Nitrosomonas sp. Is79 is a chemolithoautotrophic ammonia-oxidizing bacterium that belongs to the family Nitrosomonadaceae within the phylum Proteobacteria. Ammonia oxidation is the first step of nitrification, an important process in the global nitrogen cycle ultimately resulting in the production of nitrate. Nitrosomonas sp. Is79 is an ammonia oxidizer of high interest because it is adapted to low ammonium and can be found in freshwater environments around the world. The 3,783,444-bp chromosome with a total of 3,553 protein coding genes and 44 RNA genes was sequenced by the DOE-Joint Genome Institute Program CSP 2006.
The ammonia monooxygenase encodes the first enzyme in the oxidation of ammonia to nitrite via hydroxylamine [37]. Three amoCAB operons can be detected in the genome of Nitrosomonas sp. Is79 (Figure 3) and downstream of two of these operons the hypothetical genes (amoE and amoD [55]) were identified. The genome of Nitrosomonas sp. Is79 contains two single copies of the amoC gene. The copper resistance genes, copC and copD were not detected downstream of any of the amoCAB operons as it was identified in all other described betaproteobacterial ammonia oxidizers [9–12,56].
Figure 3.Organization of the amo gene clusters in the genome of Nitrosomonas sp. Is79.
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The hydroxylamine oxidoreductase (HAO) is the second enzyme in ammonia oxidation, catalyzing the oxidation of hydroxylamine to nitrite [37]. As all other betaproteobacterial ammonia oxidizers, Nitrosomonas sp. Is79 encodes three syntenous hao operons consisting of the genes haoA encoding the octaheme cytochrome c protein subunit that forms the functional HAO complex, haoB encoding an uncharacterized gene product, cycA encoding cytochrome c554, and cycB encoding the quinone reductase, cytochrome cM552.
A copper-containing nitrite reductase (nirK) was detected in the genome of Nitrosomonas sp. Is79. As detected in N. multiformis and Nitrosomonas sp. AL212, the nirK gene exists as a singleton in the genome, which is in contrast to its position in the genomes of N. europaea and N. eutropha where nirK is a member of a conserved multigene cluster (Table 5) [9–12,56].
The nitrite or nitric oxide responsive transcription factor nsrR [57] is missing in the genome of Nitrosomonas sp. Is79, indicating that Nitrosomonas sp. Is79 might have different response mechanisms to nitrite and nitric oxide than N. europaea or N. eutropha (Table 5) [9,10].
Table 5. Presence and absence of genes involved in nitrogen oxide metabolism based on [9–12]Full size table
Genes encoding enzymes for nitric oxide reduction to nitrous oxide (norCBQD) were found in all betaproteobacterial ammonia oxidizers except again in Nitrosomonas sp. Is79. Genes found in the genomes of most chemolithotrophic ammonia oxidizers encoding additional inventory implicated in nitric oxide detoxification to prevent nitrosative stress (cytochrome P460, cytochrome c′-beta [58];) (Table 5) were also identified in the genome Nitrosomonas sp. Is79; however, the genes encoding sNOR were absent. Based on these results it is very likely that Nitrosomonas sp. Is79 can avoid nitrosative stress caused by nitric oxide [59]. In addition, it is likely that Nitrosomonas sp. Is79 cannot reduce nitric oxide to nitrous oxide via nitrifier denitrification [60], but may use an alternate pathway as demonstrated for the nitrifying methanotroph, Methylococcus capsulatus strain Bath [61,62].
Finally and in contrast to all other betaproteobacterial ammonia oxidizers, the gene encoding the red copper protein nitrosocyanin [63] was not identified in the genome of Nitrosomonas sp. Is79. It is currently unclear what implications the absence of this gene may have on the metabolism of Nitrosomonas sp. Is79, because the function of the protein itself is still elusive.
The gene encoding an ammonia transporter (amtB type) was detected in the genome of Nitrosomonas sp. Is79. Ammonia transporters are needed for the acquisition of ammonia/ammonium for assimilation. The function of these genes in ammonia oxidizers that are adapted to low ammonium concentrations is of particular interest also because the process of nitrogen assimilation competes directly with the bacterium’s need for ammonia to sustain catabolism or the generation of energy.
The enzyme urease is responsible for hydrolyzing urea to yield ammonium and carbon dioxide, thereby increasing the substrates for N and C assimilation in the cytoplasm. While the genome of Nitrosomonas sp. Is79 lacks the gene cluster encoding urea hydrolase (ureABCDEFJ) [64]; the genes encoding biotin-containing urea carboxylase and the putative allophanate hydrolase were detected. It has been suggested that the products of these genes convert urea to ammonium and carbon dioxide while consuming metabolic energy (ATP). Incubation of Nitrosomonas sp. Is79 in the presence of urea did not result in the production of nitrite [Sedlacek and Bollmann, unpublished] indicating that urea was not degraded, and that expression of these genes might be regulated through a network controlled by the energy status of the cell.
The genome of Nitrosomonas Is79 contained most of the putative [NiFe] hydrogenase-encoding genes found in the genome of N. multiformis [11]. However, one of the hypothetical proteins is missing, and the genes are scattered over the genome instead of being members of single gene cluster as in N. multiformis [11].
As observed in all ammonia-oxidizing bacteria, Nitrosomonas sp. Is79 fixes carbon dioxide via the Calvin cycle involving the main enzyme RuBisCO (Ribulose-1,5-bisphosphate carboxylase oxygenase). The genomes of Nitrosomonas sp. Is79 and Nitrosomonas sp. AL212 [12] encoded two copies of the RuBisCO operon (Figure 4). One copy belongs to form IA (green-like) RuBisCO and is closely related to the RuBisCO in N. europaea and N. eutropha, while the other copy belongs to form IC (red-like) RuBisCO and is closely related to the enzyme in N. multiformis (Figure 4). The form A RuBisCO is not associated with the genes for the carboxysome as in N. eutropha [10]. The two RuBisCO copies differ in their kinetic properties. Bacteria with RuBisCO form IA have a higher affinity for carbon dioxide than organisms with RuBisCO form IC [65]. Therefore it is very likely that ammonia oxidizers with two different gene copies of the RuBisCO gene have a higher flexibility with respect to the carbon dioxide availability in the environment.
Figure 4.Phylogenetic tree of betaproteobacterial ammonia oxidizers inferred using the Maximum Likelihood criterion using the software package MEGA [35] based on the protein sequence of the large subunit of the RuBisCO (cbbL). The alignment was inferred by ClustalW software [35]. Numbers adjacent to the branches are support values from 1,000 ML bootstrap replicates if higher than 60% [35].
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When comparing the genome of Nitrosomonas sp. Is79 with the other available genomes of betaproteobacterial ammonia oxidizers, several genes and operons were detected that were missing in or unique to the investigated strain.
The genomes of N. europaea, N. eutropha, N. multiformis and Nitrosomonas sp. AL212 encode the genes phaABCDEFG for a NADH driven potassium (cation) proton antiporter. While this potassium transporter was not detected in the genome of Nitrosomonas sp. Is79, the genes for another high affinity ATP driven potassium transporter (kdpABC) were found. These three genes encoding the potassium transporter ATPase (Nit79A3_1970-1972) were upstream of an osmosensitive signal transduction histidine kinase (kdpD) and a two component transcriptional regulator (kdpE). The kdp operon encodes an inducible high affinity potassium transport system that will be expressed under potassium deficiency [66,67]. Nitrosomonas sp. Is79 is known to be adapted to low nutrient concentrations and oligotrophic conditions. The presence of this high affinity transport system could be an adaptation to low ion strength environments.
The genome of N. europaea was characterized by a high number of different kinds of iron transporters [9] and all other genomes including Nitrosomonas sp. Is79 contained high affinity iron transporters. In addition a low affinity iron permease was detected in the genome of Nitrosomonas sp. Is79 (Nit79A3_3148). This enzyme has been characterized well in Saccharomyces cerevisiae and is involved in transport of iron, copper and zinc [68]. These authors discuss the possibility that the high affinity transport systems are active under limiting conditions, while the iron permease becomes active under non-limiting conditions. The enzyme might have the same function in Nitrosomonas sp. Is79.