Structure and organisation 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 1904. It also describes an in vitro technique based on PCR for sequencing without cloning. This new technique was developed because it was impossible 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, pyrD, 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 Bacillus caldolyticus (Ghim et al., Microbiology 140 (1994) 479-491): the absence of pyrP encoding for uracil permease, and the absence of an open reading frame named orf2, whose function is unknown. Two mutually exclusive stem-loop structures were predicted at the 5'-end of L. plantarum pyr mRNA; this operon could be regulated by transcriptional attenuation under the control of PyrR. Complementation of E. coli pyrD, pyrF and pyrE mutants was obtained with a L. plantarum genomic DNA library. Alignment of the L. plantarum Pyr proteins with other known procaryotic Pyr proteins indicates that they display highly conserved regions in Gram-positive and Gram-negative bacteria.