Cloning and characterization of a novel gene involved in nitrogen fixation in Heliobacterium chlorum : a possible regula
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O RI G I NAL PAPE R
Cloning and characterization of a novel gene involved in nitrogen Wxation in Heliobacterium chlorum: a possible regulatory gene Jigjiddorj Enkh-Amgalan · Hiroko Kawasaki · Hirozo Oh-oka · Tatsuji Seki
Received: 9 December 2005 / Revised: 19 May 2006 / Accepted: 10 July 2006 / Published online: 24 August 2006 © Springer-Verlag 2006
Abstract In the present study, the transcriptional properties of the nitrogen Wxation gene cluster of Hbt. chlorum, a strictly anaerobic, gram-positive, phototrophic bacterium, were explored. The cluster consisted of eleven genes in the same orientation in the order nifI1, nifI2, nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB, and nifV as detected previously. An open reading frame (orf1) preceding these genes was revealed by further cloning. The orf1 was co-transcribed with downstream nif genes in a single polycistronic transcript, the transcription start site (TSS) was located upstream of the orf1, and a putative promoter was identiWed 10 bp preceding the TSS. Unlike most diazotrophs which have a 54-dependent ¡24/¡12 promoter, the promoter was similar to the ¡35/¡10 E. coli promoter. The orf1 had no nif homolog in DNA databases, and the highest level of identity (27% at amino acid level) was found with hutP, a positive regulatory gene of the histidine utilization (hut) operon in B. subtilis. Analogous to the regulatory mechanism of the hut operon in B. subtilis, it is conceivable that the orf1 product interacts with the terminator-like structure located downstream of the orf1 during N-deWcient condition and prevents transcription termination; thus, the transcription continues into the nif structural genes.
J. Enkh-Amgalan · H. Kawasaki (&) · T. Seki The International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 560-0871, Japan e-mail: [email protected] H. Oh-oka Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
Keywords Heliobacterium · Nitrogen Wxation · Nif genes · Transcriptional terminator · Anti-termination · Real-time RT-PCR
Introduction Biological nitrogen Wxation, the conversion of atmospheric nitrogen into ammonia, is catalyzed by a nitrogenase complex, which consists of two separable components: dinitrogenase reductase encoded by nifH and dinitrogenase encoded by nifD and nifK (Dean and Jacobson 1992). This is a very energy-demanding process since two ATP molecules are hydrolyzed for every electron that is transferred at each step in the catalytic cycle of nitrogenase. Moreover, nitrogenase is a relatively slow enzyme and thus nitrogen-Wxing organisms (diazotrophs) have to synthesize a large amount of nitrogenase to use N2 as a sole nitrogen source. It is therefore not surprising that diazotrophs regulate nitrogenase tightly at both the transcriptional and post-translational levels in response to environmental factors such as Wxed nitrogen and oxygen (Merrick 1992; Dixon and Kahn 2004). In the great majority of diazotrophs, the transcript
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