Cellular mechanisms of pH tolerance in Rhizobium loti O.S. Correa* and A.J. Barneix Seven strains of Rhizobium loti were tested for acid tolerance in yeast-extract mannitol (YEM) broth at pH values ranging from 4.0 to 8.0. The strains that grew at pH 4.0 showed the slowest generation time when grown at pH above 7.0 and also produced the most acid. The acid tolerance was related to the composition and structure of the membrane. pH in¯uenced protein expression in acid-tolerant strains growing at pH 4.0 or 7.0. Acid tolerant strains showed one membrane protein of 49.5 kDa and three soluble proteins of 66.0, 58.0 and 44.0 kDa; their expression increased when the cells grew at pH 4.0. It is suggested that acid tolerance in Rhizobium loti involves constitutive mechanisms, such as permeability of the outer membrane together with adaptive responses, including the state of bacterial growth and concomitant changes in protein expression. Key words: Cellular mechanisms, pH tolerance, Rhizobium loti. Soil acidity affects many areas of the world and limits legume productivity. Most leguminous plants require a neutral or slightly acidic soil for growth, especially when depending on symbiotic nitrogen ®xation (Rice et al. 1977; Brockwell et al. 1991; Bordeleau & Pre Âvost 1994). However, differences in acid soil tolerance by legumes have been reported. Some species, like lucerne (Medicago sativa), are extremely sensitive to acidity (Rice et al. 1977), while others, such as Lotus tenuis tolerate relatively low soil pH (Min Äo Ân et al. 1990). Nodule bacteria differ in their response to acidity when grown in liquid culture. Some are moderately acid tolerant (Rhizobium loti, R. tropici) (Wood et al. 1988) while others, like R. meliloti, are very sensitive to acidity (Brockwell et al. 1991; Tiwari et al. 1992). Different strains of the same species may also vary widely in their pH tolerance (Glenn & Dilworth 1994). Root nodule bacteria can be more sensitive to low pH than their legume host, directly affecting the establishment of the symbiosis (Glenn & Dilworth 1994; O'Hara & Glenn 1994), and so the selection of Rhizobium strains tolerant to low pH may improve the acid tolerance of the legume. There is still little known about the biochemical and physiological basis of acid tolerance by Rhizobium strains. Possible mechanisms involve the regulation of cyto- plasmic pH (Chen et al. 1993; Graham et al. 1994), proton exclusion and/or extrusion (Chen et al. 1993), exopoly- saccharide production (Cunningham & Munns 1984), and changes in the hydrophobicity of the plasma mem- brane (Graham et al. 1994). In several bacteria, like R. leguminosarum bv trifolii these mechanisms have been described as being an adaptive response to pH (O'Hara & Glen 1994), while in others, such as the acid tolerant R. tropici UMR 1899 these mechanisms seem to be con- stitutive (Graham et al. 1994). In the present paper we explore mechanisms of tolerance to acid pH in R. loti strains differing in their tolerance to acidity. Materials and Methods Bacterial Strains and Growth Conditions Strains of Rhizobium loti LL22, LL52, LL56, LL57, LL58, LL59 were provided by the Instituto Nacional de Tecnologõ Âa Agro- pecuaria-INTA-Castelar, Buenos Aires, Argentina, and strain Intech was provided by the Instituto Tecnolo Âgico Chascomu Á s- INTECH, Buenos Aires, Argentina. Cultures were maintained at 4 °C on yeast extract mannitol (YEM) agar slopes (Vincent 1970). World Journal of Microbiology & Biotechnology, Vol 13, 1997 153 The authors are with Ca  tedra de Microbiologõ Âa Agrõ Âcola, Facultad de Agrono- mõ Âa, Universidad de Buenos Aires, Av. San Martõ Ân 4453 1417 Buenos Aires, Argentina; fax: +54 1 523 4936. *Corresponding author. World Journal of Microbiology & Biotechnology 13, 153±157 ã 1997 Rapid Science Publishers