Small open reading frames (ORFs) (less than ~100 amino acids) in microbial genomes are typically ignored in annotation efforts, largely due to a lack of comparative sequence information and, consequently, difficulty in assigning functional roles. As such, this size fraction of genome sequences has by far the largest fraction of ORFs designated as “hypotheticals” or “unknowns”. However, encoded within this group of small ORFs are biomolecules that potentially play essential roles in cellular physiology. For example, included in this group are putative peptides produced into the secretome (or extracellular proteome) that function as anti-microbial peptides or signaling peptides. Whereas most cellular chemistry proceeds by enzymes and proteins found in the cytoplasm, the secretome plays a pivotal role in ecological intra- and interspecies interactions, meeting nutritional needs through substrate acquisition, and mediating colonization of biological and non-biological surfaces. Proteomics approaches have been used to determine the content of the secretome, although such tactics are somewhat limited at this time, given difficulties in sample acquisition and preparation, expense of the analytical instrumentation required, and restricted set of cultivation conditions that typically can be examined. Indeed, the relationship between genotype and phenotype hinges on many factors, such that the secretome can vary extensively with the cellular environmental and nutritional context. Approaches that can identify the most meaningful samples for proteome analysis are needed so that the power of this methodology can be put to its most productive use. In addition to small ORFs encoding components of the secretome, it has recently been determined that many microorganisms use a system of poisons and antidotes to regulate cellular function. Toxin-Antitoxin (TA) loci, encoding a stable poison (T) and a proteolytically labile antidote (A), are widely distributed in prokaryotic genomes and have recently been found to contribute to stress response. Chromosomally-encoded TA loci are associated with small ORFs (~100 aa) have been implicated in programmed cell death in prokaryotes, but may also act cytostatically by down-regulating protein synthesis in the face of adverse circumstances. The detailed mechanisms by which TA loci contribute to prokaryote stress management are not clear. However, strategic approaches to manipulate toxin efficacy could create opportunities for improved understanding of microbial survival and proliferation and thereby inspire development of novel anti-microbial therapies. In this presentation, we will discuss functional genomics efforts that relate to the annotation of small ORFs in microbial genomes, with particular attention to hyperthermophiles (optimal growth temperature e 80°C), which inhabit biologically extreme environments and have distinctive phylogenetic placement within the Tree of Life. A functional genomics approach has been used to probe the genomes of three model hyperthermophilic microorganisms (Thermotoga maritima, Pyrococcus furiosus, and Sulfolobus solfaraticus) with respect to the functional importance of small ORFs, and the roles of associated gene products. Among the issues to be considered is the role of TA loci in stress response (Tachdjian and Kelly, 2006) and mechanisms associated with ecologically important putative peptides (quorum sensing, anti-microbials) encoded in small ORFs (Johnson et al., 2005; Johnson et al., 2006; Montero et al., 2006; Conners et al., 2006). Extension of the approaches used here to studying small ORFs in less thermophilic microorganisms will also be considered.

REFERENCES:

Johnson, M.R., C.I. Montero, S.B. Conners, K.R. Shockley, S.L. Bridger and R.M. Kelly. 2005. Population density-dependent regulation of exopolysaccharide formation in the hyperthermophilic bacterium Thermotoga maritima. Mol. Microbiol. 55:664-674.

Johnson, M.R., S.B. Conners, C.I. Montero, K.R. Shockley, and R.M. Kelly. 2006. Thermotoga maritima phenotype is impacted by symbiotic interaction with Methanococcus jannaschii in hyperthermophilic co-culture. Appl. Environ. Microbiol. 72:811-818.

Tachdjian, S., and R.M. Kelly. 2006. Metabolic adjustments and genome plasticity are implicated in Sulfolobus solfataricus response to pH and thermal stress. J. Bacteriol. 188:4553-4559.

Conners, S.B. E.F. Mongodin, M.R. Johnson, C.I. Montero, K.E. Nelson and R.M. Kelly. 2006. Microbial biochemistry, physiology, ecology and biotechnology of hyperthermophilic Thermotoga species. FEMS Microbiol. Rev. 30:872-905.

Montero, C.I., D.L. Lewis, M.R. Johnson, S.B. Conners, E.A. Nance, J.D. Nichols, and R.M. Kelly. 2006. Co-location of genes encoding tRNA-mRNA hybrid and a putative signaling peptide on complementary strands in the genome of the hyperthermophilic bacterium Thermotoga maritima. J. Bacteriol. 188:6802-6807.

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