Characterization of the O-acetylserine(thiol)lyase gene family in Solanum lycopersicum L.

Key message

This research demonstrated the conservation and diversification of the functions of the O-acetylserine-(thiol) lyase gene family genes in Solanum lycopersicum L.

Abstract

Cysteine is the first sulfur-containing organic molecule generated by plants and is the precursor of many important biomolecules and defense compounds. Cysteine and its derivatives are also essential in various redox signaling-related processes. O-acetylserine(thiol)lyase (OASTL) proteins catalyze the last step of cysteine biosynthesis. Previously, researches focused mainly on OASTL proteins which were the most abundant or possessed the authentic OASTL activity, whereas few studies have ever given a comprehensive view of the functions of all the OASTL members in one specific species. Here, we characterized 8 genes belonging to the OASTL gene family from tomato genome (SlOAS2 to SlOAS9), including the sequence analyses, subcellular localization, enzymatic activity assays, expression patterns, as well as the interaction property with SATs. Apart from SlOAS3, all the other genes encoded OASTL-like proteins. Tomato OASTLs were differentially expressed during the development of tomato plants, and their encoded proteins had diverse compartmental distributions and functions. SlOAS5 and SlOAS6 catalyzed the biogenesis of cysteine in chloroplasts and in the cytosol, respectively, and this was in consistent with their interaction abilities with SlSATs. SlOAS4 catalyzed the generation of hydrogen sulfide, similar to its Arabidopsis ortholog, DES1. SlOAS2 also functioned as an L-cysteine desulfhydrase, but its expression pattern was very different from that of SlOAS4. Additionally, SlOAS8 might be a β-cyanoalanine synthase in mitochondria, and the S-sulfocysteine synthase activity appeared lost in tomato plants. SlOAS7 exhibited a transactivational ability in yeast; while the subcellular localization of SlOAS9 was in the peroxisome and correlated with the process of leaf senescence, indicating that these two genes might have novel roles.

     

Identification of O-acetylserine(thiol)lyase (OASTL) genes in sorghum (Sorghum bicolor) and gene expression analysis under cadmium stress

Cysteine (Cys) is the first identified molecule in plant metabolism which includes both sulfur and nitrogen. It can be synthesized in three cellular compartments, containing chloroplast, cytoplasm and mitochondrion. The final step of cysteine biosynthesis is catalyzed by the O-acetylserine(thiol)lyase enzyme (OASTL, E.C. 4.2.99). In the present study, seven members of the OASTL gene family in the sorghum (Sorghum bicolor) genome were identified at a genome-wide scale and comparative bioinformatics analyses were performed between sorghum and Arabidopsis OASTLs. In all OASTL proteins, a pyridoxal-phosphate dependent domain structure (PALP, PF00291) was identified. The gene ontology annotations also revealed that all sorghum OASTL genes have KOG1252 (Cystathionine beta-synthase and related enzyme) and K01738 (cysteine synthase A) activities. In promotor sequences of OASTL genes, diverse cis-acting elements were found, including hormone and light responsiveness, abiotic stress responsiveness, and tissue-specific ones (meristem and endosperm). Sorghum OASTL genes demonstrated medium or high level expressions in anatomical parts and developmental stages based on the digital expression data. Expression of OASTL genes were also analyzed under cadmium (Cd) stress in sorghum by Real Time-quantitative PCR (RT-qPCR). The results exclusively showed that OASTL A1-2 gene was 1.12 fold up-regulated in roots, whereas cysteine synthase 26 was 2.25 fold down-regulated in leaves. The predicted 3D structure of OASTLs indicated some structural diversities as well as variations in the secondary structures.

     

Cloning and Characterization of the Gene Encoding O-Acetylserine Lyase from Streptococcus suis

We have cloned and sequenced a gene encoding O-acetylserine lyase from Streptococcus suis. The gene encodes a protein of 309 amino acids with a calculated molecular mass of 32,038 Da. The deduced amino acid sequence showed more extensive similarities to the CysK proteins than to the CysM proteins of other bacteria. The cloned gene was inserted into a pTrcHisB histidine hexamer expression vector. A 38-kDa fusion protein was expressed in a cysMK auxotrophic mutant of Salmonella typhimurium and complemented the auxotrophic properties of the mutant. Furthermore, the transformants could grow in minimal defined media supplemented with not only sulfide but also thiosulfate as a sole sulfur source. These data indicated that the cloned gene encodes a protein that was a functional homolog of the CysM in S. typhimurium. Received: 1 July 1999 / Accepted: 10 August 1999     

Mutational analysis of O-acetylserine (thiol) lyase conducted in yeast two-hybrid system

Cysteine biosynthesis, achieved by the sequential reaction of two enzymes, serine acetyltransferase and O-acetylserine (thiol) lyase (OASTL), represents the final step of sulfur assimilation pathway in plants and bacteria. The two enzymes form a bi-enzymatic cysteine synthase complex through specific protein-protein interactions. To identify the amino acids important for cysteine synthase complex formation, several mutations in bacterial OASTL were designed. Effects of mutagenesis were verified in a yeast two-hybrid model that allowed monitoring both, protein-protein interactions and the enzymatic activity of OASTL.     

Inhibition of Arabidopsis O-acetylserine(thiol)lyase A1 by tyrosine nitration

The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.     

Molecular Cloning and Expression of the hyu Genes from Microbacterium liquefaciens AJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids,in Escherichia coli

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to α-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl α-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.     

Molecular mechanism of the regulation of expression of plasmid-encoded mouse bacteremia (mba) genes in Salmonella serovar Choleraesuis

Summary The regulation of mouse bacteremia genes (mba genes) encoded by a 6.4 kb region on the 50 kb virulence plasmid (pKDSC50) of Salmonella serovar Choleraesuis was analyzed. The genes mba1, mba2, mba3, and mba4, are arranged in this order, and form a cluster located in the 6.4 kb mba region. We prepared four antibodies, each specific for an individual Mba protein, using synthetic peptides as antigens. Their amino acid sequences were deduced from the DNA sequence of the corresponding mba genes. Each Mba peptide antiserum was able to recognize the corresponding Mba protein produced by Escherichia coli carrying a recombinant plasmid containing individual mba genes. When the recombinant plasmid contained all four mba genes (pMKD601), three Mba proteins (Mba2, Mba3, and Mba4) were identified by Western blotting analysis using Mba antisera. These proteins could not be detected when the recombinant plasmid lacked mba1 (pMKD201). Three species of mRNA for mba2, mba3, and mba4 with different chain length were detected from pMKD601 by Northern blot hybridization, and two start sites were identified by primer extension assay. Gel mobility shift assays demonstrated that Mbal specifically bound to a fragment containing the start sites of mRNAs. The amino acid sequence of Mbal had significant homology to the LysR family of DNA binding proteins, possessing a characteristic helix-turn-helix DNA binding motif. The present study provides clear evidence to show that the Mba1 protein binds to the promoter region of mba2, and positively regulates the expression of mba2, mba3, and mba4 genes.     

Impact of reduced O-acetylserine(thiol)lyase isoform contents on potato plant metabolism

Plant cysteine (Cys) synthesis can occur in three cellular compartments: the chloroplast, cytoplasm, and mitochondrion. Cys formation is catalyzed by the enzyme O-acetylserine(thiol)lyase (OASTL) using O-acetylserine (OAS) and sulfide as substrates. To unravel the function of different isoforms of OASTL in cellular metabolism, a transgenic approach was used to down-regulate specifically the plastidial and cytosolic isoforms in potato (Solanum tuberosum). This approach resulted in decreased RNA, protein, and enzymatic activity levels. Intriguingly, H(2)S-releasing capacity was also reduced in these lines. Unexpectedly, the thiol levels in the transgenic lines were, regardless of the selected OASTL isoform, significantly elevated. Furthermore, levels of metabolites such as serine, OAS, methionine, threonine, isoleucine, and lysine also increased in the investigated transgenic lines. This indicates that higher Cys levels might influence methionine synthesis and subsequently pathway-related amino acids. The increase of serine and OAS points to suboptimal Cys synthesis in transgenic plants. Taking these findings together, it can be assumed that excess OASTL activity regulates not only Cys de novo synthesis but also its homeostasis. A model for the regulation of Cys levels in plants is proposed.     

Cloning and Sequencing of Alginate Lyase Genes from Deep-Sea Strains of Vibrio and Agarivorans and Characterization of a New Vibrio Enzyme

Four alginate lyase genes were cloned and sequenced from the genomic DNAs of deep-sea bacteria, namely members of Vibrio and Agarivorans. Three of them were from Vibrio sp. JAM-A9m, which encoded alginate lyases, A9mT, A9mC, and A9mL. A9mT was composed of 286 amino acids and 57% homologous to AlxM of Photobacterium sp. A9mC (221 amino acids) and A9mL (522 amino acids) had the highest degree of similarity to two individual alginate lyases of Vibrio splendidus with 74% and 84% identity, respectively. The other gene for alginate lyase, A1mU, was shotgun cloned from Agarivorans sp. JAM-A1m. A1mU (286 amino acids) showed the highest homology to AlyVOA of Vibrio sp. with 76% identity. All alginate lyases belong to polysaccharide lyase family 7, although, they do not show significant similarity to one another with 14% to 58% identity. Among the above lyases, the recombinant A9mT was purified to homogeneity and characterized. The molecular mass of A9mT was around 28 kDa. The enzyme was remarkably salt activated and showed the highest thermal stability in the presence of NaCl. A9mT favorably degraded mannuronate polymer in alginate. We discussed substrate specificities of family 7 alginate lyases based on their conserved amino acid sequences.     

Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria

Summary The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions. A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed. The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with M_r values of 58854, 42299, 15548, 18214 and 15108 respectively. A sixth, partial ORF is also present. Between ORF1 and ORF2 is a sequence of unknown function showing 7 by duplications. The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products. In addition, the deduced amino acid sequence of ORF2 showed homology to the Pu1F and to the ComG ORF2 products. The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively. The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions. By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.The nucleotide sequence data in this paper have been deposited in the EMBL, Genbank and DDBJ nucleotide sequence databases under the accession number X59079     

Molecular organization of the mating type (Mat) locus of Exserohilum monoceras (Setosphaeria monoceras), a bioherbicide agent for Echinochloa weeds

Mating-type genes were cloned and sequenced in the heterothallic phytopathogenic fungus Exserohilum monoceras, a candidate bioherbicide for the control of Echinochloa weeds in rice fields. Using PCR-based methods, we determined both idiomorphs and flanking regions of these genes. The structural organization of the E. monoceras Mat locus was similar to that of other heterothallic ascomycetes. The MAT1-1-1 gene was 1191 bp and encoded a predicted protein of 379 amino acids with an α box domain. The MAT1-2-1 gene was 1093 bp and encoded a predicted protein of 345 amino acids with a high mobility group box domain. Expression of both MAT genes was detected under vegetative and invasive growth conditions. MAT-specific primers were developed to assess the mating-type frequency of E. monoceras field isolates. Both mating types were observed in Japanese field isolates. Analysis of mating types and sexual hybridization of E. monoceras could provide useful approaches for the conventional genetic manipulation of this fungus to produce a more efficient bioherbicide.     

The ORF5 of Grapevine virus A is involved in symptoms expression in Nicotiana benthamiana plants

Grapevine virus A (GVA), a member of the genus Vitivirus which belongs to the family Flexiviridae, has a single‐stranded RNA genome of about 7.4 kb that comprises five open reading frames (ORFs). ORF5 encodes a small 10‐kDa protein (p10), which is believed to interact with nucleic acids and to suppress the plant's RNA‐ silencing response. We obtained molecular and biological data indicating that ORF5‐encoded product, specifically its N‐terminus, affects the appearance of symptoms in Nicotiana benthamiana plants. The ORF5‐encoded products of the severe GR5 and the mild GTR1‐1 isolates were found to affect RNA silencing similarly in mesophyll cells of N. benthamiana, despite being involved in different expressions of symptoms on this host.     

Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.     

Complete Sequence of Plasmid pMP1 from the Marine Environmental Vibrio vulnificus and Location of Its Replication Origin

A novel cryptic plasmid, pMP1, from an environmental Vibrio vulnificus MP-4 isolated from Mai Po Nature Reserve in Hong Kong, has been characterized. The 7.6-kb plasmid had guanine–cytosine content of 40.03% and encoded four open reading frames (ORFs) with >100 amino acids. The predicted protein of ORF1 contained 478 amino acids showing 29% identity and 50% similarity over 309 amino acids to the integrase of Vibrio cholerae phage VP2. ORF2 encoded a putative protein of 596 amino acids, which were 23% identity and 42% similarity over 455 amino acids to the tail tape measure protein TP901 of Chromohalobacter salexigens phage. ORF3 and ORF4 encoded putative proteins of 103 and 287 amino acids, respectively, but showed no homologies to any known proteins. Further experiments indicated that a 3.2-kb fragment from EcoRI digestion could self-replicate. Analysis indicated that a sequence upstream of ORF4 had the features characteristic of theta-type replicons: AT-rich region, six potential direct repeats (iterons) spaced approximately two DNA helical turn apart (about 23 bp), two copies of 9 bp dnaA boxes, three Dam methylation sites, and five inverted repeats. Complementation experiments confirmed that the protein encoded by ORF4 was required for plasmid replication. We propose that ORF4 encode a new type of Rep protein and pMP1 is a new type of theta plasmid.