Substrate Specificities and Expression Patterns Reflect the Evolutionary Divergence of Maltose ABC Transporters in Thermotoga maritima

Annotation of the complete genome sequence of the hyperthermophilic, heterotrophic bacterium Thermotoga maritima indicates that 24% of the bacterium's open reading frames (ORFs) are most closely related to archaeal sequences (16). Subsequent studies have provided evidence that these genes were acquired by horizontal gene transfer (HGT). The closest known relative of T. maritima, Thermotoga species RQ2, acquired sugar ABC transporter and polysaccharide hydrolase genes independently of T. maritima, and this acquisition presumably provides selective nutritional advantages to this organism in its natural habitats (18). Intradomain gene acquisition or even gene duplication may confer novel selective advantages as well. This may be evident in the two putative maltose ABC transporters encoded in distant locations in the T. maritima genome (16). The amino acid sequences of the individual components of both transporters are very similar, suggesting a common evolutionary history (16). The transporter gene clusters (putative operons) may have arisen through operon duplication or horizontal acquisition of a second, orthologous operon by the ancestor of T. maritima. Evidence of a similar intradomain HGT of ABC transporter genes was found in two closely related archaea, Thermococcus litoralis and Pyrococcus furiosus (4). Regardless of their mechanism of duplication, the functions of these two T. maritima ABC transporters must confer distinct selective advantages since both have been retained. An organism will acquire a new catabolic trait after acquisition of new genes only if the genes are expressed and regulated in concert with other catabolism genes. Consequently, the evolution of the regions upstream of new genes and the binding of transcription factors to these regions are important processes that dictate the addition of novel traits. The processes by which new regulatory connections are made following gene acquisition are not well understood. An examination of the role of gene duplication in the evolution of transcriptional regulators and their regulated operons in Escherichia coli suggests that regulatory networks often evolve through duplications and that about half of those examined evolved new regulatory connections not present in the ancestral regulatory element (30). Those studies showed that new connections can arise either from new DNA binding activities of duplicated transcription factor proteins or from changes in upstream regulatory sites that allow other proteins to bind there. For operons like the T. maritima mal operons that originated either through operon duplication or horizontal acquisition of an orthologous operon, the new operon could fall under the control of a new promoter downstream of which it recombined. This phenomenon has been observed in Lactococcus lactis, where lac genes acquired from a gram-negative organism fell under the control of an apparently preexisting gal promoter (31). Acquisition of a new ABC transporter may cause selection for an altered periplasmic binding protein that responds to the same ligand as the transcription factor that controls its expressions. It has been postulated that periplasmic binding proteins and their cognate transcriptional repressors acquire their specificities for the same ligands through independent mutations rather than through domain swapping (7). We sought to learn about the divergence of these mal operons in terms of their transport functions and their transcriptional regulatory controls. To do so, we have determined the substrate affinities of their corresponding binding proteins and compared their transcriptional and translational expression patterns. Maltose transport has been the subject of previous investigations into the processes of sugar transport and its regulation in T. maritima. Maltose binding activities in periplasmic extracts of T. maritima cells grown on different sugars have been measured (15). MalE2, one of two putative periplasmic maltose binding proteins identified in the T. maritima genome sequence, has been shown to bind maltose, maltotriose, and trehalose (32). In this report, we describe how we have determined the substrate affinities of the other maltose binding protein, MalE1. We show that, although similar in sequences, MalE1 and MalE2 have different substrate binding specificities. We also present transcriptional and proteomic analyses showing that each binding protein is expressed under different conditions. Based on our findings, we conclude that these transporters have assumed different cellular functions while still retaining some features of their common origin.