Regulation Of Amino Acid Transporter Aap Under Acidic Conditions

H.L. Steele, B.U. Becker, P. Müller, P. Vinuesa, D. Werner

FB Biologie, FG Zellbiologie und Angewandte Botanik, Philipps Universität Marburg, Karl-von-Frisch-Strasse, 35032 Marburg, Germany

2D SDS-PAGE of proteins from acid tolerant R. tropici CIAT899 wild-type and its acid-sensitive derivative PY4, grown under neutral and acidic conditions, revealed a differentially expressed protein with high homology (79% identity over 24 amino acids) to AapJ from R. leguminosarum bv. viciae (Walshaw, Poole 1996). The mutant had less AapJ only under acidic conditions, indicating that acidity had disrupted regulation of expression.

The R. leguminosarum bv. viciae ABC transporter, encoded by the aapJQMP operon, is involved in the exchange of various nutrients between rhizobia and their environment (Walshaw, Poole 1996). This ABC transporter has an optimum efficiency between pH 6.0 and pH 7.0 (Poole et al. 1985). To compensate for the reduced efficiency under acidic conditions, rhizobia may produce higher numbers of transporters. It is possible that highly acid tolerant strains, such as Rhizobium tropici CIAT899, are better able to regulate their transporters under acidic conditions than less acid tolerant strains, thus giving them a competitive advantage. The aim of this research is to construct a reporter gene fusion to the intact aapJQMP operon in R. tropici CIAT899 and in the less acid tolerant R. leguminosarum TAL 1400. This should allow analysis of aap regulation, as well as comparison between the two strains in their ability to regulate expression of the aap operon under acidic conditions. We present here the strategy we are using to achieve this.

A 556 bp fragment of aapJ from R. tropici CIAT899 was PCR amplified, cloned and sequenced to confirm its identity (100% identity over 182 amino acids). The 556 bp piece is located directly downstream of the signal sequence which guides the protein to its periplasmic location. Southern blot hybridization analysis with DIG labeled aapJ indicated that it is present as a single copy. To determine whether aap genes are influenced by acidity, a reporter gene 'phoA (Rodríguez-Quiñones et al. 1994) was inserted directly downstream of the signal sequence of aapJ so that the intact ABC transporter is produced as well as the reporter-gene fusion product. To achieve this the vector pJQ200 (Quandt, Hynes 1993) was modified to include 'phoA (Rodríguez-Quiñones et al. 1994).

The region upstream of aapJ including the aapJQMP promoter and signal sequence has been PCR amplified from the two strains, ligated in the vector pJQ200/'phoA, transformed into E. coli strain DH5oc, and plated onto media containing X-phosphate. Blue clones have been obtained indicating that the DNA fragment was inserted in the correct orientation and reading frame into the vector. The hybrid plasmid now has to be sequenced to confirm that the correct insert is present. The vector will then be transformed into an E. coli donor strain and used to mutagenize the wild-type in a conjugation experiment. The transconjugants will be grown at different pHs and the expression measured by determining alkaline phosphatase activity. Our aim is to show that this operon is differently regulated under acidic conditions, and that the ability to regulate the operon under acidic conditions is one of the factors influencing acid tolerance.

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