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Actinomyces oris strain K20 was isolated from oral apical lesions. Here, we report the complete circular genome sequence of this strain, obtained by means of hybrid assembly using two next-generation sequencing datasets. The strain has a 3.1-Mb genome with 2,636 coding sequences.

Streptococcus mitisis a normal commensal of the human oral cavity and oropharynx. This microorganism is an opportunistic pathogen in immune compromised hosts and a cause of invasive diseases such as infective endocarditis. We isolated a matrix metalloprotease (MMP)-like protein ofS. mitis. The gene for the MMP-like protein was found on the genome sequence database ofS. mitis. The gene encodes a protein consisting of 240 amino acid residues with a conserved zinc-binding motif (HEXXHXXGXXH) among the matrix metallopeptidase family. The gene was PCR-amplified fromS. mitisATCC 49456 and cloned for construction of the recombinant protein. The recombinant MMP-like protein with a GST-tag was purified and the enzymatic activity was assessed. The recombinant protein showed approximately half the level of MMP-like activity as compared to human recombinant MMP-8. The MMP-like protein ofS. mitismay be involved in the pathogenesis of infection to the dentin with collagen fibers and systemic invasive diseases.&nbsp

Oral microbiome dysbiosis has important links to human health and disease. Although photodynamic therapy influences microbiome diversity, the specific effect of violet light irradiation remains largely unknown. In this study, we analyzed the effect of violet light-emitting diode (LED) irradiation on interdental plaque microbiota. Interdental plaque was collected from 12 human subjects, exposed to violet LED irradiation, and cultured in a specialized growth medium. Next-generation sequencing of the 16S ribosomal RNA genes revealed that α-diversity decreased, whereas β-diversity exhibited a continuous change with violet LED irradiation doses. In addition, we identified several operational taxonomic units that exhibited significant shifts during violet LED irradiation. Specifically, violet LED irradiation led to a significant reduction in the relative abundance of Fusobacterium species, but a significant increase in several species of oral bacteria, such as Veillonella and Campylobacter. Our study provides an overview of oral plaque microbiota changes under violet LED irradiation, and highlights the potential of this method for adjusting the balance of the oral microbiome without inducing antibiotic resistance.

Background: Oral microbiota has been linked to both health and diseases. Specifically, tongue-coating microbiota has been implicated in aspiration pneumonia and halitosis. Approaches altering one's oral microbiota have the potential to improve oral health and prevent diseases. Methods: Here, we designed a study that allows simultaneous monitoring of the salivary and tongue microbiomes during an intervention on the oral microbiota. We applied this study design to evaluate the effect of single-day use of oral care tablets on the oral microbiome of 10 healthy individuals. Tablets with or without actinidin, a protease that reduces biofilm formation in vitro, were tested. Results: Alpha diversity of the tongue microbiome was significantly lower than that of the salivary microbiome, using both the number of observed amplicon sequence variants (254 ± 53 in saliva and 175 ± 37 in tongue; P = 8.9e-7, Kruskal–Wallis test) and Shannon index (6.0 ± 0.4 in saliva and 5.4 ± 0.3 in tongue; P = 2.0e-7, Kruskal–Wallis test). Fusobacterium periodonticum, Saccharibacteria sp. 352, Streptococcus oralis subsp. dentisani, Prevotella melaninogenica, Granulicatella adiacens, Campylobacter concisus, and Haemophilus parainfluenzae were the core operational taxonomic units (OTUs) common to both sites. The salivary and tongue microbiomes of one individual tended to be more similar to one another than to those of other individuals. The tablets did not affect the alpha or beta diversity of the oral microbiome, nor the abundance of specific bacterial species. Conclusions: While the salivary and tongue microbiomes differed significantly in terms of bacterial composition, they showed inter- rather than intra-individual diversity. A one-day usage of oral care tablets did not alter the salivary or tongue microbiomes of healthy adults. Whether the use of oral tablets for a longer period on healthy people or people with greater tongue coating accumulation shifts their oral microbiome needs to be investigated.

The bacterial composition of oral samples has traditionally been determined by PCR amplicon sequencing of 16S rRNA genes. Recent amplicon sequence variant (ASV)-based analyses of 16S rRNA genes differ from that based on operational taxonomic unit (OTU) clustering in the way it deals with sequences having potential errors. However, little information is available on its application in oral microbiome studies. Here, we conducted ASV-based analysis of oral microbiome samples using QIIME 2. We investigated the optimal parameters for sequence denoising, using DADA2, and found the trimming of the first 20 nucleotides from 5′-end of both paired reads avoided excessive sequence loss during chimera removal. Truncating reads at positions 240–245 allowed the removal of low-quality sequences while maintaining sufficient length to merge matching paired ends. Taxonomic assignment, using the naïve Bayes classifier trained with the V3-V4 region of reference 16S rRNA sequences in the extended human oral microbiome database (eHOMD), resulted in bacterial compositions similar to those of OTU-based analyses. Contrary to OTU-based clustering, ASV-based analysis showed taxonomic abundance at the genus or species level to not differ significantly in tongue microbiomes, regardless of brushing. QIIME 2 can, therefore, be a standard pipeline for ASV-based analysis of oral microbiomes.

Archaeal species encode a variety of distinct lineage-specific chromosomal proteins. We have previously shown that inThermococcus kodakarensis, histone, Alba, and TrmBL2 play distinct roles in chromosome organization. Although our understanding of individual archaeal chromosomal proteins has been advancing, how archaeal chromosomes are folded into higher-order structures and how they are regulated are largely unknown. Here, we investigated the primary and higher-order structures of archaeal chromosomes from different archaeal lineages. Atomic force microscopy of chromosome spreads out ofThermoplasma acidophilumandPyrobaculum calidifontiscells revealed 10-nm fibers and 30–40-nm globular structures, suggesting the occurrence of higher-order chromosomal folding. Our results also indicated that chromosome compaction occurs toward the stationary phase. Micrococcal nuclease digestion indicated that fundamental structural units of the chromosome exist inT. acidophilumandT. kodakarensisbut not inP. calidifontisorSulfolobus solfataricus. In vitro reconstitution showed that, inT. acidophilum,the bacterial HU protein homolog HTa formed a 6-nm fiber by wrapping DNA, and that Alba was responsible for the formation of the 10-nm fiber by binding along the DNA without wrapping. Remarkably, Alba could form different higher-order complexes with histone or HTa on DNA in vitro. Mass spectrometry detected HTa in theT. acidophilumchromosome but not in other species. A putative transcriptional regulator of the AsnC/Lrp family (Pcal_1183) was detected on theP. calidifontischromosome, but not on that of other species studied. Putative membrane-associated proteins were detected in the chromosomes of the three archaeal species studied, includingT. acidophilum,P. calidifontis, andT. kodakarensis. Collectively, our data show that Archaea use different combinations of proteins to achieve chromosomal architecture and functional regulation.

Active participation of students is paramount not only for their learning experiences but also for their academic performance. Therefore, various methods have been developed and proven to help students achieve active learning. However, several shortcomings in these methods have been indicated as increasing students’ sense of burden and discomfort, eventually preventing them from benefiting sufficiently. This study aimed to determine the efficiency of a low-load web-based review quiz built by the researchers on Google Forms to enhance students’ reviewing habits and active class participation. Participants in this study were 53 first-year dental hygiene students in a 10-class microbiology course. After each class, all students were given the web-based quiz to prepare for a paper-based review test, which assessed the learning of the content covered in the previous classes. We analyzed the correlations between frequency of participation in the web-based quiz and the average scores of the weekly review tests or the final examination scores. Consequently, voluntary participation in the web-based quiz positively correlated with both short-term and long-term students’ learning outcomes. Through this web-based quiz during the first year of the dental hygiene program, students can develop the “self-learning attitude” needed to pass the national examination.

Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable bacteria number on BHI-Y agar in the oral cavities of all subjects, respectively. It was indicated that among oral Rothia species, R. mucilaginosa is most predominant in the oral cavity of humans. A novel selective medium, ORSM, was useful for isolating each oral Rothia species.

The deterioration of human oral microbiota is known to not only cause oral diseases but also to affect systemic health. Various environmental factors are thought to influence the disruption and restoration of the oral ecosystem. In this study, we focused on the effect of nitric oxide (NO) produced by denitrification and NO synthase enzymes on dental plaque microbiota. Interdental plaques collected from 10 subjects were exposed to NO donor sodium nitroprusside (SNP) and then cultured in a specialized growth medium. Depending on the concentration of exposed SNP, a decrease in &alpha, diversity and a continuous change in &beta, diversity in the dental plaque community were shown by sequencing bacterial 16S rRNA genes. We also identified eight operational taxonomic units that were significantly altered by NO exposure. Among them, the exposure of NO donors to Fusobacterium nucleatum cells showed a decrease in survival rate consistent with the results of microbiota analysis. Meanwhile, in addition to NO tolerance, an increase in the tetrazolium salt-reducing activity of Campylobacter concisus cells was confirmed by exposure to SNP. This study provides an overview of how oral plaque microbiota shifts with exposure to NO and may contribute to the development of a method for adjusting the balance of the oral microbiome.

Here, we present the complete genome sequence of Rothia aeria type strain JCM 11412, isolated from air in the Russian space laboratory Mir. Recently, there has been an increasing number of reports on infections caused by R. aeria . The genomic information will enable researchers to identify the pathogenicity of this organism.

Here, we present the complete genome sequence of Actinomyces naeslundii strain ATCC 27039, isolated from an abdominal wound abscess. This strain is genetically transformable and will thus provide valuable information related to its crucial role in oral multispecies biofilm development.

Introduction Although the production of biofilm is thought to be crucial in the pathogenesis of abscess formations caused by oral resident microorganisms, the particular mechanisms are still unknown. The aim of this study was to identify gene(s) responsible for maintaining the cell surface-associated meshwork-like structures, which are found in some biofilm-producing bacteria, in a clinical isolate of Actinomyces oris K20. Methods Random insertional mutagenesis by using transposon EZ-Tn5 was performed against the strain K20. Transposon insertion mutants were screened by scanning electron microscopy for the absence of cell surface-associated meshwork-like structures. The disrupted genes by the transposon insertion were determined by direct genome sequencing with the transposon-end primers. Results Five mutants without the meshwork-like structures were identified from 175 mutants. Sequencing of flanking regions of transposon insertion revealed that 3 mutants had a gene encoded polysaccharide deacetylase, Spo0J containing ParB-like nuclease domain, and hypothetical protein, respectively. The other 2 mutants had an insertion in a noncoding region and an unidentified region, respectively. Conclusions Our findings indicated that these genes might be involved in the formation of meshwork-like structures on Actinomyces oris K20.

Objective Dental plaque is a causative factor for oral disease and a potential reservoir for respiratory infection in the elderly. Therefore, there is a critical need for the development of effective methods to remove oral biofilm. The objective of this study was to investigate the effect of proteases on oral biofilm formation andremoval. Design The in vivo effect of actinidin, a cysteine protease, on the removal of tongue coating was assessed after orally taking a protease tablet. Effects of the proteases trypsin, papain and actinidin on Actinomyces monospecies biofilm and multispecies biofilm that was reconstructed using a plaque sample from the tongue coating were investigated using the microtiter plate method. Antimicrobial tests and limited proteolysis of fimbrial shaft proteins were also performed to clarify underlying mechanisms of oral biofilm removal. Results Tablets containing actinidin removed tongue coating in elderly subjects. Oral Actinomyces biofilm was significantly reduced by the proteases papain, actinidin and trypsin. Papain and trypsin effectively digested the major fimbrial proteins, FimP and FimA, from Actinomyces. Actinidin, papain and trypsin reduced multispecies biofilm that was reconstructed in vitro. Papain and trypsin inhibited formation of multispecies biofilm in vitro. Conclusions This study shows that proteases reduced oral biofilm in vivo in elderly subjects and in vitro, and suggests that protease digests fimbriae and inhibits biofilm formation.

Aim To demonstrate a capacity for producing exopolysaccharides (EPSs) and an ability to form biofilm on abiotic materials of Actinomyces oris strain K20. Methodology The productivity of EPSs and the ability to form biofilm of strain K20 were evaluated by measuring viscosity of spent culture media and by scanning electron microscopy (SEM) and the biofilm assay on microtitre plates, respectively. High-performance liquid chromatography was used to determine the chemical composition of the viscous materials. To examine the role of the viscous materials attributable to the pathogenicity in this organism, the ability of strain K20 to induce abscess formation was compared in mice to that of ATCC 27044. Results The viscosity of the spent culture media of K20 was significantly higher than that of ATCC 27044. Strain K20 showed dense meshwork structures around the cells and formed biofilms on microtitre plates, whereas ATCC 27044 did not. Chemical analysis of the viscous materials revealed that they were mainly composed of neutral sugars with mannose constituting 77.5% of the polysaccharides. Strain K20 induced persistent abscesses in mice lasting at least 5 days at a concentration of 108 cells mL−1, whereas abscesses induced by ATCC 27044 healed and disappeared or decreased in size at day 5. Conclusions Strain K20 produced EPSs, mainly consisting of mannose, and formed biofilms. This phenotype might play an important role for A. oris to express virulence through the progression of apical periodontitis.

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.

Here, we present the complete genome sequence of Rothia mucilaginosa NUM-Rm6536, a strain isolated from the tongue plaque of a healthy human adult. This strain is amenable to genetic manipulation by transformation and so provides a useful foundation for more detailed investigation of this species.

Prevotella intermedia , a Gram-negative black-pigmented anaerobic rod, is frequently isolated from not only periodontal pockets but also purulent infections. We report here the complete genome sequence of P. intermedia strain 17-2, which is a non-exopolysaccharide-producing variant obtained from exopolysaccharide (EPS)-producing P. intermedia strain 17 stock culture.

P ring is a periplasmic substructure of the flagellar basal body and is believed to connect with the peptidoglycan layer in Salmonella. Two flagellar genes, flgA and flgI, are known to be indispensable for P ring formation. The flgI gene encodes the component protein of the P ring. However, the role of the flgA gene product in P ring assembly remained unknown. Here, evidence is presented that FlgA is synthesized as a precursor form and exported via the Sec secretory pathway into the periplasmic space where P ring formation takes place. Overproduction of the FlgI protein led flgA mutants to form flagella with a P ring, suggesting that FlgA plays an auxiliary role in P ring assembly. Far-Western blot analysis revealed that FlgA binds in vitro to both FlgI and FlgA itself. Though a direct FlgI-FlgI interaction in the absence of FlgA could not be demonstrated, an indirect or direct interaction between the FlgI proteins was observed in the presence of FlgA. FlgA alone was very unstable in vivo, but co-expression with FlgI could stabilize FlgA. This suggests the presence of FlgA-FlgI interaction in vivo. On the basis of these results, a hypothesis is proposed that FlgA acts as a periplasmic chaperone, which assists a polymerization reaction of FlgI into the P ring through FlgA-FlgI interaction.

Rothia mucilaginosa is known as a member of commensal bacterial flora in the oral cavity and has received attention as a potential opportunistic pathogen. We previously determined the genomic sequence of R. mucilaginosa DY-18, a clinical strain with biofilm-like structures isolated from an infected root canal of a tooth with persistent apical periodontitis. We found that the DY-18 genome had only two sigma factor genes that encoded the primary and extracytoplasmic function (ECF) sigma factors. Genomic analysis on the available database of R. mucilaginosa ATCC 25296 (a type strain for R. mucilaginosa) revealed that ATCC 25296 has three sigma factors: one primary sigma factor and two ECF sigma factors, one of which was highly homologous to that of DY-18. ECF sigma factors play an important role in the response to environmental stress and to the production of virulence factors. Therefore, we first examined gene-encoding sigma factors on R. mucilaginosa genome in silico. The homologous ECF sigma factors found in strains DY-18 and ATCC 25296 formed a distinct SigH (SigR) clade in a phylogenetic tree and their cognate anti-sigma factor has a HXXXCXXC motif known to respond against disulphide stress. Quantitative reverse transcription polymerase chain reaction (PCR) and microarray analysis showed that the transcriptional levels of sigH were markedly up-regulated under disulphide stress in both strains. Microarray data also demonstrated that several oxidative-stress-related genes (thioredoxin, mycothione reductase, reductase and oxidoreductase) were significantly up-regulated under the diamide stress. On the basis of these results, we conclude that the alternative sigma factor SigH of R. mucilaginosa is a candidate regulator in the redox state.

Exopolysaccharide (EPS) productivities in many bacteria have been associated with pathogenicity in mammalian hosts as providing extracellular matrices to form biofilm (Costerton et al., 1995). Bacteria assuming biofilm-forming capacity have enormous advantages in establishing persistent infections (Costerton et al., 1999). Chronic periodontitis is caused by dental plaque known as a complex biofilm which consists of several hundred different species of bacteria (Chen, 2001; Socransky and Haffajee, 2002; Lovegrove, 2004). While sucrose-derived homopolysaccharides are well known substrates which mediate adhesion of bacteria to the tooth surface and co-aggregation interactions between species of oral bacteria in the dental plaque (Russell, 2009), recent studies suggest that each bacterium produces distinctive EPS components in a sucrose-independent manner and can form so called single species biofilm (Branda et al., 2005). In the oral cavity, several species of oral bacteria are known to produce their own EPS with this manner (Okuda et al., 1987; Dyer and Bolton, 1985; Kaplan et al., 2004; Yamane et al., 2005; Yamanaka et al., 2009; Yamanaka et al., 2010). In this chapter, we will describe a possibility that a single species biofilm in the oral cavity can cause persistent chronic periodontitis along with the importance of dental plaque formation and maturation with sucrose-derived polysaccharides.

S. Treptococcus constellatus, a member of the S. Treptococcus anginosus Group (SAG), and known as part of indigenous oral microbiota, has been described to cause abscesses in various regions of the body, despite this organism appears to be innocuous, in general at its habitat. In this communication, we report a biofilm-forming capacity of a facultative anaerobic gram-positive coccus isolated as a dominant bacterium in an odontogenic subperiosteal abscess lesiony. The clinical isolate designated as strain H39 formed dense meshwork structures around the cells that are typical for biofilm forming bacteria, and produced viscous materials in its spent culture media. The 16S rRNA gene sequence of strain H39 was 99% homologous to that of S. Treptococcus constellatus ATCC 27823, a type strain for S. Treptococcus constellatus. Phylogenetic analysis using data sets of recN, groEL, tuf and 16S rRNA genes showed a sister relationship between strain H39 and S. Treptococcus constellatus ATCC 27823. Dense meshwork-like structures found on strain H39 were observed on S. Treptococcus constellatus ATCC 27823, but not on S. intermedius ATCC 27335 and S. anginosus ATCC 33397. Biofilm assay using 96-wells polystyrene microtiter plates revealed that S. Treptococcus constellatus strains H39 and ATCC 27823 can form dense biofilm on abiotic materials consistently. S. intermedius ATCC 27335 was able to form biofilm on microtiter plates at a lesser extent to those of S. Treptococcus constellatus strains. S. anginosus ATCC 33397 did not form biofilm on an abitotic material. As conclusions, dense meshwork structures around the cells of S. Treptococcus constellatus, and the capacity of S. Treptococcus constellatus and S. intermedius to form biofilm on abiotic materials as observed in this study might be related to the pathogenicity of these two organism and the tropism of organisms in SAG. As recently suggested, phylogenetic analysis could be a powerful tool for differentiating and identifying clinical isolates belong to SAG.

Background Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built. Methods This study evaluates the role of EPS in Prevotella intermedia for the expression of virulence. This evaluation was accomplished by comparing EPS-producing P. intermedia strains 17 and OD1-16 with non-producing P. intermedia ATCC 25611 and Porphyromonas gingivalis strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis. Results EPS-producing P. intermedia strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 107 colony-forming units (CFU). In comparison, P. intermedia ATCC 25611 and P. gingivalis ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (109 CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, P. intermedia strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes. Conclusions These results demonstrate that the production of EPS by P. intermedia strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.

Escherichia hermannii, formerly classified as enteric group 11 of Escherichia coli, is considered to be nonpathogenic. In this report, we described some of the pathogenic properties of a viscous material-producing E. hermannii strain YS-11, which was clinically isolated from a persistent apical periodontitis lesion. YS-11 possessed cell surface-associated meshwork-like structures that are found in some biofilm-forming bacteria and its viscous materials contained mannose-rich exopolysaccharides. To further examine the biological effect of the extracellular viscous materials and the meshwork structures, we constructed a number of mutants using transposon mutagenesis. Strain 455, which has a transposon inserted into wzt, a gene that encodes an ATP-binding cassette transporter, lacked the expression of the cell surface-associated meshwork structures and the ability to produce extracellular materials. Complementation of the disrupted wzt in strain 455 with an intact wzt resulted in the restoration of these phenotypes. We also compared these strains in terms of their ability to induce abscess formation in mice as an indication of their pathogenicity. Strains with meshwork-like structures induced greater abscesses than those induced by strains that lacked such structures. These results suggest that the ability to produce mannose-rich exopolysaccharides and to form meshwork-like structures on E. hermannii might contribute to its pathogenicity.

Bacterial flagellar rod structure is built across the peptidoglycan (PG) layer. A Salmonella enterica flagellar protein FlgJ is believed to consist of two functional domains, the N-terminal half acting as a scaffold or cap essential for rod assembly and the C-terminal half acting as a PG hydrolase (PGase) that makes a hole in the PG layer to facilitate rod penetration. In this study, molecular data analyses were conducted on FlgJ data sets sampled from a variety of bacterial species, and three types of FlgJ homologs were identified: (i) "canonical dual-domain" type found in beta- and gamma-proteobacteria that has a domain for one of the PGases, acetylmuramidase (Acm), at the C terminus, (ii) "non-canonical dual-domain" type found in the genus Desulfovibrio (delta-proteobacteria) that bears a domain for another PGase, M23/M37-family peptidase (Pep), at the C terminus and (iii) "single-domain" type found in phylogenetically diverged lineages that lacks the Acm or Pep domain. FlgJ phylogeny, together with the domain architecture, suggested that the single-domain type was the original form of FlgJ and the canonical dual-domain type had evolved from the single-domain type by fusion of the Acm domain to its C terminus in the common ancestor of beta- and gamma-proteobacteria. The non-canonical dual-domain type may have been formed by fusion of the Pep domain to the single-domain type in the ancestor of Desulfovibrio. In some lineages of gamma-proteobacteria, the Acm domain appeared to be lost secondarily from the dual-domain type FlgJ to yield again a single-domain type one. To rationalize the underlying mechanism that gave rise to the two different types of dual-domain FlgJ homologs, we propose a model assuming the lineage-specific co-option of flagellum-specific PGase from diverged housekeeping PGases in bacteria.

The bacterial flagellum is a supramolecular structure which originates from the cell envelope and extends into the extracellular space. The individual flagellum consists of at least three substructures: a basal body, a hook, and a filament (1, 5). The basal body is embedded in the cell membrane and consists of a rod with an inner ring (MS ring) and two outer rings (L and P rings). The rod crosses the inner membrane, the periplasmic space, and the outer membrane. The MS ring is associated with the inner membrane, whereas the L ring connects with the outer membrane. The P ring resides in the periplasmic space and is believed to interact with the peptidoglycan layer (6). The MS ring is composed of a single species of protein, FliF, and is believed to be built first during the assembly of the basal body (1, 20, 36). Four proteins, FlgB, FlgC, FlgF, and FlgG, comprise the rod and are postulated to be transported from the cytoplasm into the periplasmic space via a flagellum-specific protein export pathway (12). These proteins assemble onto the MS ring to form the MS ring-rod structure. Subsequently, the FlgI and FlgH proteins, which are transported via the conventional signal sequence-dependent export pathway, assemble around the rod to form the P and L rings, respectively (16). Therefore, the rod is the earliest structure which should penetrate and assemble through the peptidoglycan layer. Because the peptidoglycan is a mechanically rigid structure (11, 31), it has been postulated that breakdown of peptidoglycan should be a prerequisite for rod formation (7, 9). Peptidoglycan hydrolases such as muramidase and amidase (10, 33) are presumed to be responsible for this process. In addition to the structural genes for the rod subunit proteins, more than 10 genes are known to be required for rod formation (19, 35). Most of them are believed to encode the component proteins of the flagellum-specific export apparatus. They include flhA, fliI, and fliH (37). Mutants defective in any one of these genes produce the MS ring lacking the rod and other flagellar substructures (19). Because flgJ mutants also produce the MS ring lacking the rod (19), FlgJ must be essential for the rod assembly. Through the comparative study of the primary structures of proteins, Joris et al. (17) demonstrated that the C-terminal half of FlgJ has homology to the catalytic domain of two species of muramidase, autolysin from Streptococcus faecalis and muramidase 2 from Enterococcus hirae. Recently, the same region of FlgJ was also shown to be homologous to the Lactococcus lactis AcmA protein, which acts as a muramidase essential for cell separation (4). Especially, two amino acids, aspartic acid and glutamic acid, which are conserved in the active center of the enzymes of the muramidase family, are also present in the corresponding positions of FlgJ (Glu-223 and Asp-248) (Fig. ​(Fig.1A).1A). According to these observations, Dijkstra and Keck (7) proposed a hypothesis that FlgJ may be responsible for the penetration of the peptidoglycan by the rod structure. However, this remained to be proven experimentally. This work was carried out to address this issue. FIG. 1 Structures of the wild-type (A), His-tagged (B), and mutant (C) FlgJ proteins. Asterisks indicate the amino acids which are conserved in the putative active center of the enzymes of the muramidase family. Numbers above the bars indicate amino acid residues ... We showed that the purified FlgJ protein has a peptidoglycan-hydrolyzing activity in its C-terminal half. The flgJ mutants with amino acid substitutions in the putative active center of muramidase produced FlgJ proteins with reduced enzymatic activity and showed poor motility. We showed further that FlgJ is exported into the periplasmic space. These results suggest that FlgJ may be the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to construct the rod structure in the periplasmic space. On the basis of this and other available information, we propose a model of rod assembly in the periplasmic space.