Construction and characterization of a recombinant ureolytic Streptococcus mutans and its use to demonstrate the relationship of urease activity to pH modulating capacity

Abstract

To begin to understand the contribution of oral microbial ureolysis to the inhibition of dental caries, we sought to construct a recombinant, ureolytic mutans streptococcus and correlate the ureolytic capacity of plaque bacteria with pH moderating ability. Streptococcus mutans GS-5 was transformed with a plasmid containing the urease genes from Streptococcus salivarius 57.I. The recombinant strain, S. mutans AC04, stably maintained the urease genes. High levels of urease activity were detected, with a maximum specific activity of 0.9 mumol of urea hydrolyzed/min/mg cell dry weight when the growth medium was supplemented with 50 microM exogenous NiCl2. Harboring the recombinant plasmid, or growth in NiCl2, did not markedly affect the glycolytic capacity of S. mutans. In vitro pH drop analysis of S. mutans AC04, metabolizing glucose and physiologically relevant concentrations of urea simultaneously, demonstrated that increasing the urease activity of plaque bacteria resulted in a corresponding reduction in the depth and the duration of the glycolytic pH fall. The results demonstrate the feasibility of engineering urease producing S. mutans and suggest that enhancing the ureolytic capacity of dental plaque, particularly cariogenic plaque, may help to offset the progression of the caries process.

Could Alkali Production Be Considered an Approach for Caries Control?

“…It has been suggested that plaque ureolysis can be almost entirely explained by the percentage of total detectable ureolytic bacteria in the plaque microbiota [Sissons and Cutress, 1988]. In line with this thinking, the results of a previous in vitro study showed that levels of urease activity produced by a recombinant S. mutans strain could be manipulated, resulting in small increases in urease activity, which significantly decreased environmental acidification [Clancy and Burne, 1997]. In the current study, the urease activity in plaque of caries-free individuals was higher than in saliva samples of the same individuals.…”

“…Urea is hydrolyzed by urease enzymes which are present in a number of oral bacteria. Ammonia, generated from ureolysis, can lead to considerable increase in plaque pH despite a diet rich in carbohydrates [Kleinberg, 1961;Stephen, 1993;Imfeld et al, 1995;Clancy and Burne, 1997]. Ammonia can also be produced from arginine through the arginine deiminase system (ADS), which has not been thoroughly studied as a source for alkali production.…”

Oral Arginine Metabolism May Decrease the Risk for Dental Caries in Children

“…We observed an extremely high degree of variability in ADS activity among plaque samples, in some cases greater than 10,000-fold. In in vitro studies, a five-fold decrease in alkaligenerating potential was shown to markedly diminish the pHmoderating potential of oral bacteria (Clancy and Burne, 1997;Clancy et al, 2000). Plaque bacteria from tooth surfaces without caries lesions showed the widest range of ADS activities.…”

Regulation of urease gene ofActinomyces naeslundiiin biofilms in response to environmental factors

“…Substantial amounts of urea are secreted continuously from saliva and gingival crevicular fluid at concentrations of 3-10 mM in healthy individuals (Epstein et al, 1980). Urea is rapidly hydrolyzed by urease from ureolytic bacteria in the oral cavity to produce two molecules of ammonia and one molecule of carbon dioxide, which could neutralize the acid end-products from carbohydrate metabolism, and may be helpful in inhibiting enamel demineralization, creating an environment less conducive to the emergence of an aciduric, acidogenic microbial communities (Clancy & Burne, 1997;Burne, 1998), and stabilizing microbial communities diversity in the face of a strong carbohydrate challenge (Shu et al, 2003). For these reasons, ureolysis is considered to be an important factor to prevent caries formation.…”