A mutant strain of Streptococcus uberis (AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO 4 but not following addition of other metal ions. The mutant contained a single insertion within mtuA, a homologue of mtsA and psaA, which encode metal-binding proteins in Streptococcus pyogenes and Streptococcus pneumoniae, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10 5 CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of S. uberis organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that mtuA was required for efficient uptake of Mn 2؉ during growth in bovine milk and infection of the lactating bovine mammary gland.

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Chromosomal DNA from two geographically distinct isolates of Streptococcus uberis was used to clone the plasminogen activator in an active form in Escherichia coli. The cloned fragments from each strain contained four potential open reading frames (ORFs). That for the plasminogen activator encoded a protein of 286 amino acids (33.4 kDa) which is cleaved between residues 25 and 26 during secretion by S. uberis. The amino acid sequence of the mature protein showed only weak homology (23.5-28%) to streptokinase. The plasminogen activator gene, pauA, in S. uberis was located between two ORFs with high homology to the DNA mismatch repair genes, hexA and hexB, and not on a DNA fragment between the genes encoding an ATP binding cassette transporter protein (abc) and a protein involved in the formation and degradation of guanosine polyphosphates (rel) as is the case for streptokinase in other streptococci.