The comparative metabolism of the mollicutes (Mycoplasmas): the utility for taxonomic classification and the relationship of putative gene annotation and phylogeny to enzymatic function in the smallest free-living cells.

Mollicutes or mycoplasmas are a class of wall-less bacteria descended from low G + C% Gram-positive bacteria. Some are exceedingly small, about 0.2 micron in diameter, and are examples of the smallest free-living cells known. Their genomes are equally small; the smallest in Mycoplasma genitalium is sequenced and is 0.58 mb with 475 ORFs, compared with 4.639 mb and 4288 ORFs for Escherichia coli. Because of their size and apparently limited metabolic potential, Mollicutes are models for describing the minimal metabolism necessary to sustain independent life. Mollicutes have no cytochromes or the TCA cycle except for malate dehydrogenase activity. Some uniquely require cholesterol for growth, some require urea and some are anaerobic. They fix CO2 in anaplerotic or replenishing reactions. Some require pyrophosphate not ATP as an energy source for reactions, including the rate-limiting step of glycolysis: 6-phosphofructokinase. They scavenge for nucleic acid precursors and apparently do not synthesize pyrimidines or purines de novo. Some genera uniquely lack dUTPase activity and some species also lack uracil-DNA glycosylase. The absence of the latter two reactions that limit the incorporation of uracil or remove it from DNA may be related to the marked mutability of the Mollicutes and their tachytelic or rapid evolution. Approximately 150 cytoplasmic activities have been identified in these organisms, 225 to 250 are presumed to be present. About 100 of the core reactions are graphically linked in a metabolic map, including glycolysis, pentose phosphate pathway, arginine dihydrolase pathway, transamination, and purine, pyrimidine, and lipid metabolism. Reaction sequences or loci of particular importance are also described: phosphofructokinases, NADH oxidase, thioredoxin complex, deoxyribose-5-phosphate aldolase, and lactate, malate, and glutamate dehydrogenases. Enzymatic activities of the Mollicutes are grouped according to metabolic similarities that are taxonomically discriminating. The arrangements attempt to follow phylogenetic relationships. The relationships of putative gene assignments and enzymatic function in My. genitalium, My. pneumoniae, and My. capricolum subsp. capricolum are specially analyzed. The data are arranged in four tables. One associates gene annotations with congruent reports of the enzymatic activity in these same Mollicutes, and hence confirms the annotations. Another associates putative annotations with reports of the enzyme activity but from different Mollicutes. A third identifies the discrepancies represented by those enzymatic activities found in Mollicutes with sequenced genomes but without any similarly annotated ORF. This suggests that the gene sequence is significantly different from those already deposited in the databanks and putatively annotated with the same function. Another comparison lists those enzymatic activities that are both undetected in Mollicutes and not associated with any ORF. Evidence is presented supporting the theory that there are relatively small gene sequences that code for functional centers of multiple enzymatic activity. This property is seemingly advantageous for an organism with a small genome and perhaps under some coding restraint. The data suggest that a concept of "remnant" or "useless genes" or "useless enzymes" should be considered when examining the relationship of gene annotation and enzymatic function. It also suggests that genes in addition to representing what cells are doing or what they may do, may also identify what they once might have done and may never do again.