Hydroxylamine Oxidoreductase Gene Research Articles

The physiological potential of anammox bacteria as revealed by their core genome structure.

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. Kuenenia stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologues. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least five paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH2OH by self-production for anammox metabolism.

Inhibition of Bacterial Ammonia Oxidation by Organohydrazines in Soil Microcosms

Hydroxylamine oxidation by hydroxylamine oxidoreductase (HAO) is a key step for energy-yielding in support of the growth of ammonia-oxidizing bacteria (AOB). Organohydrazines have been shown to inactivate HAO from Nitrosomonas europaea, and may serve as selective inhibitors to differentiate bacterial from archaeal ammonia oxidation due to the absence of bacterial HAO gene homolog in known ammonia-oxidizing archaea (AOA). In this study, the effects of three organohydrazines on activity, abundance, and composition of AOB and AOA were evaluated in soil microcosms. The results indicate that phenylhydrazine and methylhydrazine at the concentration of 100 μmol g−1 dry weight soil completely suppressed the activity of soil nitrification. Denaturing gradient gel electrophoresis fingerprinting and sequencing analysis of bacterial ammonia monooxygenase subunit A gene (amoA) clearly demonstrated that nitrification activity change is well paralleled with the growth of Nitrosomonas europaea-like AOB in soil microcosms. No significant correlation between AOA community structure and nitrification activity was observed among all treatments during the incubation period, although incomplete inhibition of nitrification activity occurred in 2-hydroxyethylhydrazine-amended soil microcosms. These findings show that the HAO-targeted organohydrazines can effectively inhibit bacterial nitrification in soil, and the mechanism of organohydrazine affecting AOA remains unclear.

PCR Amplification and Bioinformatics of a Novel Gene Coding Hydroxylamine Oxidoreductase*

A probable novel gene coding hydroxylamine oxidoreductase (HAO) was acquired by PCR and gene sequencing in Acinetobactor sp. YY-5. Four pairs of PCR primers were designed according to the conservative regions of reported HAO gene to amplify and sequence the partial sequence of the HAO gene; several primers for genome walking PCR were designed according to the partial sequence to acquire the flank region of the partial HAO sequence; and the acquired sequence was subjected to Blast analysis in NCBI and open read frame (ORF) prediction by the professional ORF -analyzing software, FGeneSB. A 462 bp DNA sequence was obtained by one of the 4 pairs of primers, and further 2 690 bp DNA sequence of the flank region of 462 bp sequence was acquired through two rounds of genome walking PCR. Four ORFs were found within the 3 152 bp DNA sequence, one of which was of 555 bp. The 555-bp ORF contained the original 462 bp sequence and corresponded to an amino acid sequence of Mr 20.2×103, and no obvious similar HAO gene was found in GenBank. Thus, a novel HAO gene was possibly acquired by PCR.

Whole‐genome analysis of the ammonia‐oxidizing bacterium, Nitrosomonas eutropha C91: implications for niche adaptation

Analysis of the structure and inventory of the genome of Nitrosomonas eutropha C91 revealed distinctive features that may explain the adaptation of N. eutropha-like bacteria to N-saturated ecosystems. Multiple gene-shuffling events are apparent, including mobilized and replicated transposition, as well as plasmid or phage integration events into the 2.66 Mbp chromosome and two plasmids (65 and 56 kbp) of N. eutropha C91. A 117 kbp genomic island encodes multiple genes for heavy metal resistance, including clusters for copper and mercury transport, which are absent from the genomes of other ammonia-oxidizing bacteria (AOB). Whereas the sequences of the two ammonia monooxygenase and three hydroxylamine oxidoreductase gene clusters in N. eutropha C91 are highly similar to those of Nitrosomonas europaea ATCC 19718, a break of synteny in the regions flanking these clusters in each genome is evident. Nitrosomonas eutropha C91 encodes four gene clusters for distinct classes of haem-copper oxidases, two of which are not found in other aerobic AOB. This diversity of terminal oxidases may explain the adaptation of N. eutropha to environments with variable O(2) concentrations and/or high concentrations of nitrogen oxides. As with N. europaea, the N. eutropha genome lacks genes for urease metabolism, likely disadvantaging nitrosomonads in low-nitrogen or acidic ecosystems. Taken together, this analysis revealed significant genomic variation between N. eutropha C91 and other AOB, even the closely related N. europaea, and several distinctive properties of the N. eutropha genome that are supportive of niche specialization.

Transcriptional Analysis of the MulticopyhaoGene Coding for Hydroxylamine Oxidoreductase inNitrosomonassp. Strain ENI-11

The nitrifying bacterium Nitrosomonas sp. strain ENI-11 has three copies of the gene encoding hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)) on its genome. Broad-host-range reporter plasmids containing transcriptional fusion genes between hao copies and lacZ were constructed to analyze the expression of each hydroxylamine oxidoreductase gene (hao) copy individually and quantitatively. beta-Galactosidase assays of ENI-11 harboring reporter plasmids revealed that all hao copies were transcribed in the wild-type strain. Promoter analysis of hao copies revealed that transcription of hao(3) was highest among the hao copies. Expression levels of hao(1) and hao(2) were 40% and 62% of that of hao(3) respectively. Transcription of hao(1) was negatively regulated, whereas a portion of hao(3) transcription was read through transcription from the rpsT promoter. When energy-depleted cells were incubated in the growth medium, only hao(3) expression increased. This result suggests that it is hao(3) that is responsible for recovery from energy-depleted conditions in Nitrosomonas sp. strain ENI-11.

Comparison of 16S rRNA, ammonia monooxygenase subunit A and hydroxylamine oxidoreductase gene, in chemolithotrophic ammonia-oxidizing bacteria.

Comparison of 16S rRNA, ammonia monooxygenase subunit A and hydroxylamine oxidoreductase gene, in chemolithotrophic ammonia-oxidizing bacteria.

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.