Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history

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.

FIG. 1.

Cartoon showing ORFs of the hao gene clusters in Nitrosococcus oceani (Nc_oce), Nitrosomonas europaea (Nm_eur), and Nitrosospira multiformis (Ns_mul).

FIG. 2.

ClustalW alignment of Hao protein sequences from Nitrosococcus oceani (NO0160), Nitrosospira multiformis (NMU1996), Nitrosomonas europaea (NE0962), Methylococcus capsulatus (MCA0956), and Silicibacter pomeroyi (SPOA0200). Also included are the deduced protein sequences of putative Hao proteins from Geobacter metallireducens (Gmet02001372), Desulfovibrio desulfuricans (Ddes02001487), Methanococcoides burtonii (Mbur03000734), and the likely nonfunctional Hao-like sequence from Magnetococcus sp. strain Mc-1 (Mmc10221065). Amino acid residues conserved in the majority of sequences are shaded. The secondary structure of HAO from N. europaea (26) is shown beneath the alignments, with the letters H designating α-helical regions and B designating β-turns. Heme-binding regions are underlined.

FIG. 3.

Southern blot of restriction digests of genomic DNA from N. oceani, probed with the fragment of N. oceani hao amplified by PCR. Lanes 1 to 5, restriction digests of N. oceani DNA (lane 1, BamHI; lane 2, EcoRI; lane 3, KpnI; lane 4, PstI; lane 5, SacI); lane 6, E. coli LE392 genomic DNA digested with EcoRI; lane 7, N. europaea genomic DNA digested with EcoRI.

FIG. 4.

ClustalW alignments of the predicted amino acid sequences of ORFs 2, 3, and 4 in the hao gene clusters from N. oceani (NO), N. multiformis (NMU), and N. europaea (NE). Because the sequences of the multiple copies in NMU and NE are identical, only one sequence is shown. (A) Alignment of the predicted sequences of Orf2 proteins listed by their accession numbers. To highlight the putative involvement of this protein in catabolic electron flow during ammonia oxidation, the sequences of Orf2 proteins found in tandem arrangement with HAO as single copies in the nucleoid of Methylococcus capsulatus (MCA0955) and on the megaplasmid of Silicibacter pomeroyi (SPOA0200) are shown. (B) Alignment of the predicted sequences of cytochrome c₅₅₄ proteins listed by their accession numbers. These cytochromes have been reported, so far, only from aAOB. Signal peptides, predicted by PSORT, have been removed from the sequences. The secondary structure of cytochrome c₅₅₄ from NE (28) is shown below the alignment, with letters H designating α-helical regions and B designating β-turns. Heme-binding regions are underlined. (C) Alignment of the predicted sequences of cytochrome c_m552 proteins from aAOB listed by their accession numbers. The result of a phylogenetic analysis of these and 53 related sequences is provided below in Fig. 6.

FIG. 4.

ClustalW alignments of the predicted amino acid sequences of ORFs 2, 3, and 4 in the hao gene clusters from N. oceani (NO), N. multiformis (NMU), and N. europaea (NE). Because the sequences of the multiple copies in NMU and NE are identical, only one sequence is shown. (A) Alignment of the predicted sequences of Orf2 proteins listed by their accession numbers. To highlight the putative involvement of this protein in catabolic electron flow during ammonia oxidation, the sequences of Orf2 proteins found in tandem arrangement with HAO as single copies in the nucleoid of Methylococcus capsulatus (MCA0955) and on the megaplasmid of Silicibacter pomeroyi (SPOA0200) are shown. (B) Alignment of the predicted sequences of cytochrome c₅₅₄ proteins listed by their accession numbers. These cytochromes have been reported, so far, only from aAOB. Signal peptides, predicted by PSORT, have been removed from the sequences. The secondary structure of cytochrome c₅₅₄ from NE (28) is shown below the alignment, with letters H designating α-helical regions and B designating β-turns. Heme-binding regions are underlined. (C) Alignment of the predicted sequences of cytochrome c_m552 proteins from aAOB listed by their accession numbers. The result of a phylogenetic analysis of these and 53 related sequences is provided below in Fig. 6.

FIG. 5.

Most parsimonious phylogenetic tree constructed from 36 ClustalX-aligned sequences of multi-heme cytochrome proteins in the proposed tetrathionate reductase (Ttr), hydroxylamine oxidoreductase (Hao), and formate-dependent nitrite reductase (Nrf) protein family. The tetrathionate reductase and hydroxylamine oxidoreductase protein subfamilies likely evolved from a common ancestral octaheme cytochrome c reductase (*) that emerged from the formate-dependent pentaheme nitrate reductase (Nrf) systems of ancestral delta-/epsilonproteobacteria. Bootstrap values for the clades are shown at the branch points. Protein accession numbers are indicated.

FIG. 6.

Unrooted most parsimonious phylogenetic tree constructed from 56 ClustalX-aligned protein sequences of the membrane-associated tetraheme cytochromes in the proposed Cm552/NrfB/H&NapC/TorC/NirT protein family. The clade with members of the NapC/TorC/NirT protein subfamily was collapsed to focus on the clade with the members of the Cm552/NrfB/H protein subfamily in the phylogram. Bootstrap values for the clades are shown at the branch points. See Materials and Methods for accession numbers not provided in the figure.