genes similar to hspR were also identified in Mycobacterium leprae, M. tuberculosis and in bacteria unrelated to the actinomycetales order, such as Helicobacter pylori and Aquifex aeolicus. HspR binding sites were found in these bacteria upstream from various heat shock genes, suggesting that these genes are regulated by HspR. The HspR binding site, here called HAIR (H spR associated inverted repeat), has the consensus sequence CTTGAGT N7 ACTCAAG.
Secretion of ATP‐utilizing enzymes, nucleoside diphosphate kinase and ATPase, by Mycobacterium bovis BCG: sequestration of ATP from macrophage P2Z receptors?
Authors: Zaborina Olga , Li Xiaoming , Cheng Guofeng , Kapatral Vinayak , Chakrabarty A. M.
Published: March 2003

Mycobacterium bovis BCG secretes two ATP‐scavenging enzymes, nucleoside diphosphate kinase (Ndk) and ATPase, during growth in Middlebrook 7H9 medium. In synthetic Sauton medium without any protein supplements, there is less secretion of these two enzymes unless proteins such as bovine serum albumin (BSA), ovalbumin or extracts of macrophages are added to the medium. There is a gradient of activity among various proteins in triggering the induction of secretion of these two enzymes. Other mycobacteria, such as M. smegmatis, primarily secrete Ndk, while M. chelonae does not appear to secrete either of these two enzymes. Purification of the enzymes from the culture filtrate of 7H9‐grown M. bovis BCG cells and determination of the N‐terminal amino‐acid sequence have demonstrated a high level of sequence identity of one of the ATPases with DnaK, a heat shock chaperone, of M. tuberculosis and M. leprae, while that of Ndk shows significant identity with the Ndk of Myxococcus xanthus. As both Ndk and ATPase use ATP as a substrate, the physiological significance of the secretion of these two ATP‐utilizing enzymes was explored. External ATP is important in the activation of macrophage surface‐associated P2Z receptors, whose activation has been postulated to allow phagosome–lysosome fusion and macrophage cell death. We demonstrate that the presence of the filtrate containing these enzymes prevents ATP‐induced macrophage cell death, as measured by the release of an intracellular enzyme, lactate dehydrogenase. In vitro complexation studies with purified Ndk/ATPase and hyperproduced P2Z receptor protein will demonstrate whether these enzymes may be used by mycobacteria to sequester ATP from the macrophage P2Z receptors, thereby preventing phagosome–lysosome fusion or macrophage apoptotic death.
Evidence for a system of general protein glycosylation in Campylobacter jejuni
Authors: Szymanski Christine M. , Yao Ruijin , Ewing Cheryl P. , Trust Trevor J. , Guerry Patricia
Published: March 2003

A genetic locus from Campylobacter jejuni 81‐176 (O:23, 36) has been characterized that appears to be involved in glycosylation of multiple proteins, including flagellin. The lipopolysaccharide (LPS) core of Escherichia coli DH5α containing some of these genes is modified such that it becomes immunoreactive with O:23 and O:36 antisera and loses reactivity with the lectin wheat germ agglutinin (WGA). Site‐specific mutation of one of these genes in the E. coli host causes loss of O:23 and O:36 antibody reactivity and restores reactivity with WGA. However, site‐specific mutation of each of the seven genes in 81‐176 failed to show any detectable changes in LPS. Multiple proteins from various cellular fractions of each mutant showed altered reactivity by Western blot analyses using O:23 and O:36 antisera. The changes in protein antigenicity could be restored in one of the mutants by the presence of the corresponding wild‐type allele in trans on a shuttle vector. Flagellin, which is known to be a glycoprotein, was one of the proteins that showed altered reactivity with O:23 and O:36 antiserum in the mutants. Chemical deglycosylation of protein fractions from the 81‐176 wild type suggests that the other proteins with altered antigenicity in the mutants are also glycosylated.
Characterization of SprA, an AraC‐like transcriptional regulator encoded within the Salmonella typhimurium pathogenicity island 1
Authors: Eichelberg Katrin , Hardt Wolf‐Dietrich , Galán Jorge E.
Published: March 2003

Pathogenicity island 1 (SPI‐1) located at centisome 63 of the Salmonella chromosome encodes a type III protein secretion system that is essential for its pathogenicity. The translocation of effector proteins through this system results in the stimulation of signalling events, leading to actin cytoskeletal rearrangements and nuclear responses. These cellular responses ultimately lead to bacterial uptake, production of proinflammatory cytokines in non‐phagocytic cells and the initiation of programmed cell death in macrophages. The regulation of expression of components and substrates of this type III secretion system is complex and involves the activity of several specific transcriptional regulatory proteins encoded within SPI‐1. Here, we describe two additional regulatory proteins, SprA and SprB, which are encoded within SPI‐1. SprA and SprB exhibit significant sequence similarity to the AraC/XylS and the LuxR/UhaP family of transcriptional regulatory proteins respectively. Insertion mutations in sprA and sprB did not significantly affect the transcription of invasion‐associated genes and, consequently, did not affect the ability of Salmonella typhimurium to gain access into host cells. However, expression of sprA from an inducible heterologous promoter resulted in increased expression of genes associated with the centisome 63 type III secretion system and increased the ability of S. typhimurium to enter into host cells. Further analysis demonstrated that SprA acts either upstream or at the same level as HilA in the SPI‐1 transcriptional regulatory cascade.
Characterization of a single‐strand origin, ssoU, required for broad host range replication of rolling‐circle plasmids
Authors: Kramer M. Gabriela , Espinosa Manuel , Misra Tapan K. , Khan Saleem A.
Published: March 2003

Single‐stranded DNA (ssDNA) promoters are the key components of the single‐strand origins (ssos) of replication of rolling‐circle (RC) replicating plasmids. The recognition of this origin by the host RNA polymerase and the synthesis of a short primer RNA are critical for initiation of lagging‐strand synthesis. This step is thought to be a limiting factor for the establishment of RC plasmids in a broad range of bacteria, because most of the ssos described are fully active only in their natural hosts. A special type of sso, the ssoU, is unique in the sense that it can be efficiently recognized in a number of different Gram‐positive hosts. We have experimentally deduced the folded structure and characterized the ssDNA promoter present within the ssoU using P1 nuclease digestion and DNase I protection assays with the Bacillus subtilis and Staphylococcus aureus RNA polymerases. We have also identified the RNA products synthesized from this ssDNA promoter and mapped the initiation points of lagging‐strand synthesis in vivo from ssoU‐containing plasmids. Through gel mobility shift experiments, we have found that ssDNA containing the ssoU sequence can efficiently interact with the RNA polymerase from two different Gram‐positive bacteria, S. aureus and B. subtilis. We have also realigned the narrow and broad host range sso sequences of RC plasmids, and found that they contain significant homology. Our data support the notion that the strength of the RNA polymerase–ssoU interaction may be the critical factor that confers the ability on the ssoU to be fully functional in a broad range of bacteria.
The channel‐forming toxin aerolysin neutralizes human immunodeficiency virus type 1
Authors: Nguyen Dzung H. , Liao Zhaohao , Buckley J. Thomas , Hildreth James E. K.
Published: March 2003

Aerolysin is a channel‐forming toxin secreted by Aeromonas spp. that binds to glycosyl phosphatidylinositol (GPI)‐anchored proteins, such as Thy‐1, on sensitive target cells. Receptor binding is followed first by oligomerization of the toxin and then by insertion of the oligomers into the membrane to form stable channels that disrupt the permeability barrier. Human immunodeficiency virus type 1 (HIV‐1) produced from T cells is known to incorporate Thy‐1 and other GPI‐anchored proteins into its membrane. Here, we show that aerolysin is capable of neutralizing HIV‐1 in a dose‐dependent manner and that neutralization depends upon the presence of these proteins in the viral envelope. Pretreatment with phosphatidylinositol‐specific phospholipase C to remove GPI‐anchored proteins greatly reduced HIV‐1 sensitivity to the toxin, and virus originating from a mutant cell line that lacks GPI‐anchored proteins was not neutralized. Aerolysin variants with single amino acid changes that prevent oligomerization or insertion of the toxin were unable to inactivate the virus, implying that channel formation is necessary for neutralization to occur. These findings represent the first evidence that a pathogenic human virus can be neutralized by a bacterial toxin.
Interspecies complementation in Saccharopolyspora erythraea : elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives
Authors: Doumith Michel , Legrand Raymond , Lang Catherine , Salas José A. , Raynal Marie‐Cécile
Published: March 2003

Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as l‐rhamnose attached at position C‐3 of an erythronolide B or a 6‐deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP‐d‐desosamine, which we have previously proposed to be a dTDP‐4‐keto‐6‐deoxy‐d‐glucose 3,4‐isomerase.
The linker peptide of the ArsA ATPase
Authors: Li Jiaxin , Rosen Barry P.
Published: March 2003

Plasmid R773 encodes an As(III)/Sb(III)‐translocating ATPase that confers resistance to those metalloids in Escherichia coli. The catalytic subunit of the pump, the ArsA ATPase, consists of homologous N‐ and C‐terminal nucleotide‐binding domains connected by a 25‐residue linker. The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues. Cells expressing arsA with the 5‐residue insertion had wild‐type arsenite resistance. Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length. Cells with five or 10 deleted residues exhibited slightly reduced resistance. Deletion of 15 or 23 residues resulted in further decreases in resistance. Each altered ArsA was purified. The enzyme with the 5‐residue insertion had the same affinity for ATP and Sb(III) as the wild‐type enzyme. Enzymes with 5‐, 10‐, 15‐ or 23‐residue deletions exhibited decreased affinity for both Sb(III) and ATP. The enzyme with a 23‐residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III). These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis.
Identification of the Escherichia coli K‐12 Nramp orthologue (MntH) as a selective divalent metal ion transporter
Authors: Makui Hortence , Roig Eva , Cole Stewart T. , Helmann John D. , Gros Philippe , Cellier Mathieu F. M.
Published: March 2003

The Escherichia coli mntH (formerly yfeP) gene encodes a putative membrane protein (MntH) highly similar to members of the eukaryotic Nramp family of divalent metal ion transporters. To determine the function of E. coli MntH, a null mutant was created and MntH was overexpressed both in wild‐type E. coli and in the metal‐dependent mutant hflB1(Ts). At the restrictive temperature 42°C, the mntH null mutation reduces the suppression of hflB1(Ts) thermosensitivity by exogenous divalent metals. Conversely, overexpression of MntH restores growth at 42°C, increases suppression of the ts phenotype by Fe(II) and Ni(II) and renders hflB1(Ts) cells hypersensitive to Mn(II). Transport studies in intact cells show that MntH selectively facilitates uptake of 54Mn(II) and 55Fe(II) in a temperature‐, time‐ and proton‐dependent manner. Competition studies in uptake assays and growth inhibition experiments in hflB1(Ts) mutants together indicate that MntH is a divalent metal cation transporter of broad substrate specificity. The functional characteristics of MntH suggest that it corresponds to the previously described manganese transporter of E. coli. This study indicates that proton‐dependent divalent metal ion uptake has been preserved in the Nramp family from bacteria to humans.
The role of tandem IS dimers in IS911 transposition
Authors: Turlan C. , Ton‐Hoang B. , Chandler M.
Published: March 2003

Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra‐IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR–IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra‐IS recombination to generate transposon circles but additionally participated in inter‐IS recombination. This also creates an active IRR–IRL junction by generating a head‐to‐tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter‐IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR–IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co‐integrate structure. It is also shown here that the IRR–IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.
Specific amino acid changes enhance the anti‐recombination activity of the UmuD′C complex
Authors: Sommer Suzanne , Coste Genevièève , Bailone Adriana
Published: March 2003

In addition to being an essential component of trans‐lesion synthesis, the UmuD′C complex is an antagonist of RecA‐mediated homologous recombination. When constitutively expressed at an elevated concentration, the UmuD′C complex sensitizes recA+ bacteria to DNA damage, whereas it has no effect on bacteria expressing a RecA [UmuR] protein that overcomes recombination inhibition. Using as a genetic screen enhanced cell killing on mitomycin plates, we isolated novel umuD′ and umuC mutations that restored mitomycin sensitivity to recA D112G [UmuR] bacteria overproducing the UmuD′C complex. The mutations were named [Rin++] because a characterization in a recA+ as well in a recA D112G background showed that they enhanced UmuD′C‐promoted recombination inhibition in two assays, conjugational recombination and recombinational repair of palindrome‐containing DNA. The [Rin++] mutations affect five amino acids, G25D, S28T, P29L, E35K, and T95R, in UmuD′ and seven, F10L, Y270C, K277E, F287L, F287S, K342Q and F351I, in UmuC. These amino acids might play a key role in the UmuD′C anti‐recombination activity. None of the [Rin++] mutations enhanced UmuD′C‐promoted mutagenic bypass of UV lesions, in contrast, several lead to a defect in this process. In this study, we discuss a few molecular mechanisms that could account for the recombination and mutagenesis phenotypes of a mutant UmuD′C [Rin++] complex.
Evidence for donor strand complementation in the biogenesis of Haemophilus influenzae haemagglutinating pili
Authors: Krasan Graham P. , Sauer Frederic G. , Cutter David , Farley Monica M. , Gilsdorf Janet R. , Hultgren Scott J. , Geme Joseph W. ST.
Published: March 2003

Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well‐characterized forms of pili, H. influenzae haemagglutinating pili are two‐stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site‐directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone–subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB–HifA periplasmic complexes, whereas mutations at the −14 glycine in HifA had no effect on HifB–HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA‐hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA‐hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD–PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure–function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.
Death effector domain of a mammalian apoptosis mediator, FADD, induces bacterial cell death
Authors: Lee Sang Won , Ko Young‐‐gyu , Bang SookHee , Kim Key‐Sun , Kim Sunghoon
Published: March 2003

FADD is a mammalian pro‐apoptotic mediator consisting of the N‐terminal death effector domain (DED) and the C‐terminal death domain (DD). The N‐terminal 88‐residue fragment of murine FADD was defined as the stable structural unit of DED, as determined by proteolytic digestion and conformational analysis. This domain induced bacterial as well as mammalian cell death, whereas the full‐length or DD of FADD did not. The Escherichia coli cells expressing FADD‐DED showed elongated cell morphology and an increased level of nicked chromosomal DNA and mutation. The lethality of FADD‐DED was abolished by co‐expression of thioredoxin and superoxide dismutase or relieved by the addition of vitamin E as a reducing agent and under anaerobic growth conditions. The toxicity of FADD‐DED was genetically suppressed by various oxidoreductases of E. coli. All these results suggest that the death effector domain of mammalian FADD induced bacterial cell death by enhancing cellular levels of reactive oxygen species (ROS).
Identification of Escherichia coli K1 genes contributing to human brain microvascular endothelial cell invasion by differential fluorescence induction
Authors: Badger Julie L. , Wass Carol A. , Kim Kwang Sik
Published: March 2003

Most cases of Escherichia coli K1 meningitis arise as a result of haematogenous spread, however there is a limited understanding of the mechanisms by which circulating E. coli K1 cross the blood–brain barrier. We have previously shown that environmental growth conditions both positively and negatively influence the capabilities of E. coli K1 to invade brain microvascular endothelial cells (BMEC), for example growth in media supplemented with 50% newborn bovine serum (NBS) increased BMEC invasion, whereas growth in media supplemented with 0.2 M NaCl repressed invasion in vitro and in vivo. In this study, differential fluorescence induction (DFI) was used to identify E. coli K1 genes involved in this differentially expressed invasion phenotype. E. coli K1 promoter libraries were constructed and screened for gfp expression in a manner analogous to the above growth conditions. Twenty‐four clones were isolated that showed fluorescence induction when grown under the invasion‐enhancing condition (i.e. NBS). Four of these clones also demonstrated repression or no induction of fluorescence when grown under the invasion‐repressing condition (i.e. 0.2 M NaCl). One such clone, containing a ygdP promoter and an open reading frame (ORF), showed significant homology to Bartonella bacilliformis IalA (invasion associated locus). Among the other NBS‐inducing loci, finPtraJ was identified as well as several clones with no homology to other known genes. When ygdP, finPtraJ and several of the unique loci were disrupted in E. coli K1, there was a significant decrease in human BMEC (HBMEC) invasion. RNA transcript analysis determined that these newly identified invasion loci were differentially regulated at the transcriptional level. This is the first demonstration of using DFI to identify E. coli K1 genes contributing to HBMEC invasion.
The use of listeriolysin to identify in vivo induced genes in the Gram‐positive intracellular pathogen Listeria monocytogenes
Authors: Gahan Cormac G. M. , Hill Colin
Published: March 2003

Listeria monocytogenes is capable of growth within the cytoplasm of infected host cells. Escape from the host cell phagosome is mediated primarily through secretion of listeriolysin, a haemolytic factor which functions to actively lyse the phagosomal membrane. Listeriolysin negative mutants of L. monocytogenes are non‐haemolytic on blood agar plates and demonstrate a significant reduction of virulence in the mouse model of infection. We have developed a system for the identification of in vivo induced genes in L. monocytogenes which utilizes the listeriolysin gene, hly, as both a reporter of gene expression and as a means of selection of promoter elements expressed in vivo. The system is analogous to in vivo expression technology (IVET) first reported for Salmonella, however, as listeriolysin functions in the environment of the host phagosome the loci identified in this study are most likely expressed during residence in the phagosome. The system was successfully tested using the promoter of the inducible virulence gene plcA. A bank was created by fusing a promoterless copy of hly to random promoter elements in a listeriolysin negative IVET host. Sequential inoculations of mice with this bank resulted in the isolation of clones with increased survival potential in the mouse model relative to a negative control, but which remained haemolysin negative on blood agar plates. Nine in vivo induced loci were identified including genes encoding a DNA topoisomerase III, a cellobiose transporter and a fumarase. Two isolates represented fusions to proteins of unknown function and three isolates contained no significant homologues in the database. A mutant in the fumarase gene demonstrated reduced virulence for mice and an inability to grow in cultured mouse phagocytes.
Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate
Authors: Murray Marlene , Greenberg Miriam L.
Published: March 2003

Inositol monophosphatase plays a vital role in the de novo biosynthesis of inositol and in the phosphoinositide second messenger signalling pathway. We cloned the Saccharomyces cerevisiae open reading frame (ORF) YHR046c (termed INM1), which encodes inositol monophosphatase, characterized the protein Inm1p and analysed expression of the INM1 gene. INM1 was expressed in bacteria under the control of the lacZ promoter. The purified protein has inositol monophosphatase activity that is inhibited by the antibipolar drug lithium, but not valproate. In the inm1Δ::URA3 null mutant, inositol monophosphatase activity was reduced but not eliminated. The disruption had little effect on growth in the presence of lithium or valproate and no effect on growth in the absence of inositol. To characterize the regulation of INM1, we examined the effects of inositol, carbon source, growth phase, and the antibipolar drugs lithium and valproate on INM1 expression using an INM1–lacZ reporter gene. Unlike all other phospholipid biosynthetic enzyme‐encoding genes studied, which contain the UASINO regulatory element, INM1 expression is increased in the presence of inositol. In addition, INM1 expression was repressed during growth in glycerol and derepressed as glucose‐grown cells entered stationary. Both lithium and valproate, which cause a decrease in intracellular inositol, effect a decrease in INM1 expression. A model is presented to account for regulation of INM1 expression.
Loss of Cmk1 Ca2+–calmodulin‐dependent protein kinase in yeast results in constitutive weak organic acid resistance, associated with a post‐transcriptional activation of the Pdr12 ATP‐binding cassette transporter
Authors: Holyoak Caroline D. , Thompson Suzanne , Ortiz Calderon Claudia , Hatzixanthis Kostas , Bauer Bettina , Kuchler Karl , Piper Peter W. , Coote Peter J.
Published: March 2003

Yeast cells display an adaptive stress response when exposed to weak organic acids at low pH. This adaptation is important in the spoilage of preserved foods, as it allows growth in the presence of weak acid food preservatives. In Saccharomyces cerevisiae, this stress response leads to strong induction of the Pdr12 ATP‐binding cassette (ABC) transporter, which catalyses the active efflux of weak acid anions from the cytosol of adapted cells. S. cerevisiae cells lacking the Cmk1 isoform of Ca2+–calmodulin‐dependent protein kinase are intrinsically resistant to weak acid stress, in that they do not need to spend a long adaptive period in lag phase before resuming growth after exposure to this stress. This resistance of the cmk1 mutant is Pdr12 dependent and, unlike with wild‐type S. cerevisiae, cmk1 cells are capable of performing Pdr12‐specific functions such as energy‐dependent cellular extrusion of fluorescein and benzoate. However, they have neither higher PDR12 gene promoter activity nor higher Pdr12 protein levels. The increased Pdr12 activity in cmk1 cells is therefore caused by Cmk1 exerting a negative post‐transcriptional influence over the activity of the Pdr12 ABC transporter, a transporter protein that is constitutively expressed in low‐pH yeast cultures. This is the first preliminary evidence that shows a protein kinase, either directly or indirectly, regulating the activity of a yeast ABC transporter.
Endocytotic uptake and retrograde transport of a virally encoded killer toxin in yeast
Authors: Eisfeld Katrin , Riffer Frank , Mentges Johannes , Schmitt Manfred J.
Published: March 2003

We demonstrate that a virally encoded yeast ‘killer’ toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer toxin is a secreted α/β heterodimer that kills sensitive yeasts in a receptor‐mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the toxin precursor results in a protein whose β‐C‐terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde toxin transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Δerd2) or mutants defective in Golgi‐to‐ER protein recycling (erd1) are toxin resistant because the toxin can no longer enter and/or retrograde pass the cell. Site‐directed mutagenesis further indicated that the toxin's β‐HDEL motif ensures retrograde transport, although in a toxin‐secreting yeast the β‐C‐terminus is initially masked by an R residue (β‐HDELR) until Kex1p cleavage uncovers the toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high‐level secretion of a biologically inactive protein incapable of re‐entering the secretory pathway. Finally, we present evidence that ER‐to‐cytosol toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.
Interaction of ResD with regulatory regions of anaerobically induced genes in Bacillus subtilis
Authors: Nakano Michiko M. , Zhu Yi , LaCelle Michael , Zhang Xiaohui , Hulett F. Marion
Published: March 2003

The two‐component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE‐dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis‐acting sequences required for ResDE‐dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE‐dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.
The Escherichia coli UVM response is accompanied by an SOS‐independent error‐prone DNA replication activity demonstrable in vitro
Authors: Al Mamun Abu Amar M. , Yadava Ramesh S. , Ren Li , Humayun M. Zafri
Published: March 2003

UVM is an SOS‐independent inducible response characterized by elevated mutagenesis at a site‐specific 3, N4‐ethenocytosine (εC) residue borne on M13 single‐stranded DNA transfected into Escherichia coli cells pretreated with DNA‐damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind–]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the ‘non‐replicative’ DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA‐damaging agents. To address the question as to whether the UVM response is accompanied by an error‐prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single‐stranded DNA bearing a site‐specific lesion is converted to circular double‐stranded replicative‐form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation‐mediated polymerase chain reaction (LM‐PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site‐specific εC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM‐induced AM124 cells, with C → A mutations predominating over C → T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error‐prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.
Substrate complexes and domain organization of the Salmonella flagellar export chaperones FlgN and FliT
Authors: Bennett John C. Q. , Thomas Joanne , Fraser Gillian M. , Hughes Colin
Published: March 2003

The flagellar proteins FlgN and FliT have been proposed to act as substrate‐specific export chaperones, facilitating incorporation of the enterobacterial hook‐associated axial proteins (HAPs) FlgK/FlgL and FliD into the growing flagellum. In Salmonella typhimurium flgN and fliT mutants, the export of target HAPs was reduced, concomitant with loss of unincorporated flagellin into the surrounding medium. Gel filtration chromatography of wild‐type S. typhimurium cell extracts identified stable pools of FlgN and FliT homodimers in the cytosol, but no chaperone–substrate complexes were evident. Nevertheless, stable unique complexes were assembled efficiently in vitro by co‐incubation of FlgN and FliT with target HAPs purified from recombinant Escherichia coli. The sizes of the chaperone–substrate complexes indicated that, in each case, a chaperone homodimer binds to a substrate monomer. FlgN prevented in vitro aggregation of FlgK monomers, generating a soluble form of the HAP. Recombinant polypeptides spanning the potentially amphipathic C‐terminal regions of FlgN or FliT could not complement in trans the chaperone deficiency of the respective flgN and fliT mutants, but efficient flagellar assembly was restored by homodimeric translational fusions of these domains to glutathione S‐transferase, which bound FlgK and FlgL like the wild‐type FlgN. These data provide further evidence for the substrate‐specific chaperone function of FlgN and FliT and indicate that these chaperones comprise common N‐ and C‐terminal domains mediating homodimerization and HAP substrate binding respectively. In support of this view, the flgN mutation was specifically complemented by a hybrid chaperone comprising the N‐terminal half of FliT and the C‐terminal half of FlgN.
A potential role for periplasmic superoxide dismutase in blocking the penetration of external superoxide into the cytosol of Gram‐negative bacteria
Authors: Korshunov Sergei S. , Imlay James A.
Published: March 2003

Superoxide is a key component of the antibacterial weaponry of phagocytes. Presumably, for this rea‐son, strains of Salmonella typhimurium express a periplasmic superoxide dismutase (SOD) that is essential for full virulence. Because most anions cannot easily penetrate lipid membranes, it is thought that the phagosomal superoxide either damages an unknown target on the bacterial surface or reacts with nitric oxide to form peroxynitrite (HOONO), a toxic oxidant that can freely enter bacteria. However, in this study, we tested whether superoxide itself could penetrate membranes. Superoxide that was generated at high pH (>7.5) very slowly reduced cytochrome c that was encapsulated inside lipid vesicles. It did so much more quickly at lower pH (<7). Under the latter conditions, more superoxide was protonated and uncharged (HO2·), and the penetrance of superoxide was proportional to the concentration of this species. The permeability coefficient of HO2· was determined to be 9 × 10–4 cm sec–1, just slightly lower than that of water and far higher than the value of the anionic form (O2–, <10–7 cm sec–1). When Escherichia coli mutants that lack periplasmic SOD were exposed to super‐oxide at pH 6.5, cytosolic fumarase B was damaged. Damage was minimal at higher pH or in strains that contained periplasmic SOD. Thus, in the acidic phagolysosome, superoxide may be able to penetrate and attack cytosolic targets of captive bacteria. This process may contribute to the potency of the oxidative burst. One role of periplasmic SOD may be to avert this damage. In contrast, periplasmic SOD was ineffective at lowering the extracellular super‐oxide concentration and, therefore, may have little impact upon HOONO formation.
Role of the ribosomal stalk components in the resistance of Aspergillus fumigatus to the sordarin antifungals
Authors: Santos Cruz , Ballesta Juan P. G.
Published: March 2003

Aspergillus fumigatus, an important human nosocomial pathogen, is resistant
to sordarin derivatives, a new family of antifungals that inhibit protein synthesis
by interaction with the EF‐2–ribosomal stalk complex. To explore the role
of the A. fumigatus ribosome in the resistance mechanism, the fungal stalk
proteins were biochemically and genetically characterized and expressed in the sensitive
Saccharomyces cerevisiae. Two acidic phosphoproteins homologous to the 12
kDa P1 and P2 proteins described in other organisms were found together with the
34 kDa P0 protein, the third stalk component. The genes encoding each fungal stalk
protein were expressed in mutant S. cerevisiae strains lacking the equivalent
proteins. Both AfP1 and AfP2 proteins interact with their yeast counterparts of the
opposite type and bind to the ribosomal particles in the presence of either the S.
cerevisiae or the A. fumigatus P0 protein. The A. fumigatus acidic
phosphoproteins did not alter the yeast ribosome sordarin sensitivity. On the contrary,
the presence of the fungal P0 induces in vivo and in vitro resistance
to sordarin derivatives when present in the yeast ribosome. The mutations A117→E,
P122→R and G124→V in A. fumigatus
P0 reduce the resistance capacity of the protein. An S. cerevisiae strain
with the complete ribosomal stalk of A. fumigatus was obtained, which could be useful for the screening of new antifungals against this pathogenic fungus.
A putative three‐dimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora
Authors: Palomäki Tiina , Pickersgill Richard , Riekki Ruusu , Romantschuk Martin , Saarilahti Hannu T.
Published: March 2003

Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram‐negative bacteria was investigated. Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C‐terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops. The regions were required together to promote secretion. Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP. To our knowledge, this is the first time that a possible three‐dimensional targeting motif has been defined. At least one of the motifs comprises a cluster on the surface of the protein. The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus. Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.
Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus
Authors: Mavris Maria , Page Anne‐Laure , Tournebize Régis , Demers Brigitte , Sansonetti Philippe , Parsot Claude
Published: March 2003

The virulence plasmid‐encoded type III secretion system of Shigella flexneri

consists of the Mxi–Spa secretion apparatus, secreted proteins IpaA–D

and IpgD involved in entry of bacteria into epithelial cells, cytoplasmic chaperones

IpgC and IpgE and 15 other secreted proteins of unknown function, including VirA

and members of the IpaH family. The activity of the Mxi–Spa apparatus is regulated

by external signals, and transcription of virA and ipaH genes is specifically

induced in conditions of active secretion. We present genetic evidence that regulation

of these genes involves both MxiE, the transcriptional activator of the AraC family

encoded by the mxi operon, and IpgC, the chaperone for IpaB and IpaC. We also

show that together MxiE and IpgC are sufficient to activate virA and ipaH9.8

promoters in Escherichia coli. In S. flexneri, increasing the expression of IpgC led to a concomitant increase in IpaH production in conditions of non‐secretion. This suggests that the activity of secretion is sensed by the presence of free IpgC, which acts as a coactivator to allow MxiE to activate transcription at its target promoters.