bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of rubrerythrin in the oxidative stress response of Porphyromonas gingivalis
Authors: Sztukowska Maryta , Bugno Marcin , Potempa Jan , Travis James , Kurtz Jr Donald M.
Published: March 2003

Rubrerythrins are non‐haem iron proteins that have been implicated in oxidative stress protection in anaerobic bacteria and archaea. However, up to now, this role has not been confirmed directly by inactivation of a rubrerythrin gene. Here we report generation of an rbr− mutant of Porphyromonas gingivalis, an obligately anaerobic gingival pathogenic bacterium. Characterization of the rbr− strain clearly showed that P. gingivalis produces a rubrerythrin‐like protein that is absent in the rbr− strain, and that the P. gingivalis rbr− strain is more dioxygen‐ and hydrogen peroxide‐sensitive than the wild type. The latter conclusion is based on two independent results, namely, deeper no‐growth zones upon diffusion of the oxidants through soft agar culture tubes and growth impairment of liquid cultures exposed to the oxidants. A same‐site rbr+ revertant showed increased hydrogen peroxide and dioxygen resistance relative to the rbr− strain. Transcription of the P. gingivalis rubrerythrin gene is induced above its constitutive anaerobic level in response to dioxygen or hydrogen peroxide exposures. Purified rubrerythrins from other organisms have been shown to catalyse reduction of hydrogen peroxide, while showing relatively sluggish reaction with dioxygen and little or no catalase or superoxide dismutase activities. Porphyromonas gingivalis contains a superoxide dismutase but lacks catalase and haem peroxidases. We therefore suggest that rubrerythrin provides oxidative stress protection via catalytic reduction of intracellular hydrogen peroxide.
Complete glycosylphosphatidylinositol anchors are required in Candida albicans for full morphogenesis, virulence and resistance to macrophages
Authors: Richard Mathias , Ibata‐Ombetta Stella , Dromer Françoise , Bordon‐Pallier Florence , Jouault Thierry , Gaillardin Claude
Published: March 2003

Glycosylphosphatidylinositol (GPI)‐anchored proteins are involved in cell wall integrity and cell–cell interactions. We disrupted the Candida albicans
homologue of the Saccharomyces cerevisiae GPI7/LAS21 gene, which encodes a
GPI anchor‐modifying activity. In the mutant and on solid media, the yeast‐to‐hyphae
transition was blocked, whereas chlamydospore formation was enhanced. However, the
morphogenetic switch was normal in liquid medium. Abnormal budding patterns, cytokinesis
and cell shape were observed in both liquid and solid media. The cell wall structure
was also modified in the mutants, as shown by hypersensitivity to Calcofluor white.
In vitro and in vivo assays revealed that the mutant interacted with
its host in a modified way, resulting in reduced virulence in mice and reduced survival
in the gastrointestinal environment of mice. The mitogen‐activated protein (MAP)
kinase pathway of macrophages was downregulated by the wild‐type cells but not by
the ΔCagpi7 null strains. In agreement with this abnormal behaviour,
mutant cells were more sensitive to the lytic action of macrophages. Our results
indicate that a functional GPI anchor is required for full hyphal formation in C.
albicans, and that perturbation of the GPI biosynthesis results in hypersensitivity to host defences.
Identification of novel hrp‐regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome
Authors: Zwiesler‐Vollick Julie , Plovanich‐Jones Anne E. , Nomura Kinya , Bandyopadhyay Sruti , Joardar Vinita , Kunkel Barbara N. , He Sheng Yang
Published: March 2003

Pseudomonas syringae
pv.
tomato
(
Pst
) strain DC3000 infects the model plants
Arabidopsis thaliana
and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by
Pst
DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in
Pst
DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of
P. syringae
are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved
cis
element (called the ‘hrp box’) in the promoters of type III secretion‐associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published
hrp
operons and putative effector genes in
Pst
DC3000. Seventy‐three predicted genes were retrieved from the January 2001 release of the
Pst
DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in
hrp
‐inducing minimal medium than in nutrient‐rich Luria–Bertani broth. Expression of all eight genes was dependent on the
hrpS
gene. Most were also dependent on the
hrpL
gene, but at least one was dependent on the
hrpS
gene, but not on the
hrpL
gene. An AvrRpt2‐based type III translocation assay provides evidence that some of the
hrpS ‐regulated novel genes encode putative effector proteins.
Polymerase chain reaction‐based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803
Authors: Wang Hong‐Liang , Postier Bradley L. , Burnap Robert L.
Published: March 2003

Primary ion pumps and antiporters exist as multigene families in the Synechocystis

sp. PCC 6803 genome and show very strong homologies to those found in higher plants.

The gene knock‐outs of five putative Na+/H+ antiporters

(slr1727, sll0273, sll0689, slr1595 and slr0415)

and seven cation ATPases (sll1614, sll1920, slr0671–72,

slr0822, slr1507–08–09, slr1728– 29 and

slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html)

were performed in this study relying on homologous recombination with mutagenenic

fragments constructed using a fusion polymerase chain reaction (PCR) approach. The

impacts of these gene knock‐outs were evaluated in terms of Na+

and pH, and light‐induced acidification and alkalization that are asso‐ciated with

inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic

interplay between the pH and Na+ dependence of growth, but only

one of the antiporters appears to be necessary for high NaCl tolerance. On the other

hand, the mutation of one of the two copper‐trafficking ATPases produces a cell line

that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+‐ATPase

and a gene cluster encoding a putative Na+‐ATPase subunit also

cause high NaCl sensitivity. The findings and possible mechanisms are discussed in

relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.
Regulation of inducible peroxide stress responses
Authors: Mongkolsuk Skorn , Helmann John D.
Published: March 2003

Bacteria adapt to the presence of reactive oxygen species (ROS) by increasing the expression of detoxification enzymes and protein and DNA repair functions. These responses are co‐ordinated by transcription factors that regulate target genes in response to ROS. We compare three classes of peroxide‐sensing regulators: OxyR, PerR and OhrR. In all three cases, peroxides effect changes in the redox status of cysteine residues, but the molecular details are distinct. OxyR is converted into a transcriptional activator by the formation of a disulphide bond between two reactive cysteine residues. PerR is a metalloprotein that functions as a peroxide‐ sensitive repressor. Oxidation is modulated by metal ion composition and may also involve disulphide bond formation. OhrR represses an organic peroxide resistance protein and mediates derepression in response to organic peroxides. Peroxide sensing in this system requires a single conserved cysteine, which is oxidized to form a cysteine–sulphenic acid derivative.
Vibrio cholerae CytR is a repressor of biofilm development
Authors: Haugo Adam J. , Watnick Paula I.
Published: March 2003

Vibrio cholerae is both a human pathogen and a natural inhabitant of aquatic environments. In the aquatic environment, microorganisms are found attached to surfaces in structures known as biofilms. We have identified a transcriptional repressor in V. cholerae that inhibits exopolysaccharide synthesis and biofilm development. Our studies show that this repressor is the V. cholerae homologue of Escherichia coli CytR, a protein that represses nucleoside uptake and catabolism when nucleosides are scarce. We propose that the role of CytR in V. cholerae biofilm development is to co‐ordinate bacterial biofilm accumulation with the presence of nucleosides. Thus, nucleosides may be a signal to planktonic cells to join the biofilm.
Control of the glycolytic gapA operon by the catabolite control protein A in Bacillus subtilis: a novel mechanism of CcpA‐mediated regulation
Authors: Ludwig Holger , Rebhan Nicole , Blencke Hans‐Matti , Merzbacher Matthias , Stülke Jörg
Published: March 2003

Glycolysis is one of the main pathways of carbon catabolism in Bacillus subtilis. Expression of the gapA gene encoding glyceraldehyde‐3‐phosphate dehydrogenase, the key enzyme of glycolysis from an energetic point of view, is induced by glucose and other sugars. Two regulators are involved in induction of the gapA operon, the product of the first gene of the operon, the CggR repressor, and catabolite control protein A (CcpA). CcpA is required for induction of the gapA operon by glucose. Genetic evidence has demonstrated that CcpA does not control the expression of the gapA operon by binding directly to a target in the promoter region. Here, we demonstrate by physiological analysis of the inducer spectrum that CcpA is required only for induction by sugars transported by the phosphotransferase system (PTS). A functional CcpA is needed for efficient transport of these sugars. This interference of CcpA with PTS sugar transport results from an altered phosphorylation pattern of HPr, a phosphotransferase of the PTS. In a ccpA mutant strain, HPr is nearly completely phosphorylated on a regulatory site, Ser‐46, and is trapped in this state, resulting in its inactivity in PTS phosphotransfer. A mutation in HPr affecting the regulatory phosphorylation site suppresses both the defect in PTS sugar transport and the induction of the gapA operon. We conclude that a low‐molecular effector derived from glucose that acts as an inducer for the repressor CggR is limiting in the ccpA mutant.
GabR, a member of a novel protein family, regulates the utilization of γ‐aminobutyrate in Bacillus subtilis
Authors: Belitsky Boris R. , Sonenshein Abraham L.
Published: March 2003

The Bacillus subtilis ycnG (gabT) and ycnH (gabD) genes were shown to encode γ‐aminobutyrate (GABA) aminotransferase and succinic semi‐aldehyde dehydrogenase, respectively, and to form a GABA‐inducible operon. Null mutations in gabT, gabD or the divergently transcribed ycnF (gabR) gene blocked the utilization of GABA as sole nitrogen source. GabR proved to be a transcriptional activator of the gabTD operon and a negative autoregulator. The target of GabR action was localized to an 87 bp region that includes both gabR and gabT promoters. GabR is a member of a novel but widespread family of chimeric bacterial proteins that have apparent DNA‐binding and aminotransferase domains. Mutations in conserved residues of the putative aminotransferase domain abolished GabR function as a transcriptional activator, but did not affect its activity as a negative autoregulator.
Action at a distance in CI repressor regulation of the bacteriophage 186 genetic switch
Authors: Dodd Ian B. , Egan J. Barry
Published: March 2003

The non‐lambdoid coliphage 186 provides an alternative model to the lytic‐lysogenic switch of phage λ. Like λ, the key switch regulator, the CI repressor, associates to octamers. Unlike λ, the lytic promoter (pR) and the lysogenic promoter (pL) are face‐to‐face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located ≈ 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to λ. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co‐operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.
Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas
Authors: Visca Paolo , Leoni Livia , Wilson Megan J. , Lamont Iain L.
Published: March 2003

A variety of bacterial species secrete and take up chelating compounds that enable acquisition of iron (siderophores). It has become clear that a common feature in regulation of different iron acquisition systems is the involvement of alternative sigma factor proteins of the extracytoplasmic function (ECF) family. Two of these proteins, PvdS from Pseudomonas aeruginosa and FecI from Escherichia coli K‐12, have been studied extensively. PvdS directs transcription of genes required for the biosynthesis of a siderophore, pyoverdine, and FecI causes ex‐pression of genes for uptake of ferric citrate. FecI forms part of a signalling system that responds to the presence of ferric citrate. Here, we review recent advances in understanding of PvdS and of the Fec signalling system. PvdS and FecI are part of a distinct subfamily of ECF sigma factors involved in iron acquisition and hence named the iron‐starvation sigmas. Analysis of microbial genome sequences shows that Fec‐like signalling systems are present in a wide range of species and many such systems may be present in a single species. The availability of tools for large‐scale genome analysis is likely to lead to rapid advances in our understanding of this expanding family of proteins.
Controlling pore assembly of staphylococcal γ‐haemolysin by low temperature and by disulphide bond formation in double‐cysteine LukF mutants
Authors: Nguyen Vananh T. , Higuchi Hideo , Kamio Yoshiyuki
Published: March 2003

Staphylococcal LukF and Hlg2 are water‐soluble monomers of γ‐haemolysin that assemble into oligomeric pores on the erythrocyte membranes. Here, we have created double‐cysteine LukF mutants, in which single disulphide bonds connect either the prestem domain and the cap domain (V12C‐T136C, Cap–Stem), or two β‐strands within the prestem domain (T117C‐T136C, Stem–Stem) to control pore assembly of γ‐haemolysin at intermediate stages. The disulphide‐trapped mutants were inactive in erythrocyte lysis, but gained full haemolytic activity if the disulphide bonds were reduced. The disulphide bonds blocked neither the membrane binding ability nor the intermediate prepore oligomerization, but efficiently inhibited the transition from prepores to pores. The prepores of Cap–Stem were dissociated into monomers in 1% SDS. In contrast, the prepores of Stem–Stem were stable in SDS and had ring‐shaped structures similar to those of wild‐type LukF, as observed by transmission electron microscopy. The transition of both mutants from prepores to pores could even be achieved by reducing disulphide bonds at low temperature (2°C), whereas prepore oligomerization was effectively inhibited by low temperature. Finally, real‐time transition of Stem–Stem from prepores to pores on ghost cells, visualized using a Ca2+‐sensitive fluorescent indicator (Rhod2), was shown by the sequential appearance of fluorescence spots, indicating pore‐opening events. Taken together, these data indicate that the prepores are legitimate intermediates during γ‐haemolysin pore assembly, and that conformational changes around residues 117 and 136 of the prestem domain are essential for pore formation, but not for membrane binding or prepore oligomerization. We propose a mechanism for γ‐haemolysin pore assembly based on the demonstrated intermediates.
A new mutation delivery system for genome‐scale approaches in Bacillus subtilis
Authors: Fabret Céline , Dusko Ehrlich S. , Noirot Philippe
Published: March 2003

In Bacillus subtilis, although many genetic tools have been developed, gene replacement remains labour‐intensive and not compatible with large‐scale appro‐aches. We have developed a new one‐step gene replacement procedure that allows rapid alteration of any gene sequence or multiple gene sequences in B. subtilis without altering the chromosome in any other way. This novel approach relies on the use of upp, which encodes uracil phosphoribosyl‐transferase, as a counter‐selectable marker. We fused the upp gene to an antibiotic‐resistance gene to create an ‘upp‐cassette’. A polymerase chain reaction (PCR)‐generated fragment, consisting of the target gene with the desired mutation joined to the upp‐cassette, was integrated into the chromosome by homologous recombination, using positive selection for antibiotic resistance. Then, the eviction of the upp‐cassette from the chromosome by recombination between short repeated chromosomal sequences, included in the design of the transforming DNA molecule, was achieved by counter‐selection of upp. This procedure was successfully used to deliver a point mutation, to generate in‐frame deletions with reduced polar effects, and to combine deletions in three paralogous genes encoding two‐component sensor kinases. Also, two chromosome regions carrying previously unrecognized essential functions were identified, and large deletions in two dispensable regions were combined. This work outlines a strategy for identifying essential functions that could be used at genome scale.
Overexpression of inhA, but not kasA, confers resistance to isoniazid and ethionamide in Mycobacterium smegmatis, M. bovis BCG and M. tuberculosis
Authors: Larsen Michelle H. , Vilchèze Catherine , Kremer Laurent , Besra Gurdyal S. , Parsons Linda , Salfinger Max , Heifets Leonid , Hazbon Manzour H. , Alland David , Sacchettini James C. , Jacobs William R.
Published: March 2003

The inhA and kasA genes of Mycobacterium tuberculosis have each been proposed to encode the primary target of the antibiotic isoniazid (INH). Previous studies investigating whether overexpressed inhA or kasA could confer resistance to INH yielded disparate results. In this work, multicopy plasmids expressing either inhA or kasA genes were transformed into M. smegmatis, M. bovis BCG and three different M. tuberculosis strains. The resulting transformants, as well as previously published M. tuberculosis strains with multicopy inhA or kasAB plasmids, were tested for their resistance to INH, ethionamide (ETH) or thiolactomycin (TLM). Mycobacteria containing inhA plasmids uniformly exhibited 20‐fold or greater increased resistance to INH and 10‐fold or greater increased resistance to ETH. In contrast, the kasA plasmid conferred no increased resistance to INH or ETH in any of the five strains, but it did confer resistance to thiolactomycin, a known KasA inhibitor. INH is known to increase the expression of kasA in INH‐susceptible M. tuberculosis strains. Using molecular beacons, quantified inhA and kasA mRNA levels showed that increased inhA mRNA levels corre­‐lated with INH resistance, whereas kasA mRNA levels did not. In summary, analysis of strains harbouring inhA or kasA plasmids yielded the same conclusion: overexpressed inhA, but not kasA, confers INH and ETH resistance to M. smegmatis, M. bovis BCG and M. tuberculosis. Therefore, InhA is the primary target of action of INH and ETH in all three species.
Glutathione regulates the expression of γ‐glutamylcysteine synthetase via the Met4 transcription factor
Authors: Wheeler Glen L. , Quinn Kathryn A. , Perrone Gabriel , Dawes Ian W. , Grant Chris M.
Published: March 2003

Our previous studies have shown that glutathione is an essential metabolite in the yeast Saccharomyces cerevisiae because a mutant deleted for GSH1, encoding the first enzyme in γ‐l‐glutamyl‐l‐cysteinylglycine (GSH) biosynthesis, cannot grow in its absence. In contrast, strains deleted for GSH2, encoding the second step in GSH synthesis, grow poorly as the dipeptide intermediate, γ‐glutamylcysteine, can partially substitute for GSH. In this present study, we identify two high copy suppressors that rescue the poor growth of the gsh2 mutant in the absence of GSH. The first contains GSH1, indicating that γ‐glutamylcysteine can functionally replace GSH if it is present in sufficiently high quantities. The second contains CDC34, encoding a ubiquitin conjugating enzyme, indicating a link between the ubiquitin and GSH stress protective systems. We show that CDC34 rescues the growth of the gsh2 mutant by inducing the Met4‐dependent expression of GSH1 and elevating the cellular levels of γ‐glutamylcysteine. Furthermore, this mechanism normally operates to regulate GSH biosynthesis in the cell, as GSH1 promoter activity is induced in a Met4‐dependent manner in a gsh1 mutant which is devoid of GSH, and the addition of exogenous GSH represses GSH1 expression. Analysis of a cis2 mutant, which cannot breakdown GSH, confirmed that GSH and not a metabolic product, serves as the regulatory molecule. However, this is not a general mechanism affecting all Met4‐regulated genes, as MET16 expression is unaffected in a gsh1 mutant, and GSH acts as a poor repressor of MET16 expression compared with methionine. In summary, GSH biosynthesis is regulated in parallel with sulphate assimilation by activity of the Met4 protein, but GSH1‐specific mechanisms exist that respond to GSH availability.
TheHaemophilus influenzaeHia autotransporter harbours two adhesive pockets that reside in the passenger domain and recognize the same host cell receptor
Authors: Laarmann Sven , Cutter David , Juehne Twyla , Barenkamp Stephen J. , St Geme Joseph W.
Published: March 2003

Haemophilus influenzaeis a human‐specific pathogen and a major source of morbidity worldwide. Infection with this organism begins with colonization of the nasopharynx, a process that probably depends on adherence to respiratory epithelium. The Hia autotransporter protein is the major adhesin ex‐pressed by a subset of non‐typeableH. influenzaestrains and promotes high‐level adherence to a variety of human epithelial cell lines. In the current study, we discovered that the Hia passenger domain contains two distinct binding pockets, including one at the C‐terminal end and a second at the N‐terminal end. Competition assays revealed that the two binding pockets interact with the same host cell receptor structure, although with differing affinities. Additional experiments demonstrated that both binding domains are required for full‐level bacterial adherence. These observations are reminiscent of eukaryotic cell adhesion molecules and highlight the first example of a bacterial adhesin with two domains that participate in a bivalent interaction with identical host cell receptors. Such an interaction increases avidity, thus stabilizing bacterial adherence to the epithelial surface, despite physical forces such as coughing, sneezing and mucociliary clearance.
Competence for natural transformation in Neisseria gonorrhoeae: components of DNA binding and uptake linked to type IV pilus expression
Authors: Aas Finn Erik , Wolfgang Matthew , Frye Stephan , Dunham Steven , Løvold Cecilia , Koomey Michael
Published: March 2003

The mechanisms by which DNA is taken up into the bacterial cell during natural genetic transformation are poorly understood. Although related components essential to the uptake of DNA during transforma‐tion have been defined in Gram‐negative species, it remains unclear whether DNA binding and uptake are dissociable events. Therefore, DNA uptake has been the earliest definable step in any Gram‐negative transformation pathway. In the human pathogen Neisseria gonorrhoeae, sequence‐specific DNA uptake requires an intact type IV pili (Tfp) biogenesis machinery along with three molecules that are dispensable for Tfp expression: ComP (a pilin subunit‐like molecule), PilT (a cytoplasmic protein involved in pilus retraction) and ComE (a periplasmic protein with intrinsic DNA‐binding activity). By conditionally altering the levels of ComP and PilT expression, we show here that DNA binding and uptake are resolvable events. Consequently, we are able to demonstrate that PilT is largely dispensable for functional DNA binding and, therefore, contributes specifically to uptake. Furthermore, sequence specificity in this system is imposed at the level of DNA binding, a process that is influenced by both ComP and PilE. However, sequence‐specific DNA binding is not attributable to an intrinsic property of the Tfp subunit protein. Finally, we demonstrate the existence of a robust, non‐specific DNA‐binding activity associated with the expression of both Tfp and PilT, which is unrelated to transformation but obscures the observation of specific binding events.
Overexpression of ccl1−2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c‐type cytochromes
Authors: Deshmukh Meenal , May Matthew , Zhang Yan , Gabbert Karen K. , Karberg Katherine A. , Kranz Robert G. , Daldal Fevzi
Published: March 2003

In Gram‐negative bacteria, including Rhodobacter capsulatus, the membrane protein CycH acts as a putative apocytochrome chaperone during the biogenesis of c‐type cytochromes. CycH‐null mutants are unable to produce various c‐type cytochromes and sustain photosynthetic (Ps) growth that requires the cytochromes c1 and c2 or cy. However, Ps+ revertants are readily obtained only on minimal, but not on enriched, medium. To obtain further information about the biogenesis of c‐type cytochromes, these suppressor mutants were studied. Complementation of a CycH‐null mutant for Ps+ growth by a genomic library constructed using DNA from a Ps+ suppressor yielded a plasmid carrying the ccl1–2 operon, the products of which, Ccl1 and Ccl2, are also involved in the biogenesis of c‐type cytochromes. DNA sequence analysis revealed that the complementing activity resulted from a single point mutation, G488A, located upstream of the coding region of ccl1–2. This mutation changed the −35 region of the ccl1–2 promoter from TTGGCC to TTGACC, improving its similarity to the consensus sequence of Escherichia coliσ70‐dependent promoters. That the G488A mutation indeed enhanced transcription of ccl1–2 was demonstrated by the use of reporter gene fusions. An appropriate ccl1‐2::lacZ transcriptional–translational fusion carrying the G488A mutation produced in R. capsulatus over 30‐fold higher β‐galactosidase activity than a wild‐type construct. Immunoblot analyses confirmed that Ccl1 and Ccl2 were overproduced in the Ps+ suppressors. Deletion of either ccl1 or ccl2, from the ccl1–2 cluster carrying the G488A mutation abolished the complementing ability, indicating that overexpression of both ccl1 and ccl2 was required to confer the Ps+ phenotype on a CycH‐null mutant. These findings therefore demonstrate that, during R. capsulatus growth on minimal medium, the requirement for CycH in c‐type cytochrome biogenesis could be bypassed by overexpressing the ccl1–2 operon.
The Zygosaccharomyces bailii antifungal virus toxin zygocin: cloning and expression in a heterologous fungal host
Authors: Weiler Frank , Rehfeldt Klaus , Bautz Frank , Schmitt Manfred J.
Published: March 2003

Zygocin, a monomeric protein toxin secreted by a virus‐infected killer strain of the osmotolerant spoilage yeast Zygosaccharomyces bailii, kills a broad spectrum of human and phytopathogenic yeasts and filamentous fungi by disrupting cytoplasmic membrane function. The toxin is encoded by a double‐stranded (ds)RNA killer virus (ZbV‐M, for Z. bailii virus M) that stably persists within the yeast cell cytosol. In this study, the protein toxin was purified, its N‐terminal amino acid sequence was determined, and a full‐length cDNA copy of the 2.1 kb viral dsRNA genome was cloned and successfully expressed in a heterologous fungal system. Sequence analysis as well as zygocin expression in Schizosaccharomyces pombe indicated that the toxin is in vivo expressed as a 238‐amino‐acid preprotoxin precursor (pptox) consisting of a hydrophobic N‐terminal secretion signal, followed by a potentially N‐glycosylated pro‐region and terminating in a classical Kex2p endopeptidase cleavage site that generates the N‐terminus of the mature and biologically active protein toxin in a late Golgi compartment. Matrix‐assisted laser desorption mass spectrometry further indicated that the secreted toxin is a monomeric 10.4 kDa protein lacking detectable post‐translational modifications. Furthermore, we present additional evidence that in contrast with other viral antifungal toxins, zygocin immunity is not mediated by the toxin precursor itself and, therefore, heterologous pptox expression in a zygocin‐sensitive host results in a suicidal phenotype. Final sequence comparisons emphasize the conserved pattern of functional elements present in dsRNA killer viruses that naturally infect phylogenetically distant hosts (Saccharomyces cerevisiae and Z. bailii) and reinforce models for the sequence elements that are in vivo required for viral RNA packaging and replication.
Expression of the glutamyl‐tRNA synthetase gene from the cyanobacterium Synechococcus sp. PCC 7942 depends on nitrogen availability and the global regulator NtcA
Authors: Luque Ignacio , Contreras Asunción , Zabulon Gérald , Herrero Antonia , Houmard Jean
Published: March 2003

We report here transcriptional analyses of a cyanobacterial gene encoding an aminoacyl‐tRNA synthetase (aaRS), the gltX gene from Synechoccocus sp. PCC 7942, coding for the glutamyl‐tRNA synthetase. We show that the transcript levels of gltX in Synechococcus depend on nitrogen availability and do not increase with the growth rate, which is at odds with observations from other bacteria. We also demonstrate the involvement of the cyanobacterial global regulator NtcA in transcriptional control of gltX according to nitrogen status. Our results support a regulatory model in which the gltX transcript level is finely tuned by a dynamic equilibrium between activation and repression relying upon the cellular concentration of NtcA.
Two homologous genes, DCW1 (YKL046c) and DFG5, are essential for cell growth and encode glycosylphosphatidylinositol (GPI)‐anchored membrane proteins required for cell wall biogenesis in Saccharomyces cerevisiae
Authors: Kitagaki Hiroshi , Wu Hong , Shimoi Hitoshi , Ito Kiyoshi
Published: March 2003

The cell wall of Saccharomyces cerevisiae consists of glucan, chitin and various kinds of mannoproteins. Major parts of mannoproteins are synthesized as glycosylphosphatidylinositol (GPI)‐anchored proteins and are then transferred to cell wall β‐1,6‐glucan. A glycosyltransferase has been hypothesized to catalyse this transfer reaction. A database search revealed that the products of YKL046c and DFG5 are homologous to bacterial mannosidase. These genes are homologous to each other and have primary structures characteristic of GPI‐anchored proteins. Although single disruptants of ykl046c and dfg5 were viable, ykl046cΔ was hypersensitive to a cell wall‐digesting enzyme (zymolyase), suggesting that this gene is involved in cell wall biosynthesis. We therefore designated this gene as DCW1 (defective cell wall). A double disruptant of dcw1 and dfg5 was synthetically lethal, indicating that the functions of these gene products are redundant, and at least one of them is required for cell growth. Cells deficient in both Dcw1p and Dfg5p were round and large, had cell walls that contained an increased amount of chitin and secreted a major cell wall protein, Cwp1p, into the medium. Biochemical analyses showed that epitope‐tagged Dcw1p is an N‐glycosylated, GPI‐anchored membrane protein and is localized in the membrane fraction including the cell surface. These results suggest that both Dcw1p and Dfg5p are GPI‐anchored membrane proteins and are required for normal biosynthesis of the cell wall.
TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm
Authors: Wadhams G. H. , Martin A. C. , Porter S. L. , Maddock J. R. , Mantotta J. C. , King H. M. , Armitage J. P.
Published: March 2003

TlpC is encoded in the second chemotaxis operon of Rhodobacter sphaeroides. This protein shows some homology to membrane‐spanning chemoreceptors of many bacterial species but, unlike these, is essential for R. sphaeroides chemotaxis to all compounds tested. Genomic replacement of tlpC with a C‐terminal gfp fusion demonstrated that TlpC localized to a discrete cluster within the cytoplasm. Immunogold electron microscopy also showed that TlpC localized to a cytoplasmic electron‐dense region. Correct TlpC–GFP localization depended on the downstream signalling proteins, CheW3, CheW4 and CheA2, and was tightly linked to cell division. Newly divided cells contained a single cluster but, as the cell cycle progressed, a second cluster appeared close to the initial cluster. As elongation continued, these clusters moved apart so that, on septation, each daughter cell contained a single TlpC cluster. The data presented suggest that TlpC is either a cytoplasmic chemoreceptor responding to or integrating global signals of metabolic state or a novel and essential component of the chemotaxis signalling pathway. These data also suggest that clustering is essential for signalling and that a mechanism may exist for targeting and localizing proteins within the bacterial cytoplasm.
Mss11p is a transcription factor regulating pseudohyphal differentiation, invasive growth and starch metabolism in Saccharomyces cerevisiae in response to nutrient availability
Authors: Gagiano Marco , Bester Michael , Van Dyk Dewald , Franken Jaco , Bauer Florian F. , Pretorius Isak S.
Published: March 2003

In Saccharomyces cerevisiae, the cell surface protein, Muc1p, was shown to be critical for invasive growth and pseudohyphal differentiation. The transcription of MUC1 and of the co‐regulated STA2 glucoamylase gene is controlled by the interplay of a multitude of regulators, including Ste12p, Tec1p, Flo8p, Msn1p and Mss11p. Genetic analysis suggests that Mss11p plays an essential role in this regulatory process and that it functions at the convergence of at least two signalling cascades, the filamentous growth MAPK cascade and the cAMP‐PKA pathway. Despite this central role in the control of filamentous growth and starch metabolism, the exact molecular function of Mss11p is unknown. We subjected Mss11p to a detailed molecular analysis and report here on its role in transcriptional regulation, as well as on the identification of specific domains required to confer transcriptional activation in response to nutritional signals. We show that Mss11p contains two independent transactivation domains, one of which is a highly conserved sequence that is found in several proteins with unidentified function in mammalian and invertebrate organisms. We also identify conserved amino acids that are required for the activation function.
Structure and function of the Mur enzymes: development of novel inhibitors
Authors: El Zoeiby Ahmed , Sanschagrin François , Levesque Roger C.
Published: March 2003

One of the biggest challenges for recent medical research is the continuous development of new antibiotics interacting with bacterial essential mechanisms. The machinery for peptidoglycan biosynthesis is a rich source of crucial targets for antibacterial chemotherapy. The cytoplasmic steps of the biosynthesis of peptidoglycan precursor, catalysed by a series of Mur enzymes, are excellent candidates for drug development. There has been growing interest in these bacterial enzymes over the last decade. Many studies attempted to understand the detailed mechanisms and structural features of the key enzymes MurA to MurF. Only MurA is inhibited by a known antibiotic, fosfomycin. Several attempts made to develop novel inhibitors of this pathway are discussed in this review. Three novel inhibitors of MurA were identified recently. 4‐Thiazolidinone compounds were designed as MurB inhibitors. Many phosphinic acid derivatives and substrate analogues were identified as inhibitors of the MurC to MurF amino acid ligases.
Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium‐metabolizing enzymes (GS, NADP‐GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum
Authors: Javelle Arnaud , Morel Mélanie , Rodríguez‐Pastrana Blanca‐Rosa , Botton Bernard , André Bruno , Marini Anne‐Marie , Brun Annick , Chalot Michel
Published: March 2003

External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP‐dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N‐starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented.
A novel regulatory gene for light‐induced carotenoid synthesis in the bacteriumMyxococcus xanthus
Authors: Fontes Marta , Galbis‐Martínez Lilian , Murillo Francisco J.
Published: March 2003

Myxococcus xanthuscells respond to blue light by producing carotenoids. Light triggers a network of regulatory actions that lead to the transcriptional activation of the carotenoid genes. By screening the colour phenotype of a collection of Tn5‐lacinsertion mutants, we have isolated a new mutant devoid of carotenoid synthesis. We map the transposon insertion, which co‐segregates with the mutant phenotype, to a previously unknown gene designated here ascarF. An in frame deletion withincarFcauses the same phenotype as the Tn5‐lacinsertion. ThecarFdeletion prevents the activation of the normally light‐inducible genes, without affecting the expression of any of the regulatory genes known to be expressed in a light‐independent manner. Until now, the switch that sets off the regulatory cascade had been identified with light‐driven inactivation of protein CarR, an antisigma factor. The exact mechanism of this inactivation has remained elusive. We show by epistatic analysis that thecarFgene product participates in the light‐dependent inactivation of CarR. The predicted CarF amino acid sequence reveals no known prokaryotic homologues. On the other hand, CarF is remarkably similar to Kua, a family of proteins of unknown function that is widely distributed among eukaryotes.
Mechanisms of iron regulation in mycobacteria: role in physiology and virulence
Authors: Rodriguez G. Marcela , Smith Issar
Published: March 2003

The role of iron in mycobacteria as in other bacteria goes beyond the need for this essential cofactor. Limitation of this metal triggers an extensive response aimed at increasing iron acquisition while coping with iron deficiency. In contrast, iron‐rich environments prompt these prokaryotes to induce synthesis of iron storage molecules and to increase mechanisms of protection against iron‐mediated oxidative damage. The response to changes in iron availability is strictly regulated in order to maintain sufficient but not excessive and potentially toxic levels of iron in the cell. This response is also linked to other important processes such as protection against oxidative stress and virulence. In bacteria, iron metabolism is regulated by controlling transcription of genes involved in iron uptake, transport and storage. In mycobacteria, this role is fulfilled by the iron‐dependent regulator IdeR. IdeR is an essential protein in Mycobacterium tuberculosis, the causative agent of human tuberculosis. It functions as a repressor of iron acquisition genes, but is also an activator of iron storage genes and a positive regulator of oxidative stress responses.
Candida albicans binds human plasminogen: identification of eight plasminogen‐binding proteins
Authors: Crowe Jonathan D. , Sievwright Isla K. , Auld Gillian C. , Moore Norma R. , Gow Neil A. R. , Booth Nuala A.
Published: March 2003

Several microbial pathogens augment their invasive potential by binding and activating human plasminogen to generate the proteolytic enzyme plasmin. Yeast cells and cell wall proteins (CWP) of the human pathogenic fungus Candida albicans bound plasminogen with a Kd of 70 ± 11 nM and 112 ± 20 nM respectively. Bound plasminogen could be activated to plasmin by mammalian plasminogen activators; no C. albicans plasminogen activator was detected. Binding of plasminogen to CWP and whole cells was inhibited by ɛACA, indicating that binding was predominantly to lysine residues. Candida albicans mutant strains defective in protein glycosylation did not show altered plasminogen binding, suggesting that binding was not mediated via a surface lectin. Binding was sensitive to digestion by basic carboxypeptidase, implicating C‐terminal lysine residues in binding. Proteomic analysis identified eight major plasminogen‐binding proteins in isolated CWP. Five of these (phosphoglycerate mutase, alcohol dehydrogenase, thioredoxin peroxidase, catalase, transcription elongation factor) had C‐terminal lysine residues and three (glyceraldehyde‐3‐phosphate dehydrogenase, phosphoglycerate kinase and fructose bisphosphate aldolase) did not. Activation of plasminogen could potentially increase the capacity of this pathogenic fungus for tissue invasion and necrosis. Although surface‐bound plasmin(ogen) degraded fibrin, no direct evidence for a role in invasion of endothelial matrix or in penetration and damage of endothelial cells was found.
The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungusMagnaporthe grisea
Authors: Wang Zheng‐Yi , Thornton Christopher R. , Kershaw Michael J. , Debao Li , Talbot Nicholas J.
Published: March 2003

We describe the isolation and characterization ofICL1from the rice blast fungusMagnaporthe grisea, a gene that encodes isocitrate lyase, one of the principal enzymes of the glyoxylate cycle.ICL1shows elevated expression during development of infection structures and cuticle penetration, and a targeted gene replacement showed that the gene is required for full virulence byM. grisea. In particular, we found that the prepenetration stage of development, before entry into plant tissue, is affected by loss of the glyoxylate cycle. There is a delay in germination, infection‐related development and cuticle penetration in Δicl1mutants. Recent reports have shown the importance of the glyoxylate cycle in the virulence of the human pathogenic fungusCandida albicansand the bacterial pathogenMycobacterium tuberculosis. Our results indicate that the glyoxylate cycle is also important in this plant pathogenic fungus, demonstrating the widespread utility of the pathway in microbial pathogenesis.
On the adequate stimulus for rectal mechanoreception and perception: a study in cat and humans
Authors: Thewißen  , Rühl  , Enck
Published: March 2003

The adequate stimulus that is specific for both rectal mechanoreceptor excitation and rectal perception is still undefined. Using a visual analogue scale, healthy male volunteer subjects rated the intensity of the non‐noxious ‘pressure’ sensation evoked by slow balloon‐induced distensions of the rectum. In a parallel study, the responses of spinal afferents originating from intramural mechanoreceptors of the rectum to the same stimulus were recorded in decerebrate cats. Both receptor activity and sensation intensity were linearly related to the diameter of the rectum, which is in turn a linear function of the tangential length of the rectal wall. In contrast, both saturated when expressed as a function of intrarectal pressure or rectal wall tension. It is concluded that the perception associated with rectal distensions in the non‐noxious range is mediated by intramural mechanoreceptors that linearly encode tangential wall length, and that the underlying information is linearly transmitted throughout the CNS.
Reversed bowel segments for the treatment of short bowel syndrome: assessment of their minimal length in correlation with electromyographic pattern in the rat
Authors: Chabert  , Pellissier  , Eribon  , Roche
Published: March 2003

Reversal of a distal intestinal loop is a surgical therapy intended to cure rapid intestinal transit in short bowel syndrome. To be active, a reversed loop must present a retrograde propagation of electromyographic patterns and must not be so long as to cause total obstruction. The aim of the current study was to propose methods to calculate the minimal length of the intestinal reversed loop taking into consideration the two previous conditions. Intestinal electromyograms were recorded in 65 rats at short‐term (4 days after surgery) and ten rats at long‐term (50 days after surgery). Control rats demonstrated that the preprandial regular spiking activity (RSA) of the migrating myoelectrical complex (MMC) extended simultaneously a definite part of the intestine which corresponds to the minimal length to reverse. A similar result can be obtained from a trigonometric representation. Whatever the method, the minimal lengths allowing the recording of RSA decreased along the rat intestine from 6 cm (proximal jejunum) to 4 cm (distal ileum). The experiments demonstrated that shorter loops did not present the preprandial RSA. In conclusion, the minimal reversed length depends on intestinal electromyographic parameters and, thereby, on the intestinal level.
Synaptic transmission induces transient Ca2+ concentration changes in cultured myenteric neurones
Authors: Berghe P. Vanden , Tack  , Coulie  , Andrioli  , Bellon  , Janssens
Published: March 2003

The enteric nervous system controls most of the gastrointestinal functions. We applied confocal microscopy and the Ca2+ indicator Fluo‐3 as an optical approach to study synaptic activation in cultures of myenteric neurones. The optical recording of [Ca2+]i (the intracellular Ca2+ concentration) was used to monitor activation, since [Ca2+]i is crucial in the coupling between neuronal excitation and the activation of several intracellular events. Extracellular fibre tract stimulation (2 s, 30 Hz) caused a transient [Ca2+]i rise in a subset of neurones (50%). These transients lasted for 5.2 s (n=36), with an average amplitude of 3.4 ± 1.3 times the basal concentration. The removal of extracellular Ca2+ (n=15) or the application of 10–6M tetrodotoxin (n=16) blocked this response. The N‐type Ca2+‐channel blocker ω‐conotoxin (5 × 10 –7M) abolished the [Ca2+]i increase, while blockade of L‐type and P/Q type Ca2+ channels had no effect. Single stimuli evoked a [Ca2+]i rise in the processes. ω‐conotoxin‐sensitive postsynaptic events required repetitive stimulation. Cholinergic blockade did not inhibit the [Ca2+]i rise in all neurones, suggesting that, besides acetylcholine, other neurotransmitters are involved. Optical imaging of [Ca2+]i can be used to study synaptic spread of activation in enteric neuronal circuits expressed in culture.