A regulatory system hitherto found only in Gram‐positive bacteria in a Gram‐negative bacterium that grows only in co‐culture with a Bacillus strain (1999)

Sir, Many aminoacyl-tRNA synthetase and amino acid biosynthesis genes in Gram-positive bacteria are regulated by an unusual transcription termination control system, designated the T box system. This mechanism operates by controlling the activity of an intrinsic terminator located in the leader region of the mRNA, upstream of the start of the coding sequence; interaction of the leader RNA with the cognate uncharged tRNA stabilizes the formation of an antiterminator conformation of the leader RNA, which prevents formation of the competing terminator stem–loop, thereby driving readthrough transcription (Grundy and Henkin, 1993, Cell74: 475–482; Henkin, 1994, Mol Microbiol13: 381–387). The T box system can be recognized on the basis of a complex set of primary sequence and secondary structural elements conserved in the leader regions of the target genes, the most prominent of which is the T box itself, a 14 bp highly conserved sequence. There are 19 transcriptional units with these features in the Bacillus subtilis genome, and many others in a variety of Gram-positive organisms. To date, no example of a T box gene has been reported in a Gram-negative organism. This raises interesting questions about the evolutionary origin of this regulatory mechanism. We have now uncovered an example of an apparent T box gene in a very unusual Gram-negative organism, Symbiobacterium thermophilum. S. thermophilum was isolated as a producer of a thermostable tryptophanase (Suzuki et al. 1988, J Gen Microbiol134: 2353–2362). A surprising observation was that the organism could be grown only in co-culture with a specific strain of Bacillus, which appeared to be related to B. stearothermophilus and B. coagulans. Neither cell-free extracts nor killed cells of the Bacillus strain could support independent growth of S. thermophilum. Co-culture was optimal under low aeration at 60°C; under these conditions, the Bacillus strain grew first, then began to lyse in late exponential phase, concomitant with growth of S. thermophilum. While the Bacillus strain could be grown in pure culture, S. thermophilum appears to be an obligate symbiont, but the requirements of the symbiosis are mysterious. The two strains could be separated by differential centrifugation, permitting isolation of S. thermophilum DNA and cloning of the tryptophanase genes (Hirahara et al. 1992, Appl Environ Microbiol58: 2633–2642). Recent data deposited in GenBank (accession number AB010832) by K. Ueda and T. Beppu include additional sequence information covering the region downstream of the tryptophanase genes. This region includes a coding sequence with high similarity to leucyl-tRNA synthetases, and we found that the region upstream of the coding sequence includes the elements characteristic of members of the T box family. The putative S. thermophilum leuS leader region is shown in Fig. 1. This leader region contains all of the primary sequence and secondary structural features normally conserved in T box leaders (Rollins et al. 1997, Mol Microbiol25: 411–421), including an appropriately placed CUC specifier sequence, corresponding to the leucine codon used as the regulatory signal in the B. subtilis leuS and ilv-leu genes. The S. thermophilum leuS leader also contains a putative intrinsic transcriptional terminator, with the capacity to fold into an alternative antiterminator structure, as found in other T box genes. The terminator precedes the translation initiation region, and the leader region is preceded by a standard housekeeping-type promoter (TTGACT–17 bp spacer–GATAAT). Although the function of these putative regulatory elements has not yet been demonstrated, it appears likely that S. thermophilum leuS is in fact a member of the T box family, the first to be uncovered in a Gram-negative organism. . Structural model of the S. thermophilum leuS leader region. Stem–loop regions I, II, IIA and III and the T box precede the putative terminator. A portion of the T box can form an alternative pairing with a conserved sequence on the 5′ side of the terminator to form the antiterminator (shown on the right). The CUC specifier sequence, which corresponds to the CUC leucine codon, is boxed. Asterisks indicate primary sequence elements conserved in T box family members (Rollins et al. 1997, Mol Microbiol 25: 411–421). As S. thermophilum lives in such close proximity to a Bacillus strain and its lifestyle includes immersion in Bacillus cell lysate, it is possible that the leuS gene may have been acquired by horizontal gene transfer. The leuS coding region, although incomplete, exhibits highest similarity to leuS from Synechocystis (64% amino acid identity), followed by Aquifex (57%), Haemophilus (56%), Escherichia coli (54%), Rickettsia (53%), Borrelia (51%), human mitochondria (50%), Chlamydia (51%) and B. subtilis (47%). The relatively low similarity to the Bacillus gene suggests that the leuS coding region was unlikely to have been acquired recently from the Bacillus strain with which S. thermophilum lives. The G + C content of the leuS coding region is 65%, which matches that of S. thermophilum, while the G + C content of the Bacillus strain is 53% (Suzuki et al. 1988, J Gen Microbiol134: 2353–2362). It remains possible that the regulatory region alone was, in fact, passed from the Bacillus strain to the S. thermophilum genome. Alternatively, another T box gene located elsewhere in the genome may have been transferred, with the regulatory region copied later to the leuS locus. A third possibility is that S. thermophilum is more closely related to a common ancestor of the Gram-positive lineage and retained the T box system when it was lost from other lineages. Further characterization of this unusual organism and its place on the evolutionary tree is likely to provide insight into the history of the T box system and the processes by which it arose. Recent analysis of S. thermophilum 16S rRNA sequence indicates that by this criterion this organism should be placed at the outermost branch of Gram-positive bacterium, at the border of Gram-positive and Gram-negative organisms (T. Beppu and K. Ueda, personal communication). It therefore appears that despite its cell wall characteristics, S. thermophilum is more closely related to the Gram-positive group, and that the T box system is indeed widely distributed in the Gram-positive lineage. This work was supported by Public Health Service grant GM47823 from the National Institute of General Medical Sciences.