The vast majority of Pseudomonas species are unable to fix atmospheric nitrogen. Although several studies have demonstrated that some strains belonging to the genus Pseudomonas sensu stricto do have the ability to fix nitrogen by the expression of horizontally acquired nitrogenase, little is known about the mechanisms of nitrogenase adaptation to the new bacterial host. Recently, we transferred the nitrogen fixation island from Pseudomonas stutzeri A1501 to the non-nitrogen-fixing bacterium Pseudomonas protegens Pf-5, and interestingly, the resulting recombinant strain Pf-5 X940 showed an uncommon phenotype of constitutive nitrogenase activity. Here, we integrated evolutionary and functional approaches to elucidate this unusual phenotype. Phylogenetic analysis showed that polyhydroxybutyrate (PHB) biosynthesis genes from natural nitrogen-fixing Pseudomonas strains have been acquired by horizontal transfer. Contrary to Pf-5 X940, its derived PHB-producing strain Pf-5 X940-PHB exhibited the inhibition of nitrogenase activity under nitrogen-excess conditions, and displayed the typical switch-on phenotype observed in natural nitrogen-fixing strains after nitrogen deficiency. This indicates a competition between PHB production and nitrogen fixation. Therefore, we propose that horizontal transfer of PHB biosynthesis genes could be an ancestral mechanism of regulation of horizontally acquired nitrogenases in the genus Pseudomonas.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Tax calculation will be finalised during checkout.
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
Tax calculation will be finalised during checkout.
Althabegoiti MJ, Ormeno-Orrillo E, Lozano L, Torres Tejerizo G, Rogel MA, Mora J, Martinez-Romero E (2014) Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia. BMC Microbiol 14:6
Ayub ND, Pettinari MJ, Ruiz JA, Lopez NI (2004) A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance. Curr Microbiol 49:170
Ayub ND, Julia Pettinari M, Mendez BS, Lopez NI (2006) Impaired polyhydroxybutyrate biosynthesis from glucose in Pseudomonas sp. 14-3 is due to a defective beta-ketothiolase gene. FEMS Microbiol Lett 264:125
Ayub ND, Pettinari MJ, Mendez BS, Lopez NI (2007) The polyhydroxyalkanoate genes of a stress resistant Antarctic Pseudomonas are situated within a genomic island. Plasmid 58:240
Ayub ND, Tribelli PM, Lopez NI (2009) Polyhydroxyalkanoates are essential for maintenance of redox state in the Antarctic bacterium Pseudomonas sp. 14-3 during low temperature adaptation. Extremophiles 13:59
Bali A, Blanco G, Hill S, Kennedy C (1992) Excretion of ammonium by a nifL mutant of Azotobacter vinelandii fixing nitrogen. Appl Environ Microbiol 58:1711
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248
Brewin B, Woodley P, Drummond M (1999) The basis of ammonium release in nifL mutants of Azotobacter vinelandii. J Bacteriol 181:7356
Catone MV, Ruiz JA, Castellanos M, Segura D, Espin G, Lopez NI (2014) High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes. PLoS ONE 9:e98873
Cevallos MA, Encarnacion S, Leija A, Mora Y, Mora J (1996) Genetic and physiological characterization of a Rhizobium etli mutant strain unable to synthesize poly-beta-hydroxybutyrate. J Bacteriol 178:1646
Desnoues N, Lin M, Guo X, Ma L, Carreno-Lopez R, Elmerich C (2003) Nitrogen fixation genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiology 149:2251
Dixon R, Kahn D (2004) Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2:621
Dos Santos PC, Fang Z, Mason SW, Setubal JC, Dixon R (2012) Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genomics 13:162
Finan TM, Weidner S, Wong K, Buhrmester J, Chain P, Vorholter FJ, Hernandez-Lucas I, Becker A, Cowie A, Gouzy J, Golding B, Puhler A (2001) The complete sequence of the 1683-kb pSymB megaplasmid from the N2-fixing endosymbiont Sinorhizobium meliloti. Proc Natl Acad Sci USA 98:9889
Fox AR, Soto G, Mozzicafreddo M, Garcia AN, Cuccioloni M, Angeletti M, Salerno JC, Ayub ND (2014) Understanding the function of bacterial and eukaryotic thiolases II by integrating evolutionary and functional approaches. Gene 533:5
Galimand M, Perroud B, Delorme F, Paquelin A, Vieille C, Bozouklian H, Elmerich C (1989) Identification of DNA regions homologous to nitrogen fixation genes nifE, nifUS and fixABC in Azospirillum brasilense Sp7. J Gen Microbiol 135:1047
Galloway JN, Townsend AR, Erisman JW, Bekunda M, Cai Z, Freney JR, Martinelli LA, Seitzinger SP, Sutton MA (2008) Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320:889
Geddes BA, Ryu MH, Mus F, Garcia Costas A, Peters JW, Voigt CA, Poole P (2015) Use of plant colonizing bacteria as chassis for transfer of N-fixation to cereals. Curr Opin Biotechnol 32:216
Kechris KJ, Lin JC, Bickel PJ, Glazer AN (2006) Quantitative exploration of the occurrence of lateral gene transfer by using nitrogen fixation genes as a case study. Proc Natl Acad Sci USA 103:9584
Kessler B, Palleroni NJ (2000) Taxonomic implications of synthesis of poly-beta-hydroxybutyrate and other poly-beta-hydroxyalkanoates by aerobic pseudomonads. Int J Syst Evol Microbiol 50(Pt 2):711
Michener JK, Camargo Neves AA, Vuilleumier S, Bringel F, Marx CJ (2014a) Effective use of a horizontally-transferred pathway for dichloromethane catabolism requires post-transfer refinement. Elife 3:e04279
Michener JK, Vuilleumier S, Bringel F, Marx CJ (2014b) Phylogeny poorly predicts the utility of a challenging horizontally transferred gene in Methylobacterium strains. J Bacteriol 196:2101
Palleroni NJ (2003) Prokaryote taxonomy of the 20th century and the impact of studies on the genus Pseudomonas: a personal view. Microbiology 149:1
Peralta H, Mora Y, Salazar E, Encarnacion S, Palacios R, Mora J (2004) Engineering the nifH promoter region and abolishing poly-beta-hydroxybutyrate accumulation in Rhizobium etli enhance nitrogen fixation in symbiosis with Phaseolus vulgaris. Appl Environ Microbiol 70:3272
Peters JW, Szilagyi RK (2006) Exploring new frontiers of nitrogenase structure and mechanism. Curr Opin Chem Biol 10:101
Rediers H, Vanderleyden J, De Mot R (2004) Azotobacter vinelandii: a pseudomonas in disguise? Microbiology 150:1117
Rehm BH (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15
San Millan A, Toll-Riera M, Qi Q, MacLean RC (2015) Interactions between horizontally acquired genes create a fitness cost in Pseudomonas aeruginosa. Nat Commun 6:6845
Setten L, Soto G, Mozzicafreddo M, Fox AR, Lisi C, Cuccioloni M, Angeletti M, Pagano E, Diaz-Paleo A, Ayub ND (2013) Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. PLoS ONE 8:e63666
Soto G, Stritzler M, Lisi C, Alleva K, Pagano ME, Ardila F, Mozzicafreddo M, Cuccioloni M, Angeletti M, Ayub ND (2011) Acetoacetyl-CoA thiolase regulates the mevalonate pathway during abiotic stress adaptation. J Exp Bot 62:5699
Soto G, Setten L, Lisi C, Maurelis C, Mozzicafreddo M, Cuccioloni M, Angeletti M, Ayub ND (2012) Hydroxybutyrate prevents protein aggregation in the halotolerant bacterium Pseudomonas sp. CT13 under abiotic stress. Extremophiles 16:455
Soto G, Fox AR, Ayub ND (2013) Exploring the intrinsic limits of nitrogenase transfer from bacteria to eukaryotes. J Mol Evol 77:3
Van Dommelen A, Keijers V, Wollebrants A, Vanderleyden J (2003) Phenotypic changes resulting from distinct point mutations in the Azospirillum brasilense glnA gene, encoding glutamine synthetase. Appl Environ Microbiol 69:5699
Yan Y, Yang J, Dou Y, Chen M, Ping S, Peng J, Lu W, Zhang W, Yao Z, Li H, Liu W, He S, Geng L, Zhang X, Yang F, Yu H, Zhan Y, Li D, Lin Z, Wang Y, Elmerich C, Lin M, Jin Q (2008) Nitrogen fixation island and rhizosphere competence traits in the genome of root-associated Pseudomonas stutzeri A1501. Proc Natl Acad Sci USA 105:7564
Zhang T, Yan Y, He S, Ping S, Alam KM, Han Y, Liu X, Lu W, Zhang W, Chen M, Xiang W, Wang X, Lin M (2012) Involvement of the ammonium transporter AmtB in nitrogenase regulation and ammonium excretion in Pseudomonas stutzeri A1501. Res Microbiol 163:332
This work was supported by Grants PICT 2011-1325, PICT-2014-1397, and PICT-2014-3659 to N.D.A.
Electronic supplementary material
Below is the link to the electronic supplementary material.
About this article
Cite this article
Pascuan, C., Fox, A.R., Soto, G. et al. Exploring the Ancestral Mechanisms of Regulation of Horizontally Acquired Nitrogenases. J Mol Evol 81, 84–89 (2015). https://doi.org/10.1007/s00239-015-9698-4
- Horizontal transfer