Transcript analysis of multiple copies of amo (encoding ammonia monooxygenase) and hao (encoding hydroxylamine oxidoreductase) in Nitrosomonas europaea

Abstract

The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative sigma(70) promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium.

FIG. 1

Physical map of the amoCAB operon, hao, and hcy. The locations of the putative promoters for amoC (P₁ and P₂), amoA, hao, and hcy are indicated. The locations of the oligonucleotide primers used in primer extension experiments are indicated: a, C23 (amoC); b, A5 (amoA); c, ph10 (hao₃); d, HB5 (hao_1,2); and e, pc12 (hcy). The dark line beneath amoC indicates the location of the amoC probe used in Northern blots.

FIG. 2

Sequence alignment of the upstream regions of amoC₁, amoC₂, hao_1,2,3 (hao₃ is from reference (20)), amoA₁ (12) (GenBank no. AF058691), and amoA₂ (GenBank no. AF058692), hcy, and the E. coli consensus ς⁷⁰ promoter sequence (5). The sequences are aligned to their mapped transcriptional start sites (underlined). The −10 and −35 regions of the putative promoters are also underlined. Two promoters for amoC (P₁ and P₂) are indicated. In the E. coli consensus sequence, nucleotides found in more than 65% of the sequences are in uppercase letters, and nucleotides found in less than 65% are in lowercase (5). The numbering is relative to the start codon of the gene.

FIG. 3

Primer extensions. Lane 1, amoC using primer C23; lane 2, amoA using primer A5; lane 3, hao using primer ph10; lane 4, hao using primer HB5; lane 5, hcy using primer pc12. Lanes M, M13 sequence as size standards.

FIG. 4

Primer extensions for amoC with and without NH₄⁺. N. europaea cells whose amo mRNA was depleted by incubation in the absence of NH₄⁺ were resuspended in fresh medium either without (−) or with (+) 25 mM (NH₄)₂SO₄. (A) Northern blots using an amoC probe. (B) The same blot as in A, stripped and reprobed with a 16s rRNA probe. (C) Primer extensions were done using an amoC primer (C23). The 219-, 167-, and 156-nt fragments correspond to transcription initiation at −166, −114, and −103 bp upstream of the start codon of amoC, respectively. Lane M, M13 sequencing ladder was used as size standards.