Physicochemical parameters influencing coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13.

The aim of this study was to explore the physicochemical parameters that influence coaggregation between the freshwater bacteria Sphingomonas natatoria 2.1 and Micrococcus luteus 2.13. Using visual coaggregation assays, the effect of different buffers, solutions of differing ionic strength, pH, temperature, and viscosity on the degree of coaggregation was assessed. Coaggregation occurred maximally in distilled water but was inhibited when coaggregates were suspended in a commonly-used oral bacterial coaggregation buffer, saline solutions, and Tris-Cl buffers. Coaggregation was weakly expressed in standard laboratory buffers. The ionic strength of inorganic salt solutions required to inhibit coaggregation depended upon the inorganic salt being tested. Coaggregation occurred at a pH of 3-10, between 5 and 80°C and was inhibited in solutions with a viscosity of 22.5 centipoises at 20°C. Inhibition of coaggregation with NaCl impaired biofilm development. When developing buffers to test for coaggregation, the natural liquid environment should be considered. Coaggregation between S. natatoria 2.1 and M. luteus 2.13 is only affected by physicochemical conditions beyond those typically found in natural freshwater ecosystems. Such a robust ability to coaggregate may enhance the ability of S. natatoria 2.1 and M. luteus 2.13 to develop a niche in freshwater biofilms.

Coaggregation by the freshwater bacterium Sphingomonas natatoria alters dual-species biofilm formation.

Coaggregation is hypothesized to enhance freshwater biofilm development. To investigate this hypothesis, the ability of the coaggregating bacterium Sphingomonas natatoria to form single- and dual-species biofilms was studied and compared to that of a naturally occurring spontaneous coaggregation-deficient variant. Attachment assays using metabolically inactive cells were performed using epifluorescence and confocal laser scanning microscopy. Under static and flowing conditions, coaggregating S. natatoria 2.1gfp cells adhered to glass surfaces to form diaphanous single-species biofilms. When glass surfaces were precoated with coaggregation partner Micrococcus luteus 2.13 cells, S. natatoria 2.1gfp cells formed densely packed dual-species biofilms. The addition of 80 mM galactosamine, which reverses coaggregation, mildly reduced adhesion to glass but inhibited the interaction and attachment to glass-surface-attached M. luteus 2.13 cells. As opposed to wild-type coaggregating cells, coaggregation-deficient S. natatoria 2.1COGgfp variant cells were retarded in colonizing glass and did not interact with glass-surface-attached M. luteus 2.13 cells. To determine if coaggregation enhances biofilm growth and expansion, viable coaggregating S. natatoria 2.1gfp cells or the coaggregation-deficient variant S. natatoria 2.1COGgfp cells were coinoculated in flow cells with viable M. luteus 2.13 cells and allowed to grow together for 96 h. Coaggregating S. natatoria 2.1gfp cells outcompeted M. luteus 2.13 cells, and 96-h biofilms were composed predominantly of S. natatoria 2.1gfp cells. Conversely, when coaggregation-deficient S. natatoria 2.1COGgfp cells were coinoculated with M. luteus 2.13 cells, the 96-h biofilm contained few coaggregation-deficient S. natatoria 2.1 cells. Thus, coaggregation promotes biofilm integration by facilitating attachment to partner species and likely contributes to the expansion of coaggregating S. natatoria 2.1 populations in dual-species biofilms through competitive interactions.

Inhibitory effects of resveratrol analogs on unopsonized zymosan-induced oxygen radical production.

Resveratrol, a natural hydroxystilbene, has been reported to have anti-inflammatory and anticarcinogenic activities. Inhibitory effects of resveratrol and its analogs on reactive oxygen species (ROS) production in unopsonized zymosan-stimulated murine macrophage Raw264.7 cells, human monocytes, and neutrophils were analyzed to investigate if the anti-inflammatory and anticarcinogenic activities of resveratrol are related to the inhibition of ROS production. Resveratrol was a potent inhibitor of ROS production in both unopsonized zymosan-stimulated Raw264.7 cells and human monocytes and neutrophils. Resveratrol exhibited 50% inhibition values (IC50) of 17 microM in activated Raw264.7 cells, 18 microM in human monocytes, and 23 microM in human neutrophils. 3,5-Dihydroxy-4'-methoxystilbene or 3,4'-dimethoxy-5-hydroxystilbene exhibited IC50 values of 63 or 73 microM in Raw264.7 cells, 51 or >100 microM in human monocytes, and 10 or 37 microM in human neutrophils, respectively. Trimethylresveratrol, piceid, and 3,5-dihydroxy-4'-methoxystilbene-3-O-beta-D-glucoside were weak inhibitors of ROS production. Thus, resveratrol was identified as a potent inhibitor of ROS production, which might be one biochemical mechanism related to its anti-inflammatory and anticarcinogenic activities. The number and position of hydroxy substituents in resveratrol analogs seem to play an important role in the inhibitory potency of ROS production.

Differential inhibitory effects of sophoricoside analogs on bioactivity of several cytokines.

Effects of sophoricoside and its analogs on proinflammatory cytokines have been investigated. Sophoricoside, genistein and orobol exhibited inhibitory effects on IL-5, IL-3, GM-CSF and IL-6 bioactivities. Genistin showed inhibitory effects on IL-5 and IL-3 bioactivities, but did not inhibit GM-CSF and IL-6 bioactivities. None of the sophoricoside analogs showed inhibitory effects on both IL-1beta and TNF-alpha bioactivities. Among the compounds, sophoricoside exhibited the highest inhibitory effects on IL-5, IL-3 and IL-6 bioactivities with IC50 values of 1.9 microM, 6.9 microM and 6.0 microM, respectively and orobol did show on GM-CSF bioactivity with an IC50 value of 18.0 microM. The result would provide an additional mechanism by which the compounds exert immunosuppressive and anti-inflammatory effects.

Anti-inflammatory effects of fangchinoline and tetrandrine.

Fangchinoline and tetrandrine are the major alkaloids from Stephania tetrandrae S. Moore which has been used traditionally for the treatment of inflammatory diseases in oriental countries including Korea. Both fangchinoline and tetrandrine showed anti-inflammatory effects on mouse ear edema induced by croton oil. In addition, the effects of fangchinoline and tetrandrine on cyclooxygenase, murine interleukin-5 (mIL-5) and human interleukin-6 (hIL-6) were examined in vitro to investigate the anti-inflammatory action mechanisms. One hundred micromolar of fangchinoline showed 35% of inhibition on cyclooxygenase, but the same concentration of tetrandrine did not show any inhibition. On the other hand, 12.5 microM of tetrandrine exhibited 95% of inhibition on mIL-5 activity, while fangchinoline did not show any effects. However, 4 microM of fangchinoline and 6 microM of tetrandrine showed 63 and 86% of inhibitions on hIL-6 activity, respectively. These results suggest that biochemical mechanisms of fangchinoline and tetrandrine on anti-inflammation are significantly different even though they are similar in chemical structure.

Suppressive effects of triterpenoids on CINC-1 induction in interleukin-1beta-stimulated rat fibroblast NRK-49F cells.

CINC-1 is a member of chemokine family with chemotactic and activating properties to neutrophils. CINC-1 induction in IL-1beta-stimulated rat fibroblast NRK-49F cells was quantitated by a sensitive ELISA. CINC-1 production was increased up to 135 ng/ml from basal 2-6 ng/ml by stimulation with IL-1beta. Steroidal anti-inflammatory drugs including dexamethasone and prednisolone exhibited potent suppressive effects on IL-1beta-induced CINC-1 production. Among 39 kinds of natural triterpenoids tested, acacigenin B exhibited the highest suppressive effects with about 10 muM to be 50% of inhibition on the CINC-1 induction. The suppressive potency of acacigenin B on IL-1beta-induced CINC-1 production was about 10-fold less than that of the steroidal anti-inflammatory drugs.

Characterization of the pcbE gene encoding 2-hydroxypenta-2,4-dienoate hydratase in Pseudomonas sp. DJ-12.

Nucleotide sequence extending 2,3-dihydroxybiphenyl 1,2-dioxygenase gene (pcbC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase gene (pcbD) of Pseudomonas sp. DJ-12 was previously analyzed and the two genes were present in the order of pcbD-pcbC preceded by a promoter from Pseudomonas sp. DJ-12. In this study, a 3.8-kb nucleotide sequence located downstream of the pcbC gene was analyzed to have three open reading frames (ORFs) that are designated as orf1, pcbE and orf2 genes. All of the ORFs were preceded by each ribosome-binding sequence of 5-GGAXA-3 (X=G or A). However, no promoter-like sequence and transcription terminator sequence were found in the analyzed region, downstream of pcbC gene. Therefore, the gene cluster appeared to be present in the order of pcbD-pcbC-orf1-pcbE-orf2 as an operon, which is unique organization characterized so far in biphenyl- and PCB-degrading bacteria. The orf1 gene was composed of 1,224 base pairs which can encode a polypeptide of molecular weight 44,950 containing 405 amino acid residues. A deduced amino acid sequence of the orf1 gene product exhibited 21-33% identity with those of indole dioxygenase and phenol hydroxylase components. The pcbE gene was composed of 783 base pairs encoding 2-hydroxypenta-2,4-dienoate hydratase involved in the 4-chlorobiphenyl catabolism. The orf2 gene was composed of 1,017 base pairs encoding a polypeptide of molecular weight 37,378 containing 338 amino acid residues. A deduced amino acid sequence of the orf2 gene product exhibited 31% identity with that of a nitrilotriacetate monooxygenase component.

Nucleotide sequence of salicylate hydroxylase gene and its 5'-flanking region of Pseudomonas putida KF715.

The salicylate hydroxylase, a flavoprotein monooxygenase, catalyzes the decarboxylative hydroxylation of salicylate to form catechol. Nucleotide sequence of a salicylate hydroxylase gene and its 5'-flanking region in chromosomal DNA of Pseudomonas putida KF715 was analyzed. The salicylate hydroxylase was encoded in an open reading frame with 1308 base pairs which can encode a polypeptide of molecular weight 48 kDa with 435 amino acids. The open reading frame was preceded by a putative ribosome-binding sequence. A predicted amino acid sequence of the salicylate hydroxylase exhibited 84% identity with corresponding enzyme encoded in NAH7 plasmid, and 20 to 30% homologies with other similar flavoprotein monooxygenases. A 5'-flanking sequence of the salicylate hydroxylase gene exhibited extensive homology with promoter and nahR-binding site of sal operon in NAH7 plasmid.

Cloning and sequencing of the catechol 2,3-dioxygenase gene of Alcaligenes sp. KF711.

The catechol 2,3-dioxygenase is an aromatic ring-fission enzyme catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde. A catechol 2,3-dioxygenase gene has been cloned from chromosomal DNA of Alcaligenes sp. KF711, and its sequence was determined. The catechol 2,3-dioxygenase gene was consisted of 927 nucleotides with ATG initiation codon and TGA termination codon, which can encode a polypeptide of molecular weight 35 kDa containing 308 amino acid residues. G+C content of the gene was 58 mol%, and a putative ribosome-binding sequence was identified at about 10 nucleotides upstream from the ATG initiation codon. The sequence of catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited 81-92% homology at nucleotide level and 84-92% homology at amino acid level with those of corresponding enzymes encoded in xylE of TOL plasmid, nahH of NAH7 plasmid, and dmpB of Pseudomonas CF600.

Anti-inflammatory action of progesterone on carrageenin-induced inflammation in rats.

Effect of progesterone (1 mg/kg) on the inflammation in rats induced by carrageenin was studied. Progesterone inhibited the vascular permeability and the formation of granulation tissue in the early phase of the inflammation. In the chronic proliferative phase, progesterone suppressed the vascular permeability and there was an active resorption of the granulation tissue. Increased degradation of noncollagen protein was observed in the treated group without apparent influence on the metabolism of collagen or on the synthesis of noncollagen protein. The mode of action of progesterone was compared with that of a potent anti-inflammatory steroid, hydrocortisone acetate.

Characterization of the gene encoding catechol 2,3-dioxygenase of Alcaligenes sp. KF711: overexpression, enzyme purification, and nucleotide sequencing.

Catechol 2,3-dioxygenase (C23O) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of catechol to form 2-hydroxymuconic semialdehyde. The C23O gene of Alcaligenes sp. KF711 was overexpressed in Escherichia coli HB101 by using the lac promoter of pUC18, and its gene product was purified by using immunoaffinity chromatography. The purified C23O exhibited a 35-kDa single band on an SDS-polyacrylamide gel, and its ring-fission activity on dihydroxylated aromatics was 4-methylcatechol > 4-chlorocatechol > catechol > 3-methylcatechol >> 2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the C23O gene revealed an open reading frame of 927 bp, which can encode a polypeptide of 308 amino acid residues. The predicted molecular mass of 35 kDa is in agreement with that of purified C23O on an SDS-polyacrylamide gel. The amino acid sequence of the C23O was compared with those of nine other extradiol-type dioxygenases, including 2,3-dihydroxybiphenyl dioxygenase (2,3-DHBD) and 1,2-dihydroxynaphthalene dioxygenase (1,2-DHND). The C23O of Alcaligenes sp. KF711 exhibited 80 to 94% identity in amino acid sequence with other C23Os, and 20 to 25% identity with 1,2-DHND and 2,3-DHBDs. Furthermore, sequence comparison of 10 extradiol-type dioxygenases has led to identifying 19 evolutionarily conserved amino acid residues whose possible catalytic roles are proposed.

Suppression of interleukin-1 alpha production by protein kinase C activators in human vascular endothelial cells.

In human umbilical endothelial cells, treatment with tumor necrosis factor (TNF)-alpha stimulated the production of cell-associated interleukin (IL)-1 alpha. Combined treatment of human umbilical endothelial cells with TNF-alpha and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed the TNF-alpha-induced production of IL-1 alpha. However, concentrations of 6-keto-prostaglandin F1 alpha in the conditioned medium were increased to a greater extent by combined treatment with TNF-alpha and TPA than by single treatment with TNF-alpha or TPA. Pretreatment with TPA for 15 min was enough to suppress the TNF-alpha-induced IL-1 alpha production. Pretreatment for 15 min with other PKC activators such as aplysiatoxin or teleocidin, also inhibited the TNF-alpha-induced IL-1 alpha production. Stimulation of cell-associated IL-1 alpha production by IL-1 beta or lipopolysaccharide was also inhibited by pretreatment with the PKC activator TPA, aplysiatoxin or teleocidin. However, treatment with the protein kinase inhibitor 1-(5-isoquinoline-sulphonyl)-2- methylpiperazine dihydrochloride (H-7) did not block the inhibitory effect of TPA, aplysiatoxin or teleocidin on the cell-associated IL-1 alpha production stimulated by TNF-alpha, IL-1 beta or lipopolysaccharide, although the PKC activator-induced stimulation of 6-keto-prostaglandin F1 alpha production was counteracted by H-7 treatment. The present work indicates that the production of cell-associated IL-1 alpha stimulated by TNF-alpha, IL-1 beta or lipopolysaccharide is inhibited by treatment with TPA, aplysiatoxin or teleocidin.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of catechol 2,3-dioxygenases.

Three catechol 2,3-dioxygenases for biphenyl, naphthalene/salicylate, and toluene/xylene oxidation were cloned from Achromobacter xylosoxidans KF701, Pseudomonas putida (NAH7), and Pseudomonas sp. (pWWO). The cloned catechol 2,3-dioxygenases were identified by enzymatic activity assay in addition to yellow bands on polyacrylamide gel after electrophoresis and activity staining. All of the cloned catechol 2,3-dioxygenases exhibited their highest activities on catechol as a substrate compared with catechol derivatives including 4-chlorocatechol, 3-methylcatechol, and 4-methylcatechol. The cloned catechol 2,3-dioxygenases are not fused proteins but were significantly different from one another in their electrophoretic mobilities on nondenaturing 7.5%-polyacrylamide gel.

Characterization of the gene encoding catechol 2,3-dioxygenase from Achromobacter xylosoxidans KF701.

Catechol 2,3-dioxygenase (C23O) catalyzes a meta cleavage of the aromatic ring in catechol to form 2-hydroxymuconic semialdehyde. A C23O gene was cloned from chromosomal DNA of A. xylosoxidans KF701, a soil bacterium degrading biphenyl, and expressed in E. coli HB101. In substrate specificity to catechol and its analogs, the C23O exhibited the highest aromatic ring-fission activity to catechol, and its relative activity to other dihydroxylated aromatics was 4-chlorocatechol > 4-methylcatechol > 3-methylcatechol >> 2, 3-dihydroxybiphenyl. Aromatic ring-fission activity of the C23O to catechol was about 40-fold higher than that to 2,3-dihydroxybiphenyl. Nucleotide sequence analysis of the C23O gene from A. xylosoxidans KF701 revealed an open reading frame consisting of 924 base pairs, and identified a putative ribosome-binding sequence (AGGTGA) at about 10 nucleotides upstream from the initiation codon. The open reading frame can encode a polypeptide chain with molecular weight of 34 kDa containing 307 amino acid residues. The deduced amino acid sequence of the C23O exhibited the highest homology with that of C23O from Pseudomonas sp. IC with 96% identity, and the least homology with that of C23O from P. putida F1 with 22% identity among reported C23O sequences. Furthermore, comparison of the C23O sequence with other extradiol dioxygenases has led to identification of evolutionally conserved amino acid residues whose possible catalytic and structural roles are proposed.

Histological and immunohistochemical studies on primary gastrointestinal lymphomas.

To study the characteristics and histogenesis of the malignant lymphomas derived from the gastrointestinal mucosa, histologic and immunohistochemical analyses were performed on a series of 28 malignant lymphomas of the gastrointestinal tract. By cytomorphologic classification, there were two small lymphocytic lymphomas, one small cleaved cell lymphoma, two mixed small cleaved and large cell lymphomas, 17 large cell lymphomas, one small noncleaved cell lymphoma, three immunoblastic lymphomas, and two lymphoblastic lymphomas. This distribution of histologic types was compatible with that of nodal lymphoma. The lymphomas with poor prognostic histology (23 cases) outnumbered those with favorable prognosis (five cases). Three of 28 cases (one in the stomach and two in the small intestine) had cytologic features consistent with centrocytoid cell lymphoma of the mucosa associated lymphoid tissue and were large cell lymphomas. Immunophenotypically, 23 cases expressed B-cell markers (82.1%) and three cases reacted with T-cell markers. Two cases did not react with either T-cell or B-cell markers. True histiocytic lymphomas were not identified. Gastric lymphomas (nine cases) and colorectal lymphomas (three cases) were of B-lymphocyte origin whereas T-cell lymphomas were noted in the small intestine (two cases) and ileocecal region (one case). Three cases of centrocytoid lymphoma were of B-lymphocyte origin. Histologically B-cell lineage lymphomas were evenly distributed on various histologic subtypes but all T-lineage lymphomas belonged to the large cell type. The two cases with undetermined phenotype were lymphoblastic lymphomas histologically. This study showed that the primary GIT lymphomas, mostly of B-cell lineage, were not cytomorphologically distinctive from the nodal lymphomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of Oriental herbal medicines on IL-8 induction in lipopolysaccharide-activated rat macrophages.

Cytokine-induced neutrophil chemoattractant (CINC), a rat interleukin-8 (IL-8), was quantitated by using a sensitive ELISA. The CINC was induced up to 20 ng/ml from basal 1-2 ng/ml in lipopolysaccharide (LPS)-activated peritoneal macrophages. This CINC induction was significantly inhibited by steroidal anti-inflammatory drugs including dexamethasone, but not by non-steroidal drugs including indomethacin at all. Nine out of 59 herbal medicines which are frequently used in Korean traditional prescriptions for inflammatory diseases exhibited more than 50% of inhibition on the CINC induction by their total methanol extracts with 0.1 mg/ml as a final concentration. The active 9 total extracts were prepared from radix of Aralia continentalis, rhizoma of Cnidium officinale, rhizoma of Coptis chinensis, tuber of Fritillaria verticillata, radix of Saussurea lappa, tuber of Sparganium stoloniferum, flower of Syzygium aromaticum, semen of Trichosanthes kirilowii, and herba of Tripterygium regelii. These total extracts were sequentially fractionated with dichloromethane, ethyl acetate, butanol, and water. Among the solvent-fractionated extracts with 0.05 mg/ml as a final concentration, more than 50% of inhibition on the CINC induction was exhibited by the dichloromethane fraction of Aralia continentalis; the water fraction of Fritillaria verticillata; the dichloromethane fraction of Saussurea lappa; the dichloromethane, ethyl acetate, and butanol fractions of Syzygium aromaticum; the dichloromethane, ethyl acetate, and water fractions of Trichosanthes kirilowii; and the dichloromethane and ethyl acetate fractions of Tripterygium regelii.

Genetic structure of the bphG gene encoding 2-hydroxymuconic semialdehyde dehydrogenase of Achromobacter xylosoxidans KF701.

2-Hydroxymuconic semialdehyde dehydrogenase catalyzes the conversion of 2-hydroxymuconic semialdehyde (HMS) to an enol form of 4-oxalocrotonate which is a step in the catechol meta-cleavage pathway. A bphG gene encoding HMS dehydrogenase of A. xylosoxidans KF701, a soil bacterium degrading biphenyl, was identified at between catechol 2,3-dioxygenase gene and HMS hydrolase gene, and its sequence was analyzed. An open reading frame (ORF) corresponding to bphG gene was consisted of 1461 nucleotides with ATG initiation codon and TGA termination codon. The ORF exhibited 66% of G + C content, and a putative ribosome-binding sequence, AGAGA, was identified at about 10 nucleotides upstream initiation codon of the bphG gene. The bphG gene can encode a polypeptide of molecular weight 52 kDa containing 486 amino acid residues. A deduced amino acid sequence of HMS dehydrogenase encoded in bphG gene from A. xylosoxidans KF701 exhibited the highest 94% homology with that of corresponding enzyme encoded in xylG from P. putida mt-2, 63% to 90% homology with those of other reported HMS dehydrogenases, and 29% to 42% homology with those of betaine aldehyde dehydrogenase, 5-carboxy-HMS dehydrogenase, aldehyde dehydrogenase, indole-3-acetaldehyde dehydrogenase, succinic semialdehyde dehydrogenase, methylmalonate semialdehyde dehydrogenase, and succinylglutamate 5-semialdehyde dehydrogenase. From an alignment of amino acid sequence of HMS dehydrogenase from A xylosoxidans KF701 with other reported dehydrogenases, putative cofactor NAD(+)-binding regions and catalytic residues were identified.

Sophoricoside analogs as the IL-5 inhibitors from Sophora japonica.

Fruits of Sophora japonica L. (Leguminosae) exhibited an inhibitory effect in the IL-5 bioassay of mIL-5-dependent Y16 proliferation. The isoflavonoids of sophoricoside, genistein, orobol, and genistin were isolated as the IL-5 inhibitors from fresh fruits of the plant by activity-guided fractionation. Among the IL-5 inhibitors, sophoricoside exhibited the highest inhibitory effect with 89% inhibition at 12.5 microM, 82% at 6.3 microM, 72% at 3.1 microM, 59% at 1.6 microM, and 24% at 0.8 microM where the 50% inhibition (IC50) was shown at the concentration of 1.5 microM. Oxyphenylbutazone as the positive control exhibited the IC50 value at the concentration of 31.7 microM. In the order of IC50 values the inhibitory potency in the IL-5 bioassay was sophoricoside > orobol (9.8 microM) approximately equal to genistin (10.6 microM) > genistein (51.9 microM). The position of O-glycosylation and number of hydroxy groups in the isoflavonoids seem to play an important role in the inhibitory effect in the IL-5 bioassay.

Molecular cloning and characterization of catechol 2,3-dioxygenases from biphenyl/polychlorinated biphenyls-degrading bacteria.

Catechol 2,3-dioxygenases were cloned from Alcaligenes sp. KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putida KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.