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Nitrosomonas europaea is a chemolithoautotrophic soil bacterium which oxidizes ammonia to nitrite in the process of nitrification. Ammonia is oxidized to nitrite by the sequential actions of ammonia monooxygenase and hydroxylamine oxidoreductase, providing reductant for the cell (22). In N. europaea, the primary nitrification genes are present in multiple copies. Ammonia monooxygenase is encoded by the genes amoC, amoA, and amoB, which are arranged contiguously and are cotranscribed as a 3.5-kb RNA (12, 19) (Fig. 1). The amoCAB operon is found in two copies in the N. europaea genome (12). These two copies of amoC have only 10 bp differences in 813 bp. In addition, there is a third copy of amoC, which is not associated with amoA or amoB (19), with about 60% sequence similarity to the other copies of amoC. The two copies of amoA only have 1 bp difference in 828 bp, and the two copies of amoB are identical. The gene for hydroxylamine oxidoreductase (hao) is present in three copies in the N. europaea genome (13, 20), and these genes are transcribed monocistronically (20). The three copies of hao only have 1 bp difference in 1,701 bp. The gene encoding cytochrome c-554 (hcy or cyc), an electron acceptor from hydroxylamine oxidoreductase, is also present in three copies which are located 950 bp downstream from each copy of hao (1, 9, 13, 20). The sequences of two copies of the gene for c-554 are known and differ by only 1 bp in 708 bp.

View larger version (11K): [in this window] [in a new window]   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 (hao1,2); and e, pc12 (hcy). The dark line beneath amoC indicates the location of the amoC probe used in Northern blots.

The physiological significance of multiple copies of the nitrification genes is still unknown. Mutagenesis experiments have shown that no single copy of amo or hao is essential (7, 8). In the case of amoA, when one copy is inactivated, transcription of the other operon increases to compensate for the loss of expression of the first copy during growth. However, while amoA₁ can completely compensate for the inactivation of amoA₂, amoA₂ can only compensate to about 65% of full transcription activity when amoA₁ is inactivated (7). This reduced level of amo transcript in the amoA₁ mutant may be the cause for the reduced growth rate observed in this mutant strain in laboratory cultures (7). Mutagenesis experiments on amoA suggest that under some conditions transcription of the two copies of amo is not identical. To better understand the basis for differences in transcription of the two copies of amoCAB, experiments were done to map the putative promoters for amoCAB and to examine the expression of amo in the presence and absence of ammonium (NH₄⁺). Transcript mapping was also done for hao and hcy.

N. europaea cells (ATCC 19178) were grown in defined medium (3). For the primer extension experiments, N. europaea cells were grown for 3 days to the late exponential growth phase. Cells were harvested, washed three times, and stored as a pellet at 4°C for at least 24 h to allow the amo mRNA to be degraded (21). The cells were then resuspended in 150 ml of fresh medium (~1.3 × 10¹⁰ cells/ml) in the presence or absence of 25 mM (NH₄)₂SO₄. The cells were shaken at 30°C for 3 h before harvesting for RNA extraction.

Following the numbering of Hirota et al. (6), the copy of hao which we had previously obtained as a genomic lambda clone (8, 20) is hao₃. The upstream regions of hao₁ and hao₂ and of the two copies of amoC were cloned using inverse PCR (18). The PCR-amplified fragments were cloned into the pGEMT-Easy cloning vector (Promega Corp, Madison, Wis.), and the DNA sequences of the promoter regions of these genes were obtained (Center for Gene Research and Biotechnology, Oregon State University). Total RNA was isolated as described (16). DNA probes were labeled by random priming using the Prime-a-Gene kit from Promega Corporation with [-³²P]dCTP (3,000 Ci/mmol; DuPont NEN Products, Wilmington, Del.). The hybridization signals were analyzed using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.).

Oligonucleotide primers were synthesized by the Center for Gene Research and Biotechnology (Table 1). The amoC primers C23 and C21 were specific to amoC_1,2 and did not bind to the related gene amoC₃. The primers were 5'-end labeled with T4 polynucleotide kinase using the Prime-a-Gene kit (Promega) with [-³²P]dATP (6,000 Ci/mmol; DuPont NEN Products). Primer extensions were done using 1 µg of total RNA and 5 × 10⁵ cpm of labeled primer as described (18). Primer extensions were done using reverse transcriptase (Perkin-Elmer, Branchburg, N.J.) following the manufacturer's directions. The resulting labeled cDNA fragments were resolved on a sequencing gel (8 M urea, 8% acrylamide) at 60 W for 2 h. An M13 sequencing ladder labeled with [-³⁵S]dATP was used as a size standard. The labeled fragments were analyzed by phosphorimagery as above.

The following sequences are available in GenBank under the indicated accession numbers: amoC₁, AF058691; amoC₂, AF058692; hao₁, U04053; hao₂, U04053; hao₃, U04053; and hcy, U05951.

DNA sequencing. Using inverse PCR, the upstream flanking DNA for amoC₁, amoC₂, hao₁, hao₂, and hcy was obtained and sequenced. The nucleotide sequence of the upstream region of hao₃ was reported previously (20). The DNA sequences upstream of the two copies of amoC were found to be identical for 334 bp upstream of the gene. The sequences upstream of hao₁ and hao₂ were found to be similar for 160 bp upstream of the genes, having 3 nucleotide (nt) differences in that span. Further upstream, the sequences had no significant similarity. For hao₃, a different upstream sequence was obtained that diverged from the other hao copies 15 bp upstream of the start codon (Fig. 2).

View larger version (44K): [in this window] [in a new window]   FIG. 2.   Sequence alignment of the upstream regions of amoC1, amoC2, hao1,2,3 (hao3 is from reference (20)), amoA1 (12) (GenBank no. AF058691), and amoA2 (GenBank no. AF058692), hcy, and the E. coli consensus 70 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.

Primer extensions on amoC, amoA, hao, and hcy. Primer extension experiments from amoC revealed three fragments 219, 167, and 156 nt in size (Fig. 3, lane 1). These fragments indicate three potential transcription start sites located at 166, 114, and 103 bp upstream relative to the start codon of amoC (Fig. 2). The 166 and 103 sites both have putative promoter sequences associated with them (designated P₁ and P₂, respectively; Fig. 4C) that are similar to consensus ⁷⁰-type Escherichia coli promoters (Fig. 2). These results were confirmed with a second amoC primer (C21, Table 1) located downstream from C23 (data not shown). Both promoter sequences were identical for each of the two copies of amoC. The origin of the 167-nt fragment, which mapped to bp 114, is unclear. The 114 site did not have an identifiable match with the E. coli consensus ⁷⁰-type sequence (5). It may be a secondary transcription start site for P₂, or alternatively, the 167-nt fragment may represent a partially degraded RNA derived from P₁.

View larger version (32K): [in this window] [in a new window]   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.

View larger version (23K): [in this window] [in a new window]   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.

Effect of NH₄⁺ on primer extensions from amoC. The production of multiple fragments in primer extension reactions could result from tandem promoters for amoC. This possibility was investigated by comparing the relative amounts of the 219-, 167-, and 156-nt fragments produced in primer extension reactions when transcription of amo was induced by the addition of NH₄⁺ (Fig. 4). N. europaea cells which had their endogenous RNA levels depleted were transferred to fresh medium either lacking NH₄⁺ or containing 25 mM (NH₄)₂SO₄. The amount of amo mRNA present after 3 h was estimated by primer extensions and by Northern blots. Primer extensions showed three fragments in samples grown with and without NH₄⁺ which responded differently to the addition of NH₄⁺ (Fig. 4C). Ratios were calculated for the amount of cDNA produced in primer extension reactions in the plus-NH₄⁺ sample relative to the minus-NH₄⁺ sample. In this experiment (Fig. 4C), the ratio for the 219-nt fragment was 4.36, the ratio for the 167-nt fragment was 2.0, and the ratio for the 156-nt fragment was 0.6. When the average ratios were calculated from four independent experiments, the ratio for the 219-nt fragment was 3.3 ± 1.6, the ratio for the 167-nt fragment was 1.3 ± 0.5, and the ratio for the 156-nt fragment was 0.42 ± 0.28.

Northern blots. In the absence of NH₄⁺, Northern blots probed with an amoC probe showed a single fragment of about 1.1 kb (Fig. 4A). Previous work has shown that this fragment is derived from amoC_1,2 but not amoC₃ (19). Northern blots from samples incubated in medium containing NH₄⁺ highlighted a 3.5-kb fragment (transcription of the full amoCAB operon) as well as the 1.1-kb fragment seen in the minus-NH₄⁺ sample. The same blot stripped and reprobed with a 16S rRNA probe indicated that the relative loading concentrations of the two samples were the same (Fig. 4B). These results are consistent with the results of the primer extensions. In the sample without NH₄⁺, the 1.1-kb RNA fragment was likely the source of the three fragments produced in the primer extensions from the sample without NH₄⁺. Although the Northern blot only showed one fragment in the sample without NH₄⁺, the size resolution of fragments visualized on Northern blots was not sufficient to detect a 70-nt difference. Therefore, the fragments seen in the Northern blots were apparently a mixed population containing the different 5' ends shown by the primer extensions. When RNA from the sample with NH₄⁺ was used, the same three fragments were observed in the primer extensions as in the sample without NH₄⁺, albeit in different relative proportions. Since both the 3.5-kb fragment in the Northern and the 219-nt fragment in the primer extension are induced in the presence of NH₄⁺, it may be that the 219-nt fragment is primarily transcribed from P₁ on the 3.5-kb RNA, while the 167- and 156-nt fragments derive mainly from the 1.1-kb RNA. However, the results presented here do not allow us to ascribe primer extension products to specific RNAs on Northern blots.