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1 Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, 1955 East-West Road, Ag. Science 218, Honolulu, HI 96822, USA
2 Complex Carbohydrate Research Center, University of Georgia, Athens, 315 Riverbend Road, GA 30602, USA
Correspondence
Dulal Borthakur
dulal{at}hawaii.edu
Exopolysaccharide (EPS) produced by Rhizobium sp. strain TAL1145 has been shown to be essential for effective nodulation on Leucaena leucocephala (leucaena). This paper reports the isolation and characterization of an alternative sigma factor gene, rpoH2, involved in the regulation of EPS synthesis in TAL1145. Disruption of this gene in TAL1145 resulted in a Calcofluor-dim mutant RUH102 that produced approximately 18 % of the amount of EPS made by TAL1145. This mutation did not affect the normal growth of RUH102 in free-living state. RUH102 induced few nitrogen-fixing nodules, resulting in a significant reduction in total nitrogen content in leucaena. It was complemented for EPS production and nodulation by a 2·0 kb HindIII fragment of TAL1145. Sequence analysis of this fragment revealed the rpoH2 ORF of 870 bp that encoded a protein of 32 kDa. Expression of the rpoH2 ORF in Escherichia coli also revealed a 32 kDa protein. A PCR-constructed clone of 1263 bp, containing the rpoH2 ORF and its upstream putative regulatory region, complemented RUH102 for EPS defects. Comparison of the RpoH2 sequence to proteins in the databases showed significant similarity to RpoH-like sigma factors of other Gram-negative bacteria. By constructing several exo : : Tn3Hogus fusions and transferring them to the backgrounds of TAL1145 and RUH102, it was demonstrated that RpoH2 positively regulates the transcription of some exo genes.
-glucuronidase; MU, 7-hydroxy-4-methylcoumarin; MUG, 4-methylumbelliferyl -D-glucuronide; TMS, trimethylsilylThe GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY536267.
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