Construction and characterization of regulated L-arabinose-inducible broadhost range expression vectors in Xanthomonas

Several versions of broad host range (BHR), L-arabinose-inducible expressio n vectors were constructed. These expression vectors were based on a high c opy number BHR pBBR1MCS-4 replicon that could replicate in both enteric and non-enteric Gram-negative bacteria, Two versions of expression cassettes c ontaining multiple cloning sites either with of without a ribosome binding site were placed under transcriptional control of the Escherichia coli BAD promoter and araC gene. Three versions of vectors containing ampicillin or kanamycin or tetracycline resistance genes as selectable markers were const ructed, In all six new L-arabinose-inducible BHR expression vectors contain ing many unique cloning sites, selectable markers were made to facilitate c loning and expression of genes in various Gram-negative bacteria. A Tn9 chl oramphenicol acetyl transferase (cat) gene was cloned into an expression ve ctor, resulting in pBBad18Acat that was used to establish optimal expressio n conditions (addition of 0.02% L-arabinose to mid-exponential phase cells for at least 1 h) in a Xanthomonas campestris pv, phaseoli. Comparison of t he Cat enzyme activities between uninduced and a 180-min L-arabinose-induce d culture showed a greater than 150-fold increased Cat specific activity. I n addition, L-arabinose induction of exponential phase cells harboring pBBa d18Acat gave a higher amount of Cat than similarly treated stationary phase cells. The usefulness of the expression vector was also demonstrated in bo th enteric and non-enteric Gram-negative bacteria. (C) 1999 Federation of E uropean Microbiological Societies. Published by Elsevier Science B.V. Ail r ights reserved.