Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

Abstract

ABSTRACT Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao , hydroxylamine oxidoreductase; orf2 , a putative protein; cycA , cytochrome c 554 ; and cycB , cytochrome c m 552 . The deduced protein sequences of HAO, c 554 , and c m 552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi- c -heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c -hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c m 552 , NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria . Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c 554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c 554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.