bluuebunny/crossref_metadata_2025_split huggingface dataset:
Defective cell wall synthesis in Streptococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator
Authors: Ng Wai‐Leung , Kazmierczak Krystyna M. , Winkler Malcolm E. 
Published: August 2004

SummaryPcsB is a protein of unknown function(s) that influences the cell morphology of several pathogenic species of streptococcus. PcsB contains a CHAP (cysteine, histidine‐dependent amidohydrolase/peptidase) domain found in bacterial murein hydrolases; however, direct links between steps in cell wall biosynthesis and PcsB function(s) have not been demonstrated. We show here that pcsB is essential in the human respiratory pathogen, Streptococcus pneumoniae, that depletion of PcsB is bacteriostatic and that alanine substitutions in the conserved cysteine and histidine residues of the CHAP domain appear to be lethal. We stained wild‐type parent and mutant bacteria deficient in expression of PcsB with fluorescent vancomycin and DAPI to determine patterns of cell wall synthesis and nucleoid segregation respectively. The wild‐type parent strain exhibited ordered, simultaneous septal and equatorial cell wall synthesis. In contrast, reduced expression of PcsB resulted in formation of long chains of cells in which peptidoglycan synthesis occurred at nearly every division septum and cell equator. Severe depletion of PcsB led to abnormal, uncontrolled cell wall synthesis at misplaced septa and around large cells. Together, these physiological properties are consistent with a role for PcsB as a murein hydrolase that balances the extent of cell wall synthesis in S. pneumoniae. Finally, we show that the defects in morphology and cell wall synthesis that result from depletion of PcsB strongly resemble those caused by depletion of the essential VicRK two component regulatory system (TCS). This result and the essentiality of pcsB support the hypothesis that the essentiality of the  VicRK TCS results from its positive regulation of PcsB expression.
GGDEF and EAL domains inversely regulate cyclic di‐GMP levels and transition from sessility to motility
Authors: Simm Roger , Morr Michael , Kader Abdul , Nimtz Manfred , Römling Ute 
Published: August 2004

SummaryCyclic nucleotides represent second messenger molecules in all kingdoms of life. In bacteria, mass sequencing of genomes detected the highly abundant protein domains GGDEF and EAL. We show here that the GGDEF and EAL domains are involved in the turnover of cyclic‐di‐GMP (c‐di‐GMP) in vivo whereby the GGDEF domain stimulates c‐di‐GMP production and the EAL domain c‐di‐GMP degradation. Thus, most probably, GGDEF domains function as c‐di‐GMP cyclase and EAL domains as phosphdiesterase. We further show that, in the pathogenic organism Salmonella enterica serovar Typhimurium, the nosocomial pathogen Pseudomonas aeruginosa and the commensal species Escherichia coli, GGDEF and EAL domains mediate similar phenotypic changes related to the transition between sessility and motility. Thus, the data suggest that c‐di‐GMP is a novel global second messenger in bacteria the metabolism of which is controlled by GGDEF and EAL domain proteins.
MicroReview: The role of Plasmodium falciparum var genes in malaria in pregnancy
Authors: Rowe J. A. , Kyes S. A. 
Published: August 2004

SummarySequestration of Plasmodium falciparum‐infected erythrocytes in the placenta is responsible for many of the harmful effects of malaria during pregnancy. Sequestration occurs as a result of parasite adhesion molecules expressed on the surface of infected erythrocytes binding to host receptors in the placenta such as chondroitin sulphate A (CSA). Identification of the parasite ligand(s) responsible for placental adhesion could lead to the development of a vaccine to induce antibodies  to  prevent  placental  sequestration.  Such a vaccine would reduce the maternal anaemia and infant deaths that are associated with malaria in pregnancy. Current research indicates that the parasite ligands mediating placental adhesion may be members of the P. falciparum variant surface antigen family PfEMP1, encoded by var genes. Two relatively well‐conserved subfamilies of var genes have been implicated in placental adhesion, however, their role remains controversial. This review examines the evidence for and against the involvement of var genes in placental adhesion, and considers whether the most appropriate vaccine candidates have yet been identified.
Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus
Authors: Colebatch Gillian , Desbrosses Guilhem , Ott Thomas , Krusell Lene , Montanari Ombretta , Kloska Sebastian , Kopka Joachim , Udvardi Michael K. 
Published: July 2004

SummaryResearch on legume nodule metabolism has contributed greatly to our knowledge of primary carbon and nitrogen metabolism in plants in general, and in symbiotic nitrogen fixation in particular. However, most previous studies focused on one or a few genes/enzymes involved in selected metabolic pathways in many different legume species. We utilized the tools of transcriptomics and metabolomics to obtain an unprecedented overview of the metabolic differentiation that results from nodule development in the model legume, Lotus japonicus. Using an array of more than 5000 nodule cDNA clones, representing 2500 different genes, we identified approximately 860 genes that were more highly expressed in nodules than in roots. One‐third of these are involved in metabolism and transport, and over 100 encode proteins that are likely to be involved in signalling, or regulation of gene expression at the transcriptional or post‐transcriptional level. Several metabolic pathways appeared to be co‐ordinately upregulated in nodules, including glycolysis, CO2 fixation, amino acid biosynthesis, and purine, haem, and redox metabolism. Insight into the physiological conditions that prevail within nodules was obtained from specific sets of induced genes. In addition to the expected signs of hypoxia, numerous indications were obtained that nodule cells also experience P‐limitation and osmotic stress. Several potential regulators of these stress responses were identified. Metabolite profiling by gas chromatography coupled to mass spectrometry revealed a distinct metabolic phenotype for nodules that reflected the global changes in metabolism inferred from transcriptome analysis.
Metabolic engineering of ketocarotenoid formation in higher plants
Authors: Ralley Louise , Enfissi Eugenia M.A. , Misawa Norihiko , Schuch Wolfgang , Bramley Peter M. , Fraser Paul D. 
Published: July 2004

SummaryAlthough higher plants synthesize carotenoids, they do not possess the ability to form ketocarotenoids. In order to generate higher plants capable of synthesizing combinations of ketolated and hydroxylated carotenoids the genes responsible for the carotene 4,4′ oxygenase and 3,3′ hydroxylase have been transformed into tomato and tobacco. The gene products were produced as a polyprotein. Subsequent cleavage of the polyprotein, targeting of the two enzymes to the plastid and enzyme activities have been shown for both gene products. Metabolite profiling has shown the formation of ketolated carotenoids from β‐carotene and its hydroxylated intermediates in tobacco and tomato leaf. In the nectary tissues of tobacco flowers a quantitative increase (10‐fold) as well as compositional changes were evident, including the presence of astaxanthin, canthaxanthin and 4‐ketozeaxanthin. Interestingly, in this tissue the newly formed carotenoids resided predominantly as esters. These data are discussed in terms of metabolic engineering of carotenoids and their sequestration in higher plant tissues.
Isolation and identification of phosphatidic acid targets from plants
Authors: Testerink Christa , Dekker Henk L. , Lim Ze‐Yi , Johns Melloney K. , Holmes Andrew B. , de Koster Chris G. , Ktistakis Nicholas T. , Munnik Teun 
Published: July 2004

SummaryPhosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress‐signalling pathways and is formed in response to wounding, osmotic stress, cold stress, pathogen elicitors, Nod factors, ethylene and abscisic acid. How PA exerts its effects is still unknown, mainly because of the lack of characterized PA targets. In an approach to isolate such targets we have used PA‐affinity chromatography. Several PA‐binding proteins were present in the soluble fraction of tomato andArabidopsiscells. Using mass spectrometric analysis, several of these proteins, including Hsp90, 14‐3‐3 proteins, an SnRK2 serine/threonine protein kinase and the PP2A regulatory subunit RCN1 could be identified. As an example, the binding of one major PA‐binding protein, phosphoenolpyruvate carboxylase (PEPC), was characterized further. Competition experiments with different phospholipids confirmed specificity for PA. Hypo‐osmotic treatment of the cells increased the amount of PEPC that bound the PA beads without increasing the absolute amount of PEPC. This suggests that PEPC's affinity for PA had increased. The work shows that PA‐affinity chromatography/mass spectrometry is an effective way to isolate and identify PA‐binding proteins from plants.
Functional interactions between a glutamine synthetase promoter and MYB proteins
Authors: Gómez‐Maldonado Josefa , Avila Concepción , de la Torre Fernando , Cañas Rafael , Cánovas Francisco M. , Campbell Malcolm M. 
Published: July 2004

SummaryIn Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis‐acting elements, known as AC elements. Pine nuclear proteins bound these AC element‐rich regions in a tissue‐specific manner. As previous experiments had shown that R2R3‐MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co‐regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.
The S haplotype‐specific F‐box protein gene, SFB, is defective in self‐compatible haplotypes of Prunus avium and P. mume
Authors: Ushijima Koichiro , Yamane Hisayo , Watari Akiko , Kakehi Eiko , Ikeda Kazuo , Hauck Nathanael R. , Iezzoni Amy F. , Tao Ryutaro 
Published: July 2004

SummaryMany Prunus species, including sweet cherry and Japanese apricot, of the Rosaceae, display an S‐RNase‐based gametophytic self‐incompatibility (GSI). The specificity of this outcrossing mechanism is determined by a minimum of two genes that are located in a multigene complex, termed the S locus, which controls the pistil and pollen specificities. SFB, a gene located in the S locus region, encodes an F‐box protein that has appropriate S haplotype‐specific variation to be the pollen determinant in the self‐incompatibility reaction. This study characterizes SFBs of two self‐compatible (SC) haplotypes, S4′ and Sf, of Prunus. S4′ of sweet cherry is a pollen‐part mutant (PPM) that was produced by X‐ray irradiation, while Sf of Japanese apricot is a naturally occurring SC haplotype that is considered to be a PPM. DNA sequence analysis revealed defects in both SFB4′ and SFBf. A 4 bp deletion upstream from the HVa coding region of SFB4′ causes a frame‐shift that produces transcripts of a defective SFB lacking the two hypervariable regions, HVa and HVb. Similarly, the presence of a 6.8 kbp insertion in the middle of the SFBf coding region leads to transcripts for a defective SFB lacking the C‐terminal half that contains HVa and HVb. As all reported SFBs of functional S haplotypes encode intact SFB, the fact that the partial loss‐of‐function mutations in SFB are present in SC mutant haplotypes of Prunus provides additional evidence that SFB is the pollen S gene in GSI in Prunus.
From pollen tubes to infection threads: recruitment of Medicago floral pectic genes for symbiosis
Authors: Rodríguez‐Llorente Ignacio D. , Pérez‐Hormaeche Javier , Mounadi Kaoutar El , Dary Mohammed , Caviedes Miguel A. , Cosson Viviane , Kondorosi Adam , Ratet Pascal , Palomares Antonio J. 
Published: July 2004

SummaryWhile the biology of nitrogen‐fixing root nodules has been extensively studied, little is known about the evolutionary events that predisposed legume plants to form symbiosis with rhizobia. We have studied the presence and the expression of two pectic gene families in Medicago, polygalacturonases (PGs) and pectin methyl esterases (PMEs) during the early steps of the Sinorhizobium meliloti–Medicago interaction and compared them with related pollen‐specific genes. First, we have compared the expression of MsPG3, a PG gene specifically expressed during the symbiotic interaction, with the expression of MsPG11, a highly homologous pollen‐specific gene, using promoter‐gus fusions in transgenic M. truncatula and tobacco plants. These results demonstrated that the symbiotic promoter functions as a pollen‐specific promoter in the non‐legume host. Second, we have identified the presence of a gene family of at least eight differentially expressed PMEs in Medicago. One subfamily is represented by one symbiotic gene (MtPER) and two pollen‐expressed genes (MtPEF1 and MtPEF2) that are clustered in the M. truncatula genome. The promoter‐gus studies presented in this work and the homology between plant PGs, together with the analysis of the PME locus structure and MtPER expression studies, suggest that the symbiotic MsPG3 and MtPER could have as ancestors pollen‐expressed genes involved in polar tip growth processes during pollen tube elongation. Moreover, they could have been recruited after gene duplication in the symbiotic interaction to facilitate polar tip growth during infection thread formation.
Transcriptional profiling by cDNA‐AFLP and microarray analysis reveals novel insights into the early response to ethylene in Arabidopsis
Authors: De Paepe Annelies , Vuylsteke Marnik , Van Hummelen Paul , Zabeau Marc , Van Der Straeten Dominique 
Published: July 2004

SummaryA comprehensive transcriptome analysis by means of cDNA‐amplified fragment length polymorphism (AFLP) and cDNA‐microarray technology was performed in order to gain further understanding of the molecular mechanisms of immediate transcriptional response to ethylene. Col‐0 plants were treated with exogenous ethylene and sampled at six different time‐points ranging from 10 min until 6 h. In order to isolate truly ethylene‐responsive genes, both the ethylene‐insensitive mutant ein2‐1 and the constitutive mutant (ctr1‐1) were analysed in parallel by cDNA‐AFLP while ein2‐1 was included for the microarray experiment. Out of the cDNA‐transcript profiling covering about 5% of the Arabidopsis transcriptome, 46 ethylene‐responsive genes were isolated, falling in different classes of expression pattern and including a number of novel genes. Out of the 6008 genes present on the chip, 214 genes were significantly (α = 0.001) differentially expressed between Col‐0 and ein2‐1 over time. Cluster analysis and functional grouping of co‐regulated genes allowed to determine the major ethylene‐regulated classes of genes. In particular, a large number of genes involved in cell rescue, disease and defence mechanisms were identified as early ethylene‐regulated genes. Furthermore, the data provide insight into the role of protein degradation in ethylene signalling and ethylene‐regulated transcription and protein fate. Novel interactions between ethylene response and responses to several other signals have been identified by this study. Of particular interest is the overlap between ethylene response and responses to abscisic acid, sugar and auxin. In conclusion, the data provide unique insight into early regulatory steps of ethylene response.
Kinetics of labelling of organic and amino acids in potato tubers by gas chromatography‐mass spectrometry following incubation in 13C labelled isotopes
Authors: Roessner‐Tunali Ute , Liu JunLi , Leisse Andrea , Balbo Ilse , Perez‐Melis Alicia , Willmitzer Lothar , Fernie Alisdair R. 
Published: July 2004

SummaryMetabolic pathways of primary metabolism of discs isolated from potato tubers were evaluated by the use of a gas chromatography‐mass spectrometry (GC‐MS) method generated specifically for this purpose. After testing several possible methods including chemical ionization, it was decided for reasons of sensitivity, reproducibility and speed to use electron impact ionization‐based GC‐MS analysis. The specific labelling and label accumulation of over 30 metabolites including a broad number of sugars, organic and amino acids was analysed following the incubation of tuber discs in [U‐13C]glucose. The reproducibility of this method was similar to that found for other GC‐MS‐based analyses and comparison of flux estimates from this method with those obtained from parallel, yet less comprehensive, radiolabel experiments revealed close agreement. Therefore, the novel method allows quantitatively evaluation of a broad range of metabolic pathways without the need for laborious (and potentially inaccurate), chemical fractionation procedures commonly used in the estimation of fluxes following incubation in radiolabelled substrates. As a first experiment the GC‐MS method has been applied to compare the metabolism of wild type and wel‐characterized transgenic potato tubers exhibiting an enhanced sucrose mobilization. The fact that this method is able to rapidly yield further comprehensive information into primary metabolism illustrates its power as a further phenotyping tool for the analysis of plant metabolism.
Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure
Authors: Stahl Yvonne , Coates Steve , Bryce James H. , Morris Peter C. 
Published: July 2004

SummaryThe barley protein limit dextrinase inhibitor (LDI), structurally related to the α‐amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, α‐ and β‐amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B‐type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10–15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.
Molecular events in senescing Arabidopsis leaves
Authors: Lin Ji‐Feng , Wu Shu‐Hsing 
Published: July 2004

SummarySenescence is the final stage of leaf development. Although it means the loss of vitality of leaf tissue, leaf senescence is tightly controlled by the development to increase the fitness of the whole plant. The molecular mechanisms regulating the induction and progression of leaf senescence are complex. We used a cDNA microarray, containing 11 500 Arabidopsis DNA elements, and the whole‐genome Arabidopsis ATH1 Genome Array to examine global gene expression in dark‐induced leaf senescence. By monitoring the gene expression patterns at carefully chosen time points, with three biological replicates each time, we identified thousands of up‐ or down‐regulated genes involved in dark‐induced senescence. These genes were clustered and categorized according to their expression patterns and responsiveness to dark treatment. Genes with different expression kinetics were classified according to different biological processes. Genes showing significant alteration of expression patterns in all available biochemical pathways were plotted to envision the molecular events occurring in the processes examined. With the expression data, we postulated an innovative biochemical pathway involving pyruvate orthophosphate dikinase in generating asparagine for nitrogen remobilization in dark‐treated leaves. We also surveyed the alteration in expression of Arabidopsis transcription factor genes and established an apparent association of GRAS, bZIP, WRKY, NAC, and C2H2 transcription factor families with leaf senescence.
Phosphate transport in Arabidopsis: Pht1;1 and Pht1;4 play a major role in phosphate acquisition from both low‐ and high‐phosphate environments
Authors: Shin Heungsop , Shin Hwa‐Soo , Dewbre Gary R. , Harrison Maria J. 
Published: July 2004

SummaryOf the mineral nutrients essential for plant growth, phosphorus plays the widest diversity of roles and a lack of phosphorus has profound effects on cellular metabolism. At least eight members of the Arabidopsis Pht1 phosphate (Pi) transporter family are expressed in roots and Pht1;1 and Pht1;4 show the highest transcript levels. The spatial and temporal expression patterns of these two genes show extensive overlap. To elucidate the in planta roles of Pht1;1 and Pht1;4, we identified loss‐of‐function mutants and also created a double mutant, lacking both Pht1;1 and Pht1;4. Consistent with their spatial expression patterns, membrane location and designation as high‐affinity Pi transporters, Pht1;1 and Pht1;4 contribute to Pi transport in roots during growth under low‐Pi conditions. In addition, during growth under high‐Pi conditions, the double mutant shows a 75% reduction in Pi uptake capacity relative to wildtype. Thus, Pht1;1 and Pht1;4 play significant roles in Pi acquisition from both low‐ and high‐Pi environments.