Analysis of urease expression in Actinomyces naeslundii WVU45

Abstract

The hydrolysis of urea by ureases of oral bacteria in dental plaque can cause a considerable increase in plaque pH, which can inhibit the development of dental caries. There is also indirect evidence that urea metabolism may promote the formation of calculus and that ammonia release from urea could exacerbate periodontal diseases. Actinomyces naeslundii, an early colonizer of the oral cavity and a numerically significant plaque constituent, demonstrates comparatively low levels of urease activity on isolation, so this organism has not been considered a major contributor to total oral urease activity. In this study it was observed that urease activity and urease-specific mRNA levels in A. naeslundii WVU45 can increase up to 50-fold during growth under nitrogen-limiting conditions. Using primer extension analysis, a putative, proximal, nitrogen-regulated promoter of the A. naeslundii urease gene cluster was identified. The functionality and nitrogen responsiveness of this promoter were confirmed using reporter gene fusions and 5' deletion analysis. The data indicated that regulation of urease expression by nitrogen availability in A. naeslundii may require a positive transcriptional activator. Plaque bacteria may experience nitrogen limitation when carbohydrates are present in excess. Therefore, based on the results of this study and in contrast to previous beliefs, strains of A. naeslundii may have the potential to be significant contributors to total plaque ureolysis, particularly during periods when there is an increased risk for caries development.

FIG. 1

Slot blot analysis of total RNA from A. naeslundii WVU45 cells grown under (A) nitrogen limitation in ADM/8 or under nitrogen excess in (B) ADM/8 supplemented with 2% tryptone or (C) ADM/8 supplemented with 2% casamino acids. The blot was probed with a 0.7-kbp DNA fragment containing the 3′ region of ureA, all of ureB, and the 5′ region of ureC. RNase tx, samples were treated with RNase before being applied to the membrane.

FIG. 2

Primer extension analysis of total RNA from A. naeslundii WVU45 cells grown under conditions of nitrogen excess (lane 1, ADM/8 supplemented with 2% casamino acids; lane 2, ADM/8 supplemented with 2% tryptone) or nitrogen limitation (lane 3, ADM/8). The nucleotide sequence of the promoter region immediately 5′ to the transcription initiation site is indicated. DNA sequences that demonstrate significant homologies to consensus recognition sequences for RNA polymerase associated with ς⁷⁰ or ς⁵⁴ are underlined.

FIG. 3

Reporter gene constructs and corresponding CAT activity levels under conditions of nitrogen limitation (ADM/8) and nitrogen excess (ADM/8 plus 2% tryptone). ND, none detected (<0.25 nmol of chloramphenicol (Cm) acetylated min⁻¹ mg of protein⁻¹. RBS, ribosome-binding site.