Kegg Pathway: Protein biosynthesis (bacteria)

Proc Natl Acad Sci U S A. 2010 Feb 1;
Küper U, Meyer C, Müller V, Rachel R, Huber H

ATP synthase catalyzes ATP synthesis at the expense of an electrochemical ion gradient across a membrane that can be generated by different exergonic reactions. Sulfur reduction is the main energy-yielding reaction in the hyperthermophilic strictly anaerobic Crenarchaeon Ignicoccus hospitalis. This organism is unusual in having an inner and an outer membrane that are separated by a huge intermembrane compartment. Here we show, on the basis of immuno-EM analyses of ultrathin sections and immunofluorescence experiments with whole I. hospitalis cells, that the ATP synthase and H(2):sulfur oxidoreductase complexes of this organism are located in the outer membrane. These two enzyme complexes are mandatory for the generation of an electrochemical gradient and for ATP synthesis. Thus, among all prokaryotes possessing two membranes in their cell envelope (including Planctomycetes, Gram-negative bacteria), I. hospitalis is a unique organism, with an energized outer membrane and ATP synthesis within the periplasmic space. In addition, DAPI staining and EM analyses showed that DNA and ribosomes are localized in the cytoplasm, leading to the conclusion that in I. hospitalis energy conservation is separated from information processing and Protein biosynthesis. This raises questions regarding the function of the two membranes, the interaction between these compartments, and the general definition of a cytoplasmic membrane.

Glycobiology. 2010 Feb 1;
Séveno M, Séveno-Carpentier E, Voxeur A, Menu-Bouaouiche L, Rihouey C, Delmas F, Chevalier C, Driouich A, Lerouge P

The structures of the pectic polysaccharide rhamnogalacturonan II (RG-II) pectin constituent are remarkably evolutionary conserved in all plant species. At least twelve different glycosyl residues are present in RG-II. Among them is the seldom eight carbon sugar 3-deoxy-d-manno-octulosonic acid (Kdo) whose biosynthetic pathway has been shown to be conserved between plants and Gram-negative bacteria. Kdo is formed in the cytosol by the condensation of phosphoenol pyruvate with d-arabinose-5-P and then activated by coupling to CMP prior to its incorporation in the Golgi apparatus by a Kdo transferase into the nascent polysaccharide RG-II. To gain new insight into RG-II biosynthesis and function, we isolated and characterized null mutants for the unique putative Kdo transferase (AtKDTA) encoded in the Arabidopsis genome. We provide evidence that, in contrast to mutants affecting the RG-II biosynthesis, the extinction of the AtKDTA gene expression does not result in any developmental phenotype in the AtkdtA plants. Furthermore, the structure of RG-II from the null mutants was not altered and contained wild-type amount of Rha-alpha(1-5)Kdo side-chain. The cellular localization of AtKDTA was investigated by using laser scaning confocal imaging of the Protein fused to green-fluorescent Protein. In agreement with its cellular prediction, the fusion Protein was demonstrated to be targeted to the mitochondria. These data, together with data deduced from sequence analyses of higher plant genomes, suggest that AtKDTA encodes a putative Kdo transferase involved in the synthesis of a mitochondrial not yet identified lipid A-like molecule rather than in the synthesis of the cell wall RG-II.

Microbiology. 2010 Jan 28;
Scorpio A, Chabot DJ, Day WA, Hoover TA, Friedlander AM

Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase (GGT) and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to heat-shock Protein 60 (hsp60), elongation factor Tu (EFTu), and capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblot in lysates from wild-type Ames but was detected in strains engineered with a consensus ribosome binding site for capD. Strains overexpressing capD at amounts detected by immunoblot were found to have less surface-associated capsule and released primarily lower molecular weight capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and macrophage phagocytosis and resulted in reduced fitness in guinea pig models of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence.

Biochemistry. 2010 Jan 26;
Nicoletti FP, Comandini A, Bonamore A, Boechi L, Boubeta FM, Feis A, Smulevich G, Boffi A

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high affinity complexes with hydrogen sulfide in their ferric state. The recombinant Proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide and even highly purified samples still contain a small but significant amount of iron bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments in order to assess the relevant sulfide binding parameters. The body of experimental data indicate that both Proteins possess a high affinity for hydrogen sulfide (K = 5.0x106 M-1 and 2.8x106 M-1 in Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9x108 M-1). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by the distal residue WG8. A set of point mutants in which these residues have been replaced by Phe indicate that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH- bound ferric Tf-trHb show that WG8 plays a key role for the stabilization of coordinated SH- whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing Phe only residues in the relevant B10, G8 and CD1 positions is endowed with higher overall affinity for sulfide characterized by a very fast second order rate constant and a two order of magnitude faster kinetics of sulfide release with respect to the wt Protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low spin derivative. In analogy with other low spin ferric sulfide adducts, the strong band at 375 cm-1 is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H2S is produced in both bacteria in at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

J Bacteriol. 2010 Jan 22;
Albrecht AG, Netz DJ, Miethke M, Pierik AJ, Burghaus O, Peuckert F, Lill R, Marahiel MA

Bacteria use three distinct systems for iron-sulfur (Fe/S) cluster biogenesis: the ISC, SUF and NIF machineries. The ISC and SUF systems are widely distributed, and many bacteria possess both of them. In E. coli, ISC is the major and constitutive system, whereas SUF is induced under iron starvation and/or oxidative stress. Genomic analysis of the Fe/S cluster biosynthesis genes in B. subtilis suggests that this bacterium only encodes a SUF system consisting of a sufCDSUB gene cluster and a distant sufA gene. Mutant analysis of the putative Fe/S scaffold genes sufU and sufA revealed that sufU is essential for growth under minimal standard conditions, but not sufA. The drastic growth retardation of a conditional mutant depleted for SufU was coupled with a severe reduction of aconitase and succinate dehydrogenase activities in total cell lysates suggesting a crucial function of SufU in Fe/S Protein biogenesis. Recombinant SufU was devoid of Fe/S clusters after aerobic purification. Upon in vitro reconstitution, SufU bound an Fe/S cluster with up to approximately 1.5 Fe and S per monomer. The assembled Fe/S cluster could be transferred from SufU to the apo-form of isopropylmalate isomerase Leu1, rapidly forming catalytically active [4Fe-4S]-containing holo-enzyme. In contrast to native SufU, its D43A variant carried a Fe/S cluster after aerobic purification, indicating that the cluster is stabilized by this mutation. Further, we show that apo-SufU is an activator of the cysteine desulfurase SufS by enhancing its activity about 40-fold in vitro. SufS dependent formation of holo-SufU suggests that SufU functions as an Fe/S cluster scaffold Protein tightly cooperating with the SufS cysteine desulfurase.

Antimicrob Agents Chemother. 2010 Jan 19;
Kumagai T, Koyama Y, Oda K, Noda M, Matoba Y, Sugiyama M

In the present study, we successfully cloned a 21-kb DNA fragment containing a D-cycloserine (DCS) biosynthetic gene cluster from a DCS-producing Streptomyces (S.) lavendulae, ATCC11924. The putative gene cluster consists of 10 open reading frames (ORFs), designated dcsA to dcsJ. This cluster includes two ORFs encoding D-alanyl-D-alanine ligase (dcsI) and a putative membrane Protein (dcsJ) as the self-resistance determinants of the producer organism, which were indicated by our previous works. When the 10 orfs were introduced into DCS-non-producing S. lividans 66 as a heterologous host cell, the transformant acquired DCS productivity. This reveals that the introduced genes are responsible for the biosynthesis of DCS. As anticipated, the disruption of dcsG, seen in the DCS-biosynthetic gene cluster, made it possible for the strain ATCC11924 to lose its DCS production. We here propose the DCS-biosynthetic pathway: first, L-serine is O-acetylated by an dcsE-encoded enzyme homologous to homoserine O-acetyltransferase. Second, the O-acetyl-L-serine accepts hydroxyurea by an O-acetylserine sulfhydrylase homolog (dcsD product) and forms O-ureido-L-serine. The hydroxyurea must be supplied by the catalysis of an dcsB-encoded arginase homolog using the L-arginine derivative, N(G)-hydroxy-L-arginine. The resulting O-ureido-L-serine is then racemized to O-ureido-D-serine by a homolog of diaminopimelate epimerase. Finally, O-ureido-D-serine is cyclized to form DCS with the release of ammonia and carbon dioxide. The cyclization must be done by the dcsG or dcsH product, which belongs to the ATP-grasp fold family of Protein.

Nat Chem Biol. 2010 Feb; 6(2): 99-101
Sun Y, Hahn F, Demydchuk Y, Chettle J, Tosin M, Osada H, Leadlay PF

The Protein phosphatase inhibitor RK-682 is one of a number of potentially valuable tetronate polyketide natural products. Understanding how the tetronate ring is formed has been frustrated by the inaccessibility of the putative substrates. We report the heterologous expression of rk genes in Saccharopolyspora erythraea and reconstitution of the RK-682 pathway using recombinant enzymes, and we show that RkD is the enzyme required for RK-682 formation from acyl carrier Protein-bound substrates.

J Biol Chem. 2010 Jan 15;
Rachid S, Revermann O, Dauth C, Mueller R

Myxobacteria are soil-dwelling bacteria notable for several unique behavioral features, such as cellular movement by gliding and the formation of multicellular fruiting bodies. More recently they have gained recognition as producers of several unique polyketide and non-ribosomal polypeptide metabolites with potential therapeutic value. The biosynthesis of these compounds often involves highly unusual mechanisms including the formation of the chloro-hydroxy-styryl moiety of the chondrochloren antibiotic produced by Chondromyces crocatus Cm c5. Here it is shown that the final product of the chondrochloren megasynthetase is the novel natural product pre-chondrochloren, a carboxylated and saturated derivative of chondrochloren. This compound was isolated from strains harbouring mutants of a hypothetical oxidative decarboxylase (CndG) identified in the chondrochloren gene cluster. CndG was heterologously expressed in Escherichia coli and shown to be a FAD dependent oxidative decarboxylase. Biochemical characterization of the Protein was achieved using the intermediate described above as the substrate, and yielded chondrochloren by oxidative decarboxylation. It was also demonstrated that the CndG post assembly line modification of pre-chondrochloren is essential for the biological activity of chondrochloren.

J Proteome Res. 2010 Feb 3;
Okoli AS, Wilkins MR, Raftery MJ, Mendz GL

Helicobacter hepaticus is an enterohepatic bacterium associated with inflammatory bowel disease in children and causes severe hepatobiliary disorders in mice. To elucidate the molecular response of H. hepaticus to bovine bile, a proteomic investigation was conducted. Bacteria were grown for 48 h in liquid media supplemented with different concentrations of bovine bile to determine its effects on bacterial growth and morphology. Protein expression profiles of bacteria grown at a bile concentration of 0.1% and in the absence of bile were obtained using two-dimensional gel electrophoresis. Gel spots with differences in intensities greater than 2-fold between both conditions were determined, and 55 differentially expressed Proteins were identified using tandem mass spectrometry. Identified Proteins participate in various biological functions including cell envelope biosynthesis, cell response to stress, iron homeostasis and transport, motility, primary and secondary metabolism, and virulence. Changes in the expression of H. hepaticus genes related to Proteins involved in virulence and oxidative stress that were differentially expressed in the presence of bile were investigated using real-time reverse transcriptase PCR. The results indicated that the effects of bile on H. hepaticus included a strong response to oxidative stress and an expression of factors that can promote host colonization.

Appl Microbiol Biotechnol. 2010 Jan 14;
Caspers M, Brockmeier U, Degering C, Eggert T, Freudl R

Due to the lack of an outer membrane, Gram-positive bacteria (e.g., Bacillus species) are considered as promising host organisms for the secretory production of biotechnologically relevant heterologous Proteins. However, the yields of the desired target Proteins were often reported to be disappointingly low. Here, we used saturation mutagenesis of the positively charged N-domain (positions 2-7) of the signal peptide of the Bacillus subtilis alpha-amylase (AmyE) as a novel approach for the improvement of the secretion of a heterologous model Protein, cutinase from Fusarium solani pisi, by the general secretory pathway of B. subtilis. Automated high-throughput screening of the resulting signal peptide libraries allowed for the identification of four single point mutations that resulted in significantly increased cutinase amounts, three of which surprisingly reduced the net charge of the N-domain from +3 to +2. Characterization of the effects of the identified mutations on Protein synthesis and export kinetics by pulse-chase analyses indicates that an optimal balance between biosynthesis and the flow of the target Protein through all stages of the B. subtilis secretion pathway is of crucial importance with respect to yield and quality of secreted heterologous Proteins.

J Microbiol Biotechnol. 2009 Dec; 19(12): 1542-6
Seo YB, Choi SS, Nam SW, Lee JH, Kim YT

Zeaxanthin glucosyltransferase (CrtX) mediates the formation of zeaxanthin to zeaxanthin diglucoside. Here, we report cloning of the crtX gene responsible for zeaxanthin diglucoside biosynthesis from Paracoccus haeundaensis and the production of the corresponding carotenoids in transformed cells carrying this gene. An expression plasmid containing the crtX gene (pSTCRT-X) was constructed, and Escherichia coli cells containing this plasmid produced the recombinant Protein of approximately 46 kDa. biosynthesis of zeaxanthin diglucoside was obtained when the plasmid pSTCRT-X was co-transformed into E. coli containing the pET-44a(+)-CrtEBIYZ carrying crtE, crtB, crtI, crtY, and crtZ genes required for zeaxanthin beta-D-diglucoside biosynthesis.

J Biol Chem. 2010 Jan 14;
Broecker MJ, Waetzlich D, Saggu M, Lendzian F, Moser J, Jahn D

Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL(2) subunit carrying a [4Fe-4S] cluster, transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlNB)(2) which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlNB)(2)(ChlL(2))(2) complex after incubation with the non hydrolyzable ATP analogs adenosine-5'(gamma thio) triphosphate, adenosine-5'(betagamma-imido)-triphosphate or MgADP in combination with AlF(4)(-). Additionally, a mutant ChlL(2) Protein, with a deleted Leucine(153) in the switch-II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL(2), indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL(2) after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced.

Biol Direct. 2010 Jan 13; 5(1): 3
Bailly M, de Crecy-Lagard V

ABSTRACT: BACKGROUND: The bacterial elongation factor P (EF-P) is strictly conserved in bacteria and essential for Protein synthesis. It is homologous to the eukaryotic translation initiation factor 5A (eIF5A). A highly conserved eIF5A lysine is modified into an unusual amino acid derived from spermidine, hypusine. Hypusine is absolutely required for eIF5A's role in translation in Saccharomyces cerevisiae. The homologous lysine of EF-P is also modified to a spermidine derivative in Escherichia coli. However, the biosynthesis pathway of this modification in the bacterial EF-P is yet to be elucidated. Presentation of the Hypothesis: Here we propose a potential mechanism for the post-translational modification of EF-P. By using comparative genomic methods based on physical clustering and phylogenetic pattern analysis, we identified two Protein families of unknown function, encoded by yjeA and yjeK genes in E. coli, as candidates for this missing pathway. Based on the analysis of the structural and biochemical properties of both Protein families, we propose two potential mechanisms for the modification of EF-P. TESTING THE HYPOTHESIS: This hypothesis could be tested genetically by constructing a bacterial strain with a tagged efp gene. The tag would allow the purification of EF-P by affinity chromatography and the analysis of the purified Protein by mass spectrometry. yjeA or yjeK could then be deleted in the efp tagged strain and the EF-P Protein purified from each mutant analyzed by mass spectrometry for the presence or the absence of the modification. This hypothesis can also be tested by purifying the different components (YjeK, YjeA and EF-P) and reconstituting the pathway in vitro. Implication of the hypothesis: The requirement for a fully modified EF-P for Protein synthesis in certain bacteria implies the presence of specific post-translational modification mechanism in these organisms. All of the 725 bacterial genomes analyzed, possess an efp gene but only 200 (28 %) possess both yjeA and yjeK genes. In the other organisms, EF-P may be modified by another pathway or the translation machinery must have adapted to the lack of EF-P modification. Our hypotheses, if confirmed, will lead to the discovery of a new post-translational modification pathway. Reviewers: This article was reviewed by Celine Brochier-Armanet, Igor B. Zhulin and Mikhail Gelfand. For the full reviews, please go to the Reviewers' reports section.

Mol Microbiol. 2010 Jan 12;
Molle V, Kremer L

SUMMARY Mycobacterium tuberculosis (M. tb) has a complex life style in different environments and involving several developmental stages. The success of M. tb results from its remarkable capacity to survive within the infected host, where it can persist in a non-replicating state for several decades. The survival strategies developed by M. tb are linked to the presence of an unusual cell envelope. However, little is known regarding its capacity to modulate and adapt production of cell wall components in response to environmental conditions or to changes in cell shape and cell division. Signal sensing leading to cellular responses must be tightly regulated to allow survival under variable conditions. Although prokaryotes generally control their signal transduction processes through two-component systems, signalling through Ser/Thr phosphorylation has recently emerged as a critical regulatory mechanism in bacteria. The genome of M. tb possesses a large family of eukaryotic-like Ser/Thr Protein Kinases (STPKs). The physiological roles of several mycobacterial STPK substrates are connected to cell shape/division and cell envelope biosynthesis. Although these regulatory mechanisms have mostly been studied in Mycobacterium, Ser/Thr phosphorylation appears also to regulate cell division and peptidoglycan synthesis in Corynebacterium and Streptomyces. This review focuses on the Proteins which have been identified as STPK substrates and involved in the synthesis of major cell envelope components and cell shape/division in actinomycetes. It is also intended to describe how phosphorylation affects the activity of peptidoglycan biosynthetic enzymes or cell division Proteins.

J Biol Chem. 2010 Jan 8;
Buser R, Lazar Z, Kaeser S, Kuenzler M, Aebi M

Coprinopsis cinerea is a model organism for fruiting body development in homobasidiomycetes. Here, we focused on N linked oligosaccharides (NLO) of cell wall Proteins in the hyphae of two developmental stages, vegetative mycelium and fruiting body. High mannose type glycans were the most commonly found structures. In addition, we observed a novel glycan, predominantly present in fruiting body. This oligosaccharide structure was of the high mannose type with at least five mannoses and a bisecting alpha1 4 N acetylglucosamine (GlcNAc) at the beta mannose of the N-glycan core. The transferase responsible for this modification, CcGnt1 (C. cinerea GlcNAc transferase 1), was identified and expressed in insect cells. In vitro activity of CcGnt1 was demonstrated. This novel glycosyltransferase belongs to the glycosyltransferase family 8 (GT8) and is predicted to be a type II membrane Protein. Expression of the CcGnt1 locus was up regulated in fruiting body, but down regulation of expression by means of RNAi decreased the level of bisected NLO; however had no apparent effect on fruiting body formation.

Antibiot Khimioter. 2009; 54(5-6): 3-7
Grishin DV

J Leukoc Biol. 2010 Jan; 87(1): 25-34
Qin Z, Kearney P, Plaisance K, Parsons CH

Macrophages are an important source of inflammatory cytokines generated during the innate immune response,but in the microenvironment of certain tumors,macrophages promote tumor progression through their preferential secretion of cytokines that support tumor cell growth and suppress antitumoral immune responses. KSHV is the causative agent of KS and lymphomas preferentially arising in immuno compromised patients, and specific cytokines, including IL-6 and IL-10, have been implicated in KSHV-associated cancer pathogenesis. However, the contribution of KSHV-infected macrophages to the cytokine milieu within KSHV-related tumors is unclear. We found that individual KSHV-encoded miRNA induce IL-6 and IL-10 secretion independently and additively by murine macrophages and human myelomonocytic cells. Bioinformatics analysis identified KSHV miRNA binding sites formiR-K12-3 and miR-K12-7 within the 3'UTR of the basic region/leucine zipper motif transcription factor C/EBPbeta, a known regulator of IL-6 and IL-10 transcriptional activation.Subsequent immunoblot analyses revealed that miR-K12-3 and miR-K12-7 preferentially reduce expression of C/EBPbeta p20 (LIP), an isoform of C/EBPbeta known to function as a negative transcription regulator. In addition,RNA interference specifically targeting LIP induced basal secretion of IL-6 and IL-10 by macrophages.Taken together, these data support a role for KSHV miRNA in the programming of macrophage cytokine responses in favor of KSHV-related tumor progression.

Biochemistry. 2010 Feb 9; 49(5): 1033-41
Taylor PL, Sugiman-Marangos S, Zhang K, Valvano MA, Wright GD, Junop MS

Lipopolysaccharide is a major component of the outer membrane of gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-alpha,beta-D-heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the Protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting gram-negative bacterial infection.

J Leukoc Biol. 2010 Jan; 87(1): 9-12
Lukac DM

J Dent Res. 2010 Feb; 89(2): 186-91
Okugawa T, Kaneko T, Yoshimura A, Silverman N, Hara Y

In bacterial infection, Nucleotide-binding Oligomerization Domain (NOD) 1 and NOD2 induce innate immune responses by recognizing fragments of the bacterial component peptidoglycan (PGN). To determine the roles of these receptors in detection of periodontal pathogens, we stimulated human embryonic kidney cells expressing NOD1 or NOD2 with heat-killed Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Fusobacterium nucleatum or their soluble PGNs (sPGNs). All bacteria and their sPGNs could stimulate activation of NF-kappaB. However, there were differences in NOD1- and NOD2-stimulatory activities among the species of bacteria. P. gingivalis showed weaker NOD1- and NOD2-stimulatory activities than did other bacteria. These differences in activities were confirmed by production of interleukin-8 from oral epithelial cells stimulated with sPGNs. These findings indicate that both NOD1 and NOD2 might be involved in the recognition of periodontal pathogens, and that the weak NOD-stimulatory property of P. gingivalis might be helpful for survival in the periodontal pocket.