GENE INACTIVATION IN LACTOCOCCUS-LACTIS - HISTIDINE BIOSYNTHESIS

Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine. Among 60 dairy s trains tested, 56 required histidine, whereas only 1 of 11 nondairy st rains had this requirement. Moreover, 10 of the 56 auxotrophic strains were able to grow in the presence of histidinol (Hol+), the immediate histidine precursor. This indicates that adaptation to milk often res ults in histidine auxotrophy. The histidine operon was detected by Sou thern hybridization in eight dairy auxotrophic strains tested. A large part of the histidine operon (8 kb, containing seven histidine biosyn thetic genes and three unrelated open reading frames [ORFs]) was clone d from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol. Complementation analysis of three ge nes, hisA, hisB, and hisG, in Escherichia coli showed that they also w ere inactive. Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed p rototrophic strain, showed the presence of frameshift mutations in thr ee his genes, hisC, hisG, and hisH, and two genes unrelated to histidi ne biosynthesis, ORF3 and ORF6. In addition, several mutations were de tected in the promoter region of the operon. Northern (RNA) hybridizat ion analysis showed a much lower amount of the his transcript in the a uxotrophic strain than in the prototrophic strain. The mutations detec ted account for the histidine auxotrophy of the analyzed strain. Certa in other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histid ine prototrophy.