Journal of Biotechnology 126 (2006) 291–294 Short communication Antibiosis by Bacillus amyloliquefaciens ribonuclease barnase expressed in Escherichia coli against symbiotic and endophytic nitrogen-fixing bacteria Humberto J.O. Ramos, Emanuel M. Souza, Juliana R.L. Soares-Ramos, F´ abio O. Pedrosa ∗ Department of Biochemistry and Molecular Biology, UFPR, C. Postal 19046, CEP 81531-990 Curitiba-PR, Brazil Received 6 September 2005; received in revised form 13 April 2006; accepted 21 April 2006 Abstract A modified antibiosis assay was used to evaluate growth inhibition of symbiotic and endophytic bacteria by E. coli strains pro- ducing Bacillus amyloliquefaciens ribonuclease, barnase. Inhibition zones were only observed when the assays were performed in minimal medium agar. However, bacterial growth inhibition was not detected when using rich medium or susceptible strains expressing the ribonuclease inhibitor protein, barstar. Our results suggest that barnase may act as a broad range bacteriocin. The ecological significance of these results is discussed. © 2006 Elsevier B.V. All rights reserved. Keywords: Barnase; Antibiosis; Symbiotic and endophytic bacteria Barnase is a potent ribonuclease, an enzyme that breaks down RNA indiscriminately. The barnase gene was isolated from the soil bacterium, Bacillus amy- loliquefaciens, which also produces the barstar protein, the specific inhibitor of barnase (Paddon and Hartley, 1985; Hartley, 1988). Barstar binds to barnase and inac- tivates the enzyme. Barnase has been widely used as a lethal gene in biological research and as a molec- ∗ Corresponding author. Tel.: +55 41 33664398; fax: +55 41 32662042. E-mail address: fpedrosa@ufpr.br (F.O. Pedrosa). ular tool in genetic engineering because it degrades RNA non-specifically. Several regulatory systems have been developed to control ribonuclease expression and activity in prokaryotic and eukaryotic cells (Beals and Goldberg, 1997; De Block et al., 1997; Kuvshinov et al., 2001; Bi et al., 2001; Leuchtenberger et al., 2001; Ramos et al., 2005). These systems include conditional expression of the barnase gene by the use of a tightly regulated promoter or the regulated co-expression of the barstar and barnase genes. When the barstar pro- tein is present, it binds tightly to barnase and inhibits the intracellular ribonuclease activity. Disproportion- 0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2006.04.020