bluuebunny/crossref_metadata_2025 huggingface dataset:
The NRAMP proteins of Salmonella typhimurium and Escherichia coli are selective manganese transporters involved in the response to reactive oxygen
Authors: Kehres David G. , Zaharik Michelle L. , Finlay B. Brett , Maguire Michael E.
Published: March 2003

NRAMPs (natural resistance‐associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens. The mechanism of pathogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp. typhimurium, and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia. The genes are designated mntH because the two enterobacterial NRAMPs encode H+‐stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each species under the conditions tested. For S. typhimurium MntH, the Km for 54Mn2+ (≈ 0.1 µM) was pH independent, but maximal uptake increased as pH decreased. Monovalent cations, osmotic strength, Mg2+ and Ca2+ did not inhibit 54Mn2+ uptake. Ni2+, Cu2+ and Zn2+ inhibited uptake with Kis greater than 100 µM, Co2+ with a Ki of 20 µM and Fe2+ with a Ki that decreased from 100 µM at pH 7.6 to 10 µM at pH 5.5. Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 µM, while Cd2+ was a potent inhibitor with a Ki of about 1 µM. E. coli MntH had a similar inhibition profile, except that Kis were three‐ to 10‐fold higher. Both S. typhimurium and E. coli MntH also transport 55Fe2+ however, the Kms are equivalent to the Kis for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli, mntH::lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters. Strains overexpressing mntH were more susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH::lacZ was induced severalfold by 3 h after invasion of the macrophages. S. typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice. Thus, the NRAMP Mn2+ transporter MntH and Mn2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.
Clinical and environmental isolates of Burkholderia cepacia exhibit differential cytotoxicity towards macrophages and mast cells
Authors: Melnikov Anatoli , Zaborina Olga , Dhiman Neelam , Prabhakar B. S. , Chakrabarty A. M. , Hendrickson William
Published: March 2003

Burkholderia cepacia is an emerging opportunistic pathogen that causes fatal infections in patients suffering from cystic fibrosis (CF) and chronic granulomatous disease. Various environmental isolates of B. cepacia are, however, capable of degrading environmental pollutants, such as trichloroethylene, 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T), etc., and are also highly effective in controlling plant diseases caused by nematodes and fungi. Such strains have therefore been proposed for environmental release to clean up toxic dump sites or as biopesticides. Various efforts to distinguish between clinical and environmental isolates of B. cepacia with regard to their virulence characteristics have produced ambiguous results, suggesting that newer methods are needed to test for the presence or absence of pathogenic potential in B. cepacia strains proposed for environmental release. We now report that several clinical strains of B. cepacia secrete cytotoxic factors that allow macrophage and mast cell death in the presence of external ATP. Several environmental strains had reduced activity in this regard. We also demonstrate that, while all the strains secrete enzymes that have nucleoside diphosphate kinase (Ndk), adenylate kinase (Ak) and 5′‐nucleotidase activity, the level of secretion of the 5′‐nucleotidase (and/or ATPase/phosphatase) appears to be lower in the environmental strains than in the clinical strains. The secretion of these enzymes is specifically activated in the presence of eukaryotic proteins such as α2‐macroglobulin. As macrophage‐ or mast cell surface‐associated P2Z receptors promote their cell death in the presence of mM concentrations of ATP, and as the secreted ATP‐using enzymes generate various phosphorylated or non‐phosphorylated adenine nucleotides that may even be better agonists than ATP in activating the P2Z receptors or may act through the activation of additional purinergic receptors, such enzymes may play an important role in allowing B. cepacia to evade host defence.
The partition system of multidrug resistance plasmid TP228 includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily
Authors: Hayes Finbarr
Published: March 2003

The segregational stability of bacterial, low‐copy‐number plasmids is promoted primarily by active partition. The plasmid‐specified components of the prototypical P1 plasmid partition system consist of two proteins, ParA (44.3 kDa) and ParB (38.5 kDa), which, in conjunction with integration host factor, form a nucleoprotein complex at the plasmid partition site, parS. This complex is the probable substrate for the directed temporal and spatial intracellular movement of plasmids before cell division. The genetic organization of the partition cassette of the multidrug resistance plasmid TP228 differs markedly from that of the P1 paradigm. The TP228 system includes a novel member (ParF; 22.0 kDa) of the ParA superfamily of ATPases, of which the P1 ParA protein is the archetype. However, the ParF protein and its immediate relatives form a discrete subgroup of the ParA superfamily, which evolutionarily is more related to the MinD subgroup of cell division proteins than to ParA of P1. The TP228 and P1 partition modules differ further in that the former does not include a parB homologue, but does specify a protein (ParG; 8.6 kDa) unrelated to ParB. Homologues of the parF gene are widely disseminated on eubacterial genomes, suggesting that ParF‐mediated partition may be a common mechanism by which plasmid segregational stability is achieved.
Overcoming the restriction barrier to plasmid transformation of Helicobacter pylori
Authors: Donahue John P. , Israel Dawn A. , Peek Richard M. , Blaser Martin J. , Miller Geraldine G.
Published: March 2003

Helicobacter pylori strains demonstrate substantial variability in the efficiency of transformation by plasmids from Escherichia coli, and many strains are completely resistant to transformation. Among the barriers to transformation are numerous strain‐specific restriction‐modification systems in H. pylori. We have developed a method to protect plasmid DNA from restriction by in vitro site‐specific methylation using cell‐free extracts of H. pylori before transformation. In two cases, plasmid DNA treated with cell‐free extracts in vitro acquired the restriction pattern characteristic of genomic DNA from the source strain. Among three strains examined in detail, the transformation frequency by treated plasmid shuttle and suicide vectors was significantly increased compared with mock‐treated plasmid DNA. The results indicate that the restriction barrier in H. pylori can be largely overcome by specific DNA methylation in vitro. The approach described should significantly enhance the ability to manipulate gene function in H. pylori and other organisms that have substantial restriction barriers to transformation.
Entamoeba histolytica cysteine proteinases with interleukin‐1 beta converting enzyme (ICE) activity cause intestinal inflammation and tissue damage in amoebiasis
Authors: Zhang Zhi ,   Le Yan,, Wang Lei , Seydel Karl B. , Li Ellen , Ankri Serge , Mirelman David , Stanley Samuel L.
Published: March 2003

The protozoan parasite Entamoeba histolytica causes intestinal inflammation and ulceration. Amoebic trophozoites activate the transcription factor NF‐κB in human intestinal epithelial cells, initiating an inflammatory response programme with resultant damage to the intestinal tissue. Amoebic cysteine proteinases have been proposed as important virulence factors for amoebiasis. To test the role of amoebic cysteine proteinases in the pathogenesis of amoebic colitis, human intestinal xenografts in SCID mice were infected with E. histolytica trophozoites expressing an antisense message to ehcp5. The cysteine proteinase‐deficient amoeba failed to induce intestinal epithelial cell production of the inflammatory cytokines interleukin (IL)‐1B and IL‐8, and caused significantly less gut inflammation and damage to the intestinal permeability barrier. The critical role of amoebic cysteine proteinases in human gut inflammation and tissue damage may be explained by our discovery that amoebic cysteine proteinases possess IL‐1B converting enzyme (ICE) activity. This ICE activity could contribute to intestinal inflammation by activating human pIL‐1B released by damaged intestinal cells. These results demonstrate for the first time that amoebic cysteine proteinases are a key virulence factor in amoebic colitis, and provide a novel mechanism for their activity.
Tyr‐326 plays a critical role in controlling SecA–preprotein interaction
Authors: Kourtz Lauralynn , Oliver Donald
Published: March 2003

SecA is an essential ATP‐dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267–340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region‐specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr‐326 was found to be critical for SecA function. secA mutants with non‐conservative substitutions at Tyr‐326 showed strong protein secretion defects in vivo and were completely defective for SecA‐dependent translocation ATPase activity in vitro. The SecA‐Y326 mutant proteins were normal in their membrane, SecYE and nucleotide‐binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr‐326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.
Control of methionine biosynthesis in Escherichia coli by proteolysis
Authors: Biran Dvora , Gur Eyal , Gollan Leora , Ron Eliora Z.
Published: March 2003

Most bacterial proteins are stable, with half‐lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis – homoserine trans‐succinylase (HTS) – is unstable and subject to energy‐dependent proteolysis. The enzyme is stable in triple mutants defective in Lon‐, HslVU‐ and ClpP‐dependent proteases. The instability of the protein is determined by the amino‐terminal part of the protein, and its removal or substitution by the N‐terminal part of β‐galactosidase confers stability. The effect of the amino‐terminal segment is not caused by the N‐end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half‐life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.
Acquisition of double‐stranded DNA‐binding ability in a hybrid protein between Escherichia coli CspA and the cold shock domain of human YB‐1
Authors: Wang Nan , Yamanaka Kunitoshi , Inouye Masayori
Published: March 2003

Escherichia coli CspA, a major cold shock protein, is dramatically induced upon temperature downshift. As it binds co‐operatively to single‐stranded DNA (ssDNA) and RNA without apparent sequence specificity, it has been proposed that CspA acts as an RNA chaperone to facilitate transcription and translation at low temperature. CspA consists of a five‐stranded β‐barrel structure containing two RNA‐binding motifs, RNP1 and RNP2. Eukaryotic Y‐box proteins, such as human YB‐1, are a family of nucleic acid‐binding proteins that share a region of high homology with CspA (43% identity), termed the cold shock domain (CSD). Their cellular functions are very diverse and are associated with growth‐related processes. Here, we replaced the six‐residue loop region of CspA between the β3 and β4 strands with the corresponding region of the CSD of human YB‐1 protein. The resulting hybrid protein became capable of binding to double‐stranded DNA (dsDNA) in addition to ssDNA and RNA. The dsDNA‐binding ability of an RNP1 point mutant (F20L) of the hybrid was almost unchanged. On the other hand, the dsDNA‐binding ability of the hybrid protein was abolished in high salt concentrations in contrast to its ssDNA‐binding ability. These results indicate that the loop region between the β3 and β4 strands of Y‐box proteins, which is a little longer and more basic than that of CspA, plays an important role in their binding to dsDNA.
Both transmembrane domains of SecG contribute to signal sequence recognition by the Escherichia coli protein export machinery
Authors: Bost Sandrine , Silva Filo , Rudaz Claude , Belin Dominique
Published: March 2003

A chimeric protein containing the uncleaved signal sequence of plasminogen activators inhibitor‐2 (PAI2) fused to alkaline phosphatase (AP) interferes with Escherichia coli protein export and arrests growth. Suppressors of this toxicity include secG mutations that define the Thr‐41–Leu‐42–Phe‐43 (TLF) domain of SecG. These mutations slow down the export of PAI2‐AP. Another construct encoding a truncated PAI2 signal sequence (hB‐AP) is also toxic. Most suppressors exert their effect on both chimeric proteins. We describe here five secG suppressors that only suppress the toxicity of hB‐AP and selectively slow down its export. These mutations do not alter the TLF domain: three encode truncated SecG, whereas two introduce Arg residues in the transmembrane domains of SecG. The shortest truncated protein only contains 13 residues of SecG, suggesting that the mutation is equivalent to a null allele. Indeed, a secG disruption selectively suppresses the toxicity of hB‐AP. However, the missense mutations are not null alleles. They allow SecG binding to SecYE, although with reduced affinity. Furthermore, these mutated SecG are functional, as they facilitate the export of endogenous proteins. Thus, SecG participates in signal sequence recognition, and both transmembrane domains of SecG contribute to ensure normal signal sequence recognition by the translocase.
Analysis of mRNA decay and rRNA processing in Escherichia coli in the absence of RNase E‐based degradosome assembly
Authors: Ow Maria C. , Liu Qi , Kushner Sidney R.
Published: March 2003

We demonstrate here that the assembly of the RNase E‐based degradosome of Escherichia coli is not required for normal mRNA decay in vivo. In contrast, deletion of the arginine‐rich RNA binding site (ARRBS) from the RNase E protein slightly impairs mRNA decay. When both the degradosome scaffold region and the ARRBS are missing, mRNA decay is dramatically slowed, but 9S rRNA processing is almost normal. An extensive RNase E truncation mutation (rneδ610) had a more pronounced mRNA decay defect at 37°C than the temperature‐sensitive rne‐1 allele at 44°C. Taken together, these data suggest that the inviability associated with inactivation of RNase E is not related to defects in either mRNA decay or rRNA processing.
Topological analysis and role of the transmembrane domain in polar targeting of PilS, a Pseudomonas aeruginosa sensor kinase
Authors: Ethier Julie , Boyd Jessica M.
Published: March 2003

In Pseudomonas aeruginosa, synthesis of pilin, the major protein subunit of the pili, is regulated by a two‐component signal transduction system in which PilS is the sensor kinase. PilS is an inner membrane protein found at the poles of the bacterial cell. It is composed of three domains: an N‐terminal hydrophobic domain; a central cytoplasmic linker region; and the C‐terminal transmitter region conserved among other sensor kinases. The signal that activates PilS and, consequently, pilin transcription remains unknown. The membrane topology of the hydrophobic domain was determined using the lacZ and phoA gene fusion approach. In this report, we describe a topological model for PilS in which the hydrophobic domain forms six transmembrane helices, whereas the N‐ and C‐termini are cytoplasmic. This topology is very stable, and the cytoplasmic C‐terminus cannot cross the inner membrane. We also show that two of the six transmembrane segments are sufficient for membrane anchoring and polar localization of PilS.
The abaA homologue of Penicillium marneffei participates in two developmental programmes: conidiation and dimorphic growth
Authors: Borneman Anthony R. , Hynes Michael J. , Andrianopoulos Alex
Published: March 2003

Penicillium marneffei is the only known species of its genus that is dimorphic. At 25°C, P. marneffei exhibits true filamentous growth and undergoes asexual development producing spores borne on complex structures called conidiophores. At 37°C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. We have cloned a homologue of the Aspergillus nidulans abaA gene encoding an ATTS/TEA DNA‐binding domain transcriptional regulator and shown that it is involved in both these developmental programs. Targeted deletion of abaA blocks asexual development at 25°C before spore production, resulting in aberrant conidiophores with reiterated terminal cells. At 37°C, the abaA deletion strain fails to switch correctly from multinucleate filamentous to uninucleate yeast cells. Both the transitional hyphal cells, which produce the yeast cells, and the yeast cells themselves contain multiple nuclei. Expression of the abaA gene is activated during both conidiation and the hyphal–yeast switch. Interestingly, the abaA gene of the filamentous monomorphic fungus A. nidulans can complement both conidiation and dimorphic switching defects in the P. marneffei abaA mutant. In addition, ectopic overexpression of abaA results in anucleate yeast cells and multinucleate vegetative filamentous cells. These data suggest that abaA regulates cell cycle events and morphogenesis in two distinct developmental programmes.
pah1: a homeobox gene involved in hyphal morphology and microconidiogenesis in the filamentous ascomycetePodospora anserina
Authors: Arnaise Sylvie , Zickler Denise , Poisier Corinne , Debuchy Robert
Published: March 2003

Homeobox‐containing genes are widely described among eukaryotic species other than filamentous ascomycetes. We describe here the isolation and characterization of the first homeobox gene (pah1) identified in a filamentous ascomycete. It encodes a putative protein of 610 amino acids containing a typical homeodomain with 60 amino acids. Deletion of thepah1gene enhances the number of male gametes (microconidia), whereas overexpression ofpah1results in a decrease in microconidia. These results led us to suppose thatpah1may be a repressor of genes involved in the microconidiation process. Moreover,pah1is involved in hyphal branching and possibly in the development of female organs.
Identification of a lipopolysaccharide α‐2,3‐sialyltransferase from Haemophilus influenzae
Authors: Hood Derek W. , Cox Andrew D. , Gilbert Michel , Makepeace Katherine , Walsh Shannon , Deadman Mary E. , Cody Alison , Martin Adele , Månsson Martin , Schweda Elke K. H. , Brisson Jean‐Robert , Richards James C. , Moxon E. Richard , Wakarchuk Warren W.
Published: March 2003

We have identified a gene for the addition of N‐acetylneuraminic acid (Neu5Ac) in an α‐2,3‐linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase‐variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N‐acetyl‐lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase‐variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non‐typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl‐α‐(2–3)‐lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an α‐2,3‐sialyltransferase activity, is the first reported phase‐variable sialyltransferase gene.
Interplay between recombination, cell division and chromosome structure during chromosome dimer resolution in Escherichia coli
Authors: Pérals Koryn , Capiaux Hervé , Vincourt Jean‐Baptiste , Louarn Jean‐Michel , Sherratt David J. , Cornet François
Published: March 2003

Chromosome dimers form in bacteria by recombination between circular chromosomes. Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum‐associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ). We analyse here how these phenomena collaborate. We show that (i) both inter‐ and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ‐specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers. Our results suggest that the formation of an active XerCD‐FtsK–dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.
Evidence for non‐enzymatic glycosylation in Escherichia coli
Authors: Mironova Roumyana , Niwa Toshimitsu , Hayashi Hideki , Dimitrova Rositsa , Ivanov Ivan
Published: March 2003

Non‐enzymatic glycosylation (glycation) is a chain of chemical reactions affecting free amino groups in proteins of long‐living eukaryotes. It proceeds in several steps leading to the consecutive formation of Schiff bases, Amadori products and advanced glycation end‐products (AGEs). To our knowledge, this process has not been observed in prokaryotes so far. However, the present study provides clear‐cut evidence that glycation takes place in bacteria despite their short life span. We have detected AGEs in recombinant human interferon gamma (rhIFN‐γ) produced in Escherichia coli as well as in total protein of the same bacterium using three different approaches: (i) Western blotting using two monoclonal antibodies raised against AGEs; (ii) fluorescent spectroscopy; and (iii) investigation of the effect of known AGE inhibitors (such as acetyl salicylic acid and thiamine) on the glycation reaction. Our study shows that non‐enzymatic glycosylation is initiated during the normal growth of E. coli and results in AGE formation even after isolation of proteins. This process seems to be tightly associated with some post‐translational modifications observed in the cysteineless rhIFN‐γ, such as covalent dimerization and truncation.
Pph1 from Myxococcus xanthus is a protein phosphatase involved in vegetative growth and development
Authors: Treuner‐Lange Anke , Ward Mandy J. , Zusman David R.
Published: March 2003

Myxococcus xanthus is a Gram‐negative bacterium with a complex life cycle that includes vegetative swarming on rich medium and, upon starvation, aggregation to form fruiting bodies containing spores. Both of these behaviours require multiple Ser/Thr protein kinases. In this paper, we report the first Ser/Thr protein phosphatase gene, pph1, from M. xanthus. DNA sequence analysis of pph1 indicates that it encodes a protein of 254 residues (Mr = 28 308) with strong homology to eukaryotic PP2C phosphatases and that it belongs to a new group of bacterial protein phosphatases that are distinct from bacterial PP2C phosphatases such as RsbU, RsbX and SpoIIE. Recombinant His‐tagged Pph1 was purified from Escherichia coli and shown to have Mn2+ or Mg2+ dependent, okadaic acid‐resistant phosphatase activity on a synthetic phosphorylated peptide, RRA(pT)VA, indicating that Pph1 is a PP2C phosphatase. Pph1‐expression was observed under both vegetative and developmental conditions, but peaked during early aggregation. A pph1 null mutant showed defects during late vegetative growth, swarming and glycerol spore formation. Under starvation‐induced developmental conditions, the mutant showed reduced aggregation and failure to form fruiting bodies with viable spores. Using the yeast two‐hybrid system, we have observed a strong interaction between Pph1 and the M. xanthus protein kinase Pkn5, a negative effector of development. These results suggest a functional link between a Pkn2‐type protein kinase and a PP2C phosphatase.
Site‐directed mutagenesis of intimin α modulates intimin‐mediated tissue tropism and host specificity
Authors: Reece Stephen , Simmons Cameron P. , Fitzhenry Robert J. , Matthews Stephen , Phillips Alan D. , Dougan Gordon , Frankel Gad
Published: March 2003

The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive ‘attaching and effacing’ lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor‐binding activity of intimin is localized to the C‐terminal 280 amino acids (Int280) and at least five distinct intimin types (α, β, γ, δ and ε) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin‐binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site‐directed mutagenesis. We used the yeast two‐hybrid system and gel overlays to study intimin–Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an I237/897A substitution (positions numbered according to Int280α/whole intimin α) in intimin α did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity.
The Cryptococcus neoformans STE11α gene is similar to other fungal mitogen‐activated protein kinase kinase kinase (MAPKKK) genes but is mating type specific
Authors: Clarke D. L. , Woodlee G. L. , McClelland C. M. , Seymour T. S. , Wickes B. L.
Published: March 2003

Partial sequence analysis of the Cryptococcus neoformans MATα mating type locus revealed the presence of a gene with substantial sequence similarity to other fungal mitogen‐activated protein (MAP) kinase kinase kinase (MAPKKK) genes. The C. neoformans gene, designated STE11α, showed the highest degree of similarity to the Neurospora crassa nrc‐1, Schizosaccharomyces pombe byr2 and Saccharomyces cerevisiae STE11 genes. A polymerase chain reaction‐mediated sib‐selection technique was successfully adapted for the purpose of disrupting STE11α. C. neoformans ste11αΔ mutants were found to be sterile, consistent with the phenotypes of ste11 and byr2 mutants in S. cerevisiae and S. pombe respectively. Haploid ste11αΔ mutants were also found to be unable to produce hyphae, suggesting that the C. neoformans gene is functionally conserved when compared with its S. cerevisiae MAPKKK counterpart. Comparison of the wild‐type STE11α strain with a ste11αΔ disruptant for virulence using the mouse model showed that the ste11αΔ strain was less virulent, but the difference was only minor. In spite of some of the conserved functions of STE11α, linkage analysis showed that STE11α is only found in mating type α strains. These results demonstrate that, although functionally conserved, the mating pathway in C. neoformans has a unique organization.
Regulation of translation elongation in cyanobacteria: membrane targeting of the ribosome nascent‐chain complexes controls the synthesis of D1 protein
Authors: Tyystjärvi Taina , Herranen Mirkka , Aro Eva‐Mari
Published: March 2003

The photosystem II reaction centre protein D1 is encoded by the psbA gene. The D1 protein is stable in darkness but undergoes rapid turnover in the light. Here, we show that, in cyanobacterium Synechocystis sp. PCC6803, the synthesis of the D1 protein is regulated at the level of translation elongation in addition to the previously known transcriptional regulation. When Synechocystis sp. PCC6803 cells were transferred from light to darkness, the psbA mRNA remained abundant for hours. Cytosolic ribosomes were attached to psbA transcripts in the dark, and translation continued up to a distinct pausing site. However, ribosome nascent D1 chain complexes were not targeted to the thylakoid membrane, and no full‐length D1 protein was produced in darkness. The arrest in translation elongation was released in the light, concomitantly with targeting of ribosome D1 nascent‐chain complexes to the thylakoid membrane, allowing the synthesis of the full‐length D1 protein. Downregulation of membrane targeting of ribosome complexes was also observed in the light if damage to the D1 protein was slow. This novel type of regulation of prokaryotic translation functions to balance the synthesis and degradation of the rapidly turning over photosystem II D1 protein in Synechocystis sp. PCC6803.
Competence development by Haemophilus influenzae is regulated by the availability of nucleic acid precursors
Authors: Macfadyen Leah P. , Chen David , Vo Hung C. , Liao David , Sinotte Ryan , Redfield Rosemary J.
Published: March 2003

DNA uptake by naturally competent bacteria provides cells with both genetic information and nucleotides. In Haemophilus influenzae, competence development requires both cAMP and an unidentified signal arising under starvation conditions. To investigate this signal, competence induction was examined in media supplemented with nucleic acid precursors. The addition of physiological levels of AMP and GMP reduced competence 200‐fold and prevented the normal competence‐induced transcription of the essential competence genes comA and rec‐2. The rich medium normally used for growth allows only limited competence. Capillary electrophoresis revealed only a subinhibitory amount of AMP and no detectable GMP, and the addition of AMP or GMP to this medium also reduced competence 20‐ to 100‐fold. Neither a functional stringent response system nor a functional phosphoenolpyruvate:glycose phosphotransferase system (PTS) was found to be required for purine‐mediated repression. Added cAMP partially restored both transcription of competence genes and competence development, suggesting that purines may reduce the response to cAMP. Potential binding sites for the PurR repressor were identified in several competence genes, suggesting that competence is part of the PUR regulon. These observations are consistent with models of competence regulation, in which depleted purine pools signal the need for nucleotides, and support the hypothesis that competence evolved primarily for nucleotide acquisition.
Transcriptional control of the GAL/MEL regulon of yeast Saccharomyces cerevisiae: mechanism of galactose‐mediated signal transduction
Authors: Bhat P. Jayadeva , Murthy T. V. S.
Published: March 2003

In the yeast Saccharomyces cerevisiae, the interplay between Gal3p, Gal80p and Gal4p determines the transcriptional status of the genes needed for galactose utilization. The interaction between Gal80p and Gal4p has been studied in great detail; however, our understanding of the mechanism of Gal3p in transducing the signal from galactose to Gal4p has only begun to emerge recently. Historically, Gal3p was believed to be an enzyme (catalytic model) that converts galactose to an inducer or co‐inducer, which was thought to interact with GAL80p, the repressor of the system. However, recent genetic analyses indicate an alternative ‘protein–protein interaction model’. According to this model, Gal3p is activated by galactose, which leads to its interaction with Gal80p. Biochemical and genetic experiments that support this model provided new insights into how Gal3p interacts with the Gal80p–Gal4p complex, alleviates the repression of Gal80p and thus allows Gal4p to activate transcription. Recently, a galactose‐independent signal was suggested to co‐ordinate the induction of GAL genes with the energy status of the cell.
Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence
Authors: Gominet Myriam , Slamti Leyla , Gilois Nathalie , Rose Matthias , Lereclus Didier
Published: March 2003

PlcR is a pleiotropic regulator of virulence factors in the insect pathogen Bacillus thuringiensis and in the opportunistic human pathogen Bacillus cereus. It activates the transcription of at least 15 genes encoding extracellular proteins, including phospholipases C, proteases and enterotoxins. Expression of the plcR gene is autoregulated and activated at the onset of stationary phase. Here, we used mini‐Tn10 transposition to generate a library of B. thuringiensis mutants, with the goal of characterizing genes involved in the expression of the plcR gene. Three mutant strains were identified carrying distinct mini‐Tn10 insertions. The mutations impaired plcR expression and caused a deficient haemolytic phenotype, similar to the phenotype of a B. thuringiensis strain in which the plcR gene had been disrupted. The insertion sites of the three mini‐Tn10 transposons mapped in a five‐gene operon encoding polypeptides homologous to the components of the oligopeptide permease (Opp) system of Bacillus subtilis, and with a similar structural organization. By analogy, the five B. thuringiensis genes were designated oppA, B, C, D and F. In vitro disruption of the B. thuringiensis oppB gene reproduced the effect of the mini‐Tn10 insertions (i.e. the loss of haemolytic activity) and reduced the virulence of the strain against insects. These phenotypes are similar to those of a ΔplcR mutant. Opp is required for the import of small peptides into the cell. Therefore, plcR expression might be activated at the onset of stationary phase by the uptake of a signalling peptide acting as a quorum‐sensing effector. The opp mutations impaired the sporulation efficiency of B. thuringiensis when the cells were cultured in LB medium. Thus, Opp is on the pathway that ultimately regulates Spo0A phosphorylation, as is the case in B. subtilis. However, analysis of plcR expression in ΔoppB, Δspo0A and ΔoppBΔspo0A mutants indicates that Opp is required for plcR expression via a Spo0A‐independent mechanism.
Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae
Authors: Jin Qiaoling , Hu Wenqi , Brown Ian , McGhee Gayle , Hart Patrick , Jones Alan L. , He Sheng Yang
Published: March 2003

Pili are required for protein and/or DNA transfer from bacteria to recipient plant or bacterial cells, based on genetic evidence. However, it has never been shown directly that the effector proteins or DNA are localized along or inside the pili in situ. Failure to visualize an association of effector proteins/DNA with pili is the central issue in the debate regarding the exact function of pili in protein and DNA transfer. In this study, a newly developed in situ immunogold labelling procedure enabled visualization of the specific localization of type III effector proteins of Erwinia amylovora and Pseudomonas syringae pv. tomato along the Hrp pilus, but not along the flagellum or randomly in the intercellular space. In contrast, PelE, a pectate lyase secreted via the type II protein secretion system, was not associated with the Hrp pilus. These results provide direct evidence that type III secretion occurs only at the site of Hrp pilus assembly and that the Hrp pilus guides the transfer of effector proteins outside the bacterial cell, favouring the ‘conduit/guiding filament’ model.
The Candida glabrata Amt1 copper‐sensing transcription factor requires Swi/Snf and Gcn5 at a critical step in copper detoxification
Authors: Koch Keith A. , Allard Stéphane , Santoro Nicholas , Côté Jacques , Thiele Dennis J.
Published: March 2003

The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper‐inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome. Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE. In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels. We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific. Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE. Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation.
An ancient player unmasked: T4 rI encodes a t‐specific antiholin
Authors: Ramanculov Erlan , Young Ry
Published: March 2003

Phage T4 effects lysis by its holin T and its endolysin E. Lysis is inhibited (LIN) if the infected cell is subjected to secondary infections by T4 phage particles. The T4 rI gene is required for LIN in all hosts tested. Here, we show that a cloned rI gene can impose a T‐specific LIN on T‐mediated lysis in the context of the phage λ infective cycle, in the absence of other T4 genes and without secondary infection by T4. Moreover, it is shown that the T holin accumulates in the membrane during LIN, forming SDS‐resistant oligomers. We show by cross‐linking experiments that a T‐RI heterodimer is formed during LIN, demonstrating that RI belongs to the functional class of antiholins, such as the S107 protein of λ, which heterodimerizes with its cognate holin, S105. Finally, we show that the addition of Ni2+ ions to the medium can block lysis by a T protein hexahistidine‐tagged at its C‐terminus, suggesting that liganding of the periplasmic domain is sufficient to impose lysis inhibition. The results are discussed in terms of a model in which the LIN‐inducing signal of the secondary infecting phage influences a conformational equilibrium assumed by RI in the periplasm.
The hgl1 gene is required for dimorphism and teliospore formation in the fungal pathogen Ustilago maydis
Authors: Dürrenberger Franz , Laidlaw R. David , Kronstad James W.
Published: March 2003

The fungal pathogen Ustilago maydis causes a dramatic disease in maize involving the induction of tumours and the formation of masses of black teliospores. In this fungus, mating between haploid, budding cells results in the formation of the infectious, filamentous cell type that invades host tissue. Mating and filamentous growth are governed by the mating‐type loci and by cAMP signalling, perhaps in response to signals from maize. To dissect the involvement of cAMP signalling further, the constitutive filamentous phenotype of a mutant with a defect in the catalytic subunit of protein kinase A was used to isolate suppressor mutations that restore budding growth. One such mutation identified the hgl1 gene, which is shown to be required for both the switch between budding and filamentous growth and teliospore formation during infection. In addition, the hgl1 gene product may be a target of phosphorylation by protein kinase A, and transcript levels for the gene are elevated during mating. Thus, the hgl1 gene provides a connection between mating, cAMP signalling and two important aspects of virulence: filamentous growth and the formation of teliospores.
MlrA, a novel regulator of curli (AgF) and extracellular matrix synthesis by Escherichia coli and Salmonella enterica serovar Typhimurium
Authors: Brown Peter K. , Dozois Charles M. , Nickerson Cheryl A. , Zuppardo Amy , Terlonge Jackie , Curtiss, Roy
Published: March 2003

Production of curli (AgF) adhesins by Escherichia coli and Salmonella enterica serovar Typhimurium (S. typhimurium) is associated with extracellular matrix production and is optimal at low temperature during stationary phase. Curli and extracellular matrix synthesis involves a complex regulatory network that is dependent on the CsgD (AgfD) regulator. We have identified a novel regulator, termed MlrA, that is required for curli production and extracellular matrix formation. Two cosmids from a genomic library of avian pathogenic E. coliχ7122 conferred mannose‐resistant haemagglutination (HA) and curli production to E. coli HB101, which is unable to produce curli owing to a defective regulatory pathway. The rpoS gene, encoding a known positive regulator of curli synthesis, and the E. coli open reading frame (ORF) of unknown function, yehV, identified on each of these cosmids, respectively, conferred curli production and HA to E. coli HB101. We have designated yehV as the mlrA gene for MerR‐like regulator A because its product shares similarities with regulatory proteins of the MerR family. HA and curli production by strain χ7122 were abolished by disruption of rpoS, mlrA or csgA, the curli subunit gene. Both csgD and csgBA transcription, required for expression of curli, were inactive in an mlrA mutant grown under conditions that promote curli production. An mlrA homologue was identified in S. typhimurium. Analysis of mlrA–lac operon fusions demonstrated that mlrA was positively regulated by rpoS. mlrA mutants of wild‐type S. typhimurium SL1344 or SR‐11 no longer produced curli or rugose colony morphology, and exhibited enhanced aggregation and extracellular matrix formation when complemented with the mlrA gene from either S. typhimurium or E. coli present on a low‐copy‐number plasmid. However, inactivation of mlrA did not affect curli production and aggregative morphology in an upregulated curli producing S. typhimurium derivative containing a temperature‐ and RpoS‐independent agfD promoter region. These results indicate that MlrA is a newly defined transcriptional regulator of csgD/agfD that acts as a positive regulator of RpoS‐dependent curli and extracellular matrix production by E. coli and S. typhimurium.
The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum‐sensing signal molecule
Authors: Schauder Stephan , Shokat Kevan , Surette Michael G. , Bassler Bonnie L.
Published: March 2003

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram‐negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram‐negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI‐1 and AI‐2). AI‐1 is a homoserine lactone. The structure of AI‐2 is not known. We have suggested previously that V. harveyi uses AI‐1 for intraspecies communication and AI‐2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram‐negative and Gram‐positive bacteria produce AI‐2 and, in every case, production of AI‐2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI‐2 synthase and that AI‐2 is produced from S‐adenosylmethionine in three enzymatic steps. The substrate for LuxS is S‐ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI‐2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI‐2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI‐2 is a unique, ‘universal’ signal that could be used by a variety of bacteria for communication among and between species.
Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects
Authors: Alonso‐Monge Rebeca , Real Eliana , Wojda Iwona , Bebelman Jan‐Paul , Mager Willem H. , Siderius Marco
Published: March 2003

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37°C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature‐induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall‐degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature‐induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high‐osmolarity response routes.