Identification of NH 4 + -regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis
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ORIGINAL PAPER
Identification of NH+4 -regulated genes of Herbaspirillum seropedicae by random insertional mutagenesis Stefan Schwab Æ Humberto J. Ramos Æ Emanuel M. Souza Æ Fa´bio O. Pedrosa Æ Marshall G. Yates Æ Leda S. Chubatsu Æ Liu U. Rigo
Received: 27 July 2006 / Revised: 9 November 2006 / Accepted: 22 November 2006 / Published online: 24 February 2007 Springer-Verlag 2007
Abstract Random mutagenesis using transposons with promoterless reporter genes has been widely used to examine differential gene expression patterns in bacteria. Using this approach, we have identified 26 genes of the endophytic nitrogen-fixing bacterium Herbaspirillum seropedicae regulated in response to ammonium content in the growth medium. These include nine genes involved in the transport of nitrogen compounds, such as the highaffinity ammonium transporter AmtB, and uptake systems for alternative nitrogen sources; nine genes coding for proteins responsible for restoring intracellular ammonium levels through enzymatic reactions, such as nitrogenase, amidase, and arginase; and a third group includes metabolic switch genes, coding for sensor kinases or transcription regulation factors, whose role in metabolism was previously unknown. Also, four genes identified were of unknown function. This paper describes their involvement in response to ammonium limitation. The results provide a preliminary profile of the metabolic response of Herbaspirillum seropedicae to ammonium stress. Keywords Herbaspirillum seropedicae Insertional mutagenesis Ammonium Nitrogen metabolism
S. Schwab H. J. Ramos E. M. Souza F. O. Pedrosa M. G. Yates L. S. Chubatsu L. U. Rigo (&) Nu´cleo de Fixac¸a˜o de Nitrogeˆnio, Departamento de Bioquı´mica e Biologia Molecular, Universidade Federal do Parana´, Caixa Postal 19046, CEP 81.531-990 Curitiba-PR, Brazil e-mail: [email protected]
Introduction Nitrogen fixation is an economically and ecologically important biological process. Evidence that nitrogen fixation is significant in gramineae such as sugarcane and rice (Urquiaga et al. 1992; Shrestha and Ladha 1996) has encouraged interest in nitrogen-fixing bacteria that associate with those plants. Herbaspirillum seropedicae, a nitrogenfixing bacterium that belongs to the subdivision b of the Proteobacteria (Baldani et al. 1986), occurs endophytically in corn, rice, sugarcane, sorghum (Baldani et al. 1986; Olivares et al. 1996) and other plants such as banana and pineapple (Cruz et al. 2001). Nitrogen fixation is tightly regulated according to the cellular redox state and nitrogen availability. The nitrogenase enzyme is sensitive to oxygen, and the process has a high demand on energy, using at least 16 ATPs per N2 reduced to ammonium (Dixon and Kahn 2004). In H. seropedicae, regulation of nitrogen fixation involves the transcription regulation factor NifA according to ammonium availability and the concentration of oxygen (Souza et al. 1991; Wassem et al. 2002). Studies have also shown ammonium-dependent expression regulation of both the H. seropedicae glnAn
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