Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia

Abstract

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream The spirochetes constitute one of the major lines of eubacterial descent (24). Although these organisms share a distinctive morphology and a unique type of motility, there exists among them a great deal of diversity with respect to ecology and metabolism (18), and 16S rRNA oligonucleotide analysis has revealed an early divergence of the two major clusters: the Spirochaeta-Treponema-Borrelia cluster and the Leptospira cluster (24). In part because there has not yet been a genetic transfer system described for any spirochete, little is known about gene organization in these bacteria. However, several spirochete genes have been cloned and sequenced (for example, see references 1, 3, 19, 23, and 29 to 31), and the chromosome of one of the spirochete species, Borrelia burgdorferi, is so far unique among procaryotes in that it is linear rather than circular (13).Because the tryptophan biosynthetic pathway represents a useful paradigm for the comparative study of gene organization and expression (5-7, 28), spirochete trp genes are of particular interest. The trpE gene and the adjacent trpG gene, which encode components I and II, respectively, of anthranilate synthase (AS), the first enzyme in the tryptophan biosynthetic pathway, were cloned from Leptospira biflexa (29, 30) and have been sequenced (31). To begin our analysis of the molecular genetics of the SpirochaetaTreponema-Borrelia cluster, we cloned Spirochaeta aurantia DNA that complemented an Escherichia coli trpE deletion (1). S. aurantia is a facultatively anaerobic bacterium isolated from freshwater sediments (4) and has received considerable attention as an easily cultivated model organism for studying various aspects of spirochete physiology and molecular biology (2,3,(14)(15)(16)(17).In E. coli, the S. aurantia trpE-complementing DNA directed the synthesis of two polypeptides with apparent molecular weights of 59,000 and 47,000. The two polypeptides appear to be encoded by a single gene, with the larger * Corresponding author. t Deceased 8 October 1989. polypeptide representing a readthrough product. A transposon insertion analysis indicated that the 47,000-dalton polypeptide was required for complementation (1). Furthermore, the results of in vitro enzyme assays and in vivo growth studies indicated that the cloned DNA encoded an ASI but not an ASII activity (1) and that the S. aurantia ASI was capable of interacting with E. coli ASII.As a next step in the analysis of S. aurantia trp genes, we have sequenced and analyzed the cloned trpE-complementing DNA and the DNA which flanks it on the S. aurantia chromosome. We have also compared the deduced amino acid sequence of the encoded TrpE polypeptide with those of polypeptides encoded by trpE genes from other organisms, including the distantly related spirochete L. biflexa.

Phylogenetic analysis of the spirochetes

“…The branching order of the six major spirochete phylogenetic groups remained stable when hypervariable regions were included in the distance matrix calculations. The phylogenetic relationships obtained from the 16S rRNA sequence analysis are in overall agreement with previous catalog studies (41) and comparative sequence analysis of the trpE gene (5). Characteristics of known genera of spirochetes with respect to physiology, ecological niche, DNA base composition, and other traits are presented in Table 3.…”