Gene inactivation in Lactococcus lactis: histidine biosynthesis

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Abstract
Lactococcus lactis strains from dairy and nondairy sources were tested for the ability to grow in the absence of histidine. Among 60 dairy strains tested, 56 required histidine, whereas only 1 of 11 nondairy strains 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 results in histidine auxotrophy. The histidine operon was detected by Southern hybridization in eight dairy auxotrophic strains tested. A large part of the histidine operon (8 kb, containing seven histidine biosynthetic genes and three unrelated open reading frames [ORFs]) was cloned from an auxotroph, which had an inactive hisD gene, as judged by its inability to grow on histidinol. Complementation analysis of three genes, hisA, hisB, and hisG, in Escherichia coli showed that they also were inactive. Sequence analysis of the cloned histidine region, which revealed 98.6% overall homology with that of the previously analyzed prototrophic strain, showed the presence of frameshift mutations in three his genes, hisC, hisG, and hisH, and two genes unrelated to histidine biosynthesis, ORF3 and ORF6. In addition, several mutations were detected in the promoter region of the operon. Northern (RNA) hybridization analysis showed a much lower amount of the his transcript in the auxotrophic strain than in the prototrophic strain. The mutations detected account for the histidine auxotrophy of the analyzed strain. Certain other dairy auxotrophic strains carry a lower number of mutations, since they were able to revert either to a Hol+ phenotype or to histidine prototrophy.

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