bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of a gene (lpt‐3) required for the addition of phosphoethanolamine to the lipopolysaccharide inner core of Neisseria meningitidis and its role in mediating susceptibility to bactericidal killing and opsonophagocytosis
Authors: Mackinnon Fiona G. , Cox Andrew D. , Plested Joyce S. , Tang Christoph M. , Makepeace Katherine , Coull Philip A. , Wright J. Claire , Chalmers Ronald , Hood Derek W. , Richards James C. , Moxon E. Richard 
Published: March 2003

SummaryWe have identified a gene, lpt‐3, that is required for the addition of phosphoethanolamine to the 3‐position (PEtn‐3) on the β‐chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn‐3 substituent is characteristic of the LPS of a majority (≈ 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface‐accessible epitope. All strains of Nm that have PEtn‐3 possess the lpt‐3 gene. In some lpt‐3‐containing strains, the 3‐position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt‐3 resulted in loss of PEtn‐3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt‐3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS‐based vaccine to protect against invasive meningococcal disease.
Protein phosphatase 2A on track for nutrient‐induced signalling in yeast
Authors: Zabrocki Piotr , Hoof Christine Van , Goris Jozef , Thevelein Johan M. , Winderickx Joris , Wera Stefaan 
Published: March 2003

SummaryEarly studies identified two bona fide protein phosphatase 2A (PP2A)‐encoding genes in Saccharo‐myces cerevisiae, designated PPH21 and PPH22. In

addition, three PP2A‐related phosphatases, encoded by PPH3, SIT4 and PPG1, have been

identified. All share as much as 86% sequence similarity at the amino acid

level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4

regulation will also be discussed. Whereas a role for PP2A in yeast morphology and

cell cycle has been readily recognized, uncovering its function in yeast signal transduction

is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A

and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP–PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient‐induced signalling in baker’s yeast.
Saccharomyces cerevisiae Ccr4–Not complex contributes to the control of Msn2p‐dependent transcription by the Ras/cAMP pathway
Authors: Lenssen E. , Oberholzer U. , Labarre J. , De Virgilio C. , Collart M. A. 
Published: March 2003

SummaryThe Ccr4–Not complex is a global regulator of transcription that affects genes positively and negatively and is thought to modulate the activity of TFIID. In the present work, we provide evidence that the Ccr4–Not complex may contribute to transcriptional regulation by the Ras/cAMP pathway. Several observations support this model. First, Msn2/4p‐dependent transcription, which is known to be under negative control of cAMP‐dependent protein kinase (PKA), is derepressed in all ccr4–not mutants. This phenotype is paralleled by specific post‐translational modification defects of Msn2p in ccr4–not mutants relative to wild‐type cells. Secondly, mutations in various NOT genes result in a synthetic temperature‐sensitive growth defect when combined with mutations that compromise cells for PKA activity and at least partially suppress the effects of both a dominant‐active RAS2Val‐19 allele and loss of Rim15p. Thirdly, Not3p and Not5p, which are modified and subsequently degraded by stress signals that also lead to increased Msn2/4p‐dependent activity, show a specific two‐hybrid interaction with Tpk2p. Together, our results suggest that the Ccr4–Not complex may function as an effector of the Ras/cAMP pathway that contributes to repress basal, stress‐ and starvation‐induced transcription by Msn2/4p.
Down‐regulation of pili and capsule of Neisseria meningitidis upon contact with epithelial cells is mediated by CrgA regulatory protein
Authors: Deghmane Ala‐Eddine , Giorgini Dario , Larribe Mireille , Alonso Jean‐Michel , Taha Muhamed‐Kheir 
Published: March 2003

SummaryThe initial attachment of Neisseria meningitidis to the target cell surface

appears to be largely pilus depend‐ent in capsulated bacteria. Intimate adhesion

subsequently occurs to permit colonization. We recently reported that insertional

inactivation of the crgA gene, which encodes a transcriptional regulator belonging

to the LysR family, decreased meningococcal adhesion to epithelial cells and abolished

intimate adhesion. In this report, we analyse expression of the pilE and sia

genes, which are involved in the biosynthesis of pili and capsule respectively, during

bacteria–host cell interactions. Western blotting, transcriptional fusion

and reverse transcriptase polymerase chain reaction (RT‐PCR) analysis showed that

the expression of these genes was downregulated during intimate adhesion. DNA‐binding

assays, footprinting and RT‐PCR analysis indicated that this downregulation was directly

mediated by the CrgA protein. The pilE and sia promoters were found

to have a CrgA binding motif in common. These results strongly suggest that N.

meningitidis displays an adaptive response upon cell contact. CrgA may play a central regulatory role in meningococcal adhesion, particularly in switching from initial to intimate adhesion by downregulating the bacterial surface structures that hinder this adhesion.
Characterization of the dihydrolipoamide dehydrogenase from Streptococcus pneumoniae and its role in pneumococcal infection
Authors: Smith Alexander W. , Roche Hazeline , Trombe Marie‐Claude , Briles David E. , Håkansson Anders 
Published: March 2003

SummaryIn the present study, we have characterized the dihydrolipoamide dehydrogenase (DLDH) of Strepto‐coccus pneumoniae and its role during pneumococcal infection. We have also demonstrated that a lack of DLDH results in a deficiency in α‐galactoside meta‐bolism and galactose transport. DLDH is an enzyme that is classically involved in the three‐step conversion of 2‐oxo acids to their respective acyl‐CoA derivatives, but DLDH has also been shown to have other functions. The dldh gene was virtually identical in three pneumococcal strains examined. Besides the functional domains and motifs associated with this enzyme, analysis of the pneumococcal dldh gene sequence revealed the presence of an N‐terminal lipoyl domain. DLDH‐negative bacteria totally lacked DLDH activity, indicating that this gene encodes the only DLDH in S. pneumoniae. These DLDH‐negative bacteria grew normally in vitro but were avirulent in sepsis and lung infection models in mice, indicating that DLDH activity is necessary for the survival of pneumococci within the host. The lack of virulence was not associated with a loss of 2‐oxo acid dehydrogenase activity, as the wild‐type pneumococcal strains did not contain activity of any of the known 2‐oxo acid enzyme complexes. Instead, studies of carbohydrate utilization demonstrated that the DLDH‐negative bacteria were impaired for α‐galactoside and galactose metabolism. The DLDH mutants lost their ability to oxidize or grow with galactose or melibiose as sole carbon source and showed reduced oxidation and growth on raffinose or stachyose. The bacteria had an 85% reduction in α‐galactosidase activity and showed virtually no transport of galactose into the cells, which can explain these phenotypic changes. The DLDH‐negative bacteria produced only 50% of normal capsular polysaccharide, a phenotype that may be associated with impaired carbohydrate metabolism.
PrnA, a Zn2Cys6 activator with a unique DNA recognition mode, requires inducer for in vivo binding
Authors: Gómez Dennis , Cubero Beatriz , Cecchetto Gianna , Scazzocchio Claudio 
Published: March 2003

SummaryThe PrnA transcriptional activator of Aspergillus nidulans binds as a dimer to CCGG‐N‐CCGG inverted repeats and to CCGG‐6/7N‐CCGG direct repeats. The binding specificity of the PrnA Zn cluster differs from that of the Gal4p/Ppr1p/UaY/Put3p group of proteins. Chimeras with UaY, a protein that strictly recognizes a CGG‐6N‐CCG motif, show that the recognition of the direct repeats necessitates the PrnA dimerization and linker elements, but the recognition of the CCGG‐N‐CCGG inverted repeats depends crucially on the PrnA Zn binuclear cluster and/or on residues amino‐terminal to it. Three high‐affinity sites in two different promoters have been visualized by in vivo methy‐lation protection. Proline induction is essential for in vivo binding to these three sites but, as shown previously, not for nuclear entry. Simultaneous repression by ammonium and glucose does not affect in vivo binding to these high‐affinity sites. PrnA differs from the isofunctional Saccharomyces cerevisiæ protein Put3p, both in its unique binding specificity and in the requirement of induction for in vivo DNA binding.
Characterization and evidence of mobilization of the LEE pathogenicity island of rabbit‐specific strains of enteropathogenic Escherichia coli
Authors: Tauschek Marija , Strugnell Richard A. , Robins‐Browne Roy M. 
Published: March 2003

SummaryWe have characterized the LEE pathogenicity islands (PAIs) of two rabbit‐specific strains of enteropathogenic E. coli (REPEC), 83/39 (serotype O15:H‐) and 84/110‐1
(O103:H2), and have compared them to homologous loci from the human enteropathogenic
and enterohaemorrhagic E. coli strains, E2348/69 and EDL933, and another REPEC
strain, RDEC‐1. All five PAIs contain a 34 kb core region that is highly conserved
in gene order and nucleotide sequence. However, the LEE of 83/39 is significantly
larger (59 540 basepairs) than those of the human strains, which are less than
44 kb, and has inserted into pheU tRNA. The regions flanking the 34 kb
core of 83/39 contain homologues of two putative virulence determinants, efa1/lifA
and senA. The LEE of 84/110‐1 is approximately 85 kb and is located at pheV
tRNA. Its core is almost identical to those of 83/39 and RDEC‐1, apart from a larger
espF gene, but its flanking regions contain trcA, a putative virulence
determinant of EPEC. All three REPEC LEE PAIs contain a gene for an integrase, Int‐phe.
The LEE PAI of 84/110‐1 is also flanked by short direct repeats (representing the
3′‐end of pheV tRNA), suggesting that it may be unstable. To
investigate this possibility, we constructed a LEE::sacB derivative of 84/110‐1
and showed that the PAI was capable of spontaneous deletion. We also showed that
Int‐phe can mediate site‐specific integration of foreign DNA at the pheU tRNA
locus of E. coli DH1. Together these results indicate possible mechanisms of mobilization and integration of the LEE PAI.
The Escherichia coli FKBP‐type PPIase SlyD is required for the stabilization of the E lysis protein of bacteriophage φX174
Authors: Bernhardt Thomas G. , Roof William D. , Young Ry 
Published: March 2003

SummaryMost bacteriophages abruptly terminate their vegetative cycle by causing lysis of the host cell. The ssDNA phage φX174 uses a single lysis gene, E, encoding a 91‐amino‐acid membrane protein that causes lysis of Escherichia coli by inhibiting MraY, a conserved enzyme of murein biosynthesis. Recessive mutations in the host gene slyD (sensitivity to lysis) absolutely block E‐mediated lysis and φX174 plaque formation. The slyD gene encodes a FKBP‐type peptidyl‐prolyl cis‐trans isomerase (PPIase). To investigate the molecular basis of this unique FKBP‐dependence, spontaneous plaque‐forming mutants of φX174 were isolated on a slyD lawn. All of these Epos (‘plates on slyD’) suppressors encode proteins with either a R3H or L19F change. The double mutant was also isolated and generated the largest plaques on the slyD lawn. A c‐myc epitope tag sequence was incorporated into the parental E and Epos genes without effect on lytic function. Western blots and pulse‐chase labelling ex‐periments showed that both Epos and E are highly unstable in a slyD background; however, Epos is synthesized at a higher rate, allowing a lysis‐sufficient level of Epos to accumulate. Our results indicate that SlyD is required for stabilizing the E protein and allowing it to accumulate to the levels required to exert its lytic effect. These data are discussed in terms of a model for the specific role of the SlyD PPIase in E folding, and of the use of the very strict SlyD‐ dependence phenotype for identifying elements of PPIase selectivity.
Spindle assembly checkpoint component CaMad2p is indispensable for Candida albicans survival and virulence in mice
Authors: Bai Chen , Ramanan Narendrakumar , Wang Yan Ming , Wang Yue 
Published: March 2003

SummaryHere, we report an indispensable role for spindle assembly checkpoint (SAC) component CaMad2p in the survival and virulence of Candida albicans in mice. We hypothesized that cell cycle checkpoint functions, especially those monitoring the integrity of DNA and chromosome segregation, might be required for the pathogen to repair damage caused by host defence. To test this idea, we created SAC‐defective mutants by deleting the CaMAD2 gene that encodes a key component of the SAC pathway. The CaMAD2 mutant appears normal in morphology, growth rate and growth mode switch in unperturbed conditions. However, it quickly loses viability when treated with nocodazole, which causes disassembly of mitotic spindles. The mutant also exhibits increased frequency of chromosome loss. The virulence of the mutant is greatly reduced in mice, presumably because of the inability of the mutant cells to stop the cell cycle when the host defence damages cellular components important for chromosome segregation. Supporting this hypothesis, unlike the wild‐type cells that can proliferate within and eventually grow out of macrophages, most of the CaMAD2 null mutant cells are unable to survive. This study suggests that SAC is required for survival of C. albicans in the host and could thus be targeted for anti‐C. albicans therapies.
The ramC gene is required for morphogenesis in Streptomyces coelicolor and expressed in a cell type‐specific manner under the direct control of RamR
Authors: O'Connor Tamara J. , Kanellis Pamela , Nodwell Justin R. 
Published: March 2003

SummaryThe bacterium Streptomyces coelicolor produces two cell types during the course of its life cycle: the aerial hyphae, which metamorphose into spores, and the substrate hyphae, which synthesize antibiotics. We show that the genes ramC and ramR are required for the production of the aerial hyphae but are dispensable for vegetative growth and antibiotic synthesis. We find that ramC is expressed in the substrate hyphae and shut off in the aerial hyphae by the time visible signs of sporulation‐associated septation are evident. Production of RamC requires the developmental regulators bldD, cprA and ramR, but not bldM or bldN, and we show that the RamR protein interacts directly with DNA in the ramC promoter region sug‐gesting that it is, at least in part, responsible for regulating ramC expression.
LrhA as a new transcriptional key regulator of flagella, motility and chemotaxis genes in Escherichia coli
Authors: Lehnen D. , Blumer C. , Polen T. , Wackwitz B. , Wendisch V. F. , Unden G. 
Published: March 2003

SummaryThe function of the LysR‐type regulator LrhA of Escherichia coli was defined by comparing whole‐genome mRNA profiles from wild‐type E. coli and an isogenic lrhA mutant on a DNA microarray. In the lrhA mutant, a large number (48) of genes involved in flagellation, motility and chemotaxis showed relative mRNA abundances increased by factors between 3 and 80. When a representative set of five flagellar, motility and chemotaxis genes was tested in lacZ reporter gene fusions, similar factors for derepression were found in the lrhA mutant. In gel retardation experiments, the LrhA protein bound specifically to flhD and lrhA promoter DNA (apparent KD≈ 20 nM), whereas the promoters of fliC, fliA and trg were not bound by LrhA. The expression of flhDC (encoding FlhD2C2) was derepressed by a factor of 3.5 in the lrhA mutant. FlhD2C2 is known as the master regulator for the expression of flagellar and chemotaxis genes. By DNase I footprinting, LrhA binding sites at the flhDC and lrhA promoters were identified. The lrhA gene was under positive autoregulation by LrhA as shown by gel retardation and lrhA expression studies. It is suggested that LrhA is a key regulator controlling the transcription of flagellar, motility and chemotaxis genes by regulating the synthesis and concentration of FlhD2C2.
Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35‐amino‐acid cell‐killing peptide and a cis‐encoded small antisense RNA in Escherichia coli
Authors: Kawano Mitsuoki , Oshima Taku , Kasai Hiroaki , Mori Hirotada 
Published: March 2003

SummaryGenome sequence analyses of Escherichia coli K‐12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR‐A, ‐B, ‐C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR‐D), 450 bp in length and nearly identical to LDR‐A, ‐B and ‐C, is located at 79.7 min, a position that is directly opposite the position of LDR‐A, ‐B and ‐C. In this study, we demonstrate that LDR‐D encodes a 35‐amino‐acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (≈ 370 nucleotides) encoding LdrD and an unstable cis‐encoded antisense RNA (≈ 60 nucleotides), which functions as a trans‐acting regulator of ldrD translation. We propose that LDR encodes a toxin–antitoxin module. LDR‐homologous sequences are not present on any known plasmids but are conserved in Salmonella and other enterobacterial species.
Imp/OstA is required for cell envelope biogenesis in Escherichia coli
Authors: Braun Martin , Silhavy Thomas J. 
Published: March 2003

SummaryIn Gram‐negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high‐molecular‐weight disulphide‐bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane.
CPMK2, an SLT2‐homologous mitogen‐activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis‐related MAP kinase cascade in phytopathogenic fungi
Authors: Mey Géraldine , Held Katrin , Scheffer Jan , Tenberge Klaus B. , Tudzynski Paul 
Published: March 2003

SummaryCpmk2
, encoding a mitogen‐activated protein (MAP) kinase from the ascomycete
Claviceps purpurea
, is an orthologue of
SLT2
from
Saccharomyces cerevisiae
, the first isolated from a biotrophic, non‐appressorium‐forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen,
Magnaporthe grisea
, that have been deprived of their MAP kinase gene
mps1
, the ability of
cpmk2
to complement the defects of Δ
mps1
was investigated. Interestingly, the
C. purpurea
gene, under the control of its own promoter, was able to complement the
M. grisea
mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2
/ MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.
Type III‐dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell
Authors: Szurek Boris , Rossier Ombeline , Hause Gerd , Bonas Ulla 
Published: March 2003

SummaryMany plant pathogenic bacteria utilize a conserved type III secretion system (TTSS) to deliver effector proteins into the host tissue. Indirect evidence has suggested that at least some effector proteins are translocated from the bacterial cytoplasm into the plant cell. Using an immunocytochemical approach, we demonstrate that the type III effector AvrBs3 from Xanthomonas campestris pv. vesicatoria localizes to nuclei of infected pepper leaves. Importantly, AvrBs3 translocation was observed in situ in native tissues of susceptible and resistant plants. AvrBs3 was detected in the nucleus as soon as 4 h post infection, which was dependent on a functional TTSS and the putative translocator HrpF. N‐terminal AvrBs3 deletion derivatives are no longer secreted by the TTSS in vitro and could not be detected inside the host cells, suggesting that the N‐terminus of AvrBs3 is important for secretion. Deletion of the nuclear localization signals in the AvrBs3 C‐terminus, which are required for the AvrBs3‐mediated induction of the hypersensitive reaction in resistant pepper plants, abolished AvrBs3 localization to the nucleus. This is the first report on direct evidence for translocation of a native type III effector protein from a plant pathogenic bacterium into the host cell.
Critical role of the N‐terminal residues of listeriolysin O in phagosomal escape and virulence of Listeria monocytogenes
Authors: Lety Marie‐Annick , Frehel Claude , Berche Patrick , Charbit Alain 
Published: March 2003

SummaryA putative PEST sequence was recently identified close to the N‐terminus of listeriolysin O (LLO), a major virulence factor secreted by the pathogenic Listeria monocytogenes. The deletion of this motif did not affect the secretion and haemolytic activity of LLO, but abolished bacterial virulence. Here, we first tested whether the replacement of the PEST motif of LLO by two different sequences, with either a very high or no PEST score, would affect phagosomal escape, protein stability and, ultimately, the virulence of L. monocytogenes. Then, we constructed LLO mutants with an intact PEST sequence but carrying mutations on either side, or on both sides, of the PEST motif. The properties of these mutants prompted us to construct three LLO mutants carrying single amino acid substitutions in the distal portion of the PEST region (P49A, K50A and P52A; preprotein numbering). Our data demonstrate that the susceptibility of LLO to intracellular proteolytic degradation is not related to the presence of a high PEST score sequence and that the insertion of two residues immediately downstream of the intact PEST sequence is sufficient to impair phagosomal escape and abolish bacterial virulence. Furthermore, we show that single amino acid substitutions in the distal portion of the PEST motif are sufficient to attenuate bacterial ­virulence significantly, unravelling the critical role of this region of LLO in the pathogenesis of L. ­monocytogenes.