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Host-specific regulation of symbiotic nitrogen fixation in Rhizobium leguminosarum biovar trifolii

Simon H. Miller

Rachel M. Elliot

Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand

Correspondence
Clive W. Ronson
Clive.Ronson{at}otago.ac.nz

Strains of Rhizobium leguminosarum bv. trifolii (Rlt) able to form effective nodules on Trifolium ambiguum (Caucasian clover, CC) form ineffective nodules on Trifolium repens (white clover, WC), whereas strains that form effective nodules on WC usually do not nodulate CC. Here, we investigate the genetic basis of the host-specific nitrogen-fixation phenotype of CC rhizobia. A cosmid library of the symbiotic plasmid from the WC rhizobium strain Rlt NZP514 was introduced into the CC rhizobium strain Rlt ICC105. An 18 kb Asp718 fragment containing the nifABHDKEN and fixABCX genes of NZP514 that imparted the Fix⁺ phenotype was identified. Tn5 mutagenesis of this region revealed that the nifHDKEN, fixABC and nifB genes were required for the Fix⁺ phenotype, but that the nifA gene was not. Introduction of several plasmids containing NZP514 nif/fix genes into an ICC105 nifA mutant strain demonstrated that the NifA protein of ICC105 was able to activate expression of the NZP514 nif/fix genes but not the ICC105 nif/fix genes in WC nodules. Reporter gene fusion studies showed that the host-specific regulation of the nif/fix genes depended on the DNA region between the promoters of the divergently transcribed nifH and fixA genes. We hypothesize that a protein acting either in response to a host-specific signal or in the absence of such a signal is able to bind upstream of the NifA-binding sites and interact with NifA to prevent it activating nif/fix gene expression.

Present address: BIOMERIT Research Centre, Microbiology Department, University College Cork, Ireland.

Present address: Research and Enterprise Office, Centre for Innovation, University of Otago, PO Box 56, Dunedin, New Zealand.

The GenBank/EMBL/DDBJ accession numbers for the Asp718 fragments containing the nif/fix genes of Rlt NZP514 and ICC105 are EF165535 and EF165526, respectively. Accession numbers of the nifH–fixA intergenic regions of Rlt 144, 162C11, ABH12-1, ARH2-1, ARR13-1, ART1-2, AZR13-1, ICMP4074b, ICC144, NZP560, NZP561 and OSS82 are EF165519, EF165520, EF165521, EF165522, EF165523, EF165524, EF165525, EF165530, EF165531, EF165532, EF165533 and EF165534, respectively.

Two supplementary figures showing electron micrographs of nodules formed on WC by the ICC105 and NZP561 strains and nucleotide sequence alignment of the nifH–fixA intergenic regions of several CC and WC rhizobia strains are available with the online version of this paper.