bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of pPS10 host range by DnaA
Authors: Maestro Beatriz , Sanz Jesús M. , Faelen Michel , Couturier Martine , Díaz‐Orejas Ramón , Fernández‐Tresguerres Elena 
Published: March 2003

SummaryNarrow‐host‐range plasmid pPS10, originally found inPseudomonas savastanoi, is unable to replicate in other strains such asEscherichia coli. Here, we report that the establishment of pPS10 inE. colican be achieved by a triple mutation in thednaAgene ofE. coli(dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As theE. colistrain used contained double amber suppressor mutations (supE,supF), the amber codon indnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon inE. coli.The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon inE. coliand provides support for the hypothesis that interactions between RepA and DnaA modulate the establish‐ment of pPS10 in that bacteria and probably in other species.
Acidic phospholipids inhibit the DNA‐binding activity of DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli
Authors: Makise Masaki , Mima Shinji , Katsu Takashi , Tsuchiya Tomofusa , Mizushima Tohru 
Published: March 2003

SummaryIn order to initiate chromosomal DNA replication in Escherichia coli, the DnaA protein must bind to both ATP and the origin of replication (oriC). Acidic phospholipids are known to inhibit DnaA binding to ATP, and here we examine the effects of various phospholipids on DnaA binding to oriC. Among the phospholipids in E. coli membrane, cardiolipin showed the strongest inhibition of DnaA binding to oriC. Synthetic phosphatidylglycerol containing unsaturated fatty acids inhibited binding more potently than did synthetic phosphatidylglycerol containing saturated fatty acids, suggesting that membrane fluidity is important. Thus, acidic phospholipids seem to inhibit DnaA binding to both oriC and adenine nucleotides in the same manner. Adenine nucleotides bound to DnaA did not affect the inhibitory effect of cardiolipin on DnaA binding to oriC. A mobility‐shift assay re‐vealed that acidic phospholipids inhibited formation of a DnaA–oriC complex containing several DnaA molecules. DNase I footprinting of DnaA binding to oriC showed that two DnaA binding sites (R2 and R3) were more sensitive to cardiolipin than other DnaA binding sites. Based on these in vitro data, the physiological relevance of this inhibitory effect of acidic phospholipids on DnaA binding to oriC is discussed.
Genetic analysis of functions involved in the late stages of biofilm development in Burkholderia cepacia H111
Authors: Huber Birgit , Riedel Kathrin , Köthe Manuela , Givskov Michael , Molin Søren , Eberl Leo 
Published: March 2003

SummaryBurkholderia cepacia
and
Pseudomonas aeruginosa
often co‐exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, we report the isolation of 13 random mini‐Tn
5
insertion mutants of
B. cepacia
H111 that are defective in biofilm formation on a polystyrene surface. We show that the screening procedure used in this study is biased towards mutants defective in the late stages of biofilm development. A detailed quantitative analysis of the biofilm structures formed by wild‐type and mutant strains revealed that the isolated mutants are impaired in their abilities to develop a typical three‐dimensional biofilm structure. Molecular investigations showed that the genes required for biofilm maturation fall into several classes: (i) genes encoding for surface proteins; (ii) genes involved in the biogenesis and maintenance of an integral outer membrane; and (iii) genes encoding regulatory factors. It is shown that three of the regulatory mutants produce greatly reduced amounts of
N
‐octanoylhomoserine lactone (C8‐HSL). This compound serves as the major signal molecule of the
cep
quorum‐sensing system. As this density‐dependent regulatory system is involved in the regulation of biofilm maturation, we investigated the interplay between the three regulatory genes and the quorum‐sensing cascade. The results of these investigations show that the identified genes encode for regulatory elements that are positioned upstream of the
cep
system, indicating that the quorum‐sensing system of
B. cepacia is a major checkpoint for biofilm formation.
The glycosylphosphatidylinositol (GPI) signal sequence of human placental alkaline phosphatase is not recognized by human Gpi8p in the context of the yeast GPI anchoring machinery
Authors: Meyer Urs , Fraering Patrick , Bosson Régine , Imhof Isabella , Benghezal Mohammed , Vionnet Christine , Conzelmann Andreas 
Published: March 2003

SummaryBiosynthesis of glycosylphosphatidylinositol (GPI)‐anchored proteins involves the action of a GPI trans‐amidase, which replaces the C‐terminal GPI signal sequence (GPI‐SS) of the primary translation product with a preformed GPI lipid. The transamidation depends on a complex of four proteins, Gaa1p, Gpi8p, Gpi16p and Gpi17p. Although the GPI anchoring pathway is conserved throughout the eukaryotic kingdom, it has been reported recently that the GPI‐SS of human placental alkaline phosphatase (hPLAP) is not recognized by the yeast transamidase, but is recognized in yeast that contain the human Gpi8p homologue . This finding suggests that Gpi8p is intimately involved in the recognition of GPI precursor proteins and may also be responsible for the subtle taxon‐specific differences in transamidase specificity that sometimes prevent the efficient GPI anchoring of heterologously expressed GPI proteins. Here, we confirm that the GPI signal sequence of hPLAP is indeed not recognized by the yeast GPI‐anchoring machinery. However, in our hands, GPI attachment cannot be restored by the co‐expression of human Gpi8p in yeast cells under any circumstances.
The VirB/VirD4 type IV secretion system of Bartonella is essential for establishing intraerythrocytic infection
Authors: Schulein Ralf , Dehio Christoph 
Published: March 2003

SummaryBartonellae are pathogenic bacteria uniquely adapted to cause intraerythrocytic infection in their human or animal reservoir host(s). Experimental infection of rats by Bartonella tribocorum revealed the initial colonization of a yet unidentified niche outside of circulating blood. This primary niche periodically seeds bacteria into the bloodstream, resulting in the invasion and persistent intracellular colonisation of erythrocytes. Here, this animal model was used for a genetic analysis of the virB locus (virB2‐11) and the downstream located virD4 gene, which together encode a putative type IV secretion system (T4SS). A generic method for marker‐less gene replacement allowed the generation of non‐polar in‐frame deletions in either virB4 or virD4. Both mutants were unable to cause bacteraemia, whereas complementation with the full‐length genes in trans completely restored infectivity. Segregation analysis of the complementation plasmids further denoted that VirB4 and VirD4 are required at an early stage of the infection course before the onset of intraerythrocytic bacteraemia. This analysis of defined mutants in an in vivo model identified components of the VirB/VirD4 T4SS as the first bona fide pathogenicity factors in Bartonella.
On the mechanism of substrate specificity by resistance nodulation division (RND)‐type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition
Authors: Mao Weimin , Warren Mark S. , Black Deborah S. , Satou Takahumi , Murata Takeshi , Nishino Takeshi , Gotoh Naomasa , Lomovskaya Olga 
Published: March 2003

SummaryTripartite efflux systems of Gram‐negative bacteria that contain an inner membrane transporter belonging to the resistance nodulation division (RND) superfamily can extrude a large variety of structurally diverse compounds. To gain an insight into the molecular mechanisms of substrate recognition by these multidrug resistance (MDR) transporters, we isolated spontaneous mutations that altered the substrate specificity of the MexCD–OprJ pump from Pseudomonas aeruginosa. These mutations enabled the pump to extrude the normally non‐transported β‐lactam antibiotic carbenicillin. All amino acid substitutions were mapped to the large periplasmic loops (LPLs) of the RND proper, MexD. Q34K, E89K, A292V and P328L were found in the first LPL, located between transmembrane domains (TMD) 1 and 2, whereas F608S and N673K were contained in the second LPL, located between TMD7 and TMD8. These mutations also had a substantial impact on the MexCD–OprJ‐mediated transport of numerous other substrates. Subsequent replacement of amino acid residues identified above by cysteines rendered MexCD–OprJ susceptible to inhibition by a thiol‐reactive agent, MIANS. Interestingly, MIANS inhibited the transport of some (pyronin, EtBr) but not other (ANS, Leu–Nap) substrates of the pump. Our results suggest that the precise structure of the periplasmic loops of MexD determines the rate of transport of individual substrates. These results are consistent with the hypothesis that, in the case of RND transporters, the LPLs are directly implicated in substrate recognition and contain multiple sites of interaction for various structurally diverse compounds.
ExsD is a negative regulator of the Pseudomonas aeruginosa type III secretion regulon
Authors: McCaw Michelle L. , Lykken Guinevere L. , Singh Pradeep K. , Yahr Timothy L. 
Published: March 2003

SummaryExpression of the Pseudomonas aeruginosa type III secretion system is induced by contact with eukaryotic cells, serum or low Ca2+ concentrations. We report that ExsD, a unique protein, is a negative regulator of the type III regulon. Localization studies indicate that ExsD is not secreted by P. aeruginosa. To determine the role of exsD, a non‐polar deletion was returned to the chromosome by allelic exchange. The ΔexsD mutant is competent for type III secretion and translocation of the ExoU cytotoxin to eukaryotic host cells. To examine the effect of ExsD on transcription, lacZ transcriptional reporter fusions were integrated into the chromosome. Promoters controlling transcription of genes encoding the type III secretory, regulatory and effector proteins demonstrated significant derepression in the ΔexsD background. Expression of ExsD from a multicopy plasmid completely repressed transcription of the regulon. Although a mutant in pscC, encoding a structural component of the type III translocase, is repressed for expression of the regulon, a ΔexsD, pscC::Ω double mutant is derepressed. Bacterial two‐hybrid data indicate that ExsD binds the transcriptional activator of the regulon, ExsA. We conclude that ExsD is a negative regulator and propose that ExsD functions as an ExsA antiactivator to regulate transcription of the regulon.
A novel apoptosis‐inducing protein from Helicobacter pylori
Authors: Shibayama Keigo , Kamachi Kazunari , Nagata Noriyo , Yagi Tetsuya , Nada Toshi , Doi Yohei , Shibata Naohiro , Yokoyama Keiko , Yamane Kunikazu , Kato Haru , Iinuma Yoshitsugu , Arakawa Yoshichika 
Published: March 2003

Summary Helicobacter pylori
infection induces apoptosis in gastric epithelial cells. Here, we report a novel apoptosis‐inducing protein that functions as a leading factor in
H. pylori
‐mediated apoptosis induction. We purified the protein from
H. pylori
by separating fractions that showed apoptosis‐inducing activity. This protein induced apoptosis of AGS cells in a dose‐dependent manner. The purified protein consisted of two protein fragments with molecular masses of about 40 and 22 kDa, which combined to constitute a single complex in their natural form. N‐terminal sequencing indicated that both these protein fragments were encoded by the HP1118 gene. The purified protein exhibited γ‐glutamyl transpeptidase activity, the inhibition of which by 6‐diazo‐5‐oxo‐
l
‐norleucine resulted in a complete loss of apoptosis‐inducing activity. To the best of our knowledge, the apoptosis‐inducing function is a newly identified physiological role for bacterial γ‐glutamyl transpeptidase. The apoptosis‐inducing activity of the isogenic mutant γ‐glutamyl transpeptidase‐deficient strain was significantly lower compared with that of the parent strain, demonstrating that γ‐glutamyl transpeptidase plays a significant role in
H. pylori
‐mediated apoptosis. Our findings provide new insights into
H. pylori pathogenicity and reveal a novel aspect of the bacterial γ‐glutamyl transpeptidase function.
Putrescine or a combination of methionine and arginine restores virulence gene expression in a tRNA modification‐deficient mutant of Shigella flexneri: a possible role in adaptation of virulence
Authors: Durand Jérôme M. B. , Björk Glenn R. 
Published: March 2003

SummaryThe wild‐type strain YSH6000 of Shigella flexneri growing in minimal medium contains the modified nucleoside epoxy‐Q (oQ) in a subset of tRNAs. This nucleoside is lacking in tRNA from a tgt mutant of this bacterium. When these bacteria are growing in minimal medium, the expression of virulence genes is 10‐fold lower in the tgt mutant than in the wild type, although only a twofold reduction in the expression of these virulence factors is observed in broth. Such a strong media‐dependant expression of virulence genes was not observed in the wild type. Accordingly, the level of the positive regulator of virulence, VirF, is much lower in the mutant than in the wild type. However, the transcription of the virF gene in minimal medium is the same in the wild type and in the tgt mutant. As the undermodification of tRNA is not affected by the quality of the growth medium, we conclude that such an environmental change in growth conditions partly restores virulence gene expression by counteracting poor translation of the virF mRNA mediated by an oQ‐deficient tRNA. Virulence gene expression is partly restored in the tgt mutant by the addition of a mixture of arginine and methionine. Addition of the polyamine putrescine, synthesis of which is metabolically related to that of arginine and methionine, has a comparable stimulatory effect on virulence gene expression. These results not only suggest a role for amino acids and polyamines in the environmental regulation of virulence gene expression in S. flexneri, but also demonstrate a strong and specific involvement of tRNA modifications, and especially oQ, in the adaptation of virulence gene expression to the nutritional quality of the growth medium.
A plasmid‐encoded regulator couples the synthesis of toxins and surface structures in Bacillus anthracis
Authors: Mignot Tâm , Mock Michèle , Fouet Agnès 
Published: March 2003

SummaryTranscription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal believed to reflect the host environment. A 180 kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation, pagR and atxA; the latter encodes a major trans‐activator. It has long been known that pXO1 genes have major effects on the physiology of B. anthracis, probably through regulatory cross‐talk between plasmid and chromosomal genes. Accordingly, we found that the chromosomal S‐layer genes, sap and eag, are regulated by pXO1 genes so that only eag is significantly expressed in the presence of CO2. This effect results from the product of pagR acting as the most downstream element of a signalling cascade initiated by AtxA. In vitro evidence showed that PagR is a transcription factor that controls the S‐layer genes by direct binding on their promoter regions. This work provides evidence that AtxA is a master regulator that co‐ordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.
Proinflammatory signalling stimulated by the type III translocation factor YopB is counteracted by multiple effectors in epithelial cells infected with Yersinia pseudotuberculosis
Authors: Viboud Gloria I. , So Stephane Shu Kin , Ryndak Michelle B. , Bliska James B. 
Published: March 2003

SummaryType III secretion systems are used by several pathogens to translocate effector proteins into host cells. Yersinia pseudotuberculosis delivers several Yop effectors (e.g. YopH, YopE and YopJ) to counteract signalling responses during infection. YopB, YopD and LcrV are components of the translocation machinery. Here, we demonstrate that a type III translocation protein stimulates proinflammatory signalling in host cells, and that multiple effector Yops counteract this response. To examine proinflammatory signalling by the type III translocation machinery, HeLa cells infected with wild‐type or Yop–Y. pseudotuberculosis strains were assayed for interleukin (IL)‐8 production. HeLa cells infected with a YopEHJ– triple mutant released significantly more IL‐8 than HeLa cells infected with isogenic wild‐type, YopE–, YopH– or YopJ– bacteria. Complementation analysis demonstrated that YopE, YopH or YopJ are sufficient to counteract IL‐8 production. IL‐8 production required YopB, but did not require YopD, pore formation or invasin‐mediated adhesion. In addition, YopB was required for activation of nuclear factor kappa B, the mitogen‐activated protein kinases ERK and JNK and the small GTPase Ras in HeLa cells infected with the YopEHJ– mutant. We conclude that interaction of the Yersinia type III translocator factor YopB with the host cell triggers a proinflammatory signalling response that is counteracted by multiple effectors in host cells.
Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis
Authors: Kawai Yoshikazu , Moriya Shigeki , Ogasawara Naotake 
Published: March 2003

SummaryA knock‐out mutant of the dinR gene that encodes the SOS regulon repressor in Bacillus subtilis was constructed. The yneA, yneB and ynzC genes transcribed divergently from the dinR gene were strongly induced in mutant cells. Northern hybridization analyses revealed that these genes collectively form an operon and belong to the SOS regulon. The simultaneous deletion of dinR and yneA suppressed the filamentous phenotype of the dinR mutant. Furthermore, although yneA is suppressed in the wild‐type cell in the absence of SOS induction, artificial expression of the YneA protein using an IPTG‐inducible promoter resulted in cell elongation. Disruption of yneA significantly reduced cell elongation after the induction of the SOS response by mitomycin C in dinR+ cells. These results indicate that the YneA protein is responsible for cell division suppression during the SOS response in B. subtilis. Localization of the FtsZ protein to the cell division site was reduced in dinR‐disrupted or yneA‐expressing cells, further suggesting that the YneA protein suppresses cell division through the suppression of FtsZ ring formation. Interestingly, the B. subtilis YneA protein is structurally and phylogenetically unrelated to its functional counterpart in Escherichia coli, SulA.
Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus
Authors: Figge Rainer M. , Easter Jesse , Gober James W. 
Published: March 2003

SummaryIn Caulobacter crescentus the partitioning proteins ParA and ParB operate a molecular switch that couples chromosome partitioning to cytokinesis. Homologues of these proteins have been shown to be important for the stable inheritance of F‐plasmids and the prophage form of bacteriophage P1. In C. crescentus, ParB binds to sequences adjacent to the origin of replication and is required for the initiation of cell division. Additionally, ParB influences the nucleotide‐bound state of ParA by acting as a nucleotide exchange factor. Here we have performed a genetic analysis of the chromosome partitioning protein ParB. We show that C. crescentus ParB, like its plasmid homologues, is composed of three domains: a carboxyl‐terminal dimerization domain; a central DNA‐binding, helix–turn–helix domain; and an amino‐terminal domain required for the interaction with ParA. In vivo expression of amino‐terminally deleted parB alleles has a dominant lethal effect resulting in the inhibition of cell division. Fluorescent in situ hybridization experiments indicate that this phenotype is not caused by a chromosome partitioning defect, but by the reversal of the amounts of ATP‐ versus ADP‐ bound ParA inside the cell. We present evidence suggesting that amino‐terminally truncated and full‐length, wild‐type ParB form heterodimers which fail to interact with ParA, thereby reversing the intracellular ParA‐ATP to ParA‐ADP ratio. We hypothesize that the amino‐terminus of ParB is required to regulate the nucleotide exchange of ParA which, in turn, regulates the initiation of cell division.
The MAPKK kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in the homothallic fungus Aspergillus nidulans
Authors: Wei Huijun , Requena Natalia , Fischer Reinhard 
Published: March 2003

SummaryEnvironmental signals can be transduced into intracellular responses by the action of MAP kinase cascades. Sequential phosphorylation results in the transient activation of a MAP kinase, which in turn activates certain transcription factors and thus a set of pathway‐specific genes. Many steps in this cascade are conserved, and homologues have been discovered from yeast to human. We have characterized the MAPKK kinase, SteC, a homologue of Saccharomyces cerevisiae Ste11, in the filamentous fungus Aspergillus nidulans. The 886‐amino‐acid‐long protein shares the highest similarity to Neurospora crassa Nrc‐1. Deletion of the gene in A. nidulans results in a slower growth rate, the formation of more branched hyphae, altered conidiophore morphology, an inhibition of heterokaryon formation and a block of cleistothecium development. The gene is transcriptionally activated during asexual development and controls the phosphorylation of two putative MAP kinases.
A role for cerebrovascular disease in determining the pattern of β‐amyloid deposition in Alzheimer's disease
Authors: Yow H. Y. , Weller R. O. 
Published: March 2003

Introduction:  The pattern of β‐amyloid (Aβ) deposition in cerebral amyloid angiopathy (CAA) in Alzheimer's disease (AD) suggests that Aβ is eliminated along perivascular interstitial fluid drainage pathways. The age at which accumulation of Aβ in the brain becomes significant in AD coincides with the development of cerebrovascular disease (CVD) and increasing rigidity of artery walls through arteriosclerosis.Hypothesis:  The distribution of Aβ deposits in Alzheimer brains is influenced by CVD.Materials and methods:  Paraffin sections of frontal, parietal, temporal and occipital lobes from six cases with Alzheimer's disease were stained for reticulin and by immunocytochemistry for Aβ and GFAP.Results:  Three distinct patterns of Aβ deposition were seen in relation to cortical blood vessels: (i) early deposition of Aβ in cortical parenchyma was related to individual cortical arterial territories; (ii) severe capillary amyloid angiopathy and an absence of Aβ plaques were associated with thromboembolic occlusion of the feeding artery; (iii) Aβ plaques were abundant in areas devoid of capillary amyloid angiopathy.Conclusions:  The pattern of Aβ deposition corresponds to individual intracortical arterial territories, and hence periarterial ISF drainage territories. Cerebrovascular disease influences the pattern of Aβ deposition within brain parenchyma and in vessel walls in individual arterial territories. Failure of periarterial drainage of Aβ from the brain may be related to cerebrovascular disease and is possibly due to altered arterial pulsations.