In silico approach for the design of a culture medium for `Candidatus Liberibacter solanacearum´

the laboratory. We are working in a genome annotation-based in silico approach for the design of a culture medium for this bacterium. The first consideration is to understand how this bacterium is provided with carbon and energy source compounds, as well as nitrogen and sulphur compounds. Thus, none of the sequences in its gene repertoire suggests that it is capable of transporting sucrose or fructose, leading to hypothesize that glucose is a major form of reduced carbon, and therefore glucose may be the main or the only source of energy and carbon. CaLsol can only synthesize de novo 6 aminoacids, and none of them is asparagine or tryptophan. Moreover, CaLsol lacks the enzymes required for incorporation of sulphur-containing inorganic compounds into amino acids, but the reduction of sulphate by a non-canonic enzymatic process or the use of an alternative terminal electron acceptor under anaerobic conditions cannot be ruled out. Also, it appears to be able to incorporate ammonia into glutamine. Concerning vitamins, no sequences matching complete transporters for riboflavin, pyridoxal phosphate, niacin, cobalamin, biotin or folate were found in the genome. Thiamine cannot be synthesized, but all three constituents of a typical prokaryotic thiamine ABC transporter are present. The source of NAD is nowadays unknown because it cannot synthesize or transport niacin. This bacterium lacks the Opp ABC oligopeptide transporters which partly accounts for the recycling of wall cell peptides, so the presence in the culture medium of cell wall components should aid in the growth of it. Culture temperature should not surpass 32.5ºC as cell membrane LpxL is absent in CaLsol. We are currently working on two approaches to try to achieve the growth of CaLsol in the laboratory, always taking into account some of the main traits of the genome of this bacterium. On the one hand, the use of complex culture media which, according to their general composition, could allow the growth of bacteria with significant deficiencies. On the other hand, the use of defined culture media supplemented with carrot phloem extract and other additives.