Chitinimonas Taiwanensis Gen. Nov., Sp. Nov., a Novel Chitinolytic Bacterium Isolated from a Freshwater Pond for Shrimp Culture

System. Appl. Microbiol. 27, 43–49 (2004) http://www.elsevier-deutschland.de/syapm

Chitinimonas taiwanensis gen. nov., sp. nov., a Novel Chitinolytic Bacterium Isolated from a Freshwater Pond for Shrimp Culture

Shu-Chen Chang3, Jih-Terng Wang3, Peter Vandamme4, Jie-Horng Hwang3, Poh-Shing Chang2, and Wen-Ming Chen1

1 Department of Seafood Science, National Kaohsiung Institute of Marine Technology, Kaohsiung , Taiwan 2 Department of Aquaculture, National Kaohsiung Institute of Marine Technology, Kaohsiung , Taiwan 3 Tajen Institute of Technology, Yen-Pu, Ping-Tung, Taiwan 4 Laboratorium voor Microbiologie, Faculteit Wetenschappen, Universiteit Gent, Gent, Belgium

Received: September 26, 2003

Summary

A bacterial strain, designated cfT was isolated from surface water of a freshwater pond for shrimp (Mac- robrachium rosenbergii) culture at Ping-Tung (Southern Taiwan). Cells of this organism were Gram-negative, slightly curved rods which were motile by means of a single polar flagellum. Strain cfT utilized chitin as the exclusive carbon, nitrogen, and energy source for growth, both under aerobic and anaerobic conditions. Optimum conditions for growth were between 25 and 37 °C, 0 and 1% NaCl and pH 6 to 8. Strain cfT secreted two chitinolytic enzymes with approximate molecular weight 52 and 64 kDa, which hydrolyzed chitin to produce chitotriose as major product. Sequence comparison of an almost complete 16S rDNA gene showed less than 92% sequence similarity with known bacterial species. Phylogenetic analysis based on the neighbour-joining and other methods indicated that the organism formed a distinct lineage within the β-subclass of Proteobacteria. The predominant cellular fatty acids of strain cfT were hexadecanoic acid (about 29%), octadecenoic acid (about 12%) and summed feature 3 (16:1 ω7c or 15 iso 2-OH or both [about 49%]). Its DNA base ratio was 62.8 mol% G+C. We propose to classify strain cfT (= CCRC 17210T = LMG 22011T) as Chitinimonas taiwanensis gen. nov., sp. nov.

Key words: Chitinimonas taiwanensis – chitinolytic enzymes – chitotriose – shrimp culture – taxonomy

Introduction

Chitin, a complex molecule of natural polymers, is a lowing carbon and nitrogen to return to the ecosystem common constituent of shells of crustaceans, cell walls of [11] Bacteria are the principal mediators of chitin degra- fungi and certain green algae, and exoskeletons of insects dation and their abilities to promote hydrolysis of chitin and is the second most abundant polysaccharide in na- play an important role in the recycling of chitin in nature ture (after cellulose). Chemically, chitin is an insoluble [7, 9]. Chitinolytic bacteria produce chitinase to hy- homopolymer and composed of linear chains of β-1,4- drolyze the glycosidic bonds between GlcNAc residues for linked N-acetyl-β-D-glucosamine (GlcNAc) residues utilization of chitin as a carbon and energy source [17]. which are highly cross-linked by hydrogen bonds. Chitin The capability to degrade chitin is widespread among var- and its partially deacetylated derivatives exhibit interest- ious groups of bacteria and archaea [4, 10]. ing properties and constitute a valuable raw material for Shrimp farming has significantly contributed to the biomedical, agricultural, and cosmetic applications [18, economics of Southern Taiwan. Shrimps have a hard shell 21]. made of chitin and will moult periodically. Therefore, Enormous amounts of chitin are synthesized in the pond water for shrimp culture contains enormous biosphere to the level about 1010 to 1011 metric tons an- nually [6], but only traces of chitin remain in the environment. The turnover rate of the polysaccharide is attribut- Accession: The DDBJ/EMBL/GenBank accession number for the ed primarily to microorganisms that degrade chitin, al- 16S rDNA sequence of the strain cfT is AY323827

0723-2020/04/27/01-43 $ 30.00/0 44 S.-C. Chang et al. amounts of suspended chitin and bacteria which rely on Antimicrobial susceptibility testing chitin for growth substrate. Some chitinolytic bacteria are The susceptibility to antimicrobial agents was determined by also been known as pathogens implicated in shell disease disk diffusion. The density of the bacterial suspension from ex- of crabs and shrimps [25]. In the present study, we de- ponential-phase culture was adjusted with sterile saline to the scribe a novel chitinolytic organism isolated from a fresh- turbidity corresponding to 0.5 of the McFarland standard and spread onto CB medium for further incubation incubated at water pond for shrimp culture, and the enzymes secreted 35 °C. The following antimicrobial disks (Dispens-O-Suscepti- from the organism were also partially characterized. bility Test Disks, Difco) were used including ampicillin (10 µg discs), penicillin G (10 U), cefotaxime (30 µg), ceftizoxime (30 Materials and Methods µg), amikacin (30 µg), gentamicin (10 µg), kanamycin (30 µg), netilmicin (30 µg), streptomycin (10 µg), erythromycin (10 µg), tetracycline (30 µg), nalidixic acid (30 µg), chloramphenicol Isolation and growth condition (30 µg), rifampin (5 µg), imipenem (10 µg) and trimethoprim In August 2000, a sample of surface water was taken from a (1.25 µg)/sulfamethoxazole (23.75 µg). Resistance was defined freshwater pond for shrimp (Macrobrachium rosenbergii) cul- as confluent growth up to the edge of a disk. ture at Ping-Tung County, Southern Taiwan. The chitin enrichment culture containing 200 ml of sample water and 0.5% (w/v) 16S rDNA sequencing and phylogenetic analysis autoclaved colloidal chitin was incubated at 25 °C with shaking Amplification and sequence analysis of the 16S rRNA gene at 125 r.p.m. The colloidal chitin was prepared from commer- was performed as described previously [1]. The sequence was cial chitin (Ohka chemical, Taiwan) using the method of Ro- compared with others available in GenBank and Ribosomal driguez-Kabana et al. [20] with minor modifications. Chitin Database Project II. The multiple-sequence alignment including powder was partially hydrolysed with 12 N HCl, followed by strain cfT and its closest relatives were using the BioEdit pro- repeated washings with tap water and then dialysis against dis- gram [8]. The phylogenetic reconstruction was inferred by using tilled water until the colloidal chitin gave a final pH of 6.0 to the neighbour-joining, UPGMA, maximum-likehood and Fitch- 6.5. After 5 days of culture, the enrichment broth was diluted with sterile distilled water and spread onto chitin basal (CB) Margoliash methods using the BioEdit software [8]. A bootstrap medium incubated at 35 °C. CB medium (l–1) contained 0.5% analysis (confidence values estimated from 1000 replications of (w/v) colloid chitin, 0.1 g of MgSO · 7H O, 0.2 g of K HPO , each sequence) was performed for the neighbour-joining analy- 4 2 2 4 sis using the CLUSTAL w 1.7 program [22]. A phylogenetic tree 0.5 g of CaCl2 · 2H2O and 1.0 ml trace element solution. The pH was adjusted to 6.5. The trace element solution (l–1) con- was drawn using the TREEVIEW program [14]. Sequence iden- tities were calculated using the BioEdit program [8]. tained 2.86 g of H3BO3, 2.03 g of MnSO4 · 4H2O, 0.22 g of ZnSO · 7H O, 0.08 g of CuSO · 5H O, 0.1 g of FeCl and 0.14 4 2 4 2 3 Determination of the DNA base composition g of Na2MoO4 · 2H2O. The agar plates were prepared by adding 1.5% (w/v) Bacto agar (Difco) to the CB media. A bacterial DNA was prepared as described by Pitcher et al. [16] and de- strain, designated cfT was isolated and shown to produce chiti- graded enzymatically into nucleosides as described by Mesbah et nolytic activity. al. [13]. The obtained nucleoside mixture was then separated by high-performance liquid chromatography using a Waters Sym- Morphological and physiological tests metry Shield C8 column thermostated at 37 °C. The solvent was Strain cfT was grown on CB medium for 3 days at 35 °C. 0.02 M NH4H2PO4 (pH 4.0) with 1.5% acetonitrille. Non- Morphology was observed by a light microscope (Zeiss Ax- methylated lambda phage DNA (Sigma) was used as the calibra- ioskop) and transmission electron microscope (Hitachi model tion reference. H600). Negative staining was performed with an aqueous solution (1%, w/v) of phosphotungstic acid adjusted to pH 7, as de- Fatty acid methyl ester analysis scribed as Robinson et al. [19]. The motility of exponential- Cells were grown for 24 h at 28 °C on Trypticase Soy Agar phase cells was examined by the hanging drop method. Poly-β- (BBL). Cellular fatty acids were analyzed as methyl esters by GC hydroxybutyrate granule accumulation was observed by light according to the instructions of Microbial Identification System microscopy after being stained with Sudan black [5]. (MIDI; Microbial ID) described elsewhere [23]. The pH range for growth was determined by adjusting the pH of CB medium to values between 4.0 and 11.0 with 1N Characterization of chitinolytic activity NaOH or 1N HCl. For the determination of the temperature In order to characterize chitinolytic enzymes produced by range for growth, cells were incubated in CB medium at temper- this organism, one ml of late-exponential-phase culture was in- atures between 4 and 45 °C. Tolerance to NaCl was determined oculated to 100 ml CB medium and incubated with shaking by adjusting the salinity of CB medium to values between 0 and (125 r.p.m) at 25 °C. The culture broth was collected periodical- 3.0% NaCl. Anaerobic cultivation was performed on CB medi- ly for cell counting and the enzyme activity in supernatant of × um under 85% N2, 5% CO2 and 10% H2 in an anaerobic cham- broth was determined after centrifugation with 10,000 g for ber (Forma Scientific). 20 min at 4 °C. Chitinase activity was measured by the extent of hydrolysis of fluorogenic substrate analogues 4-methylumbellif- Biochemical analysis eryl N-acetyl-β-D-glucosaminide (MUF-GlcNAc; Sigma), 4- Characteristics presented in the API 20NE, API ZYM methylumbelliferyl-β-D-N, N’-diacetylchitobioside (MUF-diGlc- (bioMérieux) and BBL Crystal Enteric/Nonfermenter ID (Becton NAc; Sigma) and 4-methylumbelliferyl-β-D-N, N’, N“-triacetyl- Dickinson) microtest systems were determined according to the chitotrioside (MUF-triGlcNAc; Sigma). Fluorogenic substrates recommendations of the manufacturers. For carbon substrate were made up with 1 mM stock solutions in dimethyl sulfoxide metabolisation tests, Biolog GNII microtiter test plates (Biolog) (DMSO). Enzyme activity was determined by adding 60 µl cul- were used. Early log phase cultures were used as inoculum for ture supernatant to the substrate solution which contained the test plate (150 µl well–1). Plates were incubated at 28 °C and 50 µM fluorogenic substrate in a sodium phosphate buffer examined after 24 h and 48 h to allow for the development of a (75 mM, pH 7.0; the final reaction volume was 700 µl). The purple color indicative of substrate oxidation. mixture was incubated at 37 °C for 30 min, and terminated by Chitinimonas taiwanensis gen. nov., sp. nov. 45 addition of 300 µl of 3 M sodium carbonate. The fluorescence subcultures died very quickly and colonies grown on nu- derived from 4-methylumbelliferone was determined with an ex- trient or trypticase soy agar could not be easily trans- citation wavelength of 390 nm and emission wavelength of ferred again. 485 nm using a fluorescence spectrophotometer (Hitachi Spec- Strain cfT grew at temperatures ranging from 4 to 39 °C, troPhotograph F2500). 4-Methylumbelliferone (MUF; Sigma) pH values between 4 and 10, and NaCl concentrations was used as a reference compound. N-acetylchitooligosaccharides in the culture supernatant between 0 and 1%. Optimal growth conditions (as deter- were analyzed by high performance liquid chromatography. One mined by measuring optical densities) were around ml of culture supernatant was filtrated through 0.45 µm Milli- 25–37 °C, 0–1% NaCl and pH 6–8. pore membrane to remove fine particle, and 20 µl filtrate was Light microscopic examination revealed that cells of injected into APS-2 Hypersil column (Thermo Hypersil-Key- strain cfT were Gram-negative, slightly curved rods, stone, 250 × 4.5 mm, 5 µm in particle size). The samples were 0.3–0.4 µm wide and 1.2–1.8 µm long. Poly-β-hydroxy- eluted with a concentration gradient beginning with 20/80 butyrate granules were present. Cells were motile by th (H2O/acetonitrile, v/v) and ending with 50/50 at 40 min. The means of single polar flagella. A transmission electron gradient was performed with a Hitachi L-7100 pump and eluted micrograph of strain cfT is shown in Fig. 1. N-acetylchitooligosaccharides were detected at 205 nm with a T Hitachi Diode Array detector. A mixture of authentic N-acetyl- Strain cf exhibited following biochemical characteris- chitooligosaccharides (GlcNACn, n = 1–6, sigma) was made up tics as determined using the results of API 20NE, API with 1 mg each component in 1 ml H2O as external standards. ZYM, BBL Crystal Enteric/Nonfermenter ID and Biolog A separate sample of culture supernatant collected from GNII microtest galleries: nitrate reduction; esculin hy- 7 days old culture broth was also used to determine the molecu- drolysis; oxidase, catalase, urease, β-galactosidase, N- lar weight of enzyme and hydrolyzed chitin. Estimation of acetyl-glucosaminidase, C4 esterase, C8 lipase, C14 li- molecular weight of the enzymes was performed with discontin- pase, leucine arylamidase, valine arylamidase, cystine uous SDS-PAGE according to Laemmli [12]. The culture super- arylamidase, trypsin, chymotrypsin, acid phosphatase, natant was concentrated by lyophilization, and about 20 µg of α-glucosidase, and naphthol-AS-BI-phosphohydrolase total protein was dissolved in sample buffer without 2-mercap- toethanol. Electrophoresis of proteins was conducted in a 10% activity; assimilation of dextrin, glycogen, tween 80, (w/v) polyacrylamide gel containing 0.5% (w/v) colloidal chitin cellobiose, fructose, glucose, mannitol, N-acetyl-glu- and 0.1% (w/v) SDS using a Bio-Rad Mini Protein III apparatus cosamine, maltose, methyl pyruvate, β-hydroxybutyric at a constant voltage of 140 V for 1.5 h at room temperature. acid, D-arabitol, D,L-lactic acid, L-asparagine, L-aspartic After electrophoresis, SDS was removed from the gel by soaking acid, L-glutamic acid, L-leucine, p-nitropheny α−β-gluco- in 25% (v/v) isopropanol in 10 mM acetate buffer pH 5.0 for side, proline nitroanilide and p-nitrophenyl-N-acetyl glu- 10 min, and then equilibrated in 10 mM acetate buffer pH 5.0 cosamine. for 15 min. The gel was incubated at 37 °C for 4 h in a plastic The following biochemical characteristics were absent: container under moist conditions. Proteins with enzymatic activ- indole production; glucose acidification; arginine dihy- ity were detected on gels by staining with 0.01% fluorescent drolase, tetrazolium reduction; alkaline phosphatase, α- brightener 28 (Sigma) in 50 mM Tris pH 8.3 for 5 min, followed α α by destaining in distilled water. Clear zones indicating chiti- galactosidase, -mannosidase and -fucosidase activity; nolytic activity were visualized by placing the gel on a UV tran- assimilation of caprate, adipate, malate, mannose, glu- silluminator. Proteins on gel were also stained with Coomassie conate, phenyl-acetate, α-cyclodextrin, N-acetyl-D-galac- brilliant blue G-250. The molecular weights of the chitinases tosamine, adonitol, arabinose, i-erythritol, L-fucose, D- were estimated by comparing with ProSieve protein standard galactose, gentiobiose, m-inositol, lactulose, D-melibiose, markers (FMC BioProducts).

Results and Discussion

Identification of strain cfT Strain cfT was isolated from surface water of a freshwater pond for shrimp (Macrobrachium rosenbergii) culture using the chitin enrichment culture. Strain cfT formed colonies on CB agar after 3 days of incubation at 35 °C, making large clear zones caused by degradation of chitin around the colonies. No diffusible pigments were produced on CB medium. With a limited medium containing only minerals and chitin, strain cfT still exhibited a rea- sonable growth rate under aerobic and anaerobic conditions, indicating that the organism could use chitin as the exclusive carbon, nitrogen, and energy source for growth. Vitamins and yeast extract were not needed as supple- ments in mineral medium for growth. Strain cfT could be grown on nutrient and trypticase soy agar when the Fig. 1. Electron micrograph of a negatively stained cell of Chi- colonies were transferred from CB medium. However, the tinimonas taiwanensis strain cfT. Bar, 1 µm. 46 S.-C. Chang et al.

Fig. 2. Neighbour-joining phylogenetic tree of Chitinimonas taiwanensis strain cfT and related bacteria of β-Proteobacteria based on 16S rRNA sequence comparisons. Scale bar indicates 1% sequence dissimilarity (one substitution per 100 nt). Bootstrap values (%) are indicated at the branches from 1000 replications. Only bootstrap values >50% are shown. Representative sequences in the den- drogram obtained from GenBank which were almost complete sequences were used in the phylogenetic analysis (GenBank accession numbers are shown in parentheses). *, No strain designation provided. The sequence of E. coli K-12 (L10328) was used as an out- group. Chitinimonas taiwanensis gen. nov., sp. nov. 47

β-methyl D-glucoside, D-psicose, D-raffinose, L-rham- nose, D-sorbitol, sucrose, D-trehalose, turanose, xylitol, mono-methyl succinate, acetic acid, cis-aconitic acid, cit- ric acid, formic acid, D-galactonic acid lactone, D-galac- turonic acid, D-glucosaminic acid, D-glucuronic acid, α- hydroxybutyric acid, γ-hydroxybutyric acid, ρ-hydroxy phenylacetic acid, itaconic acid, α-keto butyric acid, α- keto glutaric acid, α-keto valeric acid, malonic acid, pro- pionic acid, quinic acid, D-saccharic acid, sebacic acid, succinic acid, bromo succinic acid, succinamic acid, glu- curonamide, alaninamide, D-alanine, L-alanine, L-alanyl- glycine, glycyl-L-aspartic acid, glycyl-L-glutamic acid, L- histidine, hydroxy L-proline, L-ornithine, L-phenylala- nine, L-pyroglutamic acid, D-serine, L-serine, L-threo- nine, D,L-camitine, γ-amino butyric acid, urocanic acid, inosine, uridine, thymidine, phenyl ethylamine, pu- trescine, 2-amino ethanol, 2,3-butanediol, glycerol, D,L- α-glycerol phosphate, glucose-1-phosphate, glucose-6- phosphate. Strain cfT was susceptible to all antibiotics tested. Fig. 3. Growth curve and chitinase activity of Chitinimonas taiwanensis strain cfT during cultivation in CB medium. Chitinase activity in culture supernatant measured the rate of liberated 4- Taxonomic relationships of strain cfT Methylumbelliferone (MUF) from substrate analogues 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (MUF-GlcNAc), 4- Comparison of the 16S rRNA gene sequence of strain β D T methylumbelliferyl- - -N,N’-diacetylchitobioside (MUF-diGlc- cf (GenBank nucleotide sequence accession number is NAc) and 4-methylumbelliferyl-β-D-N,N’,N“-triacetylchitotrio- AY323827) with available 16S rRNA gene sequences in side (MUF-triGlcNAc) as substrate. , pH change in culture public databases revealed that it belonged to the β-Pro- medium; , bacterial numbers during growth; , substrate teobacteria (Fig. 2). The highest similarity values were MUF-triGlcNAc; , substrate MUF-diGlcNAc; , substrate obtained towards Burkholderia plantarii LMG 9035T MUF-GlcNAc. and Pandoraea pnomenusa LMG 18087T (91.2% and 91.0%, respectively). The similarity levels towards other bacterial species belonging to the β-Proteobacteria were of extracellular proteins in culture supernatant was mea- below 91%. The overall topologies of the phylogenetic sured by assessing the release of the fluorescent MUF in trees obtained by the neighbour-joining, UPGMA, maxi- reaction mixtures with fluorogenic analogues of disac- mum-likehood, and Fitch-Margoliash methods were simi- charide (MUF-GlcNAc), trisaccharide (MUF-diGlcNAc) lar (data not shown). and tetrasaccharide (MUF-triGlcNAc) of chitin deriva- The guanine-plus-cytosine (G+C) content of strain cfT tives. The growth curve, pH value and production of DNA was 62.8 mol%. Its major cellular fatty acid com- chitinase during cultivation in CB medium are shown in ponents were 16:0 (29.2%), 18:1 ω7c (11.6%) and a Fig. 3. There was almost no detectable activity against large amount (49.4%) of a mixed unresolved peak corre- MUF-GlcNAc and minor activity against MUF-diGlcNAc sponding to 16:1 ω7c or 15 iso 2-OH or both (summed during cell growth, indicating low exochitinases (β-N- feature 3). Other cellular fatty acid components present acetylhexosaminidase) and chitobiosidases activity in the in minor amounts were 12:0 (3.7%), 12:0 2-OH (1.8%), culture supernatant. The highest activity was against 14:0 (1.3%), 16:1 ω5c (0.6%), and 18:0 (0.6%). The MUF-triGlcNAc suggesting that the primary function of presence of a large amount of summed feature 3, and the enzyme in culture supernatant is the removal of chi- trace amounts of 17:0 cyclo, 16:0 3-OH, 19:0 cyclo ω8c totriose from chitin chains. These results suggest that the seem to differentiate strain cfT from members of the gen- chitinolytic activity exhibited by strain cfT was caused by era Burkholderia and Pandoraea [2, 3, 24], although this chitotriosidase activity. observation needs to be confirmed by the analysis of The main products of chitin cleavage after cultivation these bacteria under identical growth conditions. in CB medium were examined by HPLC. The major T These results indicate that strain cf represents a product was a chitotriose (GlcNAc)3 in the culture super- presently rather distinct phylogenetic branch within the natant as shown in Fig. 4. This indicated that the exclu- β-Proteobacteria, and that it should be classified in a sive end product hydrolyzed by chitinase of strain cfT was species belonging to a novel genus for which we propose chitotriose. Although there was still some minor activity the name Chitinimonas taiwanensis gen. nov., sp. nov. on the fluorogenic analogue MUF-diGlcNAc in the culture supernatant (Fig. 3), no corresponding product, Chitinolytic activity of strain cfT (GlcNAc)2, was revealed by HPLC analysis (Fig. 4). Patil et al. [15] have reported that the substrates with substi- In this study, the change of chitinase activity during the tuted aglyconic moieties are more prone to be hydrolyzed cell growth was also examined. The chitinolytic activity by chitinase than substrates with real sugar residue. 48 S.-C. Chang et al.

SDS-PAGE analysis revealed the presence of two chitinases with approximate molecular weights of 52 and 64 kDa as major constituents of the extracellular proteins in 7 days old culture supernatants (Fig. 5a). Chitinase ac- tivities were restored after SDS-PAGE analysis and detected by the degradation of colloidal chitin in situ in the gel (Fig. 5b).

Description of Chitinimonas gen. nov. Chitinimonas (Chi.ti.ni.mo’nas N.L. n. chitinum chitin; Gr. n. monas unit, monad; N.L. fem. n. Chitinimonas a chitin-utilizing monad) cells are Gram-negative rods motile by means of single polar flagella. Poly-β-hydroxybutyrate granules are stored as reserve material. En- dospores are not formed. Grows well by using chitin as the exclusive carbon, nitrogen and energy source both under aerobic and anaerobic conditions. Growth occurs at 4 to 39 oC, pH 4 to 10 and 0 to 1% NaCl salinity. Catalase and oxidase activity is present. Nitrate is re- duced and esculin is hydrolysed. The major fatty acid components are 16:0, 18:1 ω7c and summed feature 3 (16:1 ω7c or 15 iso 2-OH or both). The type species is Chitinimonas taiwanensis.

Description of Chitinimonas taiwanensis sp. nov. Chitinimonas taiwanensis (tai.wan.en’sis N.L. fem. Fig. 4. HPLC profile of chitin digested products of Chitinimonas adj. Taiwanensis, of Taiwan, where the type strain was taiwanensis strain cfT after cultivation in CB medium. (a) Stan- isolated) cells are Gram-negative rods with slightly dard of N-acetylchitooligosaccharides from the monosaccharide curved shape, 1.2–1.8 µm long and a diameter of 0.3–0.4 to hexasaccharide (GlcNAcn, n = 1–6). (b) Chitin digested prod- µm. They occur singly and are motile by single polar flag- ucts of Chitinimonas taiwanensis strain cfT after cultivation in ella. Colonies on CB medium display a large clear zone CB medium. around it, which is derived from degradation of chitin. No diffusible pigments are produced. Growth characteristics, biochemical and chemotaxo- nomic details are listed above. The type strain, cfT, was isolated from surface water of an aquaculture shrimp (Macrobrachium rosenbergii) pond at Ping-Tung Country in Southern Taiwan. The G+C content of its DNA is 62.8 mol%. The type strain has been deposited in the CCRC Bacte- ria Collection (Culture Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan) as CCRC 17210T and in BCCM/LMG Bacteria Collection (Laboratorium voor Microbiologie, Universiteit Gent, Gent, Belgium) as LMG 22011T.

Acknowledgement We thank Dr. Youk-Meng Choong for help in HPLC studies.

Fig. 5. Detection of chitinolytic enzymes in extracellular proteins from Chitinimonas taiwanensis strain cfT. (a) SDS-PAGE electro- pherogram; (b) Chitinolytic activity after separation by SDS- PAGE was detected in situ with the substrate of colloidal chitin. Lane 1, ProSieve SDS-PAGE standards (FMC BioProducts) were used as size markers. Lane 2 and 3, extracellular proteins from strain cfT grown in CB medium. Chitinimonas taiwanensis gen. nov., sp. nov. 49

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