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An extra-cytoplasmic function sigma factor and anti-sigma factor control carotenoid biosynthesis in Azospirillum brasilense

¹ School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221005, India
² Centre of Microbial and Plant Genetics, Department of Microbial and Molecular Systems, Faculty of Bioscience Engineering, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium
³ Departments of Microbiology and Immunology, and Cell and Tissue Biology, University of California, San Francisco, CA 94158-2517, USA

Correspondence
Anil K. Tripathi
tripathianil{at}rediffmail.com

Strains Sp7 and Cd of Azospirillum brasilense, a plant growth-promoting rhizobacterium, differ in synthesis of carotenoids. While colonies of strain Sp7 have a white–cream colour on plates, colonies of strain Cd are orange–pink coloured because of the synthesis of carotenoids. Screening of a mini-Tn5 mutant library of A. brasilense Sp7 revealed two orange–pink-coloured mutants that produced carotenoids. Cloning and sequencing of the Tn5 flanking region in both the carotenoid-producing mutants of Sp7 revealed insertion of Tn5 in an ORF encoding anti- factor, a ChrR-like protein. The upstream region of the Tn5-mutated ORF contained another ORF that encoded an extra-cytoplasmic function (ECF)-class factor (^E, RpoE). When the nucleotide sequences of the corresponding ORFs from the carotenoid-producing strain Cd were analysed, the sequence of the Cd ^E was identical to that of the carotenoid non-producing strain Sp7, but the Cd anti-^E ORF had a deletion that caused frame shifting and creation of a stop codon. This resulted in the premature termination of the protein, which was about 7 kDa smaller than the Sp7 anti-^E. Cloning of Sp7 anti-^E in a broad-host-range expression vector and expression in A. brasilense Cd and in the anti-^E knockout mutant of A. brasilense Sp7 resulted in the inhibition of carotenoid synthesis. Similarly, cloning and overexpression of A. brasilense Sp7 ^E in A. brasilense Sp7 resulted in the production of carotenoids. These observations clearly indicate that carotenoid synthesis in A. brasilense is controlled by ^E with its cognate anti-^E.

The GenBank/EMBL/DDBJ accession numbers for the ^E and anti-^E sequences of A. brasilense are DQ324853 and DQ324854, respectively.

Three supplementary figures showing phylogenetic trees for the amino acid sequences of ChrR and ECF-^E, and the effects of the expression of rpoE and chrR in Azospirillum brasilense, are available with the online version of this paper.