nif gene sequence and arrangement in the endophytic diazotroph Paenibacillus polymyxa strain P2b-2R
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nif gene sequence and arrangement in the endophytic diazotroph Paenibacillus polymyxa strain P2b-2R Richa Anand & Christopher Peter Chanway
Received: 20 December 2012 / Revised: 21 February 2013 / Accepted: 28 February 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract We characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R, an endophytic diazotroph that can provide up to 79 % of foliar N in lodgepole pine through biological nitrogen fixation. We amplified a 388-bp internal nifH gene fragment from P2b-2R using the single specific primer–polymerase chain reaction (SSPPCR) and performed a Southern blot analysis of Pst I/HindIII-digested genomic DNA to evaluate the sequence, copy number, and location in the nif gene operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and D. Phylogenetic analyses of the full nifH, partial nifB, and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus and suggest that nifH is transmitted horizontally while nifD is transmitted vertically. Our results provide the first full nifH sequence from a P. polymyxa strain and indicate that arrangement of genes in the P2b-2R nif operon is consistent with those observed in other species of the genus Paenibacillus. Keywords Paenibacillus polymyxa . Endophyte . nif genes . Sequence R. Anand : C. P. Chanway (*) Department of Forest Sciences, University of British Columbia, 3041-2424 Main Mall, Vancouver, BC V6T 1Z4, Canada e-mail: [email protected] R. Anand e-mail: [email protected] R. Anand IL-7, BC Children’s Hospital, 4480 Oak Street, Vancouver, BC V6H 3V4, Canada C. P. Chanway Faculty of Land and Food Systems, University of British Columbia, 248-2357 Main Mall, Vancouver, BC V6T 1Z4, Canada
Introduction Paenibacillus polymyxa is a widely distributed species of interest from medical, agricultural, and industrial perspectives and known to contain many strains capable of N2fixation (Lal and Tabacchioni 2009). P. polymyxa strain P2b-2R is a nitrogen-fixing bacterium that was isolated from the internal stem tissue of a lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedling naturally regenerating in central British Columbia, Canada (Bal et al. 2012). This strain is unique as it is the only microorganism that has been reported to fix substantial amounts of N2 in association with tree species. Reintroduction of P2b-2R to lodgepole pine seed under a controlled environment results in rhizospheric and endophytic colonization of resultant seedlings as well as provision of increasing amounts of foliar N as seedlings develop (Bal and Chanway 2012a; Anand and Chanway 2013a). Foliar N content and biomass of pine seedlings increased more than 2-fold 13 months after seed inoculation (Anand et al. 2013). Similar effects but of lesser magnitude were observed when seedlings generated from western red cedar (Thuja plicata Donn.) seeds inoculated wit
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