The α-aminoadipate pathway for lysine biosynthesis is widely distributed among Thermus strains

We previously reported that lysine is synthesized through the α-aminoadipate pathway in Thermus thermophilus HB27 (T. Kosuge and T. Hoshino, FEMS Microbiol. Lett., 169, 361–367, 1998), which was the first report demonstrating the synthesis of lysine through the α-aminoadipate pathway in Bacteria. LYS20 and LYS4, which respectively encode homocitrate synthase and homoaconitate hydratase have already been identified as the lysine biosynthetic genes in T. thermophilus HB27. In the present work, we examined eight other Thermus strains for the existence of genes belonging to the α-aminoadipate pathway. BamHI- or BglII-digested total DNAs from the eight strains were analyzed by Southern hybridization using LYS20 or LYS4 as a DNA probe. DNA fragments that hybridized with one or both of the genes were detected in seven of the Thermus strains but not in T. ruber. The sizes of the fragments that hybridized with the LYS20 and LYS4 probes were the same among T. thermophilus HB27, T. thermophilus HB8, “T. caldophilus” GK24, and four “T. flavus” strains. For example, a similar 4.3-kb fragment was detected in each of the above seven strains. In this fragment, four open reading frames were found downstream of the LYS4 gene in T. thermophilus HB27. Gene disruption experiments revealed that three open reading frames are involved in lysine biosynthesis in T. thermophilus HB27. These results strongly suggest that the lysine biosynthetic gene cluster for the α-aminoadipate pathway is widely distributed in the genus Thermus.     

The alpha-aminoadipate pathway for lysine biosynthesis is widely distributed among Thermus strains

We previously reported that lysine is synthesized through the alpha-aminoadipate pathway in Thermus thermophilus HB27 (T. Kosuge and T. Hoshino, FEMS Microbiol. Lett., 169, 361-367, 1998), which was the first report demonstrating the synthesis of lysine through the alpha-aminoadipate pathway in Bacteria. LYS20 and LYS4, which respectively encode homocitrate synthase and homoaconitate hydratase have already been identified as the lysine biosynthetic genes in T. thermophilus HB27. In the present work, we examined eight other Thermus strains for the existence of genes belonging to the alpha-aminoadipate pathway. BamHI- or BglII-digested total DNAs from the eight strains were analyzed by Southern hybridization using LYS20 or LYS4 as a DNA probe. DNA fragments that hybridized with one or both of the genes were detected in seven of the Thermus strains but not in T. ruber. The sizes of the fragments that hybridized with the LYS20 and LYS4 probes were the same among T. thermophilus HB27, T. thermophilus HB8, "T. caldophilus" GK24, and four "T. flavus" strains. For example, a similar 4.3-kb fragment was detected in each of the above seven strains. In this fragment, four open reading frames were found downstream of the LYS4 gene in T. thermophilus HB27. Gene disruption experiments revealed that three open reading frames are involved in lysine biosynthesis in T. thermophilus HB27. These results strongly suggest that the lysine biosynthetic gene cluster for the alpha-aminoadipate pathway is widely distributed in the genus Thermus.     

IV. Yeast sequencing reports. New open reading frames,one of which is similar to the nifV gene of Azotobacter vinelandii,Found on a 12·5 kbp fragment of chromosome IV of Saccharomyces cerevisiae

The nucleotide sequence of a 12·5 kbp segment of the left arm of chromosome IV is described. Five open reading frames (ORFs) longer than 100 amino acids were detected, all of which are completely confined to the 12·5 kbp region. Two ORFs (D1271 and D1286) correspond to previously sequenced genes (PPH22 and VMA1 or TFP1, respectively). ORF D1298 shows the characteristics of α-isopropylmalate and homocitrate synthase genes and is similar to the nifV gene of Azotobacter vinelandii. Two more ORFs have no apparent homologue in the data libraries. Conversely, two smaller ORFs of 25 and 85 amino acids encoding the ribosomal protein YL41A and an ATPase inhibitor, respectively, were detected. Although a substantial part of the 12·5 kbp fragment apparently lacks protein-coding characteristics, no other elements, such as tRNA genes or transposons, were found. The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the Accession Number X83276.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of the DNA Sequence of a 34 038 bp Region on the Left Arm of Yeast Chromosome XV

We report the DNA sequence of a 34 038 bp segment of Saccharomyces cerevisiae chromosome XV. Subsequent analysis revealed 20 open reading frames (ORFs) longer than 300 bp and two tRNA genes. Five ORFs correspond to genes previously identified in S. cerevisiae, including RPLA2, PRE6, MSE1, IFM1 and SCM2 (TAT2, TAP2, LTG3). Two putative proteins share considerable homology with other proteins in the current data libraries. ORF O2145 shows 41·2% identity with the glycophospholipid-anchored surface glycoprotein Gas1p of S. cerevisiae and ORF O2197 has 53·2% identity to chromosome segregation protein Dis3p of Schizosaccharomyces pombe. Accession Numbers for these sequences are provided in Table 1.©1997 John Wiley & Sons, Ltd.     

Functional analysis of YCL09C: Evidence for a role as the regulatory subunit of acetolactate synthase

We have analysed the function of the open reading frame (ORF) YCL09C. The deletion of this ORF from chromosome III does not affect the physiology of the corresponding yeast strain enough to give a distinct phenotype. Nevertheless a computational analysis reveals high homology between this ORF and the enterobacterial genes encoding the regulatory subunit of acetolactate synthase. We have therefore tested the possibility that ycl09cp is the regulatory subunit of yeast acetolactate synthase by in vitro enzymatic analysis. The acetolactate synthase was previously shown to be retroinhibited by its final product valine. In Escherichia coli this retro-control is assured by the regulatory subunit. Using a yeast strain carrying a complete deletion of YCL09C, we have observed the loss of such retro-inhibition. These results together with the computational predictions show that YCL09C encodes the regulatory subunit of yeast acetolactate synthase.     

Yeast sequencing reports. Genes of the linear mitochondrial DNA of Williopsis mrakii: Coding sequences for a maturase-like protein,a ribosomal protein VAR1 homologue,cytochrome oxidase subunit 2 and methionyl tRNA

The mitochondrial DNA (mtDNA) in some yeasts has a linear structure with inverted terminal repeats closed by a single-stranded loop. These mtDNAs have generally a constant gene order, beginning with a small ribosomal RNA gene at the right end and terminating with a cytochrome oxidase subunit 2 gene (COX2) at the left end, independently of the wide variation in genome size. In the mtDNAs from several species of the genus Williopsis, we found an additional open reading frame, ORF1, which was homologous to the Saccharomyces cerevisiae RF1 gene encoding a group I intron maturase-like protein. ORF1 genes from W. mrakii and W. suaveolens were mapped and sequenced. Next to ORF1, COX2 and methionyl tRNA genes were present on the opposite strand. The same relative positions of genes in the mtDNAs so far examined suggests that the constancy of gene order is generally conserved also at the level of individual tRNA genes. We identified another open reading frame, ORF2, in W. mrakii mtDNA. It was mapped next to the cytochrome oxidase subunit 3 gene. Rich in adenine-thymine bases, ORF2 appears to be a homologue of the VAR1 gene which codes for a small ribosomal subunit protein in S. cerevisiae mitochondria. Nucleotide sequences data have been deposited in the EmBL data library under the following Accession Numbers: X66594 (Apocytochrome b and ORF2 genes of W. mrakii), X66595 (ORF1, tRNA-Met and COX2 genes of W. mrakii), X73415 (tRNA-Met and COX2 genes of W. suaveolens), X73416 (ORF1 gene of W. suaveolens) and X73414 (tRNA-Met and COX2 genes of P. jadinii).     

XIV. Yeast sequencing reports. The sequence of a 44 420 bp fragment located on the left arm of chromosome XIV from Saccharomyces cerevisiae

We have determined the complete nucleotide sequence of a 44 420 bp DNA fragment from chromosome XIV of Saccharomyces cerevisiae. The sequence data revealed 23 open reading frames (ORFs) larger than 300 bp, covering 73·5% of the sequence. The ORFs N2418, N2441, N2474 and N2480 correspond to previously sequenced S. cerevisiae genes coding respectively for the mitochondrial import protein Mas5, the nucleolar protein Nop2, the outer mitochondrial membrane porin Por1, the cytochrome c oxidase polypeptide VA precursor CoxA and the yeast protein tyrosine phosphatase Msg5. Translation products of three other ORFs N2406, N2411 and N2430 exhibit similarity to previously known S. cerevisiae proteins: the ribosomal protein YL9A, the protein Nca3 involved in the mitochondrial expression of subunits 6 and 8 of the ATP synthase and actin; in addition N2505 presents strong similarity to an ORF of chromosome IX. The predicted protein products of ORFs N2417 and N2403 present similarities with domains from proteins of other organisms: the Candida maltosa cycloheximide-resistance protein, the human interleukin enhancer-binding factor (ILF-2). The 12 remaining ORFs show no significant similarity to known proteins. In addition, we have detected a DNA region very similar to the yeast transposon Ty 1–15 of which insertion has disrupted a tRNA^Asp gene. The sequence has been deposited in the GenBank database with the Accession Number U12141.     

Single-read sequence tags of a limited number of genomic DNA fragments provide an inexpensive tool for comparative genome analysis

Single-read sequences from both ends of 415 3-kb average size genomic DNA fragments of Candida albicans were compared with the complete sequence data of Saccharomyces cerevisiae. Comparison at the protein level, translated DNA against protein sequences, revealed 138 sequence tags with clear similarity to S. cerevisiae proteins or open reading frames. One case of synteny was found for the open reading frames of RAD16 and LYS2, which are adjacent to each other in S. cerevisiae and C. albicans. © 1998 John Wiley & Sons, Ltd.     

Nucleotide sequence of the Schizosaccharomyces pombe lys1 + Gene and Similarities of the lys1+ protein to peptide antibiotic synthetases

The 4·2 kbp lys1⁺ gene of Schizosaccharomyces pombe encoding the large subunit of α-aminoadipate reductase (EC1.2.1.31), an enzyme specific to lysine synthesis in higher fungi, was completely sequenced at the nucleotide level from pLYS1H. The S. pombe lys1⁺ gene product consists of 1415 amino acid residues and has a putative molecular weight of 155·8 kDa. The encoded protein converts α-aminoadipic acid to α-aminoadipate-δ-semialdehyde by an ATP-mediated adenylation. Analysis of the sequence showed that the putative protein encoded by lys1⁺ shares strong homology with the peptide antibiotic synthetases which also use an adenylation step. The sequence data reported in this paper have been submitted to GenBank database (Washington DC, USA) under the Accession Number U15923. © 1998 John Wiley & Sons, Ltd.     

Identification and Expression of the Saccharomyces cerevisiae Cytoplasmic Tryptophanyl-tRNA Synthetase Gene

The enzymes that aminoacylate tRNAs have been studied extensively and can be organized into two distinct classes based on signature sequences and the position of aminoacylation. The class I enzymes have canonical HIGH and KMSKS sequences as part of a Rossman fold nucleotide-binding site. The tryptophan-specific enzymes have been placed in class I based on analysis of the cognate genes from Escherichia coli, B. stearothermophilus, B. taurus, and Homo sapiens. An unidentified open reading frame (ORF) on Saccharomyces cerevisiae chromosome XV, HRE342, has 46% identity with the bovine tryptophanyl-tRNA synthetase and possesses the appropriate signature sequences. The predicted molecular weight of the putative HRE342 protein also closely matched the expected monomer size of the S. cerevisiae enzyme. The HRE342 ORF plus about 250 bp of 5′ and 3′ flanking sequence was amplified by polymerase chain reaction, cloned into a 2 μ based vector, and transformed into a host strain, S. cerevisiae JG369.3B. Nucleotide sequence analysis of the clone confirmed the presence of HRE342. Extracts from transformed yeast have a 30- to 100-fold increase in specific activity of the tryptophanyl-tRNA synthetase. An HRE342 locus in a diploid strain, PTY33XPTY44, was disrupted with a LEU2 insert. Sporulation and tetrad analysis of the HRE342::LEU2 strain demonstrated that HRE342 is an essential gene. We conclude that HRE342 is the S. cerevisiae gene encoding the cytoplasmic tryptophanyl-tRNA synthetase, WRS1. A search of the Saccharomyces Genome Database using amino acid sequences from other eukaryotic aminoacyl-tRNA synthetases suggests there is sufficient similarity to identify both class I and class II genes. © 1997 by John Wiley & Sons, Ltd.     

The yal017 gene on the left arm of chromosome I of Saccharomyces cerevisiae encodes a putative serine/threonine protein kinase

The DNA sequence of a region between the LTE1 and CYS3 genes on the left arm of chromosome I from Saccharomyces cerevisiae contains an open reading frame (ORF), YAL017, corresponding to the 5·0 kb FUN31 (F unction U nknown N ow) transcribed region. The predicted protein from this ORF contains 1358 amino acid residues with a molecular weight of 152 531, and an identifiable serine/threonine protein kinase catalytic domain. When compared with other yeast protein kinases, the Ya1017p kinase most resembles the SNF1 serine/threonine protein kinase which is involved in regulating sucrose fermentation genes. The Ya1017p kinase shows highest amino acid identities with two mammalian carcinoma-related serine/threonine protein kinases; PIM-1, which shows induced expression in T-cell lymphomas; and p78A1, whose expression is lost in human pancreatic carcinomas. Gene disruption of YAL017 indicates that it is non-essential for growth on glucose.     

The complete sequence of a 19,482 bp segment located on the right arm of Chromosome II from Saccharomyces cerevisiae

We report here the sequence of a 19,482 bp DNA segment of chromosome II of Saccharomyces cerevisiae. The fragment contains 16 open reading frames (ORFs) covering 74% of the sequence. Four predicted products present homology with known proteins. The ORF YBR1732 exhibits a strong homology to serine hydroxymethyl transferase; the best score is 53·1% identity in 458 amino acids overlap with the serine hydroxymethyl transferase from rabbit liver. YBR1724, which shows homology with riboflavin synthase of Bacillus subtilis, is probably the RIB5 gene implied in riboflavine synthesis and mapped in this region. YBR1733 is homologous to rab protein and YBR1728 is presumably a GTPase activating protein.     

The complete sequence of a 9037 bp DNA fragment of the right arm of Saccharomyces cerevisiae chromosome VII

We report the sequence of a 9037 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete open reading frames (ORFs), namely G7572, G7576, G7579 and G7584. The first three corresponded, respectively, to the previously cloned genes: HIP1, coding for a high-affinity histidine-specific permease, TDH1, one of the known genes coding for glyceraldehyde-3-phosphate dehydrogenase and ODPX, which encodes a precursor of protein X, a component of the pyruvate dehydrogenase complex. The ORF G7584 showed 35·8% identity with a hypothetical protein of Caenorhabditis elegans chromosome 3. The reported sequence has been deposited in the EMBL data library under Accession Number X82408.     

The isolation of a dol-P-man synthase from Ustilago maydis that functions in Saccharomyces cerevisiae

Genomic DNAs from several fungi were screened for a homologous sequence to Saccharomyces cerevisiae DPM1, an essential gene which encodes dolichyl phosphoryl mannose synthase. The fungi examined included Aspergillus nidulans, Neurospora crassa, Schizophyllum commune and Ustilago maydis. Only U. maydis gave a significant signal after Southern hybridization using DPM1 as a probe. The Ustilago homolog was subsequently cloned and sequenced. The predicted protein of 294 amino acids has 60% identity to the S. cerevisiae protein, but lacks the putative ‘dolichol recognition sequence’. RNA of ca. 900 bp is transcribed in both yeast and filamentous cells of Ustilago. In Escherichia coli, the U. maydis sequence expressed a 35 kDa protein exhibiting dolichyl phosphoryl mannose synthase activity. The sequence was also shown to complement a haploid strain of S. cerevisiae containing a deletion of the DPM1 gene. The U. maydis sequence therefore, encodes a dolichyl phosphoryl mannose synthase that can support normal vegetative growth in S. cerevisiae. The GenBank accession number is U54797.