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Identification and Characterization of the Nickel Uptake System for Urease Biogenesis in Streptococcus salivarius 57.I

Department of Oral Biology, University of Florida, Gainesville, Florida 32610

Received 8 September 2002/ Accepted 14 September 2003

Ureases are multisubunit enzymes requiring Ni²⁺ for activity. The low pH-inducible urease gene cluster in Streptococcus salivarius 57.I is organized as an operon, beginning with ureI, followed by ureABC (structural genes), and ureEFGD (accessory genes). Urease biogenesis also requires a high-affinity Ni²⁺ uptake system. By searching the partial genome sequence of a closely related organism, Streptococcus thermophilus LMG18311, three open reading frame (ORFs) homologous to those encoding proteins involved in cobalamin biosynthesis and cobalt transport (cbiMQO) were identified immediately 3' to the ure operon. To determine whether these genes were involved in urease biogenesis by catalyzing Ni²⁺ uptake in S. salivarius, regions 3' to ureD were amplified by PCRs from S. salivarius by using primers identical to the S. thermophilus sequences. Sequence analysis of the products revealed three ORFs. Reverse transcriptase PCR was used to demonstrate that the ORFs are transcribed as part of the ure operon. Insertional inactivation of ORF1 with a polar kanamycin marker completely abolished urease activity and the ability to accumulate ⁶³Ni²⁺ during growth. Supplementation of the growth medium with NiCl₂ at concentrations as low as 2.5 µM partially restored urease activity in the mutant. Both wild-type and mutant strains showed enhanced urease activity when exogenous Ni²⁺ was provided at neutral pH. Enhancement of urease activity by adding nickel was regulated at the posttranslational level. Thus, ORF1, ORF2, and ORF3 are part of the ure operon, and these genes, designated ureM, ureQ, and ureO, respectively, likely encode a Ni²⁺-specific ATP-binding cassette transporter.

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