Rodríguez-Castaño, Gina P.

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    Sequence diversity and expression of novel bacterial nitrous oxide reductase (Nosz) genes in tropical environments
    (2008) Rodríguez-Castaño, Gina P.; Massol-Deyá, Arturo A.; College of Arts and Sciences - Sciences; Loeffler, Frank E.; Montalvo-Rodríguez, Rafael R.; Muñoz, Carlos A.; Department of Biology; Corredor, Jorge E.
    Nitrous oxide (N2O) is produced in different microbial processes including denitrification, nitrification and dissimilatory nitrate reduction to ammonia (DNRA) (Kelso et al. 1999). It has been suggested that nitrification is the main source of N2O. Understanding the mechanisms that control the flux of N2O is crucial to predict and manage emissions of this powerful greenhouse gas. Amplification of genes (nosZ) coding for nitrous oxide reductases (N2ORs) from denitrifiers and N2- fixers has been obtained by PCR methods. In this thesis, we refer to these sequences as “traditional” nosZ sequences; on the other hand, the nosZ genes from microaerophilic Anaeromyxobacter spp. and Magnetospirillum spp., and other obligate anaerobic microorganisms, such as Wolinella spp., Desulfitobacterium spp and Dechloromonas spp. have not been well studied. Their primary N2OR sequences diverge from the traditional ones and therefore, different primer sets must be developed to better understand their diversity and distribution in nature. This study developed oligonucleotides for amplifying a broader range of nosZ genes to assess their diversity in soil and bioreactors by cultureindependent techniques. nosZ sequences obtained from environmental samples were different from traditional nosZ sequences. None of the clone sequences shared more than 62% amino acid similarity with traditional NosZ. Additionally, this analysis revealed the presence of conserved histidine residues essential for function of a mature N2OR protein. Through a phylogenetic analysis using Neighbor-Joining, Maximum Parsimony, Maximum Likelihood, and Bayesian Inference methods, nine clades of NosZ variants were identified. Neither of the clone sequences fell into the traditional NosZ phylogenetic clades, but grouped with Anaeromyxobacter spp., Magnetospirillum spp., Desulfitobacterium hafniense, and Dechloromonas aromatica. The detection of nosZ genes was achieved by ISRT-PCR/FISH using an internal fluorescently-labeled NosZ943 probe. This constitutes the first published report of probing nosZ amplicons inside of active microbial cells using an optimized In Situ Reverse Transcriptase-PCR/Fluorescent In Situ Hybridization (ISRT-PCR/FISH) protocol. Our results show that a clone carrying the partial Anaeromyxobacter nosZ gene emits a strong fluorescent signal when detected with NosZ943 probe, but not with Nos1527 probe; while a clone carrying the partial Pseudomonas gene did not emit any fluorescent signal with the NosZ943 probe. ISRTPCR was further applied to natural samples from an anoxic bioreactor. Approximately 4% of total cell counts were expressing the novel nosZ genes. We demonstrate the existence of many variants of nosZ gene that are not yet represented by cultured organisms. These variants could represent a high functional diversity for reducing N2O in the environment. Therefore, the diversity of nosZ genes in nature and their contribution to the N2O budget warrants further exploration.