Dataset Information

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.

SUBMITTER: Morou-Bermudez E

PROVIDER: S-EPMC97765 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Analysis of urease expression in Actinomyces naeslundii WVU45.

Morou-Bermudez E E Burne R A RA

Infection and immunity 20001201 12

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 activit ...[more]

PMID: 11083780

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Project description:Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of Actinomyces naeslundii, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the ureC gene of A. naeslundii WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the A. naeslundii urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the ureABCEFGD genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5' to the start codon of ureA. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the ureC gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by A. naeslundii via a urease-dependent pathway and that ureolysis can protect A. naeslundii against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to A. naeslundii critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.

Project description:The aim of this study was to investigate and clarify the ambiguous taxonomy of Actinomyces naeslundii and its closely related species using state-of-the-art high-throughput sequencing techniques, and, furthermore, to determine whether sub-clusters identified within Actinomyces oris and Actinomyces naeslundii in a previous study by multi locus sequence typing (MLST) using concatenation of seven housekeeping genes should either be classified as subspecies or distinct species. The strains in this study were broadly classified under Actinomyces naeslundii group as A. naeslundii genospecies I and genospecies II. Based on MLST data analysis, these were further classified as A. oris and A. naeslundii. The whole genome sequencing of selected strains of A. oris (n = 17) and A. naeslundii (n = 19) was carried out using Illumina Genome Analyzer IIxe and Roche 454 allowing paired-end and single-reads sequencing, respectively. The sequences obtained were aligned using CLC Genomic workbench version 5.1 and annotated using RAST (Rapid Annotation using Subsystem Technology) release version 59 accessible online. Additionally, genomes of seven publicly available strains of Actinomyces (k20, MG1, c505, OT175, OT171, OT170, and A. johnsonii) were also included. Comparative genomic analysis (CGA) using Mauve, Progressive Mauve, gene-by-gene, Core, and Pan Genome, and finally Digital DNA-DNA homology (DDH) analysis was carried out. DDH values were obtained using in silico genome-genome comparison. Evolutionary analysis using ClonalFrame was also undertaken. The mutation and recombination events were compared using chi-square test among A. oris and A. naeslundii isolates (analysis methods are not included in the study). CGA results were consistent with previous traditional classification using MLST. It was found that strains of Actinomyces k20, MG1, c505, and OT175 clustered in A. oris group of isolates, while OT171, OT170, and A. johnsonii appeared as separate branches. Similar clustering to MLST was observed for other isolates. The mutation and recombination events were significantly higher in A. oris than A. naeslundii, highlighting the diversity of A. oris strains in the oral cavity. These findings suggest that A. oris forms six distinct groups, whereas A. naeslundii forms three. The correct designation of isolates will help in the identification of clinical Actinomyces isolates found in dental plaque. Easily accessible online genomic sequence data will also accelerate the investigation of the biochemical characterisation and pathogenesis of this important group of micro-organisms.

Dataset Information

Analysis of urease expression in Actinomyces naeslundii WVU45.

Publications

Analysis of urease expression in Actinomyces naeslundii WVU45.

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