bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of the Escherichia coli Tat pathway in outer membrane integrity
Authors: Ize Bérengère , Stanley Nicola R. , Buchanan Grant , Palmer Tracy 
Published: May 2003

SummaryThe Escherichia coli Tat system serves to export folded proteins harbouring an N‐terminal twin‐arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin‐arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin‐arginine signal sequences in the genomes of other Gram‐negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.
The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function
Authors: Kraus Peter R. , Fox Deborah S. , Cox Gary M. , Heitman Joseph 
Published: May 2003

SummaryCell wall integrity is crucial for fungal growth, development and stress survival. In the model yeast Saccharomyces cerevisiae, the cell integrity Mpk1/Slt2 MAP kinase and calcineurin pathways monitor cell wall integrity and promote cell wall remodelling under stress conditions. We have identified the Cryptococcus neoformans homologue of the S. cerevisiae Mpk1/Slt2 MAP kinase and have characterized its role in the maintenance of cell integrity in response to elevated growth temperature and in the presence of cell wall synthesis inhibitors. C. neoformans Mpk1 is required for growth at 37°C in vitro, and this growth defect is suppressed by osmotic stabilization. C. neoformans mutants lacking Mpk1 are attenuated for virulence in the mouse model of cryptococcosis. Phosphorylation of Mpk1 is induced in response to perturbations of cell wall biosynthesis by the antifungal drugs nikkomycin Z (a chitin synthase inhibitor), caspofungin (a β‐1,3‐glucan synthase inhibitor), or FK506 (a calcineurin inhibitor), and mutants lacking Mpk1 display enhanced sensitivity to nikkomycin Z and caspofungin. Lastly, we show that calcineurin and Mpk1 play complementing roles in regulating cell integrity in C. neoformans. Our studies demonstrate that pharmacological inhibition of the cell integrity pathway would enhance the activity of antifungal drugs that target the cell wall.
RelE toxins from Bacteria and Archaea cleave mRNAs on translating ribosomes, which are rescued by tmRNA
Authors: Christensen Susanne K. , Gerdes Kenn 
Published: May 2003

SummaryRelE of Escherichia coli is a global inhibitor of translation that is activated by nutritional stress. Activation of RelE depends on Lon‐mediated degradation of RelB, the antagonist that neutralizes RelE. In vitro, RelE cleaves synthetic mRNAs positioned at the ribosomal A‐site. We show here that in vivo overexpression of RelE confers cleavage of mRNA and tmRNA in their coding regions. RelE‐mediated cleavage depended on translation of the RNAs and occurred at both sense and stop codons. RelE cleavage of mRNA and tmRNA was also induced by amino acid starvation. An ssrA deletion strain was hypersensitive to RelE, whereas overproduction of tmRNA counteracted RelE toxicity. After neutralization of RelE by RelB, rapid recovery of translation required tmRNA, indicating that tmRNA alleviated RelE toxicity by rescuing ribosomes stalled on damaged mRNAs. RelE proteins from Gram‐positive Bacteria and Archaea cleaved tmRNA with a pattern similar to that of E. coli RelE, suggesting that the function and target of RelE may be conserved across the prokaryotic domains.
The DEAD‐box RNA helicase SrmB is involved in the assembly of 50S ribosomal subunits in Escherichia coli
Authors: Charollais Julie , Pflieger Delphine , Vinh Joëlle , Dreyfus Marc , Iost Isabelle 
Published: May 2003

SummaryRibosome assembly in Escherichia coli involves 54 ribosomal proteins and three RNAs. Whereas functional subunits can be reconstituted in vitro from the isolated components, this process requires long incubation times and high temperatures compared with the in vivo situation, suggesting that non‐ribosomal factors facilitate assembly in vivo. Here, we show that SrmB, a putative DEAD‐box RNA helicase, is involved in ribosome assembly. The deletion of the srmB gene causes a slow‐growth phenotype at low temperature. Polysome profile analyses of the corresponding cells reveal a deficit in free 50S ribosomal subunits and the accumulation of a new particle sedimenting around 40S. Analysis of the ribosomal RNA and protein contents of the 40S particle indicates that it represents a large subunit that is incompletely assembled. In particular, it lacks L13, one of the five ribosomal proteins that are essential for the early assembly step in vitro. Sucrose gradient fractionation also shows that, in wild‐type cells, SrmB associates with a pre50S particle. From our results, we propose that SrmB is involved in an early step of 50S assembly that is necessary for the binding of L13. This step may consist of a structural rearrangement that, at low temperature, cannot occur without the assistance of this putative RNA helicase.
Surface localized glyceraldehyde‐3‐phosphate dehydrogenase of Mycoplasma genitalium binds mucin
Authors: Alvarez René A. , Blaylock Mark W. , Baseman Joel B. 
Published: May 2003

Summary Mycoplasma genitalium is the smallest known self‐replicating cell. It was first isolated from urethral specimens in individuals with non‐gonococcal urethritis and, more recently, from respiratory and synovial sites. Our laboratory has been interested in defining the mechanisms by which M. genitalium adheres to and colonizes host cell surfaces. In order to determine potential targets of adherence, we examined the interaction of M. genitalium with a primary component of the mucosal epithelial lining, mucin (Mn). Three Mn‐binding proteins (MnBPs) of M. genitalium were isolated by affinity chromatography. One of these proteins was identified by N‐terminal sequencing as glyceraldehyde‐3‐phosphate dehy‐drogenase (GAPDH). Antiserum raised against recombinant GAPDH blocked binding of intact biosynthetically labelled mycoplasmas to mucin by approximately 70%. Whole cell radioimmunoprecipitation indicated that GAPDH was surface‐accessible and surface localization of GAPDH was further verified by membrane fractionation and immunoelectron microscopy. The role of GAPDH as an adhesin to Mn not only provides insights into the organism's mechanisms of adherence and colonization but also into its ability to maximize its limited genome.
Proteomic studies of diauxic lag in the differentiating prokaryote Streptomyces coelicolor reveal a regulatory network of stress‐induced proteins and central metabolic enzymes
Authors: Novotna Jana , Vohradsky Jiri , Berndt Peter , Gramajo Hugo , Langen Hanno , Li Xin‐Ming , Minas Wolfgang , Orsaria Lelia , Roeder Daniel , Thompson Charles J. 
Published: May 2003

SummaryBacteria typically undergo intermittent periods of starvation and adaptation, emulated as diauxic growth in the laboratory. In association with growth arrest elicited by metabolic stress, the differentiating eubacterium Streptomyces coelicolor not only adapts its primary metabolism, but can also activate developmental programmes leading to morphogenesis and antibiotic biosynthesis. Here, we report combined proteomic and metabolomic data of S. coelicolor used to analyse global changes in gene expression during diauxic growth in a defined liquid medium. Cultures initially grew on glutamate, providing the nitrogen source and feeding carbon (as 2‐oxoglutarate) into the TCA cycle, followed by a diauxic delay allowing reorientation of metabolism and a second round of growth supported by NH4+, formed during prediauxic phase, and maltose, a glycolytic substrate. Cultures finally entered stationary phase as a result of nitrogen starvation. These four physiological states had previously been defined statistically by their distinct patterns of protein synthesis and heat shock responses. Together, these data demonstrated that the rates of synthesis of heat shock proteins are determined not only by temperature increase but also by the patterns and rates of metabolic flux in certain pathways. Synthesis profiles for metabolic‐ and stress‐induced proteins can now be interpreted by the identification of 204 spots (SWICZ database presented at http:proteom.biomed.cas.cz). Cluster analysis showed that the activity of central metabolic enzymes involved in glycolysis, the TCA cycle, starvation or proteolysis each displayed identifiable patterns of synthesis that logically underlie the metabolic state of the culture. Diauxic lag was accompanied by a structured regulatory programme involving the sequential activation of heat‐, salt‐, cold‐ and bacteriostatic antibiotic (pristinamycin I, PI)‐induced stimulons. Although stress stimulons presumably provide protection during environmental‐ or starvation‐induced stress, their identities did not reveal any coherent adaptive or developmental functions. These studies revealed interactive regulation of metabolic and stress response systems including some proteins known to support developmental programmes in S. coelicolor.
Substrate preference of stress‐activated phospholipase D in Chlamydomonas and its contribution to PA formation
Authors: Arisz Steven A. , Valianpour Fredoen , Van Gennip Albert H. , Munnik Teun 
Published: May 2003

SummaryIn response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G‐protein activation (1 µm mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization‐mass spectrometry (ESI‐MS/MS), which revealed that PE and NaCl‐induced PBut share a unique (18 : 1)2‐structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl‐induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress‐induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide‐specific fatty acids and molecular species in PA.
Sequence‐based alignment of sorghum chromosome 3 and rice chromosome 1 reveals extensive conservation of gene order and one major chromosomal rearrangement
Authors: Klein Patricia E. , Klein Robert R. , Vrebalov Julia , Mullet John E. 
Published: May 2003

SummaryThe completed rice genome sequence will accelerate progress on the identification and functional classification of biologically important genes and serve as an invaluable resource for the comparative analysis of grass genomes. In this study, methods were developed for sequence‐based alignment of sorghum and rice chromosomes and for refining the sorghum genetic/physical map based on the rice genome sequence. A framework of 135 BAC contigs spanning approximately 33 Mbp was anchored to sorghum chromosome 3. A limited number of sequences were collected from 118 of the BACs and subjected to blastx analysis to identify putative genes and blastn analysis to identify sequence matches to the rice genome. Extensive conservation of gene content and order between sorghum chromosome 3 and the homeologous rice chromosome 1 was observed. One large‐scale rearrangement was detected involving the inversion of an approximately 59 cM block of the short arm of sorghum chromosome 3. Several small‐scale changes in gene collinearity were detected, indicating that single genes and/or small clusters of genes have moved since the divergence of sorghum and rice. Additionally, the alignment of the sorghum physical map to the rice genome sequence allowed sequence‐assisted assembly of an approximately 1.6 Mbp sorghum BAC contig. This streamlined approach to high‐resolution genome alignment and map building will yield important information about the relationships between rice and sorghum genes and genomic segments and ultimately enhance our understanding of cereal genome structure and evolution.
The anticodon and the D‐domain sequences are essential determinants for plant cytosolic tRNAVal import into mitochondria
Authors: Delage Ludovic , Duchêne Anne‐Marie , Zaepfel Marlyse , Maréchal‐Drouard Laurence 
Published: May 2003

SummaryIn higher plants, one‐third to one‐half of the mitochondrial tRNAs are encoded in the nucleus and are imported into mitochondria. This process appears to be highly specific for some tRNAs, but the factors that interact with tRNAs before and/or during import, as well as the signals present on the tRNAs, still need to be identified. The rare experiments performed so far suggest that, besides the probable implication of aminoacyl‐tRNA synthetases, at least one additional import factor and/or structural features shared by imported tRNAs must be involved in plant mitochondrial tRNA import. To look for determinants that direct tRNA import into higher plant mitochondria, we have transformed BY2 tobacco cells with Arabidopsis thaliana cytosolic tRNAVal(AAC) carrying various mutations. The nucleotide replacements introduced in this naturally imported tRNA correspond to the anticodon and/or D‐domain of the non‐imported cytosolic tRNAMet–e. Unlike the wild‐type tRNAVal(AAC), a mutant tRNAVal carrying a methionine CAU anticodon that switches the aminoacylation of this tRNA from valine to methionine is not present in the mitochondrial fraction. Furthermore, mutant tRNAsVal carrying the D‐domain of the tRNAMet–e, although still efficiently recognized by the valyl‐tRNA synthetase, are not imported any more into mitochondria. These data demonstrate that in plants, besides identity elements required for the recognition by the cognate aminoacyl‐tRNA synthetase, tRNA molecules contain other determinants that are essential for mitochondrial import selectivity. Indeed, this suggests that the tRNA import mechanism occurring in plant mitochondria may be different from what has been described so far in yeast or in protozoa.
A C‐terminal sequence of soybean β‐conglycinin α′ subunit acts as a vacuolar sorting determinant in seed cells
Authors: Nishizawa Keito , Maruyama Nobuyuki , Satoh Ryohei , Fuchikami Yoshihiro , Higasa Takahiko , Utsumi Shigeru 
Published: May 2003

SummaryIn maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension‐cultured cells apart from seed cells. In this study, we examined the VSD of the α′ subunit of β‐conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild‐type α′ was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid‐like structures. Some of the wild‐type α′ was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C‐terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The C‐terminal region of α′ was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that α′ contains two VSDs: one is present in the C‐terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C‐terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.
A polyglycine stretch is necessary for proper targeting of the protein translocation channel precursor to the outer envelope membrane of chloroplasts
Authors: Inoue Kentaro , Keegstra Kenneth 
Published: May 2003

SummaryToc75 is a protein translocation channel in the outer envelope membrane of chloroplasts and its presence is essential for the biogenesis of the organelles. Toc75 is the only protein identified so far in the outer membrane of chloroplasts or mitochondria that is synthesized as a larger precursor, preToc75, with a bipartite transit peptide. Its N‐terminus targets the protein to the stroma and is removed by the stromal processing peptidase, whereas its C‐terminus mediates envelope targeting and is removed by a yet unknown peptidase. Several conserved domains have been identified in the C‐terminal portion of the preToc75 transit peptide from six plant species. We evaluated their importance in the biogenesis of Toc75 by means of deletion or site‐directed mutagenesis, followed by import experiments using isolated chlroplasts. Among the conserved domains, a polyglycine stretch was found to be necessary for envelope targeting. Substitution of this domain with other stretches of a single amino acid such as alanine caused mistargeting of the protein into the stroma, indicating an important role for this domain. Furthermore, a glutamate at +2 and two alanine residues at −3 and −1 to the second cleavage site were found to be important for processing. A potential mechanism for the biogenesis of Toc75 is discussed.
Activation tagging, a novel tool to dissect the functions of a gene family
Authors: Nakazawa Miki , Ichikawa Takanari , Ishikawa Akie , Kobayashi Hiroko , Tsuhara Yumi , Kawashima Mika , Suzuki Kumiko , Muto Shu , Matsui Minami 
Published: May 2003

SummaryIn a screen for morphological mutants from the T1 generation of approximately 50 000 activation‐tagging lines, we isolated four dominant mutants that showed hyponastic leaves, downward‐pointing flowers and decreased apical dominance. We designated them isoginchaku (iso). The iso‐1D and iso‐2D are allelic mutants caused by activation of the AS2 gene. The T‐DNAs were inserted in the 3′ downstream region of AS2. Iso‐3D and iso‐4D are the other allelic mutants caused by activation of the ASL1/LBD36 gene. These two genes belong to the AS2 family that is composed of 42 genes in Arabidopsis. The only recessive mutation isolated from this gene family was of AS2, which resulted in a leaf morphology mutant. Applying reverse genetics using a database of activation‐tagged T‐DNA flanking sequences, we found a dominant mutant that we designated peacock1‐D (pck1‐D) in which the ASL5/LBD12 gene was activated by a T‐DNA. The pck1‐D mutants have lost apical dominance, have epinastic leaves and are sterile. These results strongly suggest that activation tagging is a powerful mutant‐mining tool especially for genes that make up a gene family.
Host‐related variation among isolates of the Sirococcus shoot blight pathogen from conifers
Authors: Smith D. R. , Bronson J. J. , Stanosz G. R. 
Published: May 2003

SummaryVariation among isolates of the Sirococcus shoot blight pathogen (attributed to Sirococcus conigenus, Sirococcus sp., or one of many synonyms) was studied. Inter‐simple‐sequence‐repeat‐anchored polymerase chain reaction fingerprints, nuclear rDNA internal transcribed spacers and 5.8S sequences, and 18S rDNA sequences were analysed. In addition, culture morphology as well as sizes and shapes of conidia produced on seed cone scales in culture were compared. Two main groups were distinguished based on both genetic markers and cultural characteristics. The P group consists of isolates mostly from pine and spruce from Europe and both eastern and western North America; the T group consists of isolates mostly from hemlock from western North America. Results provide a benchmark for comparison of other isolates of these fungi and indicate the need for additional sampling and comparative research of these pathogens.
First records of soilborne Phytophthora species in Swedish oak forests
Authors: Jönsson U. , Lundberg L. , Sonesson K. , Jung T. 
Published: May 2003

SummaryThirty‐two oak stands in southern Sweden, 27 with predominantly declining trees and five with a higher proportion of healthy trees were investigated regarding the presence of soilborne Phytophthora species. Phytophthora quercina, an oak‐specific fine root pathogen, was isolated from rhizosphere soil samples in 10 of the 27 declining stands. Additionally, P. cactorum and P. cambivora were recovered from one stand each. No Phytophthora species were isolated from the healthy oak stands. The soil conditions at the sites from which Phytophthora spp. were recovered ranged from mesic sediments to moraines, with clayey to silty textures and with soil pH (BaCl2) between 3.5 and 5.0. The results show that P. quercina is geographically widespread in oak stands in southern Sweden and indicate that this pathogen may be one of the factors involved in oak decline in Northern Europe as has already been shown for western, Central and parts of southern Europe.
Resinous stem canker development during the growing season of Chamaecyparis obtusa (Hinoki cypress) inoculated with pathogenic fungus Cistella japonica
Authors: Yamada T. , Hasegawa E. , Miyashita S.‐I. 
Published: May 2003

SummarySeasonal development of resinous stem canker was determined in Chamaecyparis obtusa trees wound‐inoculated with Cistella japonica in January 1996. Samples for anatomical analysis were harvested on 20 May, 9 July and 6 August in the second year of inoculation, and the sections were observed under light microscope. Resin exudation was most abundant in the second year, decreasing in the third. All new resin exudation in the second year was recognized at the May observation. New traumatic resin canal formation was observed in the August samples, and an expansion of necrotic lesions was observed mainly in summer samples in the inoculated trees and in naturally affected trees. Cistella japonica was re‐isolated from all bark lesions or inoculated wounds examined. These results suggest that the activity of Ci. japonica in the tree affects the seasonal development of resinous stem canker during the growing season of the trees.