STRUCTURE AND ORGANIZATION OF THE PYRIMIDINE BIOSYNTHESIS PATHWAY GENES IN LACTOBACILLUS-PLANTARUM - A PCR STRATEGY FOR SEQUENCING WITHOUT CLONING

This report describes the sequence and structural organisation of the pyrimidine biosynthesis pathway genes of Lactobacillus plantarum CCM 1 904. It also describes an in vitro technique based on PCR for sequenci ng without cloning. This new technique was developed because it was im possible to clone certain parts of the L. plantarum genomic DNA in the Escherichia coli host. L. plantarum pyr genes are organised as a 9.8- kb operon with the following order: pyrR, pyrB, pyrC, pyrAA, pyrAB, py rD, pyrF and pyrE. There are two major differences from the pyrimidine operons of Bacillus subtilis (Quinn et al., J. Bacteriol. 266 (1991) 9113-9127; Turner et al., J. Bacteriol. 176 (1994) 3708-3722) and Baci llus caldolyticus (Ghim et al., Microbiology 140 (1994) 479-491): the absence of pyrP encoding for uracil permease, and the absence of an op en reading frame named orf2, whose function is unknown. Two mutually e xclusive stem-loop structures were predicted at the 5'-end of L. plant arum pyr mRNA; this operon could be regulated by transcriptional atten uation under the control of PyrR. Complementation of E. coli pyrD, pyr F and pyrE mutants was obtained with a L. plantarum genomic DNA librar y. Alignment of the L. plantarum Pyr proteins with other known procary otic Pyr proteins indicates that they display highly conserved regions in Gram-positive and Gram-negative bacteria.