MOLECULAR DISSECTION OF MINERAL PHOSPHATE SOLUBILIZATION IN Acetobacter diazotrophicus Pal5
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Date
2004
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University of Agricultural Science, Dharwad
Abstract
The genetic dissection of Mineral Phosphate Solubilization (MPS) in
Acetobacter diazotrophicus Pal5 was attempted through the development and
characterization of mutants defective in MPS activity and isolation of genes
involved in phosphate metabolism.
A. diazotrophicus released 5 2 .8 8% of Pi in TCP broth after 5 days of
incubation. TLC analysis of the culture filtrate showed the presence of
gluconic acid. Mutagenesis of A. diazotrophicus using NTG yielded six MPS"
mutants and one leaky mutant. The mutants were confirmed as derivatives of
Pal5, since the distinct 411 bp amplicon was obtained on PCR of the genomic
DNA with A D -1 4 4 0 , the species - specific primer.
PCR amplified fragments using primers for pqq, gcd and gnd (8 0 0 bp,
7 5 0 bp, 1 2 0 0 bp amplicons respectively) were cloned into pTZ57R through
11A cloning and transformed into E. coii XLIBIue. The recombinant pJKNI
containing the gnd was sequenced using M l 3 primers and the BLASTn
analysis showed 1 0 0% homology with Salmonella and E. coll g nd gene. The
recombinant pJKQI containing pqq amplicon expressed in E. coii BL21
resulting in zone of solubilization on MSM. Hence, the 8 0 0 bp amplicon was
sufficient to transcomplement apo-gdh in E. coii to release Pi from mineral
phosphates.
Azospirillum IABT-1 (MPS) showed MPS activity when PQQ was
added into MSM but was Amp'’. Hence, a construct pJSK15 containing pqq
synthase gene(s) was developed and mobilized into Azospirillum IABT-1 . The
resultant transconjugants however, showed no zone of solubilization on MSM
indicating that the pqq gene(s) in pMCG8 9 8 was not sufficient to complement
MPS activity in Azospirillum IABT-1.
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