Dual Functions of Streptococcus salivarius Urease

Abstract

A urease-deficient derivative of Streptococcus salivarius 57.I was constructed by allelic exchange at the ureC locus. The wild-type strain was protected against acid killing through hydrolysis of physiologically relevant concentrations of urea, whereas the mutant was not. Also, S. salivarius could use urea as a source of nitrogen for growth exclusively through a urease-dependent pathway.Bacterial ureases are multisubunit enzymes that require Ni 2ϩ for catalytic activity (9). Several bacterial urease gene clusters have been isolated, and high degrees of homology between species have been observed (5, 9). Most bacterial ureases consist of three subunits, ␣, ␤, and ␥, encoded by ureC, -B, and -A, respectively. Other genes are present in urease clusters, i.e., ureDEFG, which encode proteins that are required for incorporation of Ni 2ϩ into the metallocenter of the catalytic site (9). Additional gene products involved in urease biogenesis and urea metabolism include nickel and urea transporters (10,14).Urea is present in saliva and crevicular fluids at 3 to 10 mM in healthy individuals (7,8), and it is hydrolyzed by ureases to generate two molecules of ammonia and one molecule of CO 2 . Ammonia can neutralize acids generated from bacterial glycolysis, inhibiting the initiation and progression of tooth decay. Ureolysis also creates a less acidic environment, enhancing the survival of acid-sensitive species and promoting the stability of a healthy oral flora (1). Despite the abundance of urea and ureolytic activity in the oral cavity, and the impact of ureolysis on oral health and ecology, the benefits for oral microorganisms of possessing ureases are not established.In general, urease expression in enteric organisms is positively regulated and transcription is activated either in the absence of an assimilable nitrogen source or in the presence of urea (5, 9). Unlike in enteric bacteria, Streptococcus salivarius 57.I urease expression is derepressed at low pH and is further enhanced in the presence of excess carbohydrate (2). Based on this mode of regulation, ureolysis by S. salivarius may function primarily to protect the organisms against acid damage or the bacteria may use ureolysis to acquire nitrogen when carbohydrates are present in excess. To test these hypotheses, an otherwise isogenic, urease-deficient derivative of S. salivarius 57.I was constructed, and the behavior of this mutant and that of the wild-type strain under different growth conditions were compared.Construction of a urease-deficient S. salivarius strain. To construct a urease-deficient S. salivarius strain, a gene specifying erythromycin resistance (erm) was cloned within ureC, the gene encoding the ␣ subunit of urease, to generate plasmid pMC81 (Fig. 1A). Plasmid pMC81 contains an Escherichia coli replicon and thus can be used as a suicide vector in streptococcal hosts. Plasmid pMC81 was introduced into wild-type 57.I by electroporation as previously described (4), and erythromycin-resistant (Em r ) transformants were selected and subjected to S...

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

“…1B). To determine whether ORF1, ORF2, and ORF3 could be transcribed independently, presumably via a promoter embedded somewhere within the ureI-D genes, total cellular RNA was isolated from a recombinant S. salivarius strain in which ureC had been insertionally inactivated by allelic exchange (5), and the presence of ORF1-specific mRNA was determined by RT-PCR (Fig. 1C).…”

“…salivarius is one of the most abundant and highly ureolytic microorganisms in the oral cavity and can use urea as a primary source of nitrogen (5). The expression of urease in S. salivarius is subject to environmental signals, with higher levels of expression in cells grown under acidic conditions, and the induction at acidic pH values can be enhanced by growth in excess amounts of carbohydrate (6).…”

“…The expression of urease in S. salivarius is subject to environmental signals, with higher levels of expression in cells grown under acidic conditions, and the induction at acidic pH values can be enhanced by growth in excess amounts of carbohydrate (6). It is believed that regulation by low pH and carbohydrate availability allows for maximal production of the enzyme when it is most needed for survival of the organisms against lethal acidification (5). The urease gene cluster (ure) of S. salivarius is arranged as an operon, beginning with ureI, followed by ureABCEFGD (9).…”

Roles of PucR, GlnR, and TnrA in Regulating Expression of the Bacillus subtilis ure P3 Promoter

“…The expression of urease is controlled by diverse regulatory mechanisms, suggesting that this enzyme has multiple physiological roles in bacteria. For example, urease expression in Streptococcus salivarius is induced by growth at acidic pH (6). Since the degradation of urea to carbon dioxide and two molecules of ammonia produces a net increase in pH, high levels of urease can facilitate alkalization and bacterial survival in acidic environments.…”

Complete Genome Sequence of the Ureolytic Streptococcus salivarius Strain 57.I

“…When S. salivarius gains entrance to the bloodstream, it may cause severe systemic infections (13). Although not all S. salivarius strains synthesize urease, the ureolytic activity of S. salivarius strain 57.I is the major alkali generation machinery in the oral cavity that plays an essential role in maintaining oral pH homeostasis and balancing dental plaque ecology (5,17). Here we report the complete genome and annotation of the ureolytic strain 57.I and compared the genome with that of strain SK126.…”