Thermus thermophilus genome analysis: benefits and implications

Abstract

The genome sequence analysis of Thermus thermophilus HB27, a microorganism with high biotechnological potential, has recently been published. In that report, the chromosomal and the megaplasmid sequence were compared to those of other organisms and discussed on the basis of their physiological and metabolic features. Out of the 2,218 putative genes identified through the large genome sequencing project, a significant number has potential interest for biotechnology. The present communication will discuss the accumulating information on molecules participating in fundamental biological processes or having potential biotechnological importance.

Introduction

The T. thermophilus HB27 genome sequence, published on April 4 2004 [1], has already made an impact on the research community and raised intriguing questions. In the last decade a great deal of effort has been put on the biochemistry and physiology of the prokaryotes and especially the thermophilic bacteria. Since the first publication of a complete microbial genome in 1995, more than 100 microbial genomes have been completely sequenced and another 300 microbial genome sequencing projects are estimated to be in progress worldwide. Every genome that has been sequenced to date has provided new insight into the biological processes, activities and potential of these species that had not been evident before. Gene transfer, environmental applications and virulence mechanisms are only some of the many processes for which significant insight has been gained through these projects.

The genome of T. thermophilus, consists of a 1,894,877 base pair chromosome and a 232,605 base pair megaplasmid, designated pTT27 [1]. The completion of the T. thermophilus HB27 genome sequencing project resulted in the discovery of a number of new genes with potential interest for biotechnological applications [1]. Since in thermophilic bacteria, fundamental cell mechanisms and processes such as protein thermostability, replication, transcription, translation, secretion, cell-signaling etc are not well understood yet, the T. thermophilus genome analysis will greatly improve our understanding towards that direction.

Recent reviews discuss the enzymes from T. thermophilus with biotechnological interest [2] and the biochemical and molecular features of thermozymes [3-6]. All these thermozymes display higher stability and activity than their counterparts currently used in the biotechnological industry. These enzymes are not only more thermostable than their mesophilic homologues, but are also more resistant to chemical agents, properties that make them extremely attractive for industrial processes [3]. Recent structural comparisons among mesophiles (or mesozymes) and thermozymes have validated numerous protein stabilizing mechanisms including hydrophobic interactions, packing efficiency, salt bridges, hydrogen bonding, reduction of conformational strain, loop stabilization, resistance to covalent destruction [6] and binding to RNA [7]. The thermal adaptation of protein synthesis in T. thermophilus was attributed to a key enzyme, a thiolase responsible for a post-transcriptional modification of the thermophilic bacterial tRNAs [8].

Discussion

The 2,218 identified putative genes from T. thermophilus [1] were compared to those of the closest relative sequenced so far, the mesophilic bacterium Deinococcus radiodurans. Both organisms share a similar set of proteins, although their genomes lack extensive synteny. Substantial similarity to annotated data base entries allowed the authors to assign putative functions to 1,482 protein-coding genes. Of the remaining 736 open reading frames, about 488 had no substantial similarity to entries in public databases, and were therefore designated hypothetical proteins [1]. These were found more frequently on the plasmid (39%) than on the chromosome (20%). The G+C content in the T. thermophilus genome was 69.4% on average [1]. Regions with substantial lower G+C content represented ribosomal RNA clusters and at least three more gene clusters that were flanked by mobile elements. A cluster containing 15 genes with atypical codon usage showed similarities to sugar transferases, epimerases and dehydrogenases involved in lipopolysaccharide O-antigen biosynthesis.

The authors posed the question of how the thermophilic cells respond to thermal challenge at the molecular level. Surprisingly, the Ung family of proteins, coding for uracil-DNA-glycosylases, was not found in T. thermophilus. It is known that this gene family act as a general line of defense against cytosine deamination and its members are nearly ubiquitous in the bacterial and eukaryotic domains of the phylogenetic tree. However, base excision repair genes are represented in the T. thermophilus genome, while in the absence of a LexA homologue, the corresponding repair genes may be expressed constitutively.

In bacterial genomes, functionally related genes often facilitate the coordination of operons. The finding of the well studied operons in the thermophilic organisms, now in the light of the total genome analysis of T. thermophilus, will form the basis for further investigation into genome evolution and gene regulation. Recently, an unusual gene structure has been described in T. thermophilus, in which the ribosomal protein L34 coding sequence (rpmH) was found to be entirely overlapped by the unusually large RNase P protein subunit sequence (rnpA) [9]. These genes, which were part of the same operon in E. coli, are located near the origin of replication [9]. This co-localization of genes in a wide range of bacterial genomes implies an important linkage in their regulation of gene expression. The gene disruption method for deleting genes of interest [10] combined with the T. thermophilus genome sequence project will provide a unique opportunity for taking a genomics-based approach toward identifying genes encoding different biosynthetic enzymes or regulatory proteins.

Examination of all sequenced genomes reveals that almost 40% of each genome remains as hypothetical proteins. This demands that high-throughput methodologies be developed for the efficient analysis of these large data sets, including high-throughput proteomics, gene expression and protein-protein interaction studies [11]. Microarrays are rapidly becoming standard laboratory tools for investigating gene expression under different conditions, as well as for looking at the presence and absence of genes in different strains or species that are related to a reference genome.

The full genome sequence of T. thermophilus [1] will revolutionize both the character and the steps on discoveries of new biotechnological enzymes or products. Stable enzymes are useful not only for industrial applications, but also for structural biology research [1,2]. T. thermophilus cells can now be used as a host for the selection of stable enzymes. Within this context, vector systems have been developed for extreme thermophiles and the production of stabilized mutants (3-isopropylmalate dehydrogenase) has been accomplished [10]. Moreover, T. thermophilus can synthesize vitamin B12, carotens, polyhydroxyalkanoate polymers in different media, suggesting that thermophilic bacteria could be useful in producing compounds of much current interest [1,2,12]. T. thermophilus chaperonins, a kind of heat shock proteins (Hsp), demonstrate ability to facilitate folding of several enzymes [2]. In the past, T. thermophilus was characterized as a treasure house for the discovery of new naturally occurring polyamines or other secondary metabolites and now most of their biosynthetic genes are predicted [1]. Ornithine decarboxylase, the key enzyme for polyamine biosynthesis, and its antizyme were purified from T. thermophilus [7,13]. The observation that both these regulatory proteins are bound to nucleic acids indicates that this binding facilitates the stabilization of the enzymic activities of the thermophilic organisms. Fewer two-component signal transduction systems are predicted to exist in T. thermophilus compared to mesophiles [1], but a more specific alignment of the sequences of interest may reveal the presence of others. The products of atoS and atoC genes, for example, have been previously predicted to constitute a two-component signal transduction system in E. coli, regulating expression of the atoDAEB operon-encoded enzymes that control short-chain fatty acid catabolism upon acetoacetate induction and antizyme has been reported to be the atoC gene product [14,15]. The two proteins of the fore mentioned system which have been biochemically characterized in E. coli [16] as well as the corresponding regulated operon were found to have homologues in the T. thermophilus genome. The absence of chemotaxis cascades or methyl-accepting chemotaxis proteins and flagella biosynthetic genes indicates that the motility of this organism is restricted. However, type IV pili-forming genes were identified. Also, numerous proteases, lipases, pullulanases, α- and β-glucosidases, galacturonases, esterases, phosphatases, alcohol dehydrogenases and DNA/RNA processing enzymes were identified in the genome sequence as well.

The availability of the full genome sequence of T. thermophilus will immeasurably advance the re-interpretation of bacterial evolution using criteria that include the ultra structure of the cell envelope that identifies this genus as an intermediate evolutionary step between the Gram-positive (monoderms) and the Gram-negative (diderms) bacteria [17]. It has recently been reported that T. thermophilus actually has a periplasmic space, which is functionally similar to that of proteobacteria [18].

Transfer of genetic material between different individual bacterial cells (i.e. horizontal gene transfer) is mediated by plasmids, bacteriophages or conjugative elements. The latter have been named gene islands, or depending on their function, pathogenicity, symbiosis or degradation islands [19]. The possible presence of gene islands in the genome or megaplasmid of T. thermophilus will enlighten the evolutionary mechanisms that took place in order to shape the genetic content of this microorganism.

Conclusions

We have attempted to channel into the advantages coming from the genome sequence of the extreme thermophile T. thermophilus. It is evident that additional information and a lot of work are necessary. Our understanding of the function of many thermostable enzymes or proteins will improve by the analysis of the T. thermophilus genome. Nevertheless, many uncertainties must be clarified before it can be considered worthwhile to put more research endeavor in these "hot" areas. Further advances on large-scale preparation of thermophilic bacteria with high transformation efficiency, in new bioreactors working at high temperatures, could lead to significant improvements in thermophilic enzymes and other cell factory products.