Comparison of physiologically functional compounds in Sika deer Cervus nippon meats obtained from different regions in Japan.

In Japan, the promotion of effective use of many wild deer as food resource has been conducted. However, they are not necessarily utilized effectively. Thus, we focused physiologically functional compounds to find characteristics of Sika deer meats (commercially available) obtained from different regions such as Hokkaido, Wakayama, Tokushima, and Miyazaki prefectures in Japan, making it a valuable resource for future studies and applications. The amount of carnosine, anserine, and balenine in muscle of deer from Wakayama prefecture was significantly lower than that in muscle of deer from other prefectures. The differences of amount of imidazole dipeptides in different prefectures seems to be caused by feed, rearing environment, and breed. The amount of carnitine in deer meat from Hokkaido was significantly lower than that in muscle of deer from other prefectures, while the amount of acetyl-carnitine in deer meat from Miyazaki prefectures was significantly higher than that from other prefectures. The amounts of glutamine, ornithine, and 3-methylhistidine in muscles of deer from Wakayama prefectures were significantly higher than those in muscle of deer from other prefectures. These results might be caused by differences in feeding habits, habitat, the muscle types, and subspecies of deer obtained from four regions in Japan.

Acrylamide in Cooked Sprouts of Mung Bean (Vigna radiata).

We investigated the time-dependent acrylamide formation in mung bean sprouts during stir-frying under high and medium heat conditions. The acrylamide concentration range detected using the 3-mercaptobenzoic acid derivatization LC-MS/MS method was from below 29 ng/g [limit of detection (LOD)] to 6,900 ng/g. We also investigated the acrylamide levels in mung bean sprouts cooked using four methods while retaining their fresh firm texture using the thiosalicyclic acid derivatization LC-MS/MS method. The acrylamide concentration in microwave oven-cooked sprouts was below 16 ng/g (LOD). The samples cooked by stir-frying, parching, or boiling contained an acrylamide concentration above the LOD but below 42 ng/g [limit of quantification (LOQ)], except for one replicate of a stir-fried sample, whose acrylamide concentration was 42 ng/g. Bean sprouts are popular affordable vegetables, and when stir-fried, their acrylamide concentration is assumed to strongly affect the exposure of the Japanese population to acrylamide. Because the acrylamide concentration range of fried bean sprouts is as broad as mentioned above, the selection of a representative concentration value is difficult. A precise survey and data about acrylamide formation in relation to the bean sprout components before heating, their changes occurring during storage, and the cooking methods and conditions used are needed to estimate the exposure of the Japanese to acrylamide. Here, we showed that rinsing the sprouts before frying and frying them for a short time while mixing them well, while retaining the fresh firm texture to avoid burning and shriveling the sprouts is effective in decreasing the amount of acrylamide formed.

Draft Genome Sequences of Two Basidiomycetous Yeasts, Tremella yokohamensis and Tremella fuciformis.

In this study, the genome sequences of two Basidiomycetous yeasts, Tremella yokohamensis and Tremella fuciformis, which have very similar morphological characteristics, were determined. The genomic sequence data obtained will be useful for understanding the taxonomy and metabolic-related genes of basidiomycete yeasts.

Isomerization of 6-O-substituted glucose and fructose under neutral pH conditions and subsequent ß-elimination reactions.

Isomaltose (6-O-α-d-glucopyranosyl-d-glucose) and isomaltulose (palatinose; 6-O-ß-d-glucopyranosyl-d-fructose) were heated to 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Aldose-ketose isomerization between isomaltose, isomaltulose, and epi-isomaltulose was observed in the early stage of the reaction, alongside the release of a small amount of glucose. The total concentration of these disaccharides gradually decreased as the heating time increased. However, this decrease did not correlate with the amount of glucose or fructose released, suggesting that the releases of these monosaccharides were not caused by the hydrolysis of glycosidic linkages. A slight decrease in the pH of the reaction solution was attributed to the formation of two organic acids, 6-O-ß-d-glucopyranosyl-3-deoxy-d-arabino-hexonic acid (1) and 6-O-ß-d-glucopyranosyl-3-deoxy-d-ribo-hexonic acid (2). These compounds were formed from the ß-elimination of the hydroxyl group at the C-3 of fructose, leaving a substituted glucose residue at the C-6 position, followed by keto-enol tautomerization and benzilic acid rearrangement. Although approximately 30% of 1 and 2 were degraded after 360 min of heating at 90 °C in 100 mM sodium phosphate, a little release of glucose was observed, indicating no hydrolysis of the glucoside bond at C-6. Besides 1 and 2, time-dependent changes in the NMR spectra of the reaction mixture in water indicated the formation of formic acid and the presence of species possibly resulting from the ß-elimination of the hydroxyl group from 3- and 4-ulose. The glucose released by heating isomaltose and isomaltulose may be generated via tautomerizations of keto-enols between the C-4 and C-5 positions and cleavage of 6-O-glycosidic linkage via ß-elimination.

Distribution of Radioactive Cesium during Wild Boar Meat Processing.

ABSTRACT: Wild boar meat containing radioactive cesium (Cs) of approximately 1,000 Bq/kg (134Cs+137Cs) was processed into bacon, sausage, and ham. To understand the concentration and quantity change of radioactive Cs, the processing factor (Pf) and food processing retention factor (Fr) were calculated. The radioactive Cs quantity in the meat did not reduce during smoking. The dehydration-related meat mass reduction during smoking without decrease of radioactive Cs led to Cs condensation in the bacon compared with the raw rib meat before processing, resulting in a Pf of 1.18. Soaking in liquid, such as pickling in liquid and desalting or boiling in water, effectively removed radioactive Cs by leakage into water. Therefore, the Fr value of the boiled ham produced from a loin meat block through three liquid-soaking processes was 0.27. The Pf value of the boiled ham was 0.30 due to meat block mass reduction after boiling as a result of dehydration, along with protein thermal denaturation-related muscle tissue shrinkage. The steamed ham Fr value was 0.53, because the removal of radioactive Cs was less efficient by steaming than by boiling. The Pf value of the steamed ham was 0.54, almost the same as the Fr value, because the mass decrease rate was the same as the radioactive Cs decrease rate by steaming. The Fr and the Pf values of the boiled sausage, whose processing did not include soaking in the pickling liquid, were 0.64 and 0.62, respectively. Steaming the sausage meat did not remove radioactive Cs during the dehydration-related mass reduction, leading to Fr and Pf values of 1.01 and 1.17, respectively. This study found that processing into boiled ham was the most effective measure for reducing radioactive Cs quantity and concentration in raw meat. Processing into bacon and steamed sausage showed no Cs quantity reduction, with the moisture loss resulting in Cs condensation compared with the raw material.

Inhibitory effects of thiol compounds on theaflavin browning and structural analysis of the causative substances.

Theaflavin, a polyphenol responsible for the bright orange color and various bioactivities of black tea exudates, is susceptible to autoxidation at neutral and mild alkaline pH, changing its color to brown. In the presence of cysteine (Cys), glutathione (GSH), or N-acetyl cysteine (NAC), the browning of theaflavin solution was inhibited concomitantly with time-dependent decreases in the concentrations of both theaflavin and thiol group. The rank order of the decrease was Cys â‰… GSH > NAC, suggesting the relevance of the nucleophilic property of the thiol group to its reaction with theaflavin. LC-MS analysis of the reaction products indicated formation of novel derivatives that were mono- or di-molecular adducts of thiol compounds. We determined the chemical structures of theaflavin-Cys and theaflavin-GSH adducts by NMR and proposed the reaction mechanisms. It was found that the theaflavin-Cys adduct was not a simple adduct, to which a new cyclic structure was added.

Potential application of light element stable isotope ratio in crude fiber for geographical origin verification of raw and cooked kabocha pumpkin (Cucurbita maxima).

In this study, we investigated the possibility of the geographical origin discrimination between Japanese and Mexican kabocha pumpkin using δ13C and δ18O in lyophilized raw flesh. Kabocha harvested in Hokkaido, the major kabocha production area in Japan, could be discriminated against not only that grown in Mexico but also that in New Zealand. However, seasoning after cooking or processing affected the δ13C and the δ18O values. Crude fiber extraction eliminated the effect of seasonings and enabled the adoption of the δ13C and δ18O values in crude fiber for the geographical origin verification of kabocha even after cooking and processing. The usage of δ13C and the δ18O in crude fiber would extend the application possibilities of stable isotope analysis in the geographical origin determination of various cooked and processed vegetables and fruits as well as raw materials.

Generation of 3-deoxypentulose by the isomerization and ß-elimination of 4-O-substituted glucose and fructose.

Aldose-ketose isomerization is commonly used to prepare rare oligosaccharides such as maltulose (4-O-α-d-glucopyranosyl-d-fructose) and lactulose (4-O-ß-d-galactopyranosyl-d-fructose). However, both sugars are degraded under alkaline conditions via ß-elimination, while their subsequent benzylic acid rearrangement leads to the formation of isosaccharinic acids. Here, we investigated the behavior of maltose and maltulose upon heating in phosphate buffer solution at pH 7.5. Maltose was initially isomerized into maltulose. Maltulose was degraded via ß-elimination, followed by keto-enol tautomerization, which led to the formation of a 1,3-dicarbonyl intermediate bearing an aldehyde group at the C-1 position and a ketone group at the C-3 position. Subsequent hydrolysis of this intermediate afforded formic acid and 3-deoxy-d-glycero-pent-2-ulose (1) as the major products based on HPLC and NMR data. In contrast, the formation of isosaccharinic acid via benzylic acid rearrangement, not the 3-deoxypentulose, was reported under the strongly alkaline conditions (Knill and Kennedy, 2003). The heat treatment of 1→4 linked oligo- and polysaccharides possessing glucose or fructose residue at the reducing end under neutral pH conditions could be applied for the practical preparation of a 3-deoxypentulose.

Elution of Radioactive Cesium from Tofu by Water Soaking.

Elution of cesium-137 (137Cs) from tofu into water was investigated to know the behavior of 137Cs during preservation and cooking. The food processing retention factor (Fr) reached 0.55 when tofu was soaked in water at a ratio of 1:2 w/w for 24 h at 4°C. Doubling the amount of water did not further significantly decrease Fr. When tofu was held in water at a ration of 1:2 w/w at a temperature of 80℃ for 50 min, Fr was 0.72. Increasing the amount of water to 10 times the tofu weight did not further reduce Fr significantly. Cesium-137 is mostly bound to tofu and does not freely diffuse into water. Tofu was then soaked in water at a ratio of 1:2 w/w at 4℃ for 24 h, placed in new water at a ratio of 1:2 w/w, and held at 80℃ for 50 min, resulting in an Fr 0.33. This value is close to an estimated Fr calculated by multiplying the Fr of 0.55 from soaking at 4°C by the Fr of 0.72 from the hot water treatment. The calculated Fr from soybeans sequentially processing into tofu, soaking tofu at 4°C for 24 h and in hot water at 80°C for 50 min was about 0.1, indicating 90% removal of 137Cs. Degree of decrease in 137Cs during preservation and cooking of tofu demonstrated in this study will be useful for exposure assessment of 137Cs through oral intake of contaminated soybeans after processing and cooking.

Alkoxycarbonyl elimination of 3-O-substituted glucose and fructose by heat treatment under neutral pH.

3-O-Substituted reducing aldoses are commonly unstable under heat treatment at neutral and alkaline pH. In this study, to evaluate the decomposition products, nigerose (3-O-α-d-glucopyranosyl-d-glucose) and 3-O-methyl glucose were heated at 90 °C in 100 mM sodium phosphate buffer (pH 7.5). Decomposition via ß-elimination was observed that formed a mixture of 3-deoxy-arabino-hexonic acid and 3-deoxy-ribo-hexonic acid; upon further acid treatment, it was converted to their γ-lactones. Similarly, turanose (3-O-α-d-glucopyranosyl-d-fructose), a ketose isomer of nigerose, decomposed more rapidly than nigerose under the same conditions, forming the same products. These findings indicate that 3-O-substituted reducing glucose and fructose decompose via the same 1,2-enediol intermediate. The alkoxycarbonyl elimination of 3-O-substituted reducing glucose and fructose occurs readily if an O-glycosidic bond is located on the carbon adjacent to the 1,2-enediol intermediate. Following these experiments, we proposed a kinetic model for the3- decomposition of nigerose and turanose by heat treatment under neutral pH conditions. The proposed model showed a good fit with the experimental data collected in this study. The rate constant of the decomposition for nigerose was (1.2 ± 0.1) × 10-4 s-1, whereas that for turanose [(2.6 ± 0.2) × 10-4 s-1] was about 2.2 times higher.

Epimerization and Decomposition of Kojibiose and Sophorose by Heat Treatment under Neutral pH Conditions.

We evaluated the stabilities of kojibiose and sophorose when heated under neutral pH conditions. Kojibiose and sophorose epimerized at the C-2 position of glucose on the reducing end, resulting in the production of 2-O-α-D-glucopyranosyl-D-mannose and 2-O-ß-D-glucopyranosyl-D-mannose, respectively. Under weak alkaline conditions, kojibiose was decomposed due to heating into its mono-dehydrated derivatives, including 3-deoxy-2,3-unsaturated compounds and bicyclic 3,6-anhydro compounds. Following these experiments, we propose a kinetic model for the epimerization and decomposition of kojibiose and sophorose by heat treatment under neutral pH and alkaline conditions. The proposed model shows a good fit with the experimental data collected in this study. The rate constants of a reversible epimerization of kojibiose at pH 7.5 and 90 °C were (1.6 ± 0.1) × 10-5 s-1 and (3.2 ± 0.2) × 10-5 s-1 for the forward and reverse reactions, respectively, and were almost identical to those [(1.5 ± 0.1) × 10-5 s-1 and (3.5 ± 0.4) × 10-5 s-1] of sophorose. The rate constant of the decomposition reaction for kojibiose was (4.7 ± 1.1) × 10-7 s-1 whereas that for sophorose [(3.7 ± 0.2) × 10-6 s-1] was about ten times higher. The epimerization reaction was not significantly affected by the variation in the buffer except for a borate buffer, and depended instead upon the pH value (concentration of hydroxide ions), indicating that epimerization occurred as a function of the hydroxide ion. These instabilities are an extension of the neutral pH conditions for keto-enol tautomerization that are often observed under strong alkaline conditions.

Acrylamide in Stir-fried Potato and Onion for Simmered Dishes in Japan.

Acrylamide has neurotoxicity, carcinogenicity, and genotoxicity in experimental animals and cellular systems. Fried potato is one of the major intake sources of acrylamide in food, and fried onion was reported to contain up to 100 ng/g level of acrylamide. To determine acrylamide concentration in potato and onion stir-fried prior to boiling for simmered dishes such as curry, stew, and Niku-jaga, a typical Japanese meat/potato/onion cuisine, we collected samples stir-fried at homes of volunteers who intended voluntarily to cook these simmered dishes. Acrylamide level was analyzed by GC-MS after the xanthydrol derivatization. Among 53 stir-fried potato samples, median and average values of acrylamide were found to be 2.0 ng/g and 11 ng/g, respectively. Acrylamide levels of 27 samples (51%) were less than limit of detection (LOD) (4 ng/g), and those of 13 samples (25%) were less than limit of quantification (LOQ) (10 ng/g). In cases with less than LOD and less than LOQ of acrylamide levels, one-half of LOD and average of LOD and LOQ were adopted, respectively, to calculate the median and average. This median was markedly lower than those of fried potato (180 ng/g) and potato snacks including potato chips (550 ng/g) reported in monitoring in 2013 fiscal year in Japan. Among 58 stir-fried onion samples, acrylamide level of only one sample (2%) was less than LOD (3 ng/g), and those of 15 samples (26%) were less than LOQ (8 ng/g). The median and average values in the stir-fried onion were 14 ng/g and 36 ng/g, respectively. These values are comparable to those for stir-fried onion reported by Ministry of Agriculture, Forestry and Fisheries, Japan (median 19 ng/g, average 25 ng/g). But the maximum value of stir-fried onion 420 ng/g in the present study is much higher than the reported maximum value (70 ng/g).

Characterization and crystal structure determination of ß-1,2-mannobiose phosphorylase from Listeria innocua.

Glycoside hydrolase family 130 consists of phosphorylases and hydrolases for ß-mannosides. Here, we characterized ß-1,2-mannobiose phosphorylase from Listeria innocua (Lin0857) and determined its crystal structures complexed with ß-1,2-linked mannooligosaccharides. ß-1,2-Mannotriose was bound in a U-shape, interacting with a phosphate analog at both ends. Lin0857 has a unique dimer structure connected by a loop, and a significant open-close loop displacement was observed for substrate entry. A long loop, which is exclusively present in Lin0857, covers the active site to limit the pocket size. A structural basis for substrate recognition and phosphorolysis was provided.

An inverting ß-1,2-mannosidase belonging to glycoside hydrolase family 130 from Dyadobacter fermentans.

The glycoside hydrolase family (GH) 130 is composed of inverting phosphorylases that catalyze reversible phosphorolysis of ß-D-mannosides. Here we report a glycoside hydrolase as a new member of GH130. Dfer_3176 from Dyadobacter fermentans showed no synthetic activity using α-D-mannose 1-phosphate but it released α-D-mannose from ß-1,2-mannooligosaccharides with an inversion of the anomeric configuration, indicating that Dfer_3176 is a ß-1,2-mannosidase. Mutational analysis indicated that two glutamic acid residues are critical for the hydrolysis of ß-1,2-mannotriose. The two residues are not conserved among GH130 phosphorylases and are predicted to assist the nucleophilic attack of a water molecule in the hydrolysis of the ß-D-mannosidic bond.

Discovery of two ß-1,2-mannoside phosphorylases showing different chain-length specificities from Thermoanaerobacter sp. X-514.

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-ß-oligomannan using ß-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-ß-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-ß-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-ß-oligomannans containing a DP ≥3 and ß-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-ß-oligomannan. Here, we propose 1,2-ß-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-ß-oligomannan phosphorylase as the short name for Teth514_1788 and ß-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and ß-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

Potassium ion-dependent trehalose phosphorylase from halophilic Bacillus selenitireducens MLS10.

We discovered a potassium ion-dependent trehalose phosphorylase (Bsel_1207) belonging to glycoside hydrolase family 65 from halophilic Bacillus selenitireducens MLS10. Under high potassium ion concentrations, the recombinant Bsel_1207 produced in Escherichia coli existed as an active dimeric form that catalyzed the reversible phosphorolysis of trehalose in a typical sequential bi bi mechanism releasing ß-D-glucose 1-phosphate and D-glucose. Decreasing potassium ion concentrations significantly reduced thermal and pH stabilities, leading to formation of inactive monomeric Bsel_1207.

Comparative analysis of flagellin glycans among pathovars of phytopathogenic Pseudomonas syringae.

Flagellin is a principal component of the flagellum filament. Previously, we reported that the flagellin of Pseudomonas syringae pv. tabaci 6605 (Pta6605) was glycosylated by oligosaccharides composed of two or three l-rhamnosyl (l-Rha) residues and a terminal 4,6-dideoxy-4-(3-hydroxybutanamide)-2-O-methylglucopyranosyl residue. In this study, we characterized the chemical structure of flagellin glycans in P. syringae pathovars glycinea race 4 (Pgl4), phaseolicola 1448A (Pph1448A), tomato DC3000 (PtoDC3000), and syringae B728a (PsyB728a). Flagellin glycans were released by hydrazinolysis, labeled on the reducing ends with 2-aminopyridine (PA), and the PA-labeled oligosaccharides were isolated by high-performance liquid chromatography. The purified PA-labeled glycans were analyzed by mass spectrometry and NMR spectroscopy. The results showed that the glycans on flagellin of Pgl4, PtoDC3000, and Pph1448A were identical to those of Pta6605, which were characterized previously. The flagellin of PsyB728a is O-glycosylated with a novel trisaccharide identified as 2-acetamide-2-deoxy-ß-D-glucopyranosyl-(1→2)-3-O-methyl-α-L-rhamnopyranosyl-(1→2)-L-rhamnose. Our data indicate that flagellin glycosylation of P. syringae pathovars has universality with little diversity.

Defects in D-rhamnosyl residue biosynthetic genes affect lipopolysaccharide structure, motility, and cell-surface hydrophobicity in Pseudomonas syringae pathovar glycinea race 4.

D-rhamnose (D-Rha) residue is a major component of lipopolysaccharides (LPSs) in strains of the phytopathogen Pseudomonas syringae pathovar glycinea. To investigate the effects of a deficiency in GDP-D-rhamnose biosynthetic genes on LPS structure and pathogenicity, we generated three mutants defective in D-Rha biosynthetic genes, encoding proteins GDP-D-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose reductase (RMD), and a putative α-D-rhamnosyltransferase (WbpZ) in P. syringae pv. glycinea race 4. The Δgmd, Δrmd, and ΔwbpZ mutants had a reduced O-antigen polysaccharide consisting of D-Rha residues as compared with the wild type (WT). The swarming motility of the Δgmd, Δrmd, and ΔwbpZ mutant strains decreased and hydrophobicity and adhesion ability increased as compared with WT. Although the mutants had truncated O-antigen polysaccharides, and altered surface properties, they showed virulence to soybean, as WT did.

Effects of lipooligosaccharide inner core truncation on bile resistance and chick colonization by Campylobacter jejuni.

Campylobacter jejuni is the most common bacterium that causes diarrhea worldwide, and chickens are considered the main reservoir of this pathogen. This study investigated the effects of serial truncation of lipooligosaccharide (LOS), a major component of the outer membrane of C. jejuni, on its bile resistance and intestinal colonization ability in chickens. Genes encoding manno-heptose synthetases or glycosyltransferases were inactivated to generate isogenic mutants. Serial truncation of the LOS core oligosaccharide caused a stepwise increase in susceptibilities of two C. jejuni strains, NCTC 11168 and 81-176, to bile acids. Inactivation of hldE, hldD, or waaC caused severe truncation of the core oligosaccharide, which greatly increased the susceptibility to bile acids. Both wild-type strains grew normally in chicken intestinal extracts, whereas the mutants with severe oligosaccharide truncation were not detected 12 h after inoculation. These mutants attained viable bacterial counts in the bile acid-free extracts 24 h after inoculation. The wild-type strain 11-164 was present in the cecal contents at >10(7) CFU/g on 5 days after challenge infection and after this time period, whereas its hldD mutant was present at <10(3) CFU/g throughout the experimental period. Trans-complementation of the hldD mutant with the wild-type hldD allele completely restored the in vivo colonization level to that of the wild-type strain. Mutants with a shorter LOS had higher hydrophobicities. Thus, the length of the LOS core oligosaccharide affected the surface hydrophobicity and bile resistance of C. jejuni as well as its ability to colonize chicken intestines.

Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence.

Type IV pilin (PilA) is a major constituent of pilus and is required for bacterial biofilm formation, surface motility and virulence. It is known that mature PilA is produced by cleavage of the short leader sequence of the pilin precursor, followed by methylation of N-terminal phenylalanine. The molecular mass of the PilA mature protein from the tobacco bacterial pathogen Pseudomonas syringae pv. tabaci 6605 (Pta 6605) has been predicted to be 12 329 Da from its deduced amino acid sequence. Previously, we have detected PilA as an approximately 13-kDa protein by immunoblot analysis with anti-PilA-specific antibody. In addition, we found the putative oligosaccharide-transferase gene tfpO downstream of pilA. These findings suggest that PilA in Pta 6605 is glycosylated. The defective mutant of tfpO (ΔtfpO) shows reductions in pilin molecular mass, surface motility and virulence towards host tobacco plants. Thus, pilin glycan plays important roles in bacterial motility and virulence. The genetic region around pilA was compared among P. syringae pathovars. The tfpO gene exists in some strains of pathovars tabaci, syringae, lachrymans, mori, actinidiae, maculicola and P. savastanoi pv. savastanoi. However, some strains of pathovars tabaci, syringae, glycinea, tomato, aesculi and oryzae do not possess tfpO, and the existence of tfpO is independent of the classification of pathovars/strains in P. syringae. Interestingly, the PilA amino acid sequences in tfpO-possessing strains show higher homology with each other than with tfpO-nonpossessing strains. These results suggest that tfpO and pilA might co-evolve in certain specific bacterial strains.