bluuebunny/crossref_metadata_2025 huggingface dataset:
GUP1 and its close homologue GUP2, encoding multimembrane‐spanning proteins involved in active glycerol uptake in Saccharomyces cerevisiae
Authors: Holst Bjørn , Lunde Christina , Lages Fernanda , Oliveira Rui , Lucas Cândida , Kielland‐Brandt Morten C.
Published: March 2003

Many yeast species can utilize glycerol, both as a sole carbon source and as an osmolyte. In Saccharomyces cerevisiae, physiological studies have previously shown the presence of an active uptake system driven by electrogenic proton symport. We have used transposon mutagenesis to isolate mutants affected in the transport of glycerol into the cell. Here we present the identification of YGL084c, encoding a multimembrane‐spanning protein, as being essential for proton symport of glycerol into S. cerevisiae. The gene is named GUP1 (glycerol uptake) and, for growth on glycerol, is important as a carbon and energy source. In addition, in strains deficient in glycerol production it also provides osmotic protection by the addition of glycerol. Another open reading frame (ORF), YPL189w, presenting a high degree of homology to YGL084c, similarly appears to be involved in active glycerol uptake in salt‐containing glucose‐based media in strains deficient in glycerol production. Analogously, this gene is named GUP2. To our knowledge, this is the first report on a gene product involved in active transport of glycerol in yeasts. Mutations with the same phenotypes occurred in two other ORFs of previously unknown function, YDL074c and YPL180w.
The universal stress protein A of Escherichia coli is required for resistance to DNA damaging agents and is regulated by a RecA/FtsK‐dependent regulatory pathway
Authors: Diez Alfredo , Gustavsson Niklas , Nyström Thomas
Published: March 2003

The link between cell division defects and the induction of the universal stress response is demonstrated to operate via the RecA regulator of the SOS response. An insertion in the cell division gene ftsK upregulates uspA in a recA‐dependent manner. Unlike true SOS response genes, this upregulation only occurs in growth‐arrested cells and is LexA independent. Thus, besides ppGpp‐dependent starvation signals, DNA aberrations transduce RecA‐dependent signals to the uspA promoter, which only affect the promoter during stasis. Further, we show that ftsK itself, like uspA, is induced in stationary phase and that this induction requires the stringent control modulon rather than activated RecA. Thus, ftsK, like uspA, is regulated by at least two global regulators: ppGpp of the stringent control network and RecA of the SOS modulon. We suggest that UspA is a new bona fide member of the RecA‐dependent DNA protection and repair system, as mutants lacking functional UspA were found to be sensitive to UV irradiation and mitomycin C exposure. Moreover, the UV sensitivity of uspA mutants is enhanced in an additive manner by the ftsK1 mutation.
Site‐specific DNA binding and bending by the Borrelia burgdorferi Hbb protein
Authors: Kobryn Kerri , Naigamwalla Darius Z. , Chaconas George
Published: March 2003

The Borrelia burgdorferi Hbb protein shows sequence similarity to members of the Escherichia coli HU/integration host factor (IHF) family of DNA accessory factors. We have overexpressed the hbb gene product in E. coli and purified the protein to near homogeneity. Biochemical analyses have revealed that Hbb has unique properties and is neither a strict HU nor IHF analogue. Hbb was found to bind specifically to a site in the putative origin of DNA replication between dnaA and dnaN. DNA footprinting studies have shown that this site is unrelated to the consensus sequence recognized by IHF proteins. Hbb induces a dramatic bend (> 126°) at this site and was also shown to restrain negative supercoils efficiently upon DNA binding. These features of the protein suggest that Hbb may act as a DNA accessory factor that facilitates the assembly of higher order protein–DNA complexes, such as those involved in DNA replication, transcription, recombination, packaging and perhaps other DNA metabolic processes unique to Borrelia.
Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization
Authors: Graf J. , Ruby E. G.
Published: March 2003

Vibrio fischeri is the sole species colonizing the light‐emitting organ of the Hawaiian squid, Euprymna scolopes. Upon entering the nascent light organ of a newly hatched juvenile squid, the bacteria undergo morphological and physiological changes that include the loss of flagellation and the induction of bioluminescence. These and other events reveal a pattern of genetic regulation that is a response to the colonization of host tissue. In this study, we isolated and characterized a glnD::mTn5Cm mutant of V. fischeri. In addition to the predicted defects in the efficiency of nitrogen utilization, this glnD mutant had an unexpected reduction in the ability to produce siderophore and grow under iron‐limiting conditions. Although the glnD mutant could colonize juvenile squid normally over the first 24 h, it was subsequently unable to persist in the light organ to the usual extent. This persistence phenotype was more severe if the mutant was pregrown under iron‐limiting conditions before inoculation, but could be ameliorated by the presence of excess iron. These results indicate that the ability to respond to iron limitation may be an important requirement in the developing symbiosis. Supplying the glnD gene in trans restored normal efficiency of nitrogen use, iron sequestration and colonization phenotypes to the glnD::mTn5Cm mutant; thus, there appears to be a genetic and/or metabolic linkage between nitrogen sensing, siderophore synthesis and symbiosis competence in V. fischeri that involves the glnD gene.
Role of the Gp16 lytic transglycosylase motif in bacteriophage T7 virions at the initiation of infection
Authors: Moak Michael , Molineux Ian J.
Published: March 2003

The predicted catalytic glutamate residue for transglycosylase activity of bacteriophage T7 gp16 is not essential for phage growth, but is shown to be beneficial during infection of Escherichia coli cells grown to high cell density, conditions in which murein is more highly cross‐linked. In the absence of the putative transglycosylase, internalization of the phage genome is significantly delayed during infection. The lytic transglycosylase motif of gp16 is essential for phage growth at temperatures below 20°C, indicating that these growth conditions also lead to increased cross‐linking of peptidoglycan. Overexpression of sltY, E. coli soluble lytic transglycosylase, partially complements the defect in infection of mutant phage particles, allowing them to infect at higher efficiencies. Conversely, an sltY deletion increases the latent period of wild‐type phage.
Membrane fusion activity of purified SipB, a Salmonella surface protein essential for mammalian cell invasion
Authors: Hayward Richard D. , McGhie Emma J. , Koronakis Vassilis
Published: March 2003

An early event in Salmonella infection is the invasion of non‐phagocytic intestinal epithelial cells. The pathogen is taken up by macropinocytosis, induced by contact‐dependent delivery of bacterial proteins that subvert signalling pathways and promote cytoskeletal rearrangement. SipB, a Salmonella protein required for delivery and invasion, was shown to localize to the cell surface of bacteria invading mammalian target cells and to fractionate with outer membrane proteins. To investigate the properties of SipB, we purified the native full‐length protein following expression in recombinant Escherichia coli. Purified SipB assembled into hexamers via an N‐terminal protease‐resistant domain predicted to form a trimeric coiled coil, reminiscent of viral envelope proteins that direct homotypic membrane fusion. The SipB protein integrated into both mammalian cell membranes and phospholipid vesicles without disturbing bilayer integrity, and it induced liposomal fusion that was optimal at neutral pH and influenced by membrane lipid composition. SipB directed heterotypic fusion, allowing delivery of contents from E. coli‐derived liposomes into the cytosol of living mammalian cells.
Biosynthesis of the anti‐parasitic agent megalomicin: transformation of erythromycin to megalomicin in Saccharopolyspora erythraea
Authors: Volchegursky Yanina , Hu Zhihao , Katz Leonard , McDaniel Robert
Published: March 2003

Megalomicin is a therapeutically diverse compound which possesses antiparasitic, antiviral and antibacterial properties. It is produced by Micromonospora megalomicea and differs from the well‐known macrolide antibiotic erythromycin by the addition of a unique deoxyamino sugar, megosamine, to the C‐6 hydroxyl. We have cloned and sequenced a 48 kb segment of the megalomicin (meg) biosynthetic gene cluster which contains the modular polyketide synthase (PKS) and the complete pathway for megosamine biosynthesis. The similarities and distinctions between the related megalomicin and erythromycin gene clusters are discussed. Heterologous expression of the megalomicin PKS in Streptomyces lividans led to production of 6‐deoxyerythronolide B, the same macrolactone intermediate for erythromycin. A 12 kb fragment harbouring the putative megosamine pathway was expressed in Saccharopolyspora erythraea, resulting in the conversion of erythromycin to megalomicin. Considering the extensive knowledge surrounding the genetic engineering of the erythromycin PKS and the familiarity with genetic manipulation and fermentation of S. erythraea, the ability to produce megalomicin in this strain should allow the engineering of novel megalomicin analogues with potentially improved therapeutic activities.
Electrophysiological evidence for heptameric stoichiometry of ion channels formed by Staphylococcus aureus alpha‐toxin in planar lipid bilayers
Authors: Krasilnikov Oleg V. , Merzlyak Petr G. , Yuldasheva Liliya N. , Rodrigues Cláudio G. , Bhakdi Sucharit , Valeva Angela
Published: March 2003

Staphylococcal alpha‐toxin forms homo‐oligomeric channels in lipid bilayers and cell membranes. Here, we report that electrophysiological monitoring of single‐channel function using a derivatized cysteine substitution mutant allows accurate determination of the subunit stoichiometry of the oligomer in situ. The electrophysiological phenotype of channels formed in planar lipid bilayers with the cysteine replacement mutant I7C is equal to that of the wild type. When pores were formed with I7C, alterations of several channel properties were observed upon modification with SH reagents. Decreases in conductance then occurred that were seen only as negative voltage was applied. At the level of single channels, these were manifest as stepwise changes in conductance, each step most probably reflecting modification of a single SH group within the oligomer. Because seven steps were observed, the functional channel formed by alpha‐toxin in planar lipid membranes is a heptamer.
Helicobacter pylori induces but survives the extracellular release of oxygen radicals from professional phagocytes using its catalase activity
Authors: Ramarao Nalini , Gray‐Owen Scott D. , Meyer Thomas F.
Published: March 2003

Helicobacter pylori can colonize the gastric epithelium of humans, leading to the induction of an intense inflammatory response with the infiltration of mainly polymorphonuclear leucocytes (PMNs) and monocytes. These professional phagocytes appear to be a primary cause of the damage to surface epithelial layers, and probably contribute to the pathogenesis associated with persistent H. pylori infections. We have shown previously that H. pylori adheres to professional phagocytes, but is not engulfed efficiently, suggesting an antiphagocytic escape mechanism that is dependent on the pathogen's type IV secretion system. Here, we show that H. pylori induces the generation and extracellular release of oxygen metabolites as a consequence of its attachment to phagocytic cells, but is capable of surviving this response. The catalase activity of H. pylori is apparently essential for survival at the phagocytes' cell surface. Opsonization of H. pylori leads to an increased burst, and the inhibition of bacterial protein synthesis to a decreased one. Ca2+ concentration, cytoskeleton rearrangement and protein kinase C (PKC) are involved in the H. pylori‐induced oxidative burst in both monocytes and PMNs. This survival phenomenon has important implications for both the persistence of this important pathogen and the host tissue damage that accompanies persistent H. pylori infection.
Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species
Authors: Triglia Tony , Healer Julie , Caruana Sonia R. , Hodder Anthony N. , Anders Robin F. , Crabb Brendan S. , Cowman Alan F.
Published: March 2003

Apical membrane antigen 1 (AMA1) is an asexual blood‐stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using ‘knock‐out’ plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood‐stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans‐species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.
Proton motive force drives the interaction of the inner membrane TolA and outer membrane Pal proteins in Escherichia coli
Authors: Cascales Eric , Gavioli Marthe , Sturgis James N. , Lloubès Roland
Published: March 2003

The Tol–Pal system of the Escherichia coli envelope is formed from the inner membrane TolQ, TolR and TolA proteins, the periplasmic TolB protein and the outer membrane Pal lipoprotein. Any defect in the Tol–Pal proteins or in the major lipoprotein (Lpp) results in the loss of outer membrane integrity giving hypersensitivity to drugs and detergents, periplasmic leakage and outer membrane vesicle formation. We found that multicopy plasmid overproduction of TolA was able to complement the membrane defects of an lpp strain but not those of a pal strain. This result indicated that overproduced TolA has an envelope‐stabilizing effect when Pal is present. We demonstrate that Pal and TolA formed a complex using in vivo cross‐linking and immunoprecipitation experiments. These results, together with in vitro experiments with purified Pal and TolA derivatives, allowed us to show that Pal interacts with the TolA C‐terminal domain. We also demonstrate using protonophore, K+ carrier valinomycin, nigericin, arsenate and fermentative conditions that the proton motive force was coupled to this interaction.
Life in protein‐rich environments: the relA‐independent response of Streptococcus pyogenes to amino acid starvation
Authors: Steiner Kerstin , Malke Horst
Published: March 2003

Considering that group A streptococci are multiple auxotrophs that may encounter shortage of amino acids during specific stages of the infectious process, we studied their adaptive response to amino acid deprivation. We found that, in addition to the (p)ppGpp‐mediated stringent response characterized previously, Streptococcus pyogenes exhibits a relA‐independent response comprising transcriptional modulation of a specific subset of genes involved in pathogenesis. Genes/operons transcriptionally upregulated during starvation of both wild type and relA mutants included the two‐component signal transduction system covRS, the positive regulator (ropB) of the pyrogenic exotoxin B gene, speB, the oligopeptide (opp) and dipeptide (dpp) permease systems and the pepB gene putatively involved in the intracellular processing of oligopeptides. Upregulation of covRS was accompanied by downregulation of ska, one of the target genes of the negative CovR regulator, and the net effect of amino acid starvation also favoured repression of speB. A significant feature of upregulated opp expression was stimulated readthrough transcription of the operon‐internal oppA terminator, leading to increased mRNA levels for synthesis of the translocator complex relative to the substrate‐binding protein. Based on these and previous results, a stimulus–response network is proposed that counteracts the stringent response and may enable the pathogen to mount a dynamic response to the protein‐rich environment provided by its human host.
Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth
Authors: Watarai Masahisa , Andrews Helene L. , Isberg Ralph R.
Published: March 2003

Legionella pneumophila grows in human alveolar macrophages and resides within a phagosome that initially lacks proteins associated with the endocytic pathway. Required for targeting to this unique location is the Dot/Icm complex, which is highly similar to conjugative DNA transfer apparatuses. Here, we show that exposure to three distinct inducing conditions resulted in the formation of a fibrous structure on the bacterial cell surface that contained the DotH and DotO proteins. These conditions included: (i) incubation for 2 h with mouse bone marrow‐derived macrophages; (ii) incubation for 2 h in macrophage‐conditioned media; or (iii) replication of bacteria for 22 h within macrophages. Introduction of bacteria harbouring the surface‐exposed DotH and DotO onto a fresh monolayer resulted in loss of the surface localization of DotH and DotO shortly after uptake. Treatments that resulted in the production of the fibrous structure enhanced the rate at which the bacteria were internalized, but there was no corresponding increase in the efficiency of intracellular growth compared with bacteria that had been cultured in broth using conditions that resulted in maximal intracellular growth. These data indicate that the surface‐exposed DotH and DotO on L. pneumophila may act either just before lysis from the macrophage or at the earliest stages of infection, transiently relocating in a fibrous structure on the bacterial cell surface.
Identification of functional regions within invasion plasmid antigen C (IpaC) of Shigella flexneri
Authors: Picking Wendy L. , Coye Lisette , Osiecki John C. , Serfis Alexa Barnoski , Schaper Elizabeth , Picking William D.
Published: March 2003

Shigella flexneri causes bacillary dysentery with symptoms resulting from the inflammation that accompanies bacterial entry into the cells of the colonic epithelium. The effectors of S. flexneri invasion are the Ipa proteins, particularly IpaB and IpaC, which are secreted at the host–pathogen interface following bacterial contact with a host cell. Of the purified Ipa proteins, only IpaC has been shown to possess quantifiable in vitro activities that are related to cellular invasion. In this study, ipaC deletion mutants were generated to identify functional regions within the IpaC protein. From these data, we now know that the N‐terminus and an immunogenic central region are not required for IpaC‐dependent enhancement of cellular invasion by S. flexneri. However, to restore invasiveness to an ipaC null mutant of S. flexneri, the N‐terminus is essential, because IpaC mutants lacking the N‐terminus are not secreted by the bacterium. Deletion of the central hydrophobic region eliminates IpaC's ability to interact with phospholipid membranes, and fusion of this region to a modified form of green fluorescent protein converts it into an efficient membrane‐associating protein. Meanwhile, deletion of the C‐terminus eliminates the mutant protein's ability to establish protein–protein contacts with full‐length IpaC. Interestingly, the mutant form of ipaC that restores partial invasiveness to the S. flexneri ipaC null mutant also restores full contact‐mediated haemolysis activity to this bacterium. These data support a model in which IpaC possesses a distinct functional organization that is important for bacterial invasion. This information will be important in defining the precise role of IpaC in S. flexneri pathogenesis and in exploring the potential effects of purified IpaC at mucosal surfaces.
Identification and characterization of spdR mutations that bypass the BsgA protease‐dependent regulation of developmental gene expression in Myxococcus xanthus
Authors: Hager Elizabeth , Tse Hubert , Gill Ronald E.
Published: March 2003

The BsgA protease of Myxococcus xanthus is an intracellular protease closely related to the Lon protease of Escherichia coli. BsgA is required for normal levels of developmentally induced gene expression. In this report, we describe the identification of mutations that suppress the developmental defect of bsgA mutants. These mutations localized to the spdR gene (suppressor protease deficiency regulator) that appears to play a role in the regulation of early developmental gene expression. Mutations in spdR fully restored the ability of a bsgA mutant to form fruiting bodies and spores and, with one exception, restored the expression of several development‐specific lacZ fusions. spdR mutants exhibited characteristic phenotypic properties including increased expression of the development‐specific tps gene during vegetative growth, formation of fruiting bodies and spores on semi‐rich nutrient medium and completion of starvation‐induced development in a shorter time period than wild‐type strains. The spdR locus was cloned and sequenced and found to encode a member of the NtrC family of two‐component transcriptional regulators. One interpretation of these data is that SpdR acts, directly or otherwise, to regulate developmental gene expression negatively and that the BsgA protease is required to relieve this inhibitory effect at the onset of development. However, Western immunoblot analysis indicated that SpdR is present at a relatively constant level during growth and early development in both wild‐type and BsgA protease‐deficient cells. This finding suggests that BsgA does not function to degrade SpdR at the onset of development.
Osmotic stress causes a G1 cell cycle delay and downregulation of Cln3/Cdc28 activity in Saccharomyces cerevisiae
Authors: Bellí Gemma , Garí Eloi , Aldea Martí , Herrero Enrique
Published: March 2003

Moderate hyperosmotic stress on Saccharomyces cerevisiae cells produces a temporary delay at the G1 stage of the cell cycle. This is accompanied by transitory downregulation of CLN1, CLN2 and CLB5 transcript levels, although not of CLN3, which codes for the most upstream activator of the G1/S transition. Osmotic shock to cells synchronized in early G1, when Cln3 is the only cyclin present, causes a delay in cell cycle resumption. This points to Cln3 as being a key cell cycle target for osmotic stress. We have observed that osmotic shock causes downregulation of the kinase activity of Cln3–Cdc28 complexes. This is concomitant with a temporary accumulation of Cln3 protein as a result of increased stability. The effects of the osmotic stress in G1 are not suppressed in CLN3‐1 cells with increased kinase activity, as the Cln3–Cdc28 activity in this mutant is still affected by the shock. Although Hog1 is not required for the observed cell cycle arrest in hyperosmotic conditions, it is necessary to resume the cell cycle at KCl concentrations higher than 0.4 M.
Type IV secretion: intercellular transfer of macromolecules by systems ancestrally related to conjugation machines
Authors: Christie Peter J.
Published: March 2003

Bacterial conjugation systems are highly promiscuous macromolecular transfer systems that impact human health significantly. In clinical settings, conjugation is exceptionally problematic, leading to the rapid dissemination of antibiotic resistance genes and other virulence traits among bacterial populations. Recent work has shown that several pathogens of plants and mammals –Agrobacterium tumefaciens, Bordetella pertussis, Helicobacter pylori and Legionella pneumophila– have evolved secretion pathways ancestrally related to conjugation systems for the purpose of delivering effector molecules to eukaryotic target cells. Each of these systems exports distinct DNA or protein substrates to effect a myriad of changes in host cell physiology during infection. Collectively, secretion pathways ancestrally related to bacterial conjugation systems are now referred to as the type IV secretion family. The list of putative type IV family members is increasing rapidly, suggesting that macromolecular transfer by these systems is a widespread phenomenon in nature.
CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli
Authors: Yamanaka Kunitoshi , Zheng Weidong , Crooke Elliott , Wang Yuh‐Hwa , Inouye Masayori
Published: March 2003

CspD is a stationary phase‐induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of β‐strands, and the purified protein exists exclusively as a dimer and binds to single‐stranded (ss)DNA and RNA in a dose‐dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB‐coated DNA. We propose that CspD dimers, with two independent β‐sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient‐depleted cells.
Large‐scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid‐associated protein, H‐NS
Authors: Hommais Florence , Krin Evelyne , Laurent‐Winter Christine , Soutourina Olga , Malpertuy Alain , Le Caer Jean‐Pierre , Danchin Antoine , Bertin Philippe
Published: March 2003

Despite many years of intense work investigating the function of nucleoid‐associated proteins in prokaryotes, their role in bacterial physiology remains largely unknown. The two‐dimensional protein patterns were compared and expression profiling was carried out on H‐NS‐deficient and wild‐type strains of Escherichia coli K‐12. The expression of approximately 5% of the genes and/or the accumulation of their protein was directly or indirectly altered in the hns mutant strain. About one‐fifth of these genes encode proteins that are involved in transcription or translation and one‐third are known to or were in silico predicted to encode cell envelope components or proteins that are usually involved in bacterial adaptation to changes in environmental conditions. The increased expression of several genes in the mutant resulted in a better ability of this strain to survive at low pH and high osmolarity than the wild‐type strain. In particular, the putative regulator, YhiX, plays a central role in the H‐NS control of genes required in the glutamate‐dependent acid stress response. These results suggest that there is a strong relationship between the H‐NS regulon and the maintenance of intracellular homeostasis.
Escherichia coli P fimbriae utilize the Toll‐like receptor 4 pathway for cell activation
Authors: Frendéus Björn , Wachtler Caroline , Hedlund Maria , Fischer Hans , Samuelsson Patrik , Svensson Majlis , Svanborg Catharina
Published: March 2003

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae‐dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll‐like receptor 4 (TLR4)‐dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS‐CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co‐staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co‐localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4‐dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co‐receptors.
A ComGA‐dependent checkpoint limits growth during the escape from competence
Authors: Haijema B.‐J. , Hahn J. , Haynes J. , Dubnau D.
Published: March 2003

In Bacillus subtilis, competence for transformation develops in 5–10% of the cells in a stationary phase culture. These cells exhibit a prolonged lag in the resumption of growth and cell division during the escape from competence. To better understand the basis of this lag, we have characterized competent cultures microscopically. To distinguish the minority of competent cells, a translational fusion between ComK, the competence transcription factor, and the green fluorescent protein (GFP) was used as a marker. Only 5–10% of the cells in a competent culture were fluorescent, indicating that ComK synthesis is an all or nothing event. To validate the identification of competent cells, we demonstrated the coincident expression of comEA, a late competence gene, and comK‐gfp. Competent cells resemble stationary phase cells; the majority are single (not in chains), contain single nucleoids, and rarely contain FtsZ rings. Upon dilution into fresh medium, competent cells maintain this appearance for about 2 h. In contrast, the majority of non‐competent cells rapidly resume growth, exhibiting chaining, nuclear division and FtsZ‐ring formation. The late competence protein ComGA is required for the competence‐related block in chromosome replication and cell division. In the competent cells of a comGA mutant culture, chromosomal replication and FtsZ‐ring formation were no longer blocked, although competent comGA mutant cells were abnormal in appearance. It is likely that one role for ComGA is to prevent growth, chromosome replication and cell division until ComK can be eliminated by degradation. A mutation in the ATP‐binding site of comGA inactivated the protein for transformation but did not prevent it from inhibiting DNA replication and cell division. The buoyant density difference between competent and non‐competent cells depends on the competence‐specific growth arrest.
C‐signal: a cell surface‐associated morphogen that induces and co‐ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus
Authors: Kruse Thomas , Lobedanz Sune , Berthelsen Nils M. S. , Søgaard‐Andersen Lotte
Published: March 2003

In Myxococcus xanthus, morphogenesis of multicellular fruiting bodies and sporulation are co‐ordinated temporally and spatially. csgA mutants fail to synthesize the cell surface‐associated C‐signal and are unable to aggregate and sporulate. We report that csgA encodes two proteins, a 25 kDa species corresponding to full‐length CsgA protein and a 17 kDa species similar in size to C‐factor protein, which has been shown previously to have C‐signal activity. By systematically varying the accumulation of the csgA proteins, we show that overproduction of the csgA proteins results in premature aggregation and sporulation, uncoupling of the two events and the formation of small fruiting bodies, whereas reduced synthesis of the csgA proteins causes delayed aggregation, reduced sporulation and the formation of large fruiting bodies. These results show that C‐signal induces aggregation as well as sporulation, and that an ordered increase in the level of C‐signalling during development is essential for the spatial co‐ordination of these events. The results support a quantitative model, in which aggregation and sporulation are induced at distinct threshold levels of C‐signalling. In this model, the two events are temporally co‐ordinated by the regulated increase in C‐signalling levels during development. The contact‐dependent C‐signal transmission mechanism allows the spatial co‐ordination of aggregation and sporulation by coupling cell position and signalling levels.
Genome quality control: RIP (repeat‐induced point mutation) comes to Podospora
Authors: Graïa Fatima , Lespinet Olivier , Rimbault Blandine , Dequard‐Chablat Michelle , Coppin Evelyne , Picard Marguerite
Published: March 2003

RIP (repeat‐induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C‐G to T‐A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP‐like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating‐type region. The 9 kb sequenced show 106 C‐G to T‐A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP‐like event extended to single‐copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C‐methylation. Interestingly, the N. crassa zeta–eta sequence, which acts as a potent de novo C‐methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.
Acquirement of cold sensitivity by quadruple deletion of the cspA family and its suppression by PNPase S1 domain in Escherichia coli
Authors: Xia Bing , Ke Haiping , Inouye Masayori
Published: March 2003

Escherichia coli contains a large CspA family, CspA to CspI. Here, we demonstrate that E. coli is highly protected against cold‐shock stress, as these CspA homologues existed at approximately a total of two million molecules per cell at low temperature and growth defect was not observed until four csp genes (cspA, cspB, cspE and cspG) were deleted. The quadruple‐deletion strain acquired cold sensitivity and formed filamentous cells at 15°C although chromosomes were normally segregated. The cold‐sensitivity and filamentation phenotypes were suppressed by all members of the CspA family except for CspD, which causes lethality upon overexpression. Interestingly, the cold sensitivity of the mutant was also suppressed by the S1 domain of polynucleotide phosphorylase (PNPase), which also folds into a β‐barrel structure similar to that of CspA. The present results show that cold‐shock proteins and S1 domains share not only the tertiary structural similarity but also common functional properties, suggesting that these seemingly distinct protein categories may have evolved from a common primordial RNA‐binding protein.
The adhesive property of the type IV pilus‐associated component PilC1 of pathogenic Neisseria is supported by the conformational structure of the N‐terminal part of the molecule
Authors: Morand Philippe C. , Tattevin Pierre , Eugene Emmanuel , Beretti Jean‐Luc , Nassif Xavier
Published: March 2003

Neisserial PilC proteins are key elements in type IV pili biogenesis and adhesion. Two pilC alleles are usually present in Neisseria meningitidis. At least one of the PilC proteins is required for pilus assembly and competence for transformation. In addition, meningococcal PilC1, but not PilC2, modulates adhesiveness, whereas, in N. gonorrhoeae, both alleles are adhesive. The meningococcal pilC genes are differently regulated, and it was shown that the expression of pilC1, but not that of pilC2, is transiently induced by bacteria–cell contact. The aim of this work was to determine whether, besides regulation, PilC1‐mediated adhesion was conferred by some specific protein pattern not present in the meningococcal PilC2 protein. We demonstrate first that differences within the primary sequence of the meningococcal PilC1 and PilC2 are responsible for different adhesion phenotypes, thus eliminating the regulation of transcription being solely responsible for the adhesive phenotype of PilC1. To identify the regions of PilC1 responsible for adhesion, we engineered meningococcal strains expressing various PilC1–PilC2 hybrids at the pilC1 locus. Our data demonstrate that the specific PilC1 adhesion‐promoting regions are located in the amino‐terminal part of the molecule and that several domains within this region probably interact with each other to promote adhesion to human cells.
The recX gene potentiates homologous recombination in Neisseria gonorrhoeae
Authors: Stohl Elizabeth A. , Seifert H. Steven
Published: March 2003

In the pathogen Neisseria gonorrhoeae (Gc), the RecA protein is necessary for DNA repair, DNA transformation and pilus antigenic variation. Many bacteria contain a gene, recX, which has been suggested to downregulate recA through an unknown mechanism. To investigate the possible role of recX in Gc, we cloned and insertionally inactivated the recX gene. The recX loss‐of‐function mutant showed decreases in pilus phase variation, DNA transformation and DNA repair ability compared with wild type. We were able to complement all these deficiencies by supplying a functional copy of recX elsewhere in the chromosome. The recX mutant still showed increases in pilus phase variation under conditions of iron starvation, and the recX mutant showed levels of RecA protein equivalent to wild type. Although the precise role of recX in recombination remains unclear, RecX aids all RecA‐related processes in Gc, and this is the first demonstration of a role for recX in homologous recombination in any organism.
Pleiotropic transcriptional repressor CodY senses the intracellular pool of branched‐chain amino acids in Lactococcus lactis
Authors: Guédon Eric , Serror Pascale , Ehrlich S. Dusko , Renault Pierre , Delorme Christine
Published: March 2003

Proteolysis is essential for supplying Lactococcus lactis with amino acids during growth in milk. Expression of the major components of the L. lactis proteolytic system, including the cell wall proteinase (PrtP), the oligopeptide transport system (Opp) and at least four intracellular peptidases (PepO1, PepN, PepC, PepDA2), was shown previously to be controlled negatively by a rich nitrogen source. The transcription of prtP, opp–pepO1, pepN and pepC genes is regulated by dipeptides in the medium. Random insertion mutants derepressed for nitrogen control in the expression of the oligopeptide transport system were isolated using an opp–lacZ fusion. A third of the mutants were targeted in the same locus. The product of the inactivated gene shared 48% identity with CodY from Bacillus subtilis, a pleiotropic repressor of the dipeptide permease operon (dpp) and several genes including genes involved in amino acid degradation and competence induction. The signal controlling CodY‐dependent repression was searched for by analysing the response of the opp–lux fusion to the addition of 67 dipeptides with different amino acid compositions. Full correlation was found between the dipeptide content in branched‐chain amino acids (BCAA; isoleucine, leucine or valine) and their ability to mediate the repression of opp–pepO1 expression. The repressive effect resulting from specific regulatory dipeptides was abolished in L. lactis mutants affected in terms of their transport or degradation into amino acids, showing that the signal was dependent on the BCAA pool in the cell. Lastly, the repression of opp–pepO1 expression was stronger in a mutant unable to degrade BCAAs, underlining the central role of BCAAs as a signal for CodY activity. This pattern of regulation suggests that, in L. lactis and possibly other Gram‐positive bacteria, CodY is a pleiotropic repressor sensing nutritional supply as a function of the BCAA pool in the cell.
Roles of trehalose phosphate synthase in yeast glycogen metabolism and sporulation
Authors: De Silva‐Udawatta Mihiri N. , Cannon John F.
Published: March 2003

Trehalose is a major storage carbohydrate in budding yeast, Saccharomyces cerevisiae. Alterations in trehalose synthesis affect carbon source‐dependent growth, accumulation of glycogen and sporulation. Trehalose is synthesized by trehalose phosphate synthase (TPS), which is a complex of at least four proteins. In this work, we show that the Tps1p subunit protein catalyses trehalose phosphate synthesis in the absence of other TPS components. The tps1–H223Y allele (glc6–1) that causes a semidominant decrease in glycogen accumulation exhibits greater enzyme activity than wild‐type TPS1 because, unlike the wild‐type enzyme, TPS activity in tps1–H223Y cells is not inhibited by phosphate. Poor sporulation in tps1 null diploids is caused by reduced expression of meiotic inducers encoded by IME1, IME2 and MCK1. Furthermore, high‐copy MCK1 or heterozygous hxk2 mutations can suppress the tps1 sporulation trait. These results suggest that the trehalose‐6‐phosphate inhibition of hexokinase activity is required for full induction of MCK1 in sporulating yeast cells.
Role of the response regulator RssB in σS recognition and initiation of σS proteolysis in Escherichia coli
Authors: Klauck Eberhard , Lingnau Maren , Hengge‐Aronis Regine
Published: March 2003

In growing Escherichia coli cells, the master regulator of the general stress response, σS (RpoS), is subject to rapid proteolysis. In response to stresses such as sudden carbon starvation, osmotic upshift or shift to acidic pH, σS degradation is inhibited, σS accumulates and numerous σS‐dependent genes with stress‐protective functions are activated. σS proteolysis is dependent on ClpXP protease and the response regulator RssB, whose phosphorylated form binds directly to σSin vitro. Here, we show that substitutions of aspartate 58 (D58) in RssB, which result in higher σS levels in vivo, produce RssB variants unable to bind σSin vitro. Thus, RssB is the direct substrate recognition factor in σS proteolysis, whose affinity for σS depends on phosphorylation of its D58 residue. RssB does not dimerize or oligomerize upon this phosphorylation and σS binding, and RssB and σS exhibit a 1:1 stoichiometry in the complex. The receiver as well as the output domain of RssB are required for σS binding (as shown in vivo and in vitro) and for complementation of an rssB null mutation. Thus, the N‐terminal receiver domain plays an active and positive role in RssB function. Finally, we demonstrate that RssB is not co‐degraded with σS, i.e. RssB has a catalytic role in the initiation of σS turnover. A model is presented that integrates the details of RssB–σS interaction, the RssB catalytic cycle and potential stress signal input in the control of σS proteolysis.
The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae
Authors: Wysocki Robert , Chéry Cyrille C. , Wawrzycka Donata , Van Hulle Marijn , Cornelis Rita , Thevelein Johan M. , Tamás Markus J.
Published: March 2003

The Saccharomyces cerevisiae FPS1 gene encodes a glycerol channel protein involved in osmoregulation. We present evidence that Fps1p mediates influx of the trivalent metalloids arsenite and antimonite in yeast. Deletion of FPS1 improves tolerance to arsenite and potassium antimonyl tartrate. Under high osmolarity conditions, when the Fps1p channel is closed, wild‐type cells show the same degree of As(III) and Sb(III) tolerance as the fps1Δ mutant. Additional deletion of FPS1 in mutants defective in arsenite and antimonite detoxification partially suppresses their hypersensitivity to metalloid salts. Cells expressing a constitutively open form of the Fps1p channel are highly sensitive to both arsenite and antimonite. We also show by direct transport assays that arsenite uptake is mediated by Fps1p. Yeast cells appear to control the Fps1p‐mediated pathway of metalloid uptake, as expression of the FPS1 gene is repressed upon As(III) and Sb(III) addition. To our knowledge, this is the first report describing a eukaryotic uptake mechanism for arsenite and antimonite and its involvement in metalloid tolerance.
Gelatinase biosynthesis‐activating pheromone: a peptide lactone that mediates a quorum sensing in Enterococcus faecalis
Authors: Nakayama Jiro , Cao Yong , Horii Takaaki , Sakuda Shohei , Akkermans Antoon D. L. , De Vos Willem M. , Nagasawa Hiromichi
Published: March 2003

Biosynthesis of gelatinase, a virulence factor of Enterococcus faecalis, was found to be regulated in a cell density‐dependent fashion in which its production is active in late log to early stationary phase. Addition of early stationary phase culture filtrate to medium shifted the onset of gelatinase production to that of mid‐log phase, suggesting that E. faecalis secretes a gelatinase biosynthesis‐activating pheromone (GBAP). GBAP was isolated from culture supernatant of E. faecalis OG1S‐P. Structural analysis suggested GBAP to be an 11‐residue cyclic peptide containing a lactone structure, in which the α‐carboxyl group of the C‐terminal amino acid is linked to a hydroxyl group of the serine of the third residue. A synthetic peptide possessing the deduced structure showed GBAP activity at nanomolar concentrations as did natural GBAP. Database searches revealed that GBAP corresponds to a C‐terminal part of a 242‐residue FsrB protein. Northern analysis showed that GBAP slowly induces the transcription of two operons, fsrB‐fsrC encoding FsrB and a putative histidine kinase FsrC and gelE‐sprE encoding gelatinase GelE and serine protease SprE. Strains with an insertion mutation in either fsrC or a putative response regulator gene fsrA failed to respond to GBAP, suggesting that the GBAP signal is transduced by a two‐component regulatory system.
The two‐component system ArlS–ArlR is a regulator of virulence gene expression in Staphylococcus aureus
Authors: Fournier Bénédicte , Klier André , Rapoport Georges
Published: March 2003

Staphylococcus aureus is a major human pathogen that produces many virulence factors in a temporally regulated manner controlled by at least two global virulence regulatory loci (agr and sarA). We identified previously a two‐component system, ArlS–ArlR, that modifies the activity of extracellular serine protease and may be involved in virulence regulation. Here, we show that mutations in either arlR or arlS increase the production of secreted proteins [α‐toxin (Hla), β‐haemolysin, lipase, coagulase, serine protease (Ssp)] and especially protein A (Spa). Furthermore, the pattern of proteins secreted by both mutants was strikingly different from that of the wild‐type strain. Transcriptional fusions showed that expression of hla, ssp and spa was higher in both mutants than in the wild‐type strain, indicating that the arl operon decreases the production of virulence factors by downregulating the transcription of their genes. The arl mutation did not change spa expression in an agrA mutant or in a sarA mutant, suggesting that both the sarA and the agr loci are required for the action of arl on spa. Northern blot analyses indicated that the arl mutation increased the synthesis of both RNA II and RNA III, but decreased sarA transcription. Finally, arl was not autoregulated, but its expression was stimulated by agr and sarA. These results suggest that the Arl system interacts with both agr and sarA regulatory loci to modulate the virulence regulation network.