PENCIRIAN GEN PENGIKATAN NITROGEN Paenibacillus azotofixans

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Date
2004-02
Authors
CHOO, QUOK CHEONG
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Abstract
Three Paenibacillus azotojixans (synonymous to P. durus) DNA regions containing nitrogen fixation genes were cloned, sequenced and characterized. Sequence analysis of a 5.237 kb DNA fragment revealed a linkage of nifBl upstream of the structural nifgenes. These genes are designated as nifBIHIDIKI. The nifB2H2 and nifH3 genes were contained in a 6.350 kb HindIl! and a 2.840 kb EcoR!-digested DNA fragments, respectively. Comparative alignment between Nifl-II and Nifl-I2 yielded a high 97% homology. When either Nifl-II or Nifl-I2 was aligned with Nifl-I3, low 43% homology was observed. Phylogenetic analysis demonstrated a monophyletic clustering of Nifl-Il and Nifl-I2 within the cyanobacterial clade, the first of such observation in any reported NifH phylogeny. Besides this unusual clustering, P. azotofzxans NifH3 clustered with the highly divergent methanogenic NifH proteins. These anomalously placed NifHs had raised questions about the role of horizontal gene transfer and ancient gene duplications in the evolution of P. azotojixans nitrogenase. Employing NifDK and 16S rRNA phylogenies as lines of support, the horizontal transfer of nifH gene from a cyanobacterium to P. azotojixans was proposed. Determination of 5' ends in the upstream regions of P. azotojixans nifHl and nifH2 failed to detect typical SigN-dependent nif promoter recognition sites. An internal promoter within 3' end of nifBl or RNA processing phenomena was proposed as regulatory mechanism that might govern nifHl mRNA. An attempt to characterize the 5' end points of nifH3 failed, probably due to low presence or the absence of nifH3 mRNA. Meanwhile, a new nifH gene (nifH4) was elucidated in the process of determining the 5' ends of nifHI and nifH2. Alignment analyses demonstrated striking sequence conservation between NifHl, NifH2 and NifH4. The probable nifBl 5' end-point was mapped to aT-residue, 46 bp preceding the translation start site. Promoter sequence analysis demonstrated weak homology to a SigNxxi dependent nif promoter. Two putative NifA-regulated consensus sequence of TGT -N 10- ACA were found in the -70 and -120 positions of the upstream untranscribed regions of nifBl. Examination of the untranscribed DNA region of nifB2 revealed two putative promoter regions. The first putative promoter has sequences of T AAGATG and CATACGAA at the -35 and -10 positions, respectively. These sequences showed similarity with the B. subtilis SigE-dependent recognition sequence. The second nifB2 putative promoter region is located downstream from the first, with sequences of TGGAGG, TG and TCTTTT at the -35, -15 and -10 positions, respectively. The nifB2H2 genes possibly function as a single operon.
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PENCIRIAN GEN , NITROGEN
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