bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Neurospora crassa gene responsible for the cut and ovc phenotypes encodes a protein of the haloacid dehalogenase family
Authors: Youssar Loubna , Schmidhauser Thomas J. , Avalos Javier 
Published: January 2005

SummaryLight stimulation of carotenogenesis in Neurospora crassa, mediated by the White Collar proteins, is enhanced in some regulatory mutants, such as vivid and ovc. The gene responsible for the vivid mutation has been identified, but not the one responsible for the ovc phenotype. The ovc mutant is sensitive to high osmotic conditions and allelic with another mutant, cut, also osmosensitive but not affected in carotenogenesis. A phenotypic characterization of both strains is presented. Light induction of mRNA levels of the carotenoid genes al‐1 and al‐2, the regulatory gene wc‐1 or the conidiation‐specific gene con‐10 is not significantly changed in the ovc mutant when compared with the wild type. We have identified the gene affected in the ovc mutant by complementation of osmosensitivity with a cosmid library. This gene, which we call cut‐1, codes for an enzyme of the haloacid dehalogenase family, which includes different classes of phosphatases. cut‐1 is able to restore the wild‐type phenotype of the ovc and cut strains, confirming that they are affected in the same gene. DNA sequence analysis identified a point mutation in the cut mutant, leading to a truncated protein. The ovc mutant represents a deletion encompassing the entire gene and surrounding sequences. The cut‐1 promoter contains putative regulatory elements involved in osmotic or thermal stress. We show that cut‐1 transcription is low in illuminated or dark‐grown cultures, and is induced by high osmotic conditions or by heat shock.
Degradation of the HilC and HilD regulator proteins by ATP‐dependent Lon protease leads to downregulation of Salmonella pathogenicity island 1 gene expression
Authors: Takaya Akiko , Kubota Yohsuke , Isogai Emiko , Yamamoto Tomoko 
Published: January 2005

Summary Salmonella pathogenicity island 1 (SPI1) enables infecting Salmonella to cross the small intestinal barrier and to escape phagocytosis by inducing apoptosis. Several environmental signals and transcriptional regulators modulate the expression of hilA, which encodes a protein playing a central role in the regulatory hierarchy of SPI1 gene expression. We have previously shown that Lon, a stress‐induced ATP‐dependent protease, is a negative regulator of hilA, suggesting that it targets factors required for activating hilA expression. To elucidate the mechanisms by which Lon protease negatively regulates SPI1 transcription, we looked for its substrate proteins. We found that HilC and HilD, which are positive regulators of hilA expression, accumulate in Lon‐depleted cells, and that the enhancement of SPI1 expression that occurs in a lon‐disrupted mutant is not observed in the lon hilC hilD triple null mutant. Furthermore, we demonstrated that the half‐lives of HilC and HilD are, respectively, about 12 times and three times longer in the Lon‐depleted mutant, than in the Lon+ cells, suggesting that Lon targets both of HilC and HilD. In view of these findings, we suggest that the regulation of SPI1 expression is negatively controlled through degradation of the HilC and HilD transcriptional regulators by Lon.
Contact with host cells induces a DNA repair system in pathogenic Neisseriae
Authors: Morelle Sandrine , Carbonnelle Etienne , Matic Ivan , Nassif Xavier 
Published: January 2005

SummaryDNA repair systems play a major role in maintaining the integrity of bacterial genomes. Neisseria meningitidis, a human pathogen capable of colonizing the human nasopharynx, possesses numerous DNA repair genes but lacks inducible DNA repair systems such as the SOS response, present in most bacteria species. We recently identified a set of genes upregulated by contact with host cells. An open reading frame having high homology with the small subunit of Escherichia coli exonuclease VII (xseB) belongs to this regulon. The increased sensitivity of a mutant in this coding sequence to UV irradiation, alkylating agent and nalidixic acid demonstrates the participation of this gene in meningococcal DNA repair. In addition, the upregulation of the transcription of this open reading frame upon interaction of N. meningitidis with host cells increased not only the bacterial ability to repair its DNA but also the rate of phase variation by frameshifting. Together these data demonstrate that N. meningitidis possesses an inducible DNA repair system that might be used by the bacteria to adapt to its niches when it is colonizing a new host.
PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability
Authors: Roosen Johnny , Engelen Kristof , Marchal Kathleen , Mathys Janick , Griffioen Gerard , Cameroni Elisabetta , Thevelein Johan M. , De Virgilio Claudio , De Moor Bart , Winderickx Joris 
Published: January 2005

SummaryIn the yeast Saccharomyces cerevisiae, PKA and Sch9 exert similar physiological roles in response to nutrient availability. However, their functional redundancy complicates to distinguish properly the target genes for both kinases. In this article, we analysed different phenotypic read‐outs. The data unequivocally showed that both kinases act through separate signalling cascades. In addition, genome‐wide expression analysis under conditions and with strains in which either PKA and/or Sch9 signalling was specifically affected, demonstrated that both kinases synergistically or oppositely regulate given gene targets. Unlike PKA, which negatively regulates stress‐responsive element (STRE)‐ and post‐diauxic shift (PDS)‐driven gene expression, Sch9 appears to exert additional positive control on the Rim15‐effector Gis1 to regulate PDS‐driven gene expression. The data presented are consistent with a cyclic AMP (cAMP)‐gating phenomenon recognized in higher eukaryotes consisting of a main gatekeeper, the protein kinase PKA, switching on or off the activities and signals transmitted through primary pathways such as, in case of yeast, the Sch9‐controlled signalling route. This mechanism allows fine‐tuning various nutritional responses in yeast cells, allowing them to adapt metabolism and growth appropriately.
Biogenesis of a putative channel protein, ComEC, required for DNA uptake: membrane topology, oligomerization and formation of disulphide bonds
Authors: Draskovic Irena , Dubnau David 
Published: January 2005

SummaryComEC is a putative channel protein for DNA uptake in Bacillus subtilis and other genetically transformable bacteria. Membrane topology studies suggest a model of ComEC as a multispanning membrane protein with seven transmembrane segments (TMSs), and possibly with one laterally inserted amphipathic helix. We show that ComEC contains an intramolecular disulphide bond in its N‐terminal extracellular loop (between the residues C131 and C172), which is required for the stability of the protein, and is probably introduced by BdbDC, a pair of competence‐induced oxidoreductase proteins. By in vitro cross‐linking using native cysteine residues we show that ComEC forms an oligomer. The oligomerization surface includes a transmembrane segment, TMS‐G, near the cytoplasmic C‐terminus of ComEC.
The σ54‐dependent transcriptional activator SfnR regulates the expression of the Pseudomonas putida sfnFG operon responsible for dimethyl sulphone utilization
Authors: Endoh Takayuki , Habe Hiroshi , Nojiri Hideaki , Yamane Hisakazu , Omori Toshio 
Published: January 2005

Summary Pseudomonas putida DS1 is able to utilize dimethyl sulphide through dimethyl sulphoxide, dimethyl sulphone (DMSO2), methanesulphonate (MSA) and sulphite as a sulphur source. We previously demonstrated that sfnR encoding a σ54‐dependent transcriptional regulator is essential for DMSO2 utilization by P. putida DS1. To identify the target genes of SfnR, we carried out transposon mutagenesis on an sfnR disruptant (DMSO2‐utilization‐defective phenotype) using mini‐Tn5, which contains two outward‐facing constitutively active promoters; as a result, we obtained a mutant that restored the ability to utilize DMSO2. The DMSO2‐positive mutant carried a mini‐Tn5 insertion in the intergenic region between two opposite‐facing operons, sfnAB and sfnFG. Both sfnA and sfnB products were similar to acyl‐CoA dehydrogenase family proteins, whereas sfnF and sfnG encoded a putative NADH‐dependent FMN reductase (SfnF) and an FMNH2‐dependent monooxygenase (SfnG). Disruption and complementation of the sfn genes indicated that the sfnG product is essential for DMSO2 utilization by P. putida DS1. Furthermore, an enzyme assay demonstrated that SfnG is an FMNH2‐dependent DMSO2 monooxygenase that converts DMSO2 to MSA. It was revealed that the expression of the sfnFG operon is directly activated by the binding of SfnR at its upstream region. Site‐directed mutagenesis of the SfnR binding sequences allowed us to define a potential recognition sequence for SfnR. These results provided insight into regulation of sulphate starvation‐induced genes in bacteria.
Cell division in cocci: localization and properties of the Streptococcus pneumoniae FtsA protein
Authors: Lara Beatriz , Rico Ana Isabel , Petruzzelli Sabrina , Santona Antonella , Dumas Jacques , Biton Jacques , Vicente Miguel , Mingorance Jesús , Massidda Orietta 
Published: January 2005

SummaryWe studied the cytological and biochemical properties of the FtsA protein of Streptococcus pneumoniae. FtsA is a widespread bacterial cell division protein that belongs to the actin superfamily. In Escherichia coli and Bacillus subtilis, FtsA localizes to the septal ring after FtsZ, but its exact role in septation is not known. In S. pneumoniae, we found that, during exponential growth, the protein localizes to the nascent septa, at the equatorial zones of the dividing cells, where an average of 2200 FtsA molecules per cell are present. Likewise, FtsZ was found to localize with the same pattern and to be present at an average of 3000 molecules per cell. Consistent with the colocalization, FtsA was found to interact with FtsZ and with itself. Purified FtsA is able to bind several nucleotides, the affinity being highest for adenosine triphosphate (ATP), and lower for other triphosphates and diphosphates. The protein polymerizes in vitro, in a nucleotide‐dependent manner, forming long corkscrew‐like helixes, composed of 2 + 2 paired protofilaments. No nucleotide hydrolytic activity was detected. Consistent with the absence of an ATPase activity, the polymers are highly stable and not dynamic. These results suggest that the FtsA protein could also polymerize in vivo and the polymers participate in septation.
The Legionella IcmS–IcmW protein complex is important for Dot/Icm‐mediated protein translocation
Authors: Ninio Shira , Zuckman‐Cholon Deborah M. , Cambronne Eric D. , Roy Craig R. 
Published: January 2005

SummaryThe intracellular pathogen Legionella pneumophila can infect and replicate within macrophages of a human host. To establish infection, Legionella require the Dot/Icm secretion system to inject protein substrates directly into the host cell cytoplasm. The mechanism by which substrate proteins are engaged and translocated by the Dot/Icm system is not well understood. Here we show that two cytosolic components of the Dot/Icm secretion machinery, the proteins IcmS and IcmW, play an important role in substrate translocation. Biochemical analysis indicates that IcmS and IcmW form a stable protein complex. In Legionella, the IcmW protein is rapidly degraded in the absence of the IcmS protein. Substrate proteins translocated into mammalian host cells by the Dot/Icm system were identified using the IcmW protein as bait in a yeast two‐hybrid screen. It was determined that the IcmS–IcmW complex interacts with these substrates and plays an important role in translocation of these proteins into mammalian cells. These data are consistent with the IcmS–IcmW complex being involved in the recognition and Dot/Icm‐dependent translocation of substrate proteins during Legionella infection of host cells.
The svpA‐srtB locus of Listeria monocytogenes: Fur‐mediated iron regulation and effect on virulence
Authors: Newton Salete M. C. , Klebba Phillip E. , Raynaud Catherine , Shao Yi , Jiang Xiaoxu , Dubail Iharilalao , Archer Crystal , Frehel Claude , Charbit Alain 
Published: January 2005

SummaryIn Listeria monocytogenes the promoter region of the svpA‐srtB locus contains a well‐conserved Fur box. We characterized the iron‐regulation of this locus: real‐time polymerase chain reaction analyses and anti‐SvpA immunoblots showed that, in response to iron deprivation svpA transcription and SvpA production markedly increased (80‐fold and 10‐fold respectively), when initiated by either the addition of the iron chelator 2,2′‐bipyridyl to BHI media, or by growth in iron‐restricted minimal media. Green fluorescent protein (GFP) reporter constructs also showed increased activity of the svpA‐srtB promoter in Escherichia coli (37‐fold) and in L. monocytogenes (two‐ to threefold) when the bacteria were grown in iron‐deficient conditions. A Δfur mutant of L. monocytogenes constitutively synthesized SvpA, as well as GFP fused to the svpA‐srtB promoter. Cellular fractionation data revealed that in iron‐rich media wild‐type SvpA was exclusively secreted to the culture supernatant. However, both the Δfur derivative and wild‐type L. monocytogenes grown in iron‐deficient media anchored a fraction of the SvpA proteins (∼5%) to peptidoglycan, and produced a lower‐molecular weight, wholly secreted form of SvpA. Together these data establish that iron availability controls transcription of the svpA‐srtB locus (through Fur‐mediated regulation), and attachment of SvpA to the cell wall (through SrtB‐mediated covalent linkage). SvpA bears homology to IsdC, a haemin‐binding protein of Staphylococcus aureus, and haemin bound to SvpA in solution. However, site‐directed deletions of four structural genes and the promoter of the svpA‐srtB locus did not impair haemin, haemoglobin or ferrichrome utilization in nutrition tests. We did not find strong evidence to support the notion that the svpA‐srtB locus participates in haemin acquisition, as was reported for the homologous isd operon of S. aureus. Furthermore, the svpA‐srtB mutant strains showed no significant attenuation of virulence in an intravenous mouse model system, but we found that the mutations reduced the persistence of L. monocytogenes in murine liver, spleen and intestines after oral administration.
Type III secretion chaperones of Pseudomonas syringae protect effectors from Lon‐associated degradation
Authors: Losada Liliana C. , Hutcheson Steven W. 
Published: January 2005

SummaryThe hrp type III secretion system (TTSS) of Pseudomonas syringae translocates effector proteins into the cytoplasm of host cells. Proteolysis of HrpR by Lon has been shown to negatively regulate the hrp TTSS. The inability to bypass Lon‐associated effects on the regulatory system by ectopic expression of the known regulators suggested a second site of action for Lon in TTSS‐dependent effector secretion. In this study we report that TTSS‐dependent effectors are subject to the proteolytic degradation that appears to be rate‐limiting to secretion. The half‐lives of the effectors AvrPto, AvrRpt2, HopPsyA, HopPsyB1, HopPtoB2, HopPsyV1, HopPtoG and HopPtoM were substantially higher in bacteria lacking Lon. TTSS‐dependent secretion of several effectors was enhanced from Lon mutants. A primary role for chaperones  appears  to  be  protection  of  effectors  from Lon‐associated degradation prior to secretion. When coexpressed with their cognate chaperone, HopPsyB1, HopPsyV1 and HopPtoM were at least 10 times more stable in strains expressing Lon. Distinct Lon‐targeting and chaperone‐binding domains were identified in HopPtoM. The results imply that Lon is involved at two distinct levels in the regulation of the P. syringae TTSS: regulation of assembly of the secreton and modulation of effector secretion.
Environmental and genetic regulation of the phosphorylcholine epitope of Haemophilus influenzae lipooligosaccharide
Authors: Wong Sandy M. , Akerley Brian J. 
Published: January 2005

SummaryIn response to environmental signals in the host, bacterial pathogens express factors required during infection and repress those that interfere with specific stages of this process. Signalling pathways controlling virulence factors of the human respiratory pathogen, Haemophilus influenzae, are predominantly unknown. The lipooligosaccharide (LOS) outer core represents a prototypical virulence trait of H. influenzae that enhances virulence but also provides targets for innate and adaptive immunity. We report regulation of the display of the virulence‐associated phosphorylcholine (PC) epitope on the LOS in response to environmental conditions. PC display is optimal under microaerobic conditions and markedly decreased under conditions of high culture aeration. Gene expression analysis using a DNA microarray was performed to begin to define the metabolic state of the cell under these conditions and to identify genes potentially involved in PC epitope modulation. Global gene expression profiling detected changes in redox responsive genes and in genes of carbohydrate metabolism. The effects on carbohydrate metabolism led us to examine the role of the putative H. influenzae homologue of csrA, a regulator of glycolysis and gluconeogenesis in Escherichia coli. A mutant containing an in‐frame deletion of the H. influenzae csrA gene showed increased PC epitope levels under aerobic conditions. Furthermore, deletion of csrA elevated mRNA expression of galU, an essential virulence gene that is critical in generating sugar precursors needed for polysaccharide formation and LOS outer core synthesis. Growth conditions predicted to alter the redox state of the culture modulated the PC epitope and galU expression as well. The results are consistent with a multifactorial mechanism of control of LOS‐PC epitope display involving csrA and environmental signals that coordinately regulate biosynthetic and metabolic genes controlling the LOS structure.
A master regulator for biofilm formation by Bacillus subtilis
Authors: Kearns Daniel B. , Chu Frances , Branda Steven S. , Kolter Roberto , Losick Richard 
Published: January 2005

SummaryWild strains of Bacillus subtilis are capable of forming architecturally complex communities of cells known as biofilms. Critical to biofilm formation is the eps operon, which is believed to be responsible for the biosynthesis of an exopolysaccharide that binds chains of cells together in bundles. We report that transcription of eps is under the negative regulation of SinR, a repressor that was found to bind to multiple sites in the regulatory region of the operon. Mutations in sinR bypassed the requirement in biofilm formation of two genes of unknown function, ylbF and ymcA, and sinI, which is known to encode an antagonist of SinR. We propose that these genes are members of a pathway that is responsible for counteracting SinR‐mediated  repression.  We  further  propose  that  SinR is a master regulator that governs the transition between a planktonic state in which the bacteria swim as single cells in liquid or swarm in small groups over surfaces, and a sessile state in which the bacteria adhere to each other to form bundled chains and assemble into multicellular communities.
Mechanism of lipid‐body formation in prokaryotes: how bacteria fatten up
Authors: Wältermann Marc , Hinz Andreas , Robenek Horst , Troyer David , Reichelt Rudolf , Malkus Ursula , Galla Hans‐Joachim , Kalscheuer Rainer , Stöveken Tim , Von Landenberg Philipp , Steinbüchel Alexander 
Published: January 2005

SummaryNeutral lipid accumulation is frequently observed in some Gram‐negative prokaryotes like Acinetobacter sp. and most actinomycetes, including the pathogenic Mycobacterium tuberculosis and antibiotic producing streptomycetes. We examined the formation  of  wax  ester‐  and  triacylglycerol  (TAG)‐bodies in Acinetobacter calcoaceticus and Rhodococcus opacus using microscopic, immunological and biophysical methods. A general model for prokaryotic lipid‐body formation is proposed, clearly differing from the current models for the formation of lipid inclusions in eukaryotes and of poly(hydroxyalkanoic acid) (PHA) inclusions in prokaryotes. Formation of lipid‐bodies starts with the docking of wax ester synthase/acyl‐CoA:diacylglycerol acyltransferase (WS/DGAT) to the cytoplasm membrane. Both, analyses of in vivo and in vitro lipid‐body synthesis, demonstrated the formation of small lipid droplets (SLDs), which remain bound to the membrane‐associated enzyme. SLDs conglomerated subsequently to membrane‐bound lipid‐prebodies which are then released into the cytoplasm. The formation of matured lipid‐bodies in the cytoplasm occurred by means of coalescence of SLDs inside the lipid prebodies, which are surrounded by a half‐unit membrane of phospholipids.
Mig‐14 is an inner membrane‐associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection
Authors: Brodsky Igor E. , Ghori Nafisa , Falkow Stanley , Monack Denise 
Published: January 2005

Summary Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid‐like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig‐14 and virK genes of Salmonella enterica are important in bacterial resistance to anti‐microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon‐gamma (IFN‐γ) is crucial to controlling replication of mig‐14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig‐14 contributes to long‐term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig‐14 and virK contribute to the survival of Salmonella in macrophages treated with IFN‐γ and are necessary for resistance to cathelin‐related anti‐microbial peptide (CRAMP), an anti‐microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig‐14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig‐14 is an inner membrane‐associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig‐14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig‐14 in anti‐microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.