Expression of nifH Genes in Natural Microbial Assemblages in Lake George, New York, Detected by Reverse Transcriptase PCR

Abstract

A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-m pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the ␣-and ␥-subdivisions of the division Proteobacteria (␣-and ␥-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, ␣-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

NifH and NifD phylogenies: an evolutionary basis for understanding nitrogen fixation capabilities of methanotrophic bacteria

“…The cultivation-independent retrieval of nif sequences from different habitats has become a widely used approach to analyse the diversity of dinitrogen-fixing bacteria in ecosystems (Ueda et al, 1995a, b;Zehr et al, 1995Zehr et al, , 1998Widmer et al, 1999;Zani et al, 2000;Lovell et al, 2001;Rösch et al, 2002). However, with very few exceptions (Ueda et al, 1995b), most of the environmental studies so far have focused on the retrieval and comparative sequence analysis of nifH.…”

Effects of nitrogen fertilization on diazotrophic activity of microorganisms associated with Sphagnum magellanicum

“…A fragment of the nifH gene (360 bp) was amplified by polymerase chain reaction (PCR) in a nested approach: the primary step included the forward primer 19F (5′-GCIWTYTAYGGIAARGGIGG-3′; Ueda et al 1 9 9 5 ) a n d t h e r e v e r s e p r i m e r n i f H 3 (5′-ATRTTRTTNGCNGCRTA-3; Zani et al 2000); in the second step, the forward primer nifH1 (5′-TGYGAYCCNAARGCNGA-3) and the reverse primer nifH2 (5′-ANDGCCATCATYTCNCC-3; Zehr and McReynolds 1989) were used. For PCR, PerfCTa Quanta master mix (Quanta Biosciences, Gaithersburg, USA) was used.…”

High levels of heterogeneity in diazotroph diversity and activity within a putative hotspot for marine nitrogen fixation

“…nifH PCR amplification and amplicon pyrosequencing A nested PCR protocol was used to amplify an~359-bp region of the nitrogenase gene, using the degenerate primers: nifH3, nifH4, nifH1, and nifH2 (Zani et al, 2000;Zehr and Turner, 2001). Equal volumes of DNA or cDNA were used as template (2 μl) in the first stage of the reaction, and 1 μl of PCR product was used as template in the second stage, using previously described reaction conditions (Messer et al, 2015;see Supplementary Information).…”