The lithotrophic capacity of the betaproteobacteria Sphaerotilus natans subsp. sulfidivorans was confirmed at genetic level: functional genes of sulfur metabolism were detected (aprBA, soxB, and sqr, coding for adenylyl phosphosulfate reductase, thiosulfate-cleaving enzyme, and sulfide:quinone oxidoreductase, respectively), and the expression of aprA and soxB genes was demonstrated. An evolutionary scenario for soxB genes in Sphaerotilus representatives is suggested based on comparative analysis of codon occurrence frequency, DNA base composition (G + C content), and topology of phylogenetic trees. The ancestor bacterium of the Sphaerotilus-Leptothrix group was capable of lithotrophic growth in the presence of reduced sulfur compounds. However, in the course of further evolution, the sulfur metabolism genes, including the soxB gene, were lost by some Sphaerotilus strains. As a result, the lithotrophic Sphaerotilus-Leptothrix group split into two phylogenetic lineages, lithotrophic and organotrophic ones.