bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipopolysaccharide O antigen status of Yersinia enterocolitica O:8 is essential for virulence and absence of O antigen affects the expression of other Yersinia virulence factors
Authors: Bengoechea José Antonio , Najdenski Hristo , Skurnik Mikael 
Published: April 2004

SummaryLipopolysaccharide (LPS) is the major component of the outer membrane of Gram‐negative bacteria. Although much attention has been given to the biological effects of its lipid A portion, a great body of evidence indicates that its O chain polysaccharide (O antigen) portion plays an important role in the bacterium–host interplay. In this work we have studied in‐depth the role of the O antigen in Yersinia enterocolitica serotype O:8 pathogenesis. We made a detailed virulence analysis of three mutants having different O antigen phenotypes: (i) LPS with no O antigen (rough mutant); (ii) LPS with one O unit (semirough mutant) and (iii) LPS with random distribution of O antigen chain lengths. We demonstrated that these LPS O antigen mutants were attenuated in virulence regardless of the infection route used. Co‐infection experiments revealed that the rough and semirough mutants were severely impaired in their ability to colonize the Peyer's patches and in contrast to the wild‐type strain they did not colonize spleen and liver. The mutant with random distribution of O antigen chain lengths, however, survived better but started to be cleared from mouse organs after 8 days. As an explanation to this attenuation we present here evidence that other Yersinia virulence factors depend on the presence of O antigen for their proper function and/or expression. We demonstrated that in the rough mutant: (i) the YadA function but not its expression was altered; (ii) Ail was not expressed and (iii) inv expression was downregulated. On the other hand, expression of flhDC, the flagellar master regulatory operon, was upregulated in this mutant with a concomitant increase in the production of flagellins. Finally, expression of yplA, encoding for the Yersinia phospholipase A, was also upregulated accompanied by an increased flagellar type III secretion system mediated secretion of YplA to culture medium. Together these findings suggest that the absence of O antigen in the outer membrane of Yersinia either directly or indirectly, for example through a cellular or membrane stress, could act as a regulatory signal.
Identification of Campylobacter jejuni genes involved in commensal colonization of the chick gastrointestinal tract
Authors: Hendrixson David R. , DiRita Victor J. 
Published: April 2004

Summary Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans in developed countries throughout the world. This bacterium frequently promotes a commensal lifestyle in the gastrointestinal tracts of many animals including birds and consumption or handling of poultry meats is a prevalent source of C. jejuni for infection in humans. To understand how the bacterium promotes commensalism, we used signature‐tagged transposon mutagenesis and identified 29 mutants representing 22 different genes of C. jejuni strain 81–176 involved in colonization of the chick gastrointestinal tract. Among the determinants identified were two adjacent genes, one encoding a methyl‐accepting chemotaxis protein (MCP), presumably required for proper chemotaxis to a specific environmental component, and another gene encoding a putative cytochrome c peroxidase that may function to reduce periplasmic hydrogen peroxide stress during in vivo growth. Deletion of either gene resulted in attenuation for growth throughout the gastrointestinal tract. Further examination of 10 other putative MCPs or MCP‐domain containing proteins of C. jejuni revealed one other required for wild‐type levels of caecal colonization. This study represents one of the first genetic screens focusing on the bacterial requirements necessary for promoting commensalism in a vertebrate host.
Structural characterization of extracellular polysaccharides of Azorhizobium caulinodans and importance for nodule initiation on Sesbania rostrata
Authors: D’Haeze Wim , Glushka John , De Rycke Riet , Holsters Marcelle , Carlson Russell W. 
Published: April 2004

SummaryDuring lateral root base nodulation, the microsymbiont Azorhizobium caulinodans enters its host plant, Sesbania rostrata, via the formation of outer cortical infection pockets, a process that is characterized by a massive production of H2O2. Infection threads guide bacteria from infection pockets towards nodule primordia. Previously, two mutants were constructed that produce lipopolysaccharides (LPSs) similar to one another but different from the wild‐type LPS, and that are affected in extracellular polysaccharide (EPS) production. Mutant ORS571‐X15 was blocked at the infection pocket stage and unable to produce EPS. The other mutant, ORS571‐oac2, was impaired in the release from infection threads and was surrounded by a thin layer of EPS in comparison to the wild‐type strain that produced massive amounts of EPS. Structural characterization revealed that EPS purified from cultured and nodule bacteria was a linear homopolysaccharide of α‐1,3‐linked 4,6‐O‐(1‐carboxyethylidene)‐d‐galactosyl residues. In situ H2O2 localization demonstrated that increased EPS production during early stages of invasion prevented the incorporation of H2O2 inside the bacteria, suggesting a role for EPS in protecting the microsymbiont against H2O2. In addition, ex planta assays confirmed a positive correlation between increased EPS production and enhanced protection against H2O2.
Origins and evolution of isoprenoid lipid biosynthesis in archaea
Authors: Boucher Yan , Kamekura Masahiro , Doolittle W. Ford 
Published: April 2004

SummaryA characteristic feature of the domain archaea are the lipids forming the hydrophobic core of their cell membrane. These unique lipids are composed of iso‐prenoid side‐chains stereospecifically ether linked to sn‐glycerol‐1‐phosphate. Recently, considerable progress has been made in characterizing the enzymes responsible for the synthesis of archaeal lipids. However, little is known about their evolution. To better understand how this unique biosynthetic apparatus came to be, large‐scale database surveys and phylogenetic analyses were performed. All characterized enzymes involved in the biosynthesis of isoprenoid side‐chains and the glycerol phosphate backbone along with their assembly in ether lipids were included in these analyses. The sequence data available in public databases was complemented by an in‐depth sampling of isoprenoid lipid biosynthesis genes from multiple genera of the archaeal order Halobacteriales, allowing us to look at the evolution of these enzymes on a smaller phylogenetic scale. This investigation of the isoprenoid biosynthesis apparatus of archaea on small and large phylogenetic scales reveals that it evolved through a combination of evolutionary processes, including the co‐option of ancestral enzymes, modification of enzymatic specificity, orthologous and non‐orthologous gene displacement, integration of components from eukaryotes and bacteria and lateral gene transfer within and between archaeal orders.
Requirement for RecFOR‐mediated recombination in priA mutant
Authors: Grompone Gianfranco , Sanchez Nicolas , Dusko Ehrlich S. , Michel Bénédicte 
Published: April 2004

SummaryRestart of arrested replication forks is an important process and PriA, the main Escherichia coli replication restart protein, is essential for viability under any condition that increases the frequency of fork arrest. In priA mutant, replication forks are arrested by spontaneously occurring roadblocks and blocked replication forks persist as a result of the defect in replication restart. In the present work, we analysed how recombination proteins contribute to the viability of the priA mutant. RecFOR‐mediated homologous recombination occurs in a large fraction of priA mutant cells, indicating a frequent formation of DNA single strand gaps and their recombinational repair. This high level of homologous recombination renders the proteins that resolve Holliday junctions recombination intermediates essential for viability. When homologous recombination is blocked at early steps by recFOR or recA inactivation, exonuclease V‐mediated DNA degradation is required for full viability of priA mutants, indicating that unrepaired gaps are broken and that DNA degradation of the broken DNA allows the formation of viable cells. Models for the formation of single strand DNA gaps consequently to a replication restart defect and for gap processing are proposed.
A lysR‐type regulator is involved in the negative regulation of genes encoding selenium‐free hydrogenases in the archaeon Methanococcus voltae
Authors: Sun Junsong , Klein Albrecht 
Published: April 2004

SummaryThe archaeon Methanococcus voltae encodes two pairs of NiFe‐hydrogenase isoenzymes. One hydrogenase of each pair contains selenium in the active site, whereas the other one is selenium‐free. The gene groups for the selenium‐free hydrogenases, called vhc and frc, are linked by a common intergenic region. They are only transcribed under selenium limitation. A protein binding to a negative regulatory element involved in the regulation of the two operons was purified by DNA‐affinity chromatography. Through the identification of the corresponding gene the protein was found to be a LysR‐type regulator. It was named HrsM (hydrogenase gene regulator, selenium dependent in M. voltae). hrsM knockout mutants constitutively transcribed the vhc and frc operons in the presence of selenium. A putative HrsM binding site was also detected in the intergenic region in front of the hrsM gene. Northern blot analysis indicated that the hrsM gene might be autoregulated.
Bacterial mitosis: partitioning protein ParA oscillates in spiral‐shaped structures and positions plasmids at mid‐cell
Authors: Ebersbach Gitte , Gerdes Kenn 
Published: April 2004

SummaryThe par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis‐acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells with a single plasmid focus, the focus located preferentially at mid‐cell. In cells with two foci, these located at quarter‐cell positions. In the absence of ParB and parC1/parC2, ParA‐GFP formed stationary helices extending from one end of the nucleoid to the other. In the presence of ParB and parC1/parC2, ParA‐GFP oscillated in spiral‐shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid‐cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid and subsequently separate them into daughter cells.
Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor
Authors: Ermolaeva Svetlana , Novella Susana , Vega Yolanda , Ripio Maria‐Teresa , Scortti Mariela , Vázquez‐Boland José A. 
Published: April 2004

SummaryVirulence genes from the facultative intracellular pathogen Listeria monocytogenes are controlled by the transcriptional regulator PrfA. Although PrfA synthesis is activated at 37°C, PrfA‐dependent expression remains low in rich medium. However, a strong induction of the PrfA regulon is observed when L. monocytogenes is cultured in the presence of activated charcoal. Here, we show that the ‘charcoal effect’ results from the adsorption of a diffusible autorepressor substance released by L. monocytogenes during exponential growth. Analyses using an L. monocytogenes strain in which the prfA gene is expressed constitutively at 37°C from a plasmid indicate that the autoregulatory substance represses PrfA‐dependent expression by inhibiting PrfA activity. PrfA presumably functions via an allosteric activation mechanism. The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA. Our data indicate that the listerial autorepressor is a low‐molecular‐weight hydrophobic substance. We suggest that this diffusible substance mediates a quorum‐sensing mechanism by which L. monocytogenes restricts the expression of its PrfA virulence regulon. This autoregulatory pathway could serve L. monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37°C. It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA‐dependent virulence factors in the PrfA‐activating cytosolic compartment.
Dissection of the host range of the fungal plant pathogen Alternaria alternata by modification of secondary metabolism
Authors: Ito Kaoru , Tanaka Takayoshi , Hatta Rieko , Yamamoto Mikihiro , Akimitsu Kazuya , Tsuge Takashi 
Published: April 2004

SummaryThe filamentous fungus Alternaria alternata contains seven pathogenic variants (pathotypes), which produce different host‐specific toxins and cause diseases on different plants. The strawberry pathotype produces host‐specific AF‐toxin and causes Alternaria black spot of strawberry. This pathotype is also pathogenic to Japanese pear cultivars susceptible to the Japanese pear pathotype that produces AK‐toxin. The strawberry pathotype produces two related molecular species, AF‐toxins I and II: toxin I is toxic to both strawberry and pear, and toxin II is toxic only to pear. Previously, we isolated a cosmid clone pcAFT‐1 from the strawberry pathotype that contains three genes involved in AF‐toxin biosynthesis. Here, we have identified a new gene, designated AFTS1, from pcAFT‐1. AFTS1 encodes a protein with similarity to enzymes of the aldo‐ketoreductase superfamily. Targeted mutation of AFTS1 diminished the host range of the strawberry pathotype: ΔaftS1 mutants were pathogenic to pear, but not to strawberry, as is the Japanese pear pathotype. These mutants were found to produce AF‐toxin II, but not AF‐toxin I. These data represent a novel example of how the host range of a plant pathogenic fungus can be restricted by modification of secondary metabolism.
A single‐amino acid substitution in the sixth leucine‐rich repeat of barley MLA6 and MLA13 alleviates dependence on RAR1 for disease resistance signaling
Authors: Halterman Dennis A. , Wise Roger P. 
Published: April 2004

SummaryInteractions between barley and the powdery mildew pathogen, Blumeria graminis f. sp. hordei, (Bgh) are determined by unique combinations of host resistance genes, designated Mildew‐resistance locus (Ml), and cognate pathogen avirulence genes. These interactions occur both dependent and independent of Rar1 (required for Mla12 resistance) and Sgt1 (Suppressor of G‐two allele of skp1), which are differentially required for diverse plant disease‐resistance pathways. We have isolated two new functional Mla alleles, Rar1‐independent Mla7 and Rar1‐dependent Mla10, as well as the Mla paralogs, Mla6‐2 and Mla13‐2. Utilizing the inherent diversity amongst Mla‐encoded proteins, we identified the only two amino acids exclusively conserved in RAR1‐dependent MLA6, MLA10, MLA12, and MLA13 that differ at the corresponding position in RAR1‐independent MLA1 and MLA7. Two‐ and three‐dimensional modeling places these residues on a predicted surface of the sixth leucine‐rich repeat (LRR) domain at positions distinct from those within the β‐sheets hypothesized to determine resistance specificity. Site‐directed mutagenesis of these residues indicates that RAR1 independence requires the presence of an aspartate at position 721, as mutation of this residue to a structurally similar, but uncharged, asparagine did not alter RAR1 dependence. These results demonstrate that a single‐amino acid substitution in the sixth MLA LRR can alter host signaling but not resistance specificity to B. graminis.
Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development
Authors: Ye Rui , Yao Quan‐Hong , Xu Zhi‐Hong , Xue Hong‐Wei 
Published: April 2004

SummaryAn efficient yeast‐based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co‐activators). The system consists of two vectors: (i) a reporter vector (pG221) harboring the iso‐1‐cytochrome c (CYC1) core promoter and the β‐galactosidase (lacZ) gene; and (ii) a cDNA library construction vector (pYF503), which yields a library of plant peptides fused to the GAL4‐binding domain (GAL4‐BD). Expression of a peptide harboring the characteristics of a transcriptional activator leads to expression of lacZ, allowing for selection of relevant colonies. TFs during rice embryo development were isolated through this system. Approximately 200 confirmed positive colonies were obtained from screening 106 yeast colonies, and sequence analysis of conserved domains identified 75 independent cDNAs, 20 of which encoded plant TFs or co‐activators, including members of the APETALA2 (AP2)/ethylene‐responsive element‐binding protein (EREBP), MYB and growth‐regulating factor (GRF) families. Peptides encoded by 13 of the isolated cDNAs were classified as potential TFs or co‐activators because of the presence of conserved TF‐like domains. Additionally, 2, 11, and 13 clones encoded kinases, chromosome‐related proteins, and unknown proteins, respectively, while the remaining 16 cDNAs were associated with specific functions seemingly unrelated to TFs. Expression pattern analysis of selected TF‐encoding genes via RT‐PCR revealed that these genes were expressed during seed development, with differential transcription observed during various stages. This work provides informative hints for further study of the regulatory mechanism of rice seed development and illustrates an identification strategy that will be of practical value for the isolation of TFs and co‐activators associated with specific plant developmental processes.
Cytokinins play opposite roles in lateral root formation, and nematode and Rhizobial symbioses
Authors: Lohar Dasharath Prasad , Schaff Jennifer E. , Laskey James G. , Kieber Joseph J. , Bilyeu Kristin D. , Bird David McK. 
Published: April 2004

SummaryWe used the cytokinin‐responsive Arabidopsis response regulator (ARR)5 gene promoter fused to a β‐glucuronidase (GUS) reporter gene, and cytokinin oxidase (CKX) genes from Arabidopsis thaliana (AtCKX3) and maize (ZmCKX1) to investigate the roles of cytokinins in lateral root formation and symbiosis in Lotus japonicus. ARR5 expression was undetectable in the dividing initial cells at early stages of lateral root formation, but later we observed high expression in the base of the lateral root primordium. The root tip continues to express ARR5 during subsequent development of the lateral root. These results suggest a dynamic role for cytokinin in lateral root development. We observed ARR5 expression in curled/deformed root hairs, and also in nodule primordia in response to Rhizobial inoculation. This expression declined once the nodule emerged from the parent root. Root penetration and migration of root‐knot nematode (RKN) second‐stage larvae (L2) did not elevate ARR5 expression, but a high level of expression was induced when L2 reached the differentiating vascular bundle and during early stages of the nematode–plant interaction. ARR5 expression was specifically absent in mature giant cells (GCs), although dividing cells around the GCs continued to express this reporter. The same pattern was observed using a green fluorescent protein (GFP) reporter driven by the ARR5 promoter in tomato. Overexpression of CKX genes rendered the transgenic hairy roots resistant to exogenous application of the cytokinin [N6‐(Δ2 isopentenyl) adenine riboside] (iPR). CKX roots have significantly more lateral roots, but fewer nodules and nematode‐induced root galls per plant, than control hairy roots.
A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis
Authors: Schrick Kathrin , Fujioka Shozo , Takatsuto Suguru , Stierhof York‐Dieter , Stransky Harald , Yoshida Shigeo , Jürgens Gerd 
Published: April 2004

SummaryA crucial role for sterols in plant growth and development is underscored by the identification of three Arabidopsis sterol biosynthesis mutants that exhibit embryonic defects: fackel (fk), hydra1 (hyd1), and sterol methyltransferase 1/cephalopod (smt1/cph). We have taken a dual approach of sterol profiling and ultrastructural analysis to investigate the primary defects underlying the mutant phenotypes. Comprehensive gas chromatography (GC)–MS analysis of hyd1 in comparison to fk reveals an abnormal accumulation of unique sterol intermediates in each case. Sterol profiling of the fk hyd1 double mutant provides genetic evidence that FK C‐14 reductase acts upstream of HYD1 C‐8,7 isomerase. Despite distinct differences in sterol profiles, fk and hyd1 as well as smt1/cph share ultrastructural features such as incomplete cell walls and aberrant cell wall thickenings in embryonic and post‐embryonic tissues. The common defects are coupled with ectopic callose and lignin deposits. We show that all three mutants exhibit a deficiency in cellulose, but are not reduced in pectin and sugars of the cell wall and cytosol. The sterol biosynthesis inhibitors 15‐azasterol and fenpropimorph also cause cell wall gaps in dividing root cells and a reduction in bulk cellulose, corroborating that the cell wall abnormalities are due to altered sterol composition. Our results demonstrate that sterols are crucial for cellulose synthesis in the building of the plant cell wall.
The victorin‐induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity
Authors: Curtis Marc J. , Wolpert Thomas J. 
Published: April 2004

SummaryIn this study, we determined the timing of events associated with cell death induced by the host‐selective toxin, victorin. We show that the victorin‐induced collapse in mitochondrial transmembrane potential (Δψm), indicative of a mitochondrial permeability transition (MPT), on a per cell basis, did not occur simultaneously in the entire mitochondrial population. The loss of Δψm in a predominant population of mitochondria preceded cell shrinkage by 20–35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage. During and following cell shrinkage, tonoplast rupture did not occur, and membranes, including the plasma membrane and tonoplast, retained integrity. Ethylene signaling was implicated upstream of a victorin‐induced loss in mitochondrial motility and the collapse in Δψm. Results suggest that the victorin‐induced collapse in Δψm is a consequence of an MPT and that the timing of the victorin‐induced MPT is poised to influence the cell death response. The retention of plasma membrane and tonoplast integrity during cell shrinkage supports the interpretation that victorin induces an apoptotic‐like cell death response.