Expression of the Rhizobium leguminosarum biovar phaseoli melA Gene in Other Rhizobia Does Not Require the Presence of the nifA Gene

Abstract

Many different Rhizobium strains produce melanin (Mel⁺) when grown on solid media supplemented with L-tyrosine. The composition of the media and the culture conditions are of great importance for pigment production. Previous reports showed that some Rhizobium leguminosarum biovar phaseoli strains that produce the pigment in complete solid media (TY) failed to produce the pigment in minimal media (SY) supplemented with L-tyrosine or in TY liquid media. In this paper we have investigated different R. fredii, R. meliloti, R. etli and R. leguminosarum bv. trifolii and phaseoli strains (all of them Mel⁺ in solid media) for their ability to produce the pigment in liquid media. All Rhizobium species tested, except Rhizobium etli, were Mel⁺ in liquid media and in all cases the pigment yielded maximum absorption peaks at 280 and 315 nm. Melanin production by other bacteria (such as Vibrio, Streptomyces or Azospirillum) is enhanced by the presence of amino acids other that tyrosine. In this paper we show that the addition of L-methionine, which is not a precursor of rhizobial melanins, stimulated pigment production by Rhizobium cultures supplemented with L-tyrosine. The role of melanin production by Rhizobium strains is unclear. One hypothesis is that the Rhizobium tyrosinase, a bifunctional copper-containing enzyme that is essential for melanin biosynthesis, could detoxify polyphenolic compounds which might accumulate in senescing nodules. We show here that R. etli and R. fredii bacteroids produced melanin, which supports the idea that bacteroids contain the enzyme tyrosinase. Previous reports showed that, in R. leguminosarum bv. phaseoli strain 8002, the expression of the tyrosinase gene (melA) is dependent on the presence of nifA, a regulatory gene that is located in the symbiotic plasmid. However, transfer of R. leguminosarum bv. phaseoli melA gene to pSym-cured derivatives of R. leguminosarum bv. trifolii and viciae, R. fredii and Rhizobium sp. (Hedysarum) produced Mel⁺ transconjugants. DNA-hybridisation experiments showed that the pSym-cured strains did not contain any copy of nifA. Therefore, in contrast to the results reported on R. leguminosarum bv. phaseoli strain 8002, the expression of the melA gene in other rhizobia is not nifA-dependent.

Key words: Rhizobium, melanin.