Studies of the Hyperthermophile Thermotoga maritima by Random Sequencing of cDNA and Genomic Libraries: Identification and Sequencing of the trpEG (D) Operon

Random sequencing of cDNA and genomic libraries has been used to study the genome of the hyperthermophile Thermotoga maritima. To date, 175 unique clones have been analyzed by comparing short sequence tags with known proteins in the PIR and GenBank databases. We find that a significant proportion of sequences can be matched to previously identified proteins from non-Thermotoga sources. A high match rate was obtained from an oligo(dT)-primed cDNA library, where one-third of all unique sequences analyzed (21/65) shared high amino acid sequence similarity with proteins in the PIR and GenBank databases. Also, approximately one-third of the unique sequences from a second cDNA library (28/89), constructed with random oligo primers, could be snatched to sequences in PIR and GenBank. Identification of genes from the oligo(dT)-primed cDNA library indicates that some Thermotoga mRNAs are polyadenylated. Genes have also been identified from a 1 to 2 kb genomic DNA library. Here, (3/21) of genomic sequences analyzed could be matched to proteins in PIR and GenBank. One of the genomic clones had high sequence similarity to the tryptophan synthesis gene anthranilate synthase component I (trpE). Using this sequence tag, the Thermotoga trp operon was isolated and sequenced. The Thermotoga maritima trp operon is arranged with trpE forming an overlapping transcript with a second protein consisting of a fusion of anthranilate synthase component II (trpG) and anthranilate phosphoribosyltransferase (trpD). With regard to the fusion, the operon organization is similar to Escherichia coli and Salmonella typhimurium , but lacks the classic attenuation system of enteric bacteria. Amino acid sequence comparisons with 19 trpE, 18 trpG and 14 trpD genes from other organisms suggest that the Thermotoga trp genes resemble corresponding genes from other thermophiles more closely than expected.