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Journal of Bacteriology, March 1999, p. 1544-1554, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Bradyrhizobium japonicum nolA Gene Encodes Three Functionally Distinct Proteins

John Loh,1,2 Minviluz G. Stacey,3 Michael J. Sadowsky,4 and Gary Stacey1,2,5,*

Center for Legume Research,1 Department of Microbiology,2 Department of Botany,3 and Department of Ecology and Evolutionary Biology,5 The University of Tennessee, Knoxville, Tennessee 37996, and Department of Soil, Water and Climate, The University of Minnesota, St. Paul, Minnesota 551084

Received 27 August 1998/Accepted 18 December 1998

Examination of nolA revealed that NolA can be uniquely translated from three ATG start codons. Translation from the first ATG (ATG1) predicts a protein (NolA1) having an N-terminal, helix-turn-helix DNA-binding motif similar to the DNA-binding domains of the MerR-type regulatory proteins. Translation from ATG2 and ATG3 would give the N-terminally truncated proteins NolA2 and NolA3, respectively, lacking the DNA-binding domain. Consistent with this, immunoblot analyses of Bradyrhizobium japonicum extracts with a polyclonal antiserum to NolA revealed three distinct polypeptides whose molecular weights were consistent with translation of nolA from the three ATG initiation sites. Site-directed mutagenesis was used to produce derivatives of nolA in which ATG start sites were sequentially deleted. Immunoblots revealed a corresponding absence of the polypeptide whose ATG start site was removed. Translational fusions of the nolA mutants to a promoterless lacZ yielded functional fusion proteins in both Escherichia coli and B. japonicum. Expression of NolA is inducible upon addition of extracts from 5-day-old etiolated soybean seedlings but is not inducible by genistein, a known inducer of the B. japonicum nod genes. The expression of both NolA2 and NolA3 requires the presence of NolA1. NolA1 or NolA3 is required for the genotype-specific nodulation of soybean genotype PI 377578.

* Corresponding author. Mailing address: Center for Legume Research, Department of Microbiology, M409 Walters Life Science Building, The University of Tennessee, Knoxville, TN 37996-0845. Phone: (423) 974-4041. Fax: (423) 974-4007. E-mail: GSTACEY{at}utk.edu.

Journal of Bacteriology, March 1999, p. 1544-1554, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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