Perceived Cost of Auxotrophic Amino Acids in Two Bacterial Species Esley M. Heizer Jr.*, Michael L. Raymer † , Dan E. Krane* *Department of Biological Sciences, † Department of Computer Science and Engineering Wright State University Fairborn Ohio, USA Douglas W. Raiford Department of Computer Science and Engineering Southern Methodist University Dallas Texas, USA Abstract—Amino acid biosynthetic pathways are highly conserved throughout all domains of life. Biosynthesis of amino acid requires the diversion of resources from energy production to amino acid production. The consequent energy-cost of producing an individual amino acid is can be estimated by adding the amount of ATP expended in production itself to the amount of potential energy lost. Some organisms lack the metabolic pathways required for the synthesis of some or all of their amino acids and must obtain them from their environment or their host organism. The energetic costs associated with this means of obtaining amino acids are largely a matter of speculation at the present time. This study examines the perceived cost of auxotrophic amino acids (amino acids an organism is unable to synthesize) in two bacteria (Bacillus cereus ATCC 10987 and Vibrio fischeri ES114). Auxotrophic amino acids in both organisms were found to be used preferentially in highly expressed genes and are therefore likely to be energetically inexpensive relative to those the organisms are capable of synthesizing themselves. A regression approach was used to computationally estimate the perceived costs to the organism. Keywords-auxotrophy, bacteria, biosyntheist, perceived cost I. INTRODUCTION Given that the energy expended to biosynthesize amino acids varies considerably, ranging from 11.7 (glycine and alanine) to >74 (tryptophan) high energy phosphate bonds (~PO4) [1] and that energy availability commonly limits prokaryotic growth, it is reasonable that natural selection would favor substitutions that resulted in the utilization of less energetically costly amino acids where possible. Manifestation of such a substitution bias should be greatest in highly expressed genes in much the same way that adherence to codon usage biases tend to be greatest in genes that are expressed at high levels [2,3,4,5,6,7]. Indeed, Akashi and Gojobori (2002) have demonstrated that genes that adhere to organismal codon usage biases most strongly (and, by inference, are most highly expressed) tend to incorporate lower cost amino acids in Escherichia coli and Bacillus subtilis. A study that confirmed the work of Akashi and Gojoboi (2002), [8] also examined the way that auxotrophic amino acids were utilized in two organisms: Chlamydia trachomatis and Chlamydophila pneumoniae. Interestingly, one, Chlamydia trachomatis, preferentially utilized amino acids it could not synthesize itself while the other, Chlamydophila pneumoniae, avoided their use in highly expressed genes. This disparity in usage raises questions about the cost of utilizing these amino acids. It is possible that the cost of the amino acid is associated with its availability in the environment or how the organism facilitates uptake of the amino acid. Examination of auxotrophic mutants of Vibrio fischeri has raised interesting questions [9]. These bacteria are responsible for the luminescence of some squid species and live in close association with them. Nine mutants that were each auxotrophic for a single amino acid were found to have slower growth rates than the wild type bacteria but were able to obtain the needed amino acids from their host. The absence of free amino acids in the space these bacteria live led to the conclusion that either the free amino acids are used too quickly to measure or they are presented to the bacteria as peptides [9].Uptake of amino acid precursors in peptidal form could cause a change in cost relative to other uptake pathways. Many prokaryotic proteases are ATP-dependent and thus could cause the perceived cost of these amino acids to increase [10]. Reliance upon multiple lifestyles can also make inferring amino acid biosynthetic costs challenging. A recent paper [11] explored amino acid biosynthetic costs in Saccharomyces cerevisiae. This organism can live in both aerobic and anaerobic environments – each with significant differences in the energetic cost of amino acid biosynthesis. For example, producing tryptophan aerobically costs 75.5 ATP but anaerobic biosynthesis costs only 14 ATP [12]. Are the expensive amino acids produced anaerobically so that they are cheaper or is amino acid biosynthesis governed more by the conditions in which the organism finds itself? The cost varies so greatly because intermediate molecules are no longer reduced to produce energy. Three amino acids (valine, glycine, and alanine) were largely responsible for these trends [11] and the question arises whether the observed correlation is truly based upon these three amino acids or is it possible that the accepted energetic costs for the amino acids are not accurate? With the multiple lifestyles and two sets of costs for each amino it is possible that neither cost is truly correct. Here we present a method for identifying the perceived cost of amino acids. Two organisms known to be auxotrophic for only one amino acid are analyzed with a linear regression approach to estimate the energetic cost of those amino acids.