Volume 2010 (2010), Article ID 457236, 6 pages
Complete Genome Sequence of Rothia mucilaginosa DY-18: A Clinical Isolate with Dense Meshwork-Like Structures from a Persistent Apical Periodontitis Lesion
1Department of Bacteriology, Osaka Dental University, 8-1 Kuzuhahanazono-cho, Hirakata-shi, Osaka 573-1121, Japan
2Microbiology Branch, U.S. Army Dental and Trauma Research Detachment, Walter Reed Army Institute of Research, Great Lakes, IL 60088, USA
Received 3 June 2010; Revised 26 August 2010; Accepted 25 September 2010
Academic Editor: Fengfeng Zhou
Copyright © 2010 Kazuyoshi Yamane et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Rothia mucilaginosa is an opportunistic pathogen in the human oral cavity and pharynx. We found that R. mucilaginosa DY-18, a clinical isolate from a persistent apical periodontitis lesion, had biofilm-like structures. Similar structures were also observed on R. mucilaginosa ATCC25296. To further study these structures, we determined the complete genome sequence of DY-18 and found it a 2.26-Mb chromosome. Regarding stress responsive systems known to affect biofilm formation in many bacteria, DY-18 genome possessed only two sigma factor genes. One of these encoded an additional sigma factor whose promoter-binding activity may be regulated in response to environmental stimuli. Additionally, several genes assigned to two-component signal transduction systems were presented in this genome. To the best of our knowledge, this is the first complete genome of R. mucilaginosa species and our data raise the possibility that this organism regulates the biofilm phenotype through these stress responsive systems.
R. mucilaginosa, formerly classified as Stomatococcus mucilaginosus , is a gram-positive, coagulase-negative, non-spore-forming coccus considered as a part of the commensal flora of the oral cavity and the upper respiratory tract in humans . There have been an increasing number of infections reported for R. mucilaginosa, especially among immunocompromised patients [3, 4]. Therefore, this organism has received attention recently as a potential emerging opportunistic pathogen. This organism is also known to act as an initial colonizer on the tooth surface in the oral cavity . Contrarily, several studies suggested that R. mucilaginosa belongs to the healthy oral microbial community  or constitutes a microbial community whose productivity of malodor is lower than those of other communities without R. mucilaginosa . So, the factors making a divide for this organism as an opportunistic pathogen or as a member of the healthy oral microbiota are still obscure. A clinical strain, R. mucilaginosa strain DY-18 (DY-18), was isolated from the bacteria that were present in a persistent apical periodontitis lesion . Apical periodontitis is a relatively common inflammatory disease in dentistry, and a wide variety of bacterial genera have been implicated as putative pathogens [9–11]. If allowed to progress, apical periodontitis can cause the destruction of supporting connective tissues and bones, ultimately resulting in tooth loss. The ability to produce extracellular matrix and to form biofilms is recently considered to be crucial for microorganisms that are present in a root canal to resist the intraroot canal procedures of disinfection, to occupy apical foramina of teeth, and to cause persistent chronic inflammatory lesions [9, 12–14]. For future studies to genetically clarify the mechanism of matrix production, we have determined a complete genome sequence of DY-18 with a combined strategy of pyrosequencing and paired-end sequencing.
2.1. Bacterial Strains and Culture Conditions
DY-18 was from our stock culture collection isolated from a persistent apical periodontitis lesion, where the presence of bacteria was noted in root canals after numerous treatments . R. mucilaginosa ATCC25296 (ATCC25296), a reference strain for R. mucilaginosa, was obtained from American Type Culture Collection (Manassas, VA, USA). All strains were grown on blood agar plates (BBL Microbiology Systems, Cockeysville, MD, USA) at for 24 hours aerobically.
2.2. Scanning Electron Microscopy
To examine cell surface structures, scanning electron microscopy (SEM) was performed. Bacteria grown on blood agar plates for 24 hours were collected on a piece of filter paper (Glass fiber GA55, Toyo Roshi, Tokyo, Japan), fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 hours and 1% OsO4 in 0.1 M phosphate buffer for 1 hour at , and dehydrated through an ethanol series and 2-methyl-2-propanol followed by platinum ion coating (E-1030, Hitachi, Tokyo, Japan). Specimens were examined with a scanning electron microscope (S-4800, Hitachi) at an accelerating voltage of 3 kV.
2.3. Genome DNA Extraction
To extract genomic DNA, DY-18 was grown in a trypticase soy broth (BBL microbiology systems) with 0.5% yeast extract (Difco Laboratories, Detroit, MI, USA) for 24 hours. Genomic DNA was extracted using MagExtractor (Toyobo, Osaka, Japan) according to the manufacture’s protocol.
2.4. Whole Genome Sequencing
The complete genome sequence of DY-18 was determined using a combination of 454 pyrosequencing and paired-end sequencing  performed using the Roche Genome Sequencer FLX Systems (Roche Diagnostics, Tokyo, Japan) and Applied Biosystem 3730 DNA Analyzer (Applied Biosystems, Foster, CA, USA). The sequence data were assembled with GS de novo Assembler Software (version 1.1.03.24) (Roche Diagnostics). The putative gaps between contigs were closed by primer walking and sequencing of PCR amplicon over the gaps. Sanger reads were incorporated into the assembly using Sequencher (version 4.7, Gene Codes, Ann Arbor, MI, USA). Contig editing and primer design were performed using the in silico Molecular Cloning Genomics Edition (version 4.1, in silico biology, Yokohama, Japan). Bases in the assembled contigs carried a Phred-equivalent quality score of 40 or above (error rate 0.010%).
The sequence was annotated using the coding region identification tool invoking comparative analysis (CRITICA) (version 1.05b) and the Gene Locator and Interpolated Markov ModelER (Glimmer) 2 (version 2.10).
2.5. Bioinformatics Analysis
Since sigma factors play a critical role in regulating biofilm formation in some bacteria [16–20], we also attempted to identify homologs of sigma factors on the genome. Amino acid sequence of Escherichia coli K-12 RpoD (GenBank ID (GI): 1789448), RpoE (GI: 1788926), FliA (GI: 1788231), RpoS (GI: 1789098), RpoH (GI: 1789871), FecI (GI: 1790746), and RpoN (GI: 1789594) on GenBank database (National Center for Biotechnology Information (NCBI), USA) were used as queries, and BLASTP searches  were performed using the in silico Molecular Cloning Genomics Edition. NCBI conserved domain (CD) searches  were performed using NCBI server (version 2.18). Pfam domain searches  were performed using Pfam program (version 24.0).
3. Results and Discussion
3.1. Cell Surface Structures of DY-18
Scanning electron microscopy revealed that DY-18 possesses dense meshwork-like structures around cells (Figure 1(a)) which is a typical phenotype among bacteria with biofilms [20, 24, 25]. ATCC25296, a type strain for R. mucilaginosa, also showed the same structures around cells (Figure 1(b)). In both strains, this phenotype was maintained after repetitive passages (data not shown). Many oral bacteria, such as cariogenic Streptococcus mutans, are known to form biofilms in a sucrose-dependent manner . It is important to note that DY-18 possesses dense meshwork-like structures in a sucrose-independent manner. Considering the limited nutritional condition in apical periodontitis lesions, this behavior might be important for this organism to survive in a severe condition.
3.2. Complete Genome Sequence of DY-18
In order to understand the regulatory system behind the maintaining of this phenotype, the complete genome sequence of DY-18 was determined using a combination of 454 pyrosequencing and paired-end sequencing . A total of 193,024 reads from standard sequencing and 83,610 reads from paired-end sequencing were assembled. Seven scaffolds containing 64 contigs were obtained with 99.71% of the bases having a quality score of 40 and above. As the results of Gap closing by primer walking PCR and sequencing, the DY-18 genome sequence contains 2,264,603 bp and had an average sequencing depth of 18-fold. The one circular chromosome had a high GC content (59.6%). No plasmids were found.
The sequence was annotated using the CRITICA and Glimmer 2. The entire genome contains 2,052 predictive protein-coding genes including 49 tRNA genes and 11 rRNA operons (Figure 2). Genome information for the chromosome of DY-18 was deposited in DDBJ/EMBL/GenBank under the accession number AP011540. The allocation of main gene function is described in Table 1.
3.3. Sigma Factors and Two-Component Systems on DY-18 Chromosome
As described above, the biofilm formation is tightly linked to the stress responsive system in many bacteria [16–20]. So, we bioinformatically determined the stress responsive genes on the chromosome of DY-18. This organism possessed two sigma factors, one of which is a primary sigma factor that recognizes housekeeping promoters and the other is an ECF sigma factor whose promoter-binding activity may be regulated in response to physiological and environmental cues (Figure 3(a)). ATCC25296 has one primary and two ECF sigma factors (accession number: ROTMU0001_0778, ROTMU0001_0903 and ROTMU0001_0927). Furthermore, the gene encoding the ECF sigma factor is arranged in an operon-like structure with its cognate antisigma factor gene (RMDY18_07080) (Figure 3(b)).
Sigma factors are involved in promoter recognition and transcription initiation  and plays key roles on bacterial stress response systems. In Staphylococcus aureus, the sigma factor influences the expression of several virulence factors and is associated with an increased ability to adhere, invade and persist within host cells . Moreover, the activity of sigma factor positively influences the expression of several cell surface proteins . These results suggest that the sigma factor plays an important role in the establishment of chronic and intractable S. aureus infections . In Pseudomonas aeruginosa, ECF sigma factor AlgU responds to environmental stresses with the production of alginate, a component of its meshwork-like structures . The antisigma factor MucA is a negative regulator of alginate production that sequesters AlgU to the inner membrane . In this study, we attempted to find genes on DY-18 genome highly homologous to well-known biofilm-associated genes of S. aureus and P. aeruginosa. But our trial failed to hunt out homologous genes with certain accuracy. Instead, BLAST analysis of the RMDY18_07080 gene showed that the antisigma factor of DY-18 has a motif responding to oxidative stress. In Mycobacterium avium, addition of hydrogen peroxide, a known stimulus of the oxidative stress response resulted in elevated biofilm formation . Alkyl hydroperoxide reductase genes (RMDY18_18480 and RMDY18_18490) were also identified in DY-18. As for a future study, we will examine whether these oxidative stress response genes affect the biofilm formation of DY-18.
Bacteria also exploit the two-component systems for controlling gene expression in stressful environments . This signal transduction system consists of a sensor kinase together with a response regulator and is highly conserved among bacteria . In response to stimuli, following the autophosphorylation of the sensor kinase, the phosphate is transferred to a cognate response regulator to control the transcription of target genes. In Escherichia coli strains ATCC25404, DH5 and MG1655, the QseBC two-component system induced by autoinducer-2 is known to control biofilm growth . We found that five genes in the DY-18 chromosome were assigned to two-component systems on a database of Kyoto Encyclopedia of Genes and Genomes (KEGG)/PATHWAY (http://www.kegg.jp/kegg/) . Although the functions of downstream genes are still obscure, we found that DY-18 possesses orthologs of two-component system genes responding to low temperature (RMDY18_05700 and RMDY18_05710), cell envelope stress (RMDY18_06650 and RMDY18_06660), and phosphate limitation (RMDY18_17310).
Pathways built on KEGG/PATHWAY (http://www.kegg.jp/kegg-bin/show_organism?org=rmu) also suggested that there are several genes relating to biosynthesis of sugars. The pathway included the genes involved in the synthesis of mannose (RMDY18_00300, RMDY18_00310, RMDY18_00320, RMDY18_00610, and RMDY18_17420), which is a major component of meshwork-like structures of biofilm-forming bacteria, such as Prevotella intermedia/nigrescens [20, 36] and P. aeruginosa . Furthermore, twenty-eight putative glycosyltransferases were found on DY-18 genome. These genes might contribute to the exopolysaccharide biosynthesis resulting in the formation of meshwork-like structures of R. mucilaginosa DY-18.
The genes constructing ABC-type multidrug transport system were also found on DY-18 genome (RMDY18_05690 and RMDY18_05680). It is likely that the biofilm formation and the multidrug transporter contribute to the therapy resistance of this organism; however, further thorough studies on both systems are necessary to fully explain the mechanism of therapy resistance of R. mucilaginosa. In immunocompromised patients, antibiotic treatment against R. mucilaginosa is difficult .
4. Conclusive Remarks
As far as we retrieved, this is the first report of the complete genome sequence of a clinical isolate of Rothia from an apical periodontitis lesion. This organism has a unique meshwork-like phenotype. Additionally, we found that DY-18 possessed only two sigma factors and one of these was the ECF sigma factor responding to environmental stresses. Two-component systems also exist as stress responding systems in DY-18 genome. These systems seem likely to be crucial for the biofilm formation of this organism to survive unfavorable condition. However, further studies using microarray constructed with the data obtained in this study and pathway analysis  are needed to figure out how R. mucilaginosa regulates biofilm structures and causes opportunistic infections.
The author would like to acknowledge the excellent assistance of Mr. Hideaki Hori with the electron microscope. A part of this study was performed at the Institute of Dental Research, Osaka Dental University and was supported by the Osaka Dental University Joint Research Funds (B08-01).
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