Strain K4 Research Articles

Exploration of the catabolic plasmid genes profile of crude oil degrading bacteria isolated from aged oil contaminated soils of Anambra State

This study was undertaken to explore the catabolic plasmid genes profile and molecular identification of crude oil degrading bacteria isolated from aged oil contaminated soils of Anambra State. Enrichment technique, turbidometric test, plasmid profiling and curing tests, biodegradation capability testing as well asmolecular identification method were carried out using standard procedures.A total of sixty (60) bacterial isolates were encountered in the four different soil samples. Of the total bacterial isolates, 22 strains demonstrated high crude oil degrading potentials (A600nm > 0.3). The plasmid DNA was present in 8 out of the 22 bacterial strains such that bacteria strains G2, H4, and K4 had one plasmid each with molecular weight of 9416 base pairs while bacteria strains C3, D1, J3 and L2 had two plasmids each with molecular weight of 2111 base pairs. The bacterial strains C1 and J3 recorded the highest growths on the solid media after the plasmid removal. The isolates belong to various species which are Bacillus cereus C12 (100 % similarity), Pseudomonas aeruginosa KAVK01 (100% similarity), Bacillus licheniformis 126 (96 % similarity), Ochrobacteriumintermedium E85b (97% similarity), Bacillus subtillis SDDlas (100 % similarity), Bacillus subtillis LK4.5 (97 % similarity),Enterobacter cloacae GEBRI III (97 % similarity) and Bacillus cereus So24 (100 % similarity). Therefore, plasmid-borne Pseudomonas aeruginosa and Ochrobacteriumintermediumare recommended for use in bioremediation because of their resilience to the crude oil contaminant and degradation capacities.

Effective biodeterioration of a common endocrine disruptor 17β-estradiol using mixed microbial cultures isolated from waste water

Twelve bacteria were isolated from wastewater for the degradation of 17β-estradiol (strain K1, K2, K3, K4, L5, L6, L7, L8, M9, M10, M11 and M12) by enrichment method. Initial screening revealed maximum degradation (78 ± 1.1%, and 86 ± 4.2%) of 17β-estradiol by strains L7, and M10, respectively. The production of manganese peroxidase by bacteria gets involved in the degradation of various contaminants. Almost all strains isolated from the enriched medium produced manganese peroxidase. Based on initial screening on 17β-estradiol degradation and manganese peroxidase production, two bacteria (Flavobacterium longum L7 and Pseudomonas aeruginosa M10) were selected to analyze 17β-estradiol degradation in co-culture system. The co-cultured strains could rapidly remove 17β-estradiol (91.3%) from the culture medium at 10 mg/L 17β-estradiol initial concentration. Moreover, degradation of 17β-estradiol was only 82% and 75% when the strains L7 and M10 were treated individually. Initial 17β-estradiol concentration was 10 mg/L and 0.3% inoculum were optimum and achieved >90% degradation. Alkaline pH was optimum (pH 7.5) and reached 98.4 ± 7.5% degradation in co-culture. Glucose and trehalose improved the growth and degradation of 17β-estradiol. The organic nitrogen sources such as, yeast extract and peptone improved the removal of 17β-estradiol. The selected bacterial strains were resistant against penicillin, erythromycin, oxytetracycline and tetracycline. The present findings demonstrated the application of F. longum L7 and P. aeruginosa M10 co-culture for the removal of steroid hormones in the wastewater.

Partial characterization of probiotic lactic acid bacteria isolated from Chinese dairy products

ABSTRACT A total of 246 bacterial isolates were collected from various food and animal sources within Nanning city, China. Sequencing of the 16S rRNA gene revealed that 24.4% of the isolates were lactic acid bacteria (LAB). Initial screens identified three isolates from semi-hard cheese: Lactobacillus plantarum subsp. plantarum strains K3 and K4 and Lb. paracasei subsp. tolerans K8; and one from raw buffalo milk: Lb. plantarum subsp. plantarum E41 as having the highest levels of cell surface hydrophobicity and auto-aggregation. Further characterization of these strains showed that all four isolates were completely stable after exposure to the pepsin and trypsin for 6 hours, and strains K8 and E41 remained viable after 6 hours of exposure to acidic conditions (pH 2.5) and bile salts (0.3%). In addition, strains K3 and K4 were shown to inhibit the growth of potential human pathogens, including methicillin resistant Staphylococcus aureus, Escherichia coli and Salmonella typhi, with inhibition zones being >14 mm for each bacterial target. Results from this study suggested that the four characterized LAB strains could survive passage through the gastrointestinal tract, thus supporting the need for additional studies to assess their potential as probiotics.

The effect of Cu-resistant plant growth-promoting rhizobacteria and EDTA on phytoremediation efficiency of plants in a Cu-contaminated soil.

Remediation of heavy metal-contaminated soils is essential for safe agricultural or urban land use, and phytoremediation is among the most effective methods. The success of phytoremediation relies on the size of the plant biomass and bioavailability of the metal for plant uptake. This research was carried out to determine the effect of Ethylenediaminetetraacetic acid (EDTA) ligand and Cu-resistant plant growth-promoting rhizobacteria (PGPR) on phytoremediation efficiency of selected plants as well as fractionation and bioavailability of copper (Cu) in a contaminated soil. The test conditions included three plant species (maize: Zea mays L., sunflower: Helianthus annuus L., and pumpkin: Cucurbita pepo L.) and six treatments, comprising two PGPR strains (Pseudomonas cedrina K4 and Stenotrophomonas sp. A22), two PGPR strains with EDTA, EDTA, and control (without PGPR and EDTA). The combination of EDTA and PGPR enhanced the Cu concentration in both shoot and root tissues and increased the plant biomass. The Cu specific uptake was at a maximum level in the shoots of pumpkin plants when treated with the PGPR strain K4 + EDTA (202μgpot-1), and the minimum amount of Cu was recorded for sunflower with no PGPR or EDTA addition (29.6μgpot-1). The result of the PGPR-EDTA treatments showed that the combined application of EDTA and PGPR increased the shoot Cu-specific uptake approximately fourfold in pumpkin. Pumpkin with the highest shoot Cu specific uptake and maize with the highest root Cu specific uptake were the most effective plants in phytoextraction and phytostabilization, respectively. The effectiveness of different PGPR-EDTA treatments in increasing Cu specific uptake by crop plants was assessed by measuring the amount of Cu extracted from the rhizosphere soil adhering to the roots of crop species, by the use of the single extractants Diethylenetriamine pentaacetic acid (DTPA), H2O, NH4NO3, and NH4OAc. PGPR-EDTA treatments increased the amount of water-extractable Cu from rhizosphere soils more than ten times that of the control. The combined application of the EDTA and PGPR reduced the carbonated Fe and Mn oxide-bound Cu in the contaminated soil, and increased the soluble and exchangeable concentration of Cu. Pumpkin, with high shoot biomass and the highest shoot Cu specific uptake was found to be the most effective field crop in phytoextraction of Cu from the contaminated soil. The results of this pot study demonstrated that the EDTA+PGPR treatment could play an important role in increasing the Cu bioavailability and specific uptake by plants, and thus increasing the phytoremediation efficiency of plants in Cu-contaminated areas.

Draft Genome Sequence of Halomonas elongata Strain K4, an Endophytic Growth-Promoting Bacterium Enhancing Salinity Tolerance In Planta.

Halomonas elongata strain K4 is an endophytic bacterial strain that was isolated from roots of Cyperus conglomeratus collected at the Red Sea coast in Thuwal, Saudi Arabia. Here, we present a draft genome sequence of this strain, highlighting a number of pathways involved in plant growth promotion under salt stress.

Impact of donor binding on polymerization catalyzed by KfoC by regulating the affinity of enzyme for acceptor

BackgroundCurrently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme. MethodsStructurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor–acceptor pairs for KfoC. ResultsKfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro. In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase. Conclusion and general significanceThese results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals.

Draft Genome Sequence of Cryophilic Basidiomycetous Yeast Mrakia blollopis SK-4, Isolated from an Algal Mat of Naga-ike Lake in the Skarvsnes Ice-Free Area, East Antarctica.

Mrakia blollopis strain SK-4 was isolated from an algal mat of Naga-ike, a lake in Skarvsnes, East Antarctica. Here, we report the draft genome sequence of M. blollopis SK-4. This is the first report on the genome sequence of any cold-adapted fungal species.

Draft Genome Sequence of Cupriavidus sp. Strain SK-4, a di-ortho-Substituted Biphenyl-Utilizing Bacterium Isolated from Polychlorinated Biphenyl-Contaminated Sludge.

Cupriavidus sp. strain SK-4 is a bacterium capable of growing aerobically on monochlorobiphenyls and dichlorobiphenyls as the sole carbon sources for growth. Here, we report its draft genome sequence with the aim of facilitating an understanding of polychlorinated biphenyl biodegradation mechanisms.

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6g/l, and 14.6g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.

Production of kertinases using chicken feathers as substrate by a novel multifunctional strain of Pseudomonas stutzeri and its dehairing application

Abstract In this study, keratinolytic potential of a Sodium Dodecyl Sulfate (SDS) degrading strain of Pseudomonas stutzeri strain K4 was evaluated. Strain K4 completely metabolized 1% (w/v) chicken feathers after 72 h of incubation with production of 784.2 µg/ml of soluble protein and 42 U/ml of keratinase. Scanning Electron Microscopy (SEM) analysis of chicken feathers indicated damage on the surface of feathers. Activity staining for keratinase showed presence of five keratinases (26–76 kDa) during growth with chicken feathers. Optimum pH and temperature for crude keratinase was found to be pH-8 and 40 °C respectively. Crude keratinase showed activation in presence of metal ions such as Mg2+ (39%), Mn2+ (28%), Na+ (20.8%), solvents such as isopropanol (30.7%), glycerol (28.2%), reducing agents such as β- mercaptoethanol (70%), chelating agents such as EDTA (25.3%) and anionic detergent SDS (37.8%), respectively. Crude keratinases showed high substrate specificity for keratin and chicken feathers, low specificity for collagen and hair, respectively. Dehairing of goat skin by employing crude ketainase preparation (40 U/ml) indicated complete dehairing after 20 h of incubation. Enzymatic dehairing was superior over chemical dehairing, as no damage to skin and hairs were observed. This is the first report of a novel strain of P. stutzeri exhibiting excellent dehairing activity; results indicate that purification of keratinases prior to dehairing was not required.

Catabolic plasmid specifying polychlorinated biphenyl degradation in Cupriavidus sp. strain SK‐4: Mobilization and expression in a pseudomonad

Strain SK-4, a polychlorinated biphenyl (PCB) degrader previously reported to utilize di-ortho-substituted biphenyl, was genotypically re-characterized as a species of Cupriavidus. The bacterium harbored a single plasmid (pSK4), which resisted curing and which, after genetic marking by a transposon (SK4Tn5), could be mobilized into a pseudomonad. Analysis of pSK4 in both the transconjugant and the wild type revealed that it specifies the genes coding for 2-hydroxy-2,4-pentadienoate degradation in addition to those of the upper biphenyl pathway. Expression of the benzoate metabolic pathway in the transconjugant is evidence suggesting that the benzoate catabolic genes are also localized on the plasmid. This implies that pSK4 codes for all the genes involved in biphenyl mineralization. It is therefore reasonable to propose that the plasmid is the determinant for the unique metabolic capabilities known to exist in Cupriavidus sp. strain SK-4.

Kinetics of Iterative Carbohydrate Transfer to Polysaccharide Catalyzed by Chondroitin Polymerase on a Highly Sensitive Flow‐Type 27 MHz Quartz‐Crystal Microbalance

Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 μM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.

Deinobacterium chartae gen. nov., sp. nov., an extremely radiation-resistant, biofilm-forming bacterium isolated from a Finnish paper mill

A rod-shaped, non-spore-forming, non-motile, aerobic, oxidase and catalase-positive and radiation-resistant bacterium (designated strain K4.1(T)) was isolated from biofilm collected from a Finnish paper mill. The bacterium grew as pale pink colonies on oligotrophic medium at 12 to 50 °C (optimum 37 to 45 °C) and at pH 6 to 10.3. The DNA G+C content of the strain was 66.8 l%. According to 16S rRNA gene sequence analysis, strain K4.1(T) was distantly related to the genus Deinococcus, sharing highest similarity with Deinococcus pimensis (90.0 %). In the phylogenetic tree, strain K4.1(T) formed a separate branch in the vicinity of the genus Deinococcus. The peptidoglycan type was A3β with L-Orn-Gly-Gly and the quinone system was determined to be MK-8. The polar lipid profile of strain K4.1(T) differed markedly from that of the genus Deinococcus. The predominant lipid of strain K4.1(T) was an unknown aminophospholipid and it did not contain the unknown phosphoglycolipid predominant in the polar lipid profiles of deinococci analysed to date. Two of the predominant fatty acids of the strain, 15 : 0 anteiso and 17 : 0 anteiso, were lacking or present in small amounts in species of the genus Deinococcus. Phylogenetic distinctness and significant differences in the polar lipid and fatty acid profiles suggest classification of strain K4.1(T) as a novel genus and species in the family Deinococcaceae, for which we propose the name Deinobacterium chartae gen. nov., sp. nov. The type strain is K4.1(T) (=DSM 21458(T) =HAMBI 2721(T)).

Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with a mosquitocidal activity

The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS–PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.

Sphingomonas aestuarii sp. nov., isolated from tidal flat sediment

A novel bacterium (strain K4(T)) belonging to the genus Sphingomonas was isolated from tidal flat sediment in Korea. Its morphology, physiology, biochemical features and 16S rRNA gene sequence were characterized. Colonies of this strain are yellow in colour and the cells are rod-shaped, exhibiting negative Gram staining. The strain grows at 0-5 % (w/v) NaCl and 20-35 degrees C, with optimal growth occurring at 0 % (w/v) NaCl and 30 degrees C. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain K4(T) is associated with the genus Sphingomonas. Within the phylogenetic tree, this novel strain shares a branching point with Sphingomonas asaccharolytica Y-345(T), with which it shares 97.3 % 16S rRNA gene sequence similarity. The polyamine pattern predominantly contains the Sphingomonas-specific triamine sym-homospermidine. Combined analysis of 16S rRNA gene sequences, DNA-DNA relatedness, physiological and biochemical test results identified genotypic and phenotypic differences between strain K4(T) and other Sphingomonas species. On the basis of these differentiating features, it is concluded that strain K4(T) (=KCTC 22050(T)=DSM 19475(T)) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas aestuarii sp. nov. is proposed.

Sequential synthesis of chondroitin oligosaccharides by immobilized chondroitin polymerase mutants

Escherichia coli strain K4 expresses a chondroitin (CH)-polymerizing enzyme (K4CP) that contains two glycosyltransferase active domains. K4CP alternately transfers glucuronic acid (GlcA) and N-acetyl-galactosamine (GalNAc) residues using UDP-GlcA and UDP-GalNAc donors to the nonreducing end of a CH chain acceptor. Here we generated two K4CP point mutants substituted at the UDP-sugar binding motif (DXD) in the glycosyltransferase active domains, which showed either glycosyltransferase activity of the intact domain and retained comparable activity after immobilization onto agarose beads. The mutant enzyme-immobilized beads exhibited an addition of GlcA or GalNAc to GalNAc or GlcA residue at the nonreducing end of CH oligosaccharides and sequentially elongated pyridylamine-conjugated CH (PA-CH) chain by the alternate use. The sequential elongation up to 16-mer was successfully achieved as assessed by fluorescent detection on a gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI potential lift tandem TOF mass spectrometry (MALDI-LIFT-TOF/TOF MS/MS) analyses in the negative reflection mode. This method provides exactly defined CH oligosaccharide derivatives, which are useful for studies on glycosaminoglycan functions.

Nitrate Removal by Pseudomonas fluorescens K4 Isolated from a Municipal Sewage Treatment Plant

The removal of nitrogen compounds from a wastewater is essential and it is often accomplished by bio-logical process. An aerobic nitrate-removing bacterium was isolated from a municipal sewage treatment plant and soil. On the basis of its morphological, cultural and physiological characteristics and 16S rRNA sequencing data, this strain was identified as Pseudomonas fluorescens, and named as P. fluorescens K4. The optimal conditions of the initial pH and temperature of media for its growth were <TEX>$7.0{\sim}8.0$</TEX> and <TEX>$30^{\circ}C$</TEX>, respectively. P. fluorescens K4 was able to remove 99.9% of nitrate after 24 h in a culture. The strain could grow with a nitrate concentration up to 800 mg/l and was able to remove 99.9% of nitrate after 104 h of incubation. The optimal electron donor was sodium citrate for a nitrate removal. The strain K4 showed a capability of a complete nitrate removal when the initial C/N ratio was 1.0. An effect of the initial seed concentration was observed for a cell of 10% (v/v) for a nitrate removal. Especially P. fluorescens K4 could completely remove 200 mg/l ammonium for 3 days.

MS analysis of chondroitin polymerization: Effects of Mn 2+ ions on the stability of UDP-sugars and chondroitin synthesis

Chondroitin polymerase from Escherichia coli strain K4 (K4CP) synthesizes chondroitin (CH) polysaccharides by the alternate addition of N-acetyl- d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) to acceptor CH oligosaccharides in the presence of Mn 2+ ions. In this study, we applied matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI–TOF MS) for the further characterization of the products synthesized by K4CP from CH hexasaccharide as an initial acceptor and UDP-GalNAc and UDP-GlcA as donors. The analysis identified individual CH chains of various lengths and enabled the calculation of their average molecular weights. The ion peaks of the CH chains synthesized in the short-time reactions demonstrated not only the alternate addition of GlcA and GalNAc but also the more frequent transfer of GlcA and GalNAc, consistent with our previous kinetic data. In contrast, the MS spectra of the chains synthesized in the long-time reaction showed that CH chains containing GalNAc at the nonreducing ends were more abundant than those containing GlcA. We found that this inconsistency was due to the preferential decomposition of UDP-GlcA by Mn 2+ ions. We defined the optimal conditions to yield further elongation of the CH chains that have nearly equal numbers of GlcA and GalNAc residues at the nonreducing ends.

Diphenolases from Anoxybacillus kestanbolensis strains K1 and K4 T

Diphenolases from Anoxybacillus kestanbolensis strains K1 and K4T, highly active against 4-methylcatechol were characterized in terms of pH- and temperature-optima, pH- and temperature-stability, kinetic parameters, and inhibition/activation behaviour towards some general polyphenol oxidase (PPO) inhibitors and metal ions. The temperature-activity optima, for Anoxybacillus kestanbolensis K1 and K4T catecholases in the presence of 4-methylcatechol, were 80 and 70 °C, respectively. Although catecholase from A. kestanbolensis K4T lost no activity after a period of 1 h incubation at its optimum temperature, the enzyme pH from K1 was stimulated by keeping at 80 °C. Both of the enzymes possessed pH optima at 9.5, and the pH-stability profiles showed that cathecholases from both preparations retained their activities at alkaline pH values. Both A. kestanbolensis K1 and K4T catecholase activities were totally inhibited by addition of 0.01 mM sodium metabisulphite, ascorbic acid and l-cysteine. 1 mM Mn2+ increased the activities of A. kestanbolensis K1 and K4T catecholases by 6.4- and 5.3-fold, respectively. These results indicate that both A. kestanbolensis K1 and K4T strains possess thermo- and alkalostable catecholases.

Molecular Cloning and Characterization of Chondroitin Polymerase from Escherichia coli Strain K4

Escherichia coli strain K4 produces the K4 antigen, a capsule polysaccharide consisting of a chondroitin backbone (GlcUA beta(1-3)-GalNAc beta(1-4))(n) to which beta-fructose is linked at position C-3 of the GlcUA residue. We molecularly cloned region 2 of the K4 capsular gene cluster essential for biosynthesis of the polysaccharide, and we further identified a gene encoding a bifunctional glycosyltransferase that polymerizes the chondroitin backbone. The enzyme, containing two conserved glycosyltransferase sites, showed 59 and 61% identity at the amino acid level to class 2 hyaluronan synthase and chondroitin synthase from Pasteurella multocida, respectively. The soluble enzyme expressed in a bacterial expression system transferred GalNAc and GlcUA residues alternately, and polymerized the chondroitin chain up to a molecular mass of 20 kDa when chondroitin sulfate hexasaccharide was used as an acceptor. The enzyme exhibited apparent K(m) values for UDP-GlcUA and UDP-GalNAc of 3.44 and 31.6 microm, respectively, and absolutely required acceptors of chondroitin sulfate polymers and oligosaccharides at least longer than a tetrasaccharide. In addition, chondroitin polymers and oligosaccharides and hyaluronan polymers and oligosaccharides served as acceptors for chondroitin polymerization, but dermatan sulfate and heparin did not. These results may lead to elucidation of the mechanism for chondroitin chain synthesis in both microorganisms and mammals.