DIRECTED GENOMIC INTEGRATION, GENE REPLACEMENT, AND INTEGRATIVE GENE-EXPRESSION IN STREPTOCOCCUS-THERMOPHILUS

Several pGEM5- and pUC19-derived plasmids containing a selectable eryt hromycin resistance marker were integrated into the chromosome of Stre ptococcus thermophilus at the loci of the lactose-metabolizing genes. Integration occurred via homologous recombination and resulted in coin tegrates between plasmid and genome, flanked by the homologous DNA use d for integration. Selective pressure on the plasmid-located erythromy cin resistance gene resulted in multiple amplifications of the integra ted plasmid. Release of this selective pressure, however, gave way to homologous resolution of the cointegrate structures. By integration an d subsequent resolution, we were able to replace the chromosomal lacZ gene with a modified copy carrying an in vitro-generated deletion. In the same way, we integrated a promoterless chloramphenicol acetyltrans ferase (cat) gene between the chromosomal lacS and lacZ genes of the l actose operon. The inserted cat gene became a functional part of the o peron and was expressed and regulated accordingly. Selective pressure on the essential lacS and lacZ genes under normal growth conditions in milk ensures the maintenance and expression of the integrated gene. A s there are only minimal repeated DNA sequences (an NdeI site) flankin g the inserted cat gene, it was stably maintained even in the absence of lactose, i.e., when grown on sucrose or glucose. The methodology re presents a stable system in which to express and regulate foreign gene s in S. thermophilus, which could qualify in the future for an applica tion with food.