Saccharomyces cerevisiae sigma 1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance

Abstract

We discovered on the chromosome of Saccharomyces cerevisiae Sigma 1278b novel genes involved in L-proline analogue L-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the L-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Sigma 1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for L-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for L-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Sigma 1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for L-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to L-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

FIG. 1

Restriction map of the cloned DNA fragment and deletion analysis to identify the region required for AZC resistance. The predicted size and transcriptional orientation of each deduced open reading frame (ORF) is shown by an arrow. Restriction enzymes: Ba, BamHI; Bg, BglII; Ec, EcoRI; Hi, HindIII; Ml, MluI; Sa, SacI; Su, Sau3AI; Xh, XhoI. (A) Restriction map of the cloned DNA fragment in pMH1 and pMH2. Two plasmids had the overlapping 5.4-kb Sau3AI insert (open box). The region in each plasmid matching the sequence on chromosome XIV (pMH1) or X (pMH2) of S. cerevisiae S288C is indicated by a shaded box. The hatched box in pMH2 represents the unknown, partially sequenced 4.6-kb fragment. (B) Analysis of MPR1 deletion mutants. Each DNA fragment was subcloned into pYES2, and the resultant plasmids were introduced into strain CKY2. AZC resistance of the Ura⁺ transformants was examined on SD agar plates containing AZC (0.3 mg/ml) after incubation at 30°C for 3 days. +, growth; −, no growth.

FIG. 2

Nucleotide and predicted amino acid sequences of the MPR1 gene. +1 and the asterisk refer to the putative translational initiation site and the termination codon, respectively. One base change which leads to Gly and Glu at position 85 in MPR1 and MPR2, respectively, is boxed. Matches to known consensus sequences are marked as follows: TATA box (underline) and transcription termination (wavy underline).

FIG. 3

Amino acid sequence deduced from the nucleotide sequence of the MPR1 gene (MPR1) and its alignment with S. cerevisiae Spt10p (SPT10) and the S. pombe hypothetical 23.8-kDa protein (S. pombe). Numbers above the sequences refer to the MPR1 gene. An amino acid residue at position 85 in the MPR1 gene product (Gly for the MPR1 gene product and Glu for the MPR2 gene product) is boxed. Amino acids with identity or similarity are shown in shaded boxes. A horizontal line indicates the absence of the corresponding amino acid residue at this position. Filled dots under the sequences indicate the highly conserved positions in consensus motifs (A to D) of the N-acetyltransferase superfamily (23). The GenBank accession numbers for S. cerevisiae Spt10p and the S. pombe hypothetical 23.8-kDa protein are L24435 and Z67999, respectively.

FIG. 4

Southern blot analysis of genomic DNAs from S. cerevisiae strains. (A) Construction used to identify the PRO1 or MPR1 gene. Locations of the EcoRI sites are marked. The PRO1 and MPR1 genes are indicated by shaded boxes; arrows show the direction of transcription. (B) Southern hybridization. Five micrograms of yeast genomic DNA from each strain was digested with EcoRI, electrophoresed on an 0.8% agarose gel, transferred onto a nylon membrane, and hybridized with a 1,161-bp fragment for the PRO1 gene (left) and 1,636 bp fragment for the MPR1 gene (right). Lane 1, Σ1278b; lane 2, FH506; lane 3, MB329-17C; lane 4, S288C; lane 5, XU-I. An EcoT14I-BglII digest of λDNA was used as the DNA size standard.

FIG. 5

Chromosomal locations of the MPR1 and MPR2 genes in S. cerevisiae strains. (A) Pulsed-field gel electrophoresis of genomic DNAs from S. cerevisiae strains. Electrophoresis was carried out as described in Materials and Methods, and the gel was stained with ethidium bromide. CHR, chromosome. (B) Southern hybridization. After electrophoresis, each genomic DNA was transferred onto a nylon membrane and hybridized with a 1,636-bp fragment for the MPR1 gene labeled using the ECL direct nucleic acid labeling system. Lane 1, Σ1278b; lane 2, FH506; lane 3, MB329-17C; lane 4, S288C; lane 5, XU-1. (C) Schematic map of the MPR1 gene on chromosome XIV (left) and the MPR2 gene on chromosome X (right) of strain Σ1278b. Predicted transcriptional orientation of MPR1 or MPR2 is shown by an arrow.

FIG. 6

Growth phenotype of S. cerevisiae strains on minimal medium containing AZC. Approximately 10⁶ cells of each strain and serial dilutions of 10⁻¹ to 10⁻³ (from left to right) were spotted onto SD plates with appropriate amino acids in the absence (−AZC) and presence (+AZC) of AZC. Plates were incubated at 30°C for 3 days. (A) Function of the MPR1 and MPR2 genes in Σ1278b background strains. The MPR1 and MPR2 disruptants derived from strain FH506 are represented by FH506D1 and FH506D2, respectively. The MPR1 MPR2 double disruptant is represented by FH506D12. (B) Function of the MPR1 gene in the other S. cerevisiae strains. Plasmids pMH1 and pMH3 were constructed from vectors pYES2 and pRS406, respectively.