BLUE WHITE SCREENING OF RECOMBINANT PLASMIDS IN GRAM-POSITIVE BACTERIA BY INTERRUPTION OF ALKALINE-PHOSPHATASE GENE (PHOZ) EXPRESSION/

The process of screening bacterial transformants for recombinant plasm ids is made more rapid and simple by the use of vectors with visually detectable reporter genes. In such systems, an alteration in colony ph enotype occurs when a vector-borne indicator gene is interrupted with exogenous DNA. Although the lacZ system has been used extensively for this purpose in E. coli, analogous systems for use in Gram-positive ba cteria remain uncommon. We have developed a Gram-positive cloning vect or that utilizes the interruption of an alkaline phosphatase gene, pho Z, to identify recombinant plasmids. To facilitate introduction of for eign DNA, a multiple cloning site (MCS) was inserted distal to the reg ion coding for the putative signal peptide of phoZ Alkaline phosphatas e expressed from the derivative phoZ gene (phoZMCS) retained activity similar to that of the native protein. The phoZMCS was transferred to pJS3, a well-characterized, high-copy number, and broad-host-range pla smid, to produce pDC123. In pDC123, phoZMCS was transcriptionally link ed to the chloramphenicol acetyl transferase (cat) gene under the cont rol of the constitutively expressed tetM and cat promoters that drive cat expression in pJS3. S. agalactiae (Group B streptococci, GBS), E. faecalis, S. pyogenes, S. gordonii, and E. coli containing pDC123 disp layed a blue colonial phenotype on agar containing 5-bromo-4-chloro-3- indolyl phosphate (X-p), which was readily distinguished from that of colonies containing the parent plasmid pJS3. Introduction of foreign D NA into the MCS of phoZMCS produced a white colonial phenotype in E. c oli and GBS on agar containing X-p and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-annealed or uncut vector. We have used pDC123 to subclone the cp sE gene from the plasmid pCER111, which carries a 9.0-kb fragment of t he GBS capsular polysaccharide synthesis locus. The plasmid pDC123 con taining cpsE was isolated by direct electroporation into GBS strain A9 09 with selection of transformants containing recombinant plasmids ach ieved by 'blue/white' screening, without the use of an intermediate ho st. This new cloning vector should improve the efficiency of performin g recombinant DNA experiments in Gram-positive bacteria. (C) 1998 Else vier Science B.V. All rights reserved.