Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer. So far, more than 10 genes have been shown to be required for rod formation inSalmonella typhimurium. Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins. Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood. Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria. In this study, we showed that the purified FlgJ protein fromS. typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half. Through oligonucleotide-directed mutagenesis, we constructed flgJmutants with amino acid substitutions in the putative active center of the muramidase. The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility. These results indicate that the muramidase activity of FlgJ is essential for flagellar formation. Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized. On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.

Multiple protein components in gingival crevicular fluid were analysed at the same time using mass spectrometry, and this approach may be useful for the diagnosis of periodontal diseases.

BackgroundPrevotella intermedia (P. intermedia), a gram-negative, black-pigmented anaerobic rod, has been implicated in the development of chronic oral infection. P. intermedia strain 17 was isolated from a chronic periodontitis lesion in our laboratory and described as a viscous material producing strain. The stock cultures of this strain still maintain the ability to produce large amounts of viscous materials in the spent culture media and form biofilm-like structures. Chemical analyses of this viscous material showed that they were mainly composed of neutral sugars with mannose constituting 83% of the polysaccharides. To examine the biological effect of the extracellular viscous materials, we identified and obtained a naturally-occurring variant strain that lacked the ability to produce viscous materials in vitro from our stock culture collections of strain 17, designated as 17-2. We compared these two strains (strains 17 versus 17-2) in terms of their capacities to form biofilms and to induce abscess formation in mice as an indication of their pathogenicity. Further, gene expression profiles between these two strains in planktonic condition and gene expression patterns of strain 17 in solid and liquid cultures were also compared using microarray assays.ResultsStrain 17 induced greater abscess formation in mice as compared to that of the variant. Strain 17, but not 17-2 showed an ability to interfere with the phagocytic activity of human neutrophils. Expression of several genes which including those for heat shock proteins (DnaJ, DnaK, ClpB, GroEL and GroES) were up-regulated two to four-fold with statistical significance in biofilm-forming strain 17 as compared to the variant strain 17-2. Strain 17 in solid culture condition exhibited more than eight-fold up-regulated expression levels of several genes which including those for levanase, extracytoplasmic function-subfamily sigma factor (σE; putative) and polysialic acid transport protein (KpsD), as compared to those of strain 17 in liquid culture media.ConclusionThese results demonstrate that the capacity to form biofilm in P. intermedia contribute to their resistance against host innate defence responses.

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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.

BackgroundEvidence 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.MethodsThis 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.ResultsEPS-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.ConclusionsThese 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.

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 β-and γ-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 (δ-proteobacteria) that bears a domain for another PGase, M23/M37-family peptidase (Pep), at the C terminus and (iii) "singledomain" 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 dualdomain type had evolved from the single-domain type by fusion of the Acm domain to its C terminus in the common ancestor of β-and γ-proteobacteria. The noncanonical 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 γ-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.

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.