Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

Black Hills State University, SPF, South Dakota, United States
Applied and Environmental Microbiology (Impact Factor: 3.67). 10/2005; 71(9):5371-82. DOI: 10.1128/AEM.71.9.5371-5382.2005
Source: PubMed


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 c554; and cycB, cytochrome cm552. The deduced protein sequences of HAO, c554, and cm552 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 cm552, 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 c554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches
to detect aAOB.

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Available from: Martin G Klotz
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    • "Earlier results indicated that the haoA and cycAB genes are under control of different promoters in N. europaea (Sayavedra-Soto et al., 1994; Arp et al., 2002; Hommes et al., 2002), however, gammaproteobacterial AOB such as N. oceani appear to produce distinct haoAB, cycAB and haoAB-cycAB transcripts indicating complex regulation similar to what was reported for the regulation of genes in the amo gene cluster (El Sheikh and Klotz, 2008; El Sheikh et al., 2008). Cytochrome c M 552 belongs to a large superfamily of membraneassociated cytochrome c proteins (NapC/NrfH) that exchange electrons with the quinone/quinol pool (Bergmann et al., 2005; Simon and Klotz, 2013). Generally in bacterial genomes, the cycB gene encoding c M 552 is clustered with other genes encoding catalytic periplasmic proteins that facilitate reduction of nitrogen oxides such as nitrate reductase (nap), nitrite reductase (nrf ), and/or homologues of cytochrome c554 that function as nitric oxide reductases (Upadhyay et al., 2006; Klotz and Stein, 2011; Simon and Klotz, 2013). "
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    ABSTRACT: Ammonia serves as the source of energy and reductant and as a signaling molecule that regulates gene expression in obligate ammonia-oxidizing chemolithotrophic microorganisms. The gammaproteobacterium, Nitrosococcus oceani, was the first obligate ammonia-oxidizer isolated from seawater and is one of the model systems for ammonia chemolithotrophy. We compared global transcriptional responses to ammonium and the catabolic intermediate, hydroxylamine, in ammonium-starved and non-starved cultures of N. oceani to discriminate transcriptional effects of ammonium from a change in overall energy and redox status upon catabolite availability. The most highly expressed genes from ammonium- or hydroxylamine-treated relative to starved cells are implicated in catabolic electron flow, carbon fixation, nitrogen assimilation, ribosome structure and stress tolerance. Catabolic inventory-encoding genes, including electron flow-terminating Complexes IV, FoF1 ATPase, transporters, and transcriptional regulators were among the most highly expressed genes in cells exposed only to ammonium relative to starved cells, although the differences compared to steady-state transcript levels were less pronounced. Reduction in steady-state mRNA levels from hydroxylamine-treated relative to starved-cells were less than five-fold. In contrast, several transcripts from ammonium-treated relative to starved cells were significantly less abundant including those for forward Complex I and a gene cluster of cytochrome c encoding proteins. Identified uneven steady-state transcript levels of co-expressed clustered genes support previously reported differential regulation at the levels of transcription and transcript stability. Our results differentiated between rapid regulation of core genes upon a change in cellular redox status vs. those responsive to ammonium as a signaling molecule in N. oceani, both confirming and extending our knowledge of metabolic modules involved in ammonia chemolithotrophy.
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    • "A novel dual N assimilation and respiratory mechanism employing the reverse hydroxylamine-ubiquinone redox module (HURM; Klotz and Stein, 2008) pathway (haoA'þcycB) has been reported recently (Campbell et al., 2009). Interestingly, the nrfAH and haoA'þcycB inventories are homologues (Bergman et al., 2005; Kim et al., 2008; Klotz et al., 2008). Ammonia, whether available in the environment, obtained by nitrogen fixation or by ammonification from NOx, is another important pool of reactive nitrogen. "
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    ABSTRACT: Recent advances in DNA sequencing have greatly accelerated our ability to obtain the raw information needed to recognize both known and potential novel modular microbial genomic capacity for nitrogen metabolism. With PCR-based approaches to quantifying microbial mRNA expression now mainstream in most laboratories, researchers can now more efficiently propose and test hypotheses on the contributions of individual microbes to the biological accessibility of nitrogen upon which all other life depends. We review known microbial roles in these key nitrogen transformations, and describe the necessary steps in carrying out relevant gene expression studies. An example experimental design is then provided characterizing Nitrosococcus oceani mRNA expression in cultures responding to ammonia. The approach described, that of assessing microbial genome inventory and testing putative modular gene expression by mRNA quantification, is likely to remain an important tool in understanding individual microbial contributions within microbial community activities that maintain the Earth's nitrogen balance.
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    • "Some Gamma-MOB encode haoAB genes but not the adjoining cytochromes c as in the AOB (Hou et al., 2008; Klotz et al., 2008; Nyerges and Stein, 2009; Pol et al., 2007; Poret-Peterson et al., 2008), hence ammonia oxidation is not an energy-generating pathway for these bacteria. The haoAB genes with uncharacterized function have been identified in a number of other proteobacterial genomes (Bergmann et al., 2005). Interestingly, the HAO enzyme shares an evolutionary origin with pentaheme nitrite reductase, NrfA, that is used in dissimilatory reduction of nitrite to ammonia via hydroxylamine (Einsle et al., 1999). "
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