Cloning and Mutagenesis of a Cytochrome P-450 Locus from Bradyrhizobium japonicum That Is Expressed Anaerobically and Symbiotically

Author:

Tully Raymond E.1,Keister Donald L.1

Affiliation:

1. Soybean and Alfalfa Research Laboratory, USDA Agricultural Research Service, Building 011, HH-19, Beltsville Agricultural Research Center-West, Beltsville, Maryland 20705

Abstract

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of Bradyrhizobium japonicum. Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of B. japonicum USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in Escherichia coli produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown B. japonicum cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the CYP112 locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

Reference26 articles.

1. A soluble haemoprotein P 450 from nitrogen-fixing Rhizobium bacteroids;Appleby C. A.;Biochim. Biophys. Acta,1967

2. Haemoprotein P450, other CO-reactive pigments, cytochromes and oxidases in bacteroids from N2-fixing root nodules;Appleby C. A.;Biochim. Biophys. Acta,1969

3. Appleby C. A. and R. M. Daniel. 1973. Rhizobium cytochrome P-450: a family of soluble separable hemoproteins p. 415-528. In T. E. King S. Mason and M. Morrison (ed.) Oxidases and related redox systems. University Park Press Baltimore.

4. Involvement of oxyleghaemoglobin and cytochrome P-450 in an efficient oxidative phosphorylation pathway which supports nitrogen fixation;Appleby C. A.;Rhizobium. Biochim. Biophys. Acta,1975

5. Leghaemoglobin and the supply Of 02 to nitrogen-fixing root nodule bacteroids: presence of two oxidase systems and ATP production at low free 02 concentration;Bergersen F. J.;J. Gen. Microbiol.,1975

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