EXPRESSION OF THE RHIZOBIUM-LEGUMINOSARUM BIOVAR PHASEOLI MELA GENE IN OTHER RHIZOBIA DOES NOT REQUIRE THE PRESENCE OF THE NIFA GENE

Many different Rhizobium strains produce melanin (Mel(+)) when grown o n solid media supplemented with L-tyrosine. The composition of the med ia and the culture conditions are of great importance for pigment prod uction. Previous reports showed that some Rhizobium leguminosarum biov ar phaseoli strains that produce the pigment in complete solid media ( TY) failed to produce the pigment in minimal media (SY) supplemented w ith L-tyrosine or in TY liquid media. In this paper we have investigat ed different R. fredii, R. meliloti, R. etli and R. leguminosarum by. trifolii and phaseoli strains (all of them Mel(+) in solid media) for their ability to produce the pigment in liquid media. All Rhizobium sp ecies tested, except Rhizobium etli, were Mel(+) in liquid media and i n all cases the pigment yielded maximum absorption peaks at 280 and 31 5 nm. Melanin production by other bacteria (such as Vibrio, Streptomyc es or Azospirillum) is enhanced by the presence of amino acids other t hat tyrosine. In this paper we show that the addition of L-methionine, which is not a precursor of rhizobial melanins, stimulated pigment pr oduction 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 enz yme that is essential for melanin biosynthesis, could detoxify polyphe nolic compounds which might accumulate in senescing nodules. We show h ere that R. etli and R. fredii bacteroids produced melanin, which supp orts 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-c ured derivatives of R. leguminosarum by. trifolii and viciae, R. fredi i and Rhizobium sp. (Hedysarum) produced Mel(+) transconjugants. DNA-h ybridisation experiments showed that the pSym-cured strains did not co ntain any copy of nifA. Therefore, in contrast to the results reported on R. leguminosarum bv. phaseoli strain 8002, the expression of the m elA gene in other rhizobia is not nifA-dependent.