Identification of the gene encoding the tryptophan synthase beta-subunit from Chlamydomonas reinhardtii

Abstract

We report the isolation of a Chlamydomonas reinhardtii cDNA that encodes the beta-subunit of tryptophan synthase (TSB). This cDNA was cloned by functional complementation of a trp-operon-deleted strain of Escherichia coli. Hybridization analysis indicated that the gene exists in a single copy. The predicted amino acid sequence showed the greatest identity to TSB polypeptides from other photosynthetic organisms. With the goal of identifying mutations in the gene encoding this enzyme, we isolated 11 recessive and 1 dominant single-gene mutation that conferred resistance to 5-fluoroindole. These mutations fell into three complementation groups, MAA2, MAA7, and TAR1. In vitro assays showed that mutations at each of these loci affected TSB activity. Restriction fragment-length polymorphism analysis suggested that MAA7 encodes TSB. MAA2 and TAR1 may act to regulate the activity of MAA7 or its protein product.

Figure 1

cDNA sequence encoding TSB. The predicted translation product is shown below the DNA sequence. Highlighted amino acids are part of a putative chloroplast transit peptide.

Figure 2

Phylogenetic tree of TSB amino acid sequences from C. reinhardtii and 23 other organisms. The tree was generated by the Fitch-Margoliash method (see Methods for details). Included sequences are from Thermus aquaticus (GenBank accession no. X58673; Koyama and Furukawa, 1990), Synechocystis sp. (GenBank accession no. D64006; Zhao et al., 1994), Z. mays TSB1 (GenBank accession no. M76684; Wright et al., 1992), Oryza sativa (GenBank accession no. AB003491), Arabidopsis TSB1 (upper) and TSB2 (lower) (GenBank accession nos. M23872 and M81620; Berlyn et al., 1989; Last et al., 1991), Mycobacterium tuberculosis (GenBank accession no. Z95554; Philipp et al., 1996), Acinetobacter calcoaceticus (GenBank accession no. M34485; Kishan and Hillen, 1990), Pseudomonas syringae (GenBank accession no. M95710; Auerbach et al., 1993), Caulobacter crescentus (GenBank accession no. M19129; Ross and Winkler, 1988), Methanococcus jannaschii (GenBank accession no. U67546; Bult et al., 1996), Lactococcus lactis (GenBank accession no. M87483; Bardowski et al., 1992), Bacillus subtilis (GenBank accession no. D14069; Henner et al., 1985), Haloferax volcanii (GenBank accession no. M36177; Lam et al., 1990), Buchnera aphidicola (GenBank accession no. Z21938; Lai et al., 1995), Haemophilus influenzae (GenBank accession no. P43760; Fleischmann et al., 1995), Vibrio parahaemolyticus (GenBank accession no. X17149; Crawford et al., 1991), Salmonella typhimurium (GenBank accession no. J01810; Crawford et al., 1980), E. coli (GenBank accession no. V00372; Crawford et al., 1980), S. cerevisiae (GenBank accession no. V01342; Zalkin and Yanofsky, 1982), Schizosaccharomyces pombe (GenBank accession no. D89113), and Neurospora crassa (GenBank accession no. P13228; Burns and Yanofsky, 1989). For N. crassa and S. cerevisiae, the first 300 amino acids were removed because they encode the α-subunit of Trp synthase as part of a single protein.

Figure 3

Wild-type and maa7-8 genomic DNA probed with the TSB-encoding cDNA insert from pCU-100. Genomic DNA from wild-type (lanes 2 and 4) and maa7-8 (lanes 1 and 3) cultures were digested with the indicated enzymes and probed with the 1.8-kb cDNA. A restriction fragment-length polymorphism is obvious in hybridizations to NcoI-digested DNA (lanes 3 and 4).

Figure 4

Genetic maps of 5-FI-resistance mutations. The maa7 and maa2 mutations map approximately 20 and 23.5 map units (mu) centromere-distal to NIT2 on linkage group III (top). The tar1 mutations map between PF14 and ACT2 on linkage group VI (bottom). LG, Linkage group.

Figure 6

Slow-growth phenotypes associated with 5-FI-resistance mutations. The cells were plated onto acetate medium and grown for 6 d at 21°C. The two single-mutant strains produced smaller colonies than the wild-type strain. However, after 9 d of growth, the tar1-3 strain produced colonies with a size comparable to wild-type colonies. The colonies from the double-mutant, tar1-3 maa2-8, were always smaller than either the single-mutant or wild-type colonies. The same phenotypes were observed in medium supplemented with 1.5 mm l-Trp.

Figure 5

TSB activity of wild-type and 5-FI-resistant strains. Each column represents the weighted mean units of TSB activity per milligram of protein in extracts of the indicated strains. One unit consumes 1 nmol of indole per min. The error bars indicate two sd above and below the mean. A, TSB activity in wild-type and single-mutant strains. B, TSB activity in double-mutant strains.