Minimal bacterial gene set comprises the genetic elements needed for survival of 
engineered bacterium on a rich medium. This set is estimated to include 300-350 
protein-coding genes. One way of simplifying an organism with such a minimal 
genome even further is to constrain the amino acid content of its proteins. In 
this study, comparative genomics approaches and the results of gene knockout 
experiments were used to extrapolate the minimal gene set of mollicutes, and 
bioinformatics combined with the knowledge-based analysis of the 
structure-function relationships in these proteins and their orthologs, paralogs 
and analogs was applied to examine the challenges of completely replacing the 
rarest residue, cysteine. Among several known functions of cysteine residues, 
their roles in the active centers of the enzymes responsible for 
deoxyribonucleoside synthesis and transfer RNA modification appear to be 
crucial, as no alternative chemistry is known for these reactions. Thus, drastic 
reduction of the content of the rarest amino acid in a minimal proteome appears 
to be possible, but its complete elimination is challenging.