Comparative Phenotype and Genome Analysis of Cellvibrio sp ...downloads.hindawi.com/journals/bmri/2017/6304248.pdf · sp. PR1 was isolated from a freshwater sample collected fromthePearlRiver(23∘8Nand113∘17E).Thestrainwas

Research ArticleComparative Phenotype and Genome Analysis ofCellvibrio sp PR1 a Xylanolytic and Agarolytic Bacterium fromthe Pearl River

Zhangzhang Xie Weitie Lin and Jianfei Luo

Guangdong Key Laboratory of Fermentation and Enzyme Engineering College of Bioscience and BioengineeringSouth China University of Technology Guangzhou 510006 China

Correspondence should be addressed to Jianfei Luo ljfjf2002scuteducn

Received 28 February 2017 Revised 3 June 2017 Accepted 18 June 2017 Published 17 July 2017

Academic Editor Nikolai V Ravin

Copyright copy 2017 Zhangzhang Xie et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cellvibrio sp PR1 is a xylanolytic and agarolytic bacterium isolated from the Pearl River Strain PR1 is closely related to Cellvibriofibrivorans and C ostraviensis (identity gt 98) The xylanase and agarase contents of strain PR1 reach up to 154 and 259UmLrespectively The major cellular fatty acids consisted of C160 (367) C180 (88) C200 (68) C

150iso 2-OH orand C

1611205967c

(174) and C1811205967c orand C

1811205966c (67) A total of 251 CAZyme modules (63 CBMs 20 CEs 128 GHs 38 GTs and 2 PLs)

were identified from 3730 predicted proteins Genomic analysis suggested that strain PR1 has a complete xylan-hydrolyzing (5120573-xylanases 16 120573-xylosidases 17 120572-arabinofuranosidases 9 acetyl xylan esterases 4 120572-glucuronidases and 2 ferulic acid esterases)and agar-hydrolyzing enzyme system (2 120573-agarases and 2 120572-neoagarooligosaccharide hydrolases) In addition the main metabolicpathways of xylose arabinose and galactose are established in the genome-wide analysisThis study shows that strain PR1 containsa large number of glycoside hydrolases

1 Introduction

Polysaccharides (eg cellulose hemicellulose starch chitinagar and pectin) are themost abundant source of organic car-bon in the biosphere Sugars released from polysaccharidesare usually used for the production of value-added productssuch as biofuels [1 2] antioxidants [3 4] and medicines[5] However the complex chemical structures and extensiveinterconnections of these polysaccharides in the plant cellwall prevent physical chemical and enzymatic degradationsFor example endo-14-120573-glucanases exo-14-120573-glucanasesand 120573-glucosidases are essential in the enzymatic degrada-tion of cellulose [6] meanwhile xylanase xylosidase ara-binofuranosidase acetyl xylan esterase glucuronidase andferulic acid esterase are important in the complete hydrolysisof hemicellulose [7] Highly active enzymes from naturalmicroorganisms are desirable given the growing demand ofaffordable and environmentally friendly methods for the useof polysaccharide as feedstock

The genus Cellvibrio from the Pseudomonadaceae familywas first discussed by Blackall et al in 1986 [8] Eight

other species namely Cellvibrio ostraviensis C vulgarisC mixtus C fibrivorans C gandavensis C japonicus Cfulvus and C diazotrophicus have been identified to date(LPSN httpwwwbacterionet-allnamesachtml) Bacteriafrom the genus Cellvibrio are usually Gram-negative andaerobic and these bacteria are known cellulose xylan starchand chitin degraders [8ndash10] For example C japonicusdegrades all of the major plant cell wall polysaccharides(including crystalline cellulose mannan and xylan) by theactivities of approximately 130 possible glycoside hydrolases[9] C mixtus J3-8 is a xylanolytic bacterium with no cellu-lolytic activity and has a large number of genes that are notannotated [10] A cyanobacterial syntrophic bacterium Cel-lvibrio sp PR1was isolated fromawater sample from the PearlRiver Xylanolytic and agarolytic activities are observed instrain PR1 Based on the bacterial-algal interactions betweenstrain PR1 and microalgae the microalgal growths wereenhanced by coculturing with strain PR1 and by using xylanor xylose as feedstock [2] To better understand polysac-charide hydrolysis activities and possible syntrophic patterns

HindawiBioMed Research InternationalVolume 2017 Article ID 6304248 10 pageshttpsdoiorg10115520176304248

2 BioMed Research International

with microalgae we comparatively studied the phenotypesand genome of strain PR1

2 Materials and Methods

21 Bacterial Strains and Culture Medium Strain Cellvibriosp PR1 was isolated from a freshwater sample collectedfrom the Pearl River (23∘81015840N and 113∘171015840E) The strain wasdeposited in the China General Microbiological CultureCollection Center (Beijing China) and the NITE BiologicalResource Center (Tokyo Japan) under the accession numbersCGMCC 114955 and NBRC 110968 respectively

The medium used for strain PR1 cultivation containsthe following 15 g Lminus1 NaNO

3 004 g Lminus1 K

2HPO4

0075 g Lminus1 MgSO4sdot7H2O 0036 g Lminus1 CaCl

2sdot2H2O 1mg

of glucose and 1ml of trace element solution The traceelement solution contains the following 286 g Lminus1H

3BO3

186 g Lminus1 MnCl2sdot4H2O 022 g Lminus1 ZnSO

4sdot7H2O 039 g Lminus1

Na2MoO4sdot2H2O 008 g Lminus1 CuSO


Co(NO3)2sdot6H2O 6 g Lminus1 citric acid 6 g Lminus1 ferric ammonium

citrate and 1 g Lminus1 EDTANa2 pH was adjusted to 72 Xylan

(Sigma-Aldrich USA) cellulose (Avicel PH-101 and CMCSigma-Aldrich USA) starch (Sigma-Aldrich USA) andagarose (Sigma USA) were used as the primary carbonsource for testing xylanase cellulase amylase and agaraseactivities respectively

22 Morphology and Physiological and Biochemical AnalysesThe cells of strain PR1 growing in exponential phase werecollected and used for the examination of cell morphology bytransmission electron microscopy (H-7650 Hitachi Japan)at 80 kV NaCl tolerance was tested in culture medium withadditional salt concentration ranging from 0 to 5 (wv)Growth was tested at different temperatures (4 10 15 2025 30 37 45 and 55∘C) and pH levels (4ndash10) The pHswere adjusted by using disodium hydrogen phosphate-citratebuffer solution (for pH 4ndash8) and borax-sodium hydrox-ide buffer solution (for pH 9-10) Anaerobic growth wasdetermined at 30∘C in an anaerobic chamber for 7 daysGram staining was performed following the standard Gramprocedure Motility was examined on a semisolid culturemedium supplemented with 05 (wv) of agar Catalase andoxidase activities were investigated following the proceduresby Barrow and Feltham [11] H

2S production was studied by

growing the strain in a tube that contains culture mediumsupplemented with 5 gL sodium thiosulfate and detectedby using a filter-paper strip saturated with lead acetate [12]Starch hydrolysis was determined on an agar plate supple-mented with 2 gL soluble starch and detected by floodingthe plate with Lugolrsquos iodine solution and gelatin hydrolysiswas performed by growing colonies on agar plates with5 gL gelatin and detected by filling the plates with Frazierrsquosreagent [13]The substrate-utilization profile was determinedin triplicate in a nonglucose medium that contains 1 gL ofeach substrate Nitrate reduction was tested by API 20NE(bioMerieux) according to the manufacturerrsquos instructionsThe enzyme activities were examined by using API ZYM(bioMerieux) The strips were incubated at 28∘C for 48 h

23 Genomic DNA GC Content and Fatty Acid Analysis ThegenomicDNAof strain PR1was isolated and purified by usinga TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit(TaKaRa Dalian China)The genomic DNAGC content wasdetermined by HPLC according to the method of Mesbahet al [14] with Escherichia coli K-12 as a reference Cellularfatty acids were extracted according to theMIDI protocol [15]and identified by using the standardMIDI SherlockMicrobialIdentification System (version 60)

24 16S rRNA Gene Sequence Analysis The complete 16SrDNA sequence of strain PR1 was obtained from its genomicsequence by using theDNAMANprogramandwas depositedin the GenBank under the accession number KT149658 The16S rRNA gene sequence of strain PR1 and related sequenceswere aligned by using CLUSTAL X [16] The phylogenetictreeswere reconstructed by theMEGAprogramversion 5 [17]using the neighbor-joining algorithms Bootstrap values werecalculated based on 1000 replicates

25 Genome Annotation and CAZyme Family IdentificationThe genome of strain PR1 was sequenced by using Illu-mina HiSeq 2000 sequencer the methods of data process-ing and assembly were presented in our previous work[18] The genomic sequence was deposited in the Gen-Bank database under the accession number JZSC00000000The predicted genes were annotated by comparing pro-tein sequences against public databases including KyotoEncyclopedia of Genes and Genomes (KEGG) Cluster ofOrthologous Groups of proteins (COG) Gene Ontology(GO) and Swiss-Prot by using BLASTp with an 119890-valuelt 000001 Two genomes from the closely related speciesthat include Cellvibrio sp BR (GenBank accession num-ber AICM000000001) and C japonicus Ueda107 (GenBankaccession number CP000934) were selected for the compar-ative analysis of functional genes that involve carbohydratemetabolism Gene-encoding glycoside hydrolases (GHs)carbohydrate esterases (CEs) glycosyltransferases (GTs)polysaccharide lyases (PLs) carbohydrate-binding modules(CBMs) and auxiliary activities (AAs) were identified byusing theCarbohydrate-Active EnZyme (CAZy) database [19]and dbCAN database [20] In brief CAZymes were analyzedbased on HMMer searches against the dbCAN database andBLASTp searches against the CAZy database using defaultparameters and an 119864-value cutoff of 1119890 minus 20 the annotationwas confirmed only when the two database searches yieldedpositive results the positive CAZymeswere assigned to an ECnumber

26 Enzyme Activity Assays The activity assays of xylanaseand agarase were performed by using the fermentation broththat was cultured in the medium with xylan and agarose asthe primary carbon source The fermentation was performedin a 1000mL Erlenmeyer flask containing 300mL of culturemedia and supplied with 1 g Lminus1 of each carbon source Thecultivation was maintained at 30∘C on a rotary shaker witha speed of 150 rpm Fermented broths were collected after24 hoursrsquo cultivation The supernatant from culture medium

BioMed Research International 3

(a)

Pseudomonas moorei Asd MY-A1 (FM9558891)Pseudomonas sp PSB8 (EU1840851)

Pseudomonas frederiksbergensis BZ21 (HQ5888321)Pseudomonas putida IHBB 1071 (KF4758341)

Pseudomonas abietaniphila HMGU118 (HF9525411)Pseudomonas fluorescens HZN1 (GU3580731)Pseudomonas fluorescens BIT-18 (GU3678701)Pseudomonas fluorescens TC222 (DQ4020531)Pseudomonas putida CM5002 (EF5295171)

Pseudomonas sp WAB1930 (AM1842691)Pseudomonas benzenivorans DSM 8628 (NR 1169041)Cellvibrio japonicus Ueda107 (NR 0288361)

Cellvibrio gandavensis R-4069 (NR 0254191)

Cellvibrio ostraviensis LMG 19434 (NR 0255521)Cellvibrio fibrivorans R-4079 (NR 0254201)

Cellvibrio vulgaris NCIMB 8633 (NR 1254821)Cellvibrio mixtus J3-8 (KC3299161)Cellvibrio mixtus ACM 2601 (NR 0418841)Cellvibrio mixtus ACM 2603 (AJ2891601)

Escherichia coli BL21 (AJ6051151)98

9999

96

57

81

72

100

100 5474

9598

62

81

001

Cellvibrio sp PR1 (KT1496581)

(b)

Figure 1 Transmission electron microscope profile (a) and phylogenetic analysis (b) of strain PR1 Neighbor-joining tree was created aftercomputing the evolutionary distances via Kimurarsquos 2-parameter method Bootstrap values over 50 based on 1000 replicates are shown

was collected by centrifugation at 12000timesg and 4∘C for10min 1ml of supernatant was added to the phosphatebuffer solution (PBS pH 70) and modified with 10 (wv)of the related substrate for 60min The reducing sugarwas measured by using the 35-dinitrosalicylic acid (DNS)method [21] One unit of enzyme activity is defined as theamount of enzyme that is released from 1 120583mol of reducingsugar (as xylose or glucose equivalent) per min The proteinconcentrations were determined by the Lowry method andBSA as the standardThe optimal temperatures of the enzymeactivities were studied by subjecting reactions to differenttemperatures ranging from 30∘C to 50∘C in a PBS (pH 70)The NaCl tolerance of related enzyme was tested under extraNaCl concentrations ranging from 0 to 5 (wv) and theinfluence of pH on enzyme activity was tested under differentpHs (4ndash11) the pHs (pH 3ndash8) were adjusted by disodiumhydrogen phosphate-citrate buffer solution the pHs (pH 9-10) were adjusted by borax-sodiumhydroxide buffer solutionand the pH 11 was adjusted by sodium phosphate-disodiumhydrogen phosphate buffer solution

3 Results

31 Morphology and Phylogenetic Analysis Strain PR1 wasGram-negative and nonspore-forming Cells are straight rodor spiral in shape and are about 15ndash25 120583m long and 05 120583mwide The cells have single polar flagella with a length ofapproximately 2-3120583m (Figure 1(a)) Phylogenetic analysissuggested that strain PR1 is under the genus Cellvibrio andthis strain is closely related to C fibrivorans R-4079 and Costraviensis LMG 19434 (Figure 1(b)) The 16S rRNA genesequence similarities of strain PR1 with C fibrivorans R-4079

and C ostraviensis LMG 19434 are more than 98 and lessthan 96 with C vulgaris NCIMB 8633 (Table 1)

32 Comparative Physiological and Biochemical AnalysisStrain PR1 was able to grow at temperatures of 15ndash45∘C(optimum at 30ndash37∘C) additional salt of 0ndash25 NaCl(optimum at 0-1) and pH of 6ndash10 (optimum at pH 7-8)The strain is unable to grow under an anaerobic conditionResults show that the strain has a motility capability It wasfound that strain PR1 was positive for catalase oxidase andstarch hydrolysis activities and negative for H

2S production

and gelatin hydrolysis activities The main physiological andbiochemical characteristics of strain PR1 and related speciesare shown inTable 1 Strain PR1 utilizesmaltose and arabinose(the same as the other five Cellvibrio species) N-acetyl D-glucosamine (except for the C ostraviensis LMG 19434) andchitin (the same as the C fulvus LMG 2847 and C vulgarisNCIMB 8633) for growth Different from the other fiveCellvibrio species strain PR1 utilizes agar and carboxymethylcellulose for the growth and shows 120573-galactosidase and 120572-fucosidase activities but leucine arylamidase activity wasnot observed Moreover acid phosphatase 120573-glucosidase 120572-galactosidase and 120573-Glucuronidase activities were observedin strain PR1 but valine arylamidase and naphthol-AS-BI-phosphohydrolase activities were not detected

The fatty acid compositions of strain PR1 and relatedstrains are summarized in Table 1 The main fatty acidsin strain PR1 were C160 C180 C200 C

150iso 2-OH

orand C1611205967c (summed in feature 3) and C

1811205967c orand

C1811205966c (summarized in feature 8) Different from the other

five species saturated C160 was the most abundant fatty acidin strain PR1 that accounted for 3674 of the total fatty acids


Table 1 Phenotype and fatty acid characteristics of Cellvibrio species

1 2 3 4 5 616S rDNA similarity to PR1 () 100 986 98 974 973 957G+C content (mol) 4998 48 474ndash484 526 446 449Nitrate reduced to nitrite + + + minus + +Utilization of

Maltose + + + + + +Arabinose + + + + + +Mannose minus + + + minus minus

N-Acetyl-D-glucosamine + + minus + + +Carboxymethyl cellulose minus + + + + +Agar + minus minus minus minus minus

Chitin + minus minus minus + +Valine arylamidase minus minus + minus minus minus

Acid phosphatase + + + minus minus minus

120573-Galactosidase + minus minus minus minus minus

120573-Glucosidase + + + + + minus

120572-Galactosidase + + + minus + +120573-Glucuronidase + minus minus minus + +120572-Fucosidase + minus minus minus minus minus

Leucine arylamidase minus + + + + +Naphthol-AS-BI-phosphohydrolase minus minus + + + +Growth at 4∘C (14 days) minus + + minus + minus

Growth at 37∘C + minus minus minus minus +Mucoid growth on TSA minus minus minus + minus +Yellow pigment on TSA + minus + minus minus +Fatty acid

100 233 48ndash71 41ndash82 26ndash58 420 43ndash69100 3-OH 257 77ndash107 55ndash134 1300 650 61ndash109120 199 54ndash77 40ndash72 tr 440 44ndash58110 3-OH mdash 00ndash11 tr tr mdash mdash120 2-OH mdash 33ndash54 28ndash60 78ndash110 360 26ndash39121 3-OH 406 mdash mdash mdash 130 mdash120 3-OH mdash 10ndash15 tr tr mdash mdash140 197 16ndash25 12ndash24 13ndash17 tr 09ndash12150 mdash 00ndash13 tr 09ndash19 220 tr160 3674 157ndash200 185ndash278 143ndash180 1970 176ndash209170 240 16ndash45 00ndash19 22ndash37 570 13ndash201711205968c mdash tr mdash tr tr tr180 878 00ndash28 00ndash16 tr 310 tr1811205966c mdash mdash 00ndash28 00ndash33 mdash 00ndash431811205967c mdash 114ndash190 73ndash153 113ndash179 1680 129ndash1981811205969c 287 mdash mdash mdash mdash mdash190 290 mdash mdash mdash mdash mdash200 681 mdash mdash mdash mdash mdashECL 11799 mdash mdash mdash mdash mdash trECL 18814 mdash tr mdash mdash mdash mdashSummed feature 2 247 mdash mdash mdash mdash mdashSummed feature 3 1737 256ndash340 344ndash381 304ndash349 3090 341ndash382Summed feature 8 675 mdash mdash mdash mdash mdash

Strains 1 PR1 2 Cellvibrio fibrivorans LMG 18561T 3 Cellvibrio ostraviensis LMG 19434T 4 Cellvibrio mixtus ACM 2601T 5 Cellvibrio fulvus LMG 2847T 6Cellvibrio vulgarisNCIMB 8633T mdash not detected tr trace (le10 of total) Unknown fatty acids are designated by their equivalent chain-length (ECL) relativeto the chain lengths of known saturated fatty acids Summed feature 2 comprises iso-C161 I C140 3-OH andor an unknown fatty acid Summed feature 3comprises C150 iso 2-OH C1611205967c or both Summed feature 8 comprises C1811205967c andor C1811205966c Data for related Cellvibrio species came from Humphryet al [22] and Mergaert et al [23]


30 35 40 45 5002468

1012141618

3 4 5 6 7 8 9 10 110

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

Enzy

me a

ctiv

ity (U

mL)

pH0 05 1 15 2 3 4 5

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

Extra NaCl ()Temperature (∘C)

(a)

Enzy

me a

ctiv

ity (U

mL)

3 4 5 6 7 8 9 10 11pH

0 05 1 15 2 3 4 5Extra NaCl ()

35 40 45 500

5

10

15

20

25

30

Temperature (∘C)

0

20

40

60

80

100

120En

zym

e act

ivity

( m

ax)

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

(b)

Figure 2 Enzyme activities of xylanase (a) and agarase (b) of strain PR1 at different temperatures pHs and NaCl concentrations

The C1811205969c C190 C200 and summarized features 2 and 8that were not detected in the other five strains were found instrain PR1 These fatty acids accounted for 218 of the totalfatty acids Additionally the C120 2-OH and unsaturatedC1811205967c that are abundant in other Cellvibrio species werenot identified in strain PR1

33 EnzymeActivities Theextracellular xylanase and agaraseactivities of strain PR1 were assessed after 48 h incubation at30∘C (Figure 2) The optimal xylanase activity from strainPR1 was determined to be 154UmL under the condition of45∘C pH 7 and no extra NaCl 806 of xylanase activitywas observed to remain at 50∘C When compared to themaximum at pH 7 the xylanase activities continued to have80 under pH 6ndash8 1677 under pH 3 and 2011 under pH11 NaCl was determined to have influence on the xylanaseactivity however the xylanase from strain PR1 was NaCltolerant and it was detected that 6861 of enzyme activitywas retained under 5 (wv) of extra NaCl (Figure 2(a))

The optimal agarase activity was determined to be260UmL under the condition of 40∘C pH 7 and 05 extraNaCl (Figure 2(b)) It was detected that more than 90 ofagarase activities remained under pH 6ndash8 1369 under pH3 and 1707 under pH 11 Agarase from strain PR1 showeda high tolerance to NaCl retaining more than 80 of activityunder 5 of NaCl

34 Comparative CAZyme Family and Functional GenesThat Are Involved in Carbohydrate Hydrolysis The genomic

features and CAZyme families of strain PR1 C japonicusUeda107 Cellvibrio sp BR Cellvibrio sp OA-2007 Cellvibriosp PSBB023 C mixtus and Cellulomonas gilvus were sum-marized in Table 2 A total of 3730 protein-coding genesout of 3844 ORFs were identified and annotated by BLASTpsearch with the sequences in databases CAZyme modulesincluding 63 CBMs 20 CEs 128 GHs 38 GTs and 2 PLs wereidentified from strain PR1rsquos genome which was described inour previous report [2] The distribution of CAZyme familyin strain PR1 is similar to the genomes of related strainsCBM6 involved in cellulosexylanglucan hydrolysis CE2and CE4 involved in xylan hydrolysis GH13 involved instarch hydrolysis GH16 involved in agar hydrolysis GH43involved in xylan hydrolysis and GT2 and GT4 involvedin UDP-glucose metabolism are dominant in each CAZymefamily (Tables S1 and S2 in SupplementaryMaterial availableonline at httpsdoiorg10115520176304248)

Strain PR1 has an incomplete cellulose hydrolysis systemwhich has 4 120573-glucosidases and 12 endoglucanase-encodinggenes but a lack of cellobiohydrolase-encoding gene in thegenome was observed (Figure 3(a)) The same situationwas found in the strain Cellvibrio mixtus that possesses9 endoglucanases and 12 120573-glucosidases but no cellobio-hydrolase Because of the lack strain PR1 and Cellvibriomixtus could not grow on cellulose In contrast theCellvibriojaponicus Ueda107 Cellvibrio sp BR Cellvibrio sp OA-2007andCellvibrio sp PSBB023 genomes encode a single cellobio-hydrolase that is located in GH6 (the release of disaccharidecellobiose from the reducing ends of the 120573-glucan)


Table 2 Genomic feature and CAZyme family of strain PR1 and six related strains

PR1 Cellvibrio japonicus Cellvibrio sp BR Cellvibrio sp OA-2007 Cellvibrio sp PSBB023 Cellvibrio mixtusGenome size (Mb) 443 458 485 459 47 518GC (mol) 4756 52 488 477 494 4666Total genes 3844 3679 4144 4094 4062 4916Predict proteins 3730 3577 4047 3872 3910 2845Annotation percentage () 970 9723 9766 9458 9626 5787CAZyme familya

CBMs 63 95 105 118 49 121CEs 20 24 33 24 21 27GHs 128 132 159 132 97 135GTs 38 49 54 49 54 58PLs 2 14 0 5 0 0AAs 0 0 3 6 3 9Total 251 314 354 334 224 350

aCBMs carbohydrate-binding modules CEs carbohydrate esterases GHs glycoside hydrolases GTs glycosyl transferases PLs polysaccharide lyases AAsauxiliary activities

Xylanase xylosidase arabinofuranosidase acetyl xylanesterase glucuronidase and ferulic acid esterase are essentialto complete xylan hydrolysis The xylanase attacks the back-bone of xylan and generates oligosaccharides the xylosidasereleases xylose from the oligosaccharides along with theactivities of the other four enzymes by attacking the sidechains (Figure 3(b)) There are at least 2 GH10 and 2GH11 and 1 GH 30 120573-xylanases 16 GH43 120573-xylosidases17 120572-arabinofuranosidases 9 acetyl xylan esterases 2 120572-glucuronidases and 2 ferulic acid esterases in strain PR1

The complete hydrolysis of agaragarose includes twokinds of pathways (1) 120572-Agarase attacks 120572-13-glucosidicbonds to generate agarooligosaccharides which are hydro-lyzed to galactose by the 120573-14-36-anhydro-L-galacopyra-nose hydrolase (2) 120573-Agarase attacks 120572-14-glucosidic bondsto generate neoagarooligosaccharides which are hydrolyzedto galactose by the 120572-13-L-neoagarooligosaccharide hydro-lase The strain PR1 contains 3 GH50 and 1 GH86 120573-agarases along with 2 GH117 120572-neoagarooligosaccharidehydrolases whereas only the Cellvibrio sp BR contains2 GH50 and 1 GH86 120573-agarases along with 1 GH117 120572-neoagarooligosaccharide hydrolase (Figure 3(c))

35The ProposedMetabolic Pathways of Xylan and Agarose onGenome-Wide Analysis Xylan and agarose are hydrolyzed toxylose arabinose galactose and so forth in vitro by extra-cellular enzymes These monosaccharides are assimilatedby cells and take part in energy generation and substancetransformationThe main metabolic pathways of xylose ara-binose and galactose in strain PR1 are proposed on a genome-wide analysis As shown in Figure 4 xylose is convertedinto xylulose-5-phoshate by the activities of xylose isomerase(XI) and xylulokinase (XK) whereas arabinose is convertedinto ribulose-5-phosphate by the arabinose isomerase (AI)and ribulokinase (AK) Xylulose-5-phoshpate and ribulose-5-phosphate are interconversed by the ribulose-phosphate3-epimerase (RPE) and involved in the pentose phosphate

pathway (PPP) The products of PPP glyceraldehydes-3-phosphate and fructose-6-phosphate are catalyzed to pyru-vate via the glycolysis pathway The genome of strain PR1has 5 XI-encoding 1 XK-encoding 1 AI- encoding 1 RK-encoding and 1 RPE-encoding genes (Figure 4) Galactoseis the hydrolysate of agaragarose and is utilized by strainPR1 via the enzyme catalytic activities of galactokinase(GalK) UDP-glucose 4-epimerase (GalE) UDP-glucose-hexose-1-phosphate uridylyltransferase (GalT) UTP-glu-cose-1-phosphate uridylyltransferase (GalU) and phospho-glucomutase (PGM) The genome of strain PR1 also has 2GalK-encoding 2 GalE-encoding 2 GalT-encoding 1 GalU-encoding and 2 PGM-encoding genes (Figure 4)

4 Discussion

In this work we comparatively studied the phenotypesand extracellular enzyme activities of strain PR1 and thegenomic features and carbohydrate hydrolysis pathways of Cjaponicus Cellvibrio sp BR Cellvibrio sp OA-2007 Cellvibriosp PSBB023 C mixtus and Cellulomonas gilvus were alsoanalyzed C fibrivorans R-4079 and C ostraviensis LMG19434 were the closely related strains of strain PR1 and thecomparative phenotypes and genomes on these strains werealso studied however only the genome information of Cjaponicus Ueda107 Cellvibrio sp BR Cellvibrio sp OA-2007Cellvibrio sp PSBB023 and C mixtus J3-8 is available inpublic databasesThiswork is still helpful for the investigationof strain PR1 characteristics in a natural ecosystem

Bacteria from the genus Cellvibrio are known to degradecellulose xylan starch chitin and so forth the members ofCellvibrio species including C ostraviensis C fibrivorans Cfulvus C vulgaris C mixtus C gandavensis C japonicusand C diazotrophicus were isolated from soils rhizospheresand giant snails [10 22 23] Strain PR1 of Cellvibrio was firstisolated from freshwater and has xylanolytic and agarolyticactivities 120573-Agarases were also observed in Cellvibrio strains


048

12

훽-Glucosidase Cellobiohydrolase

048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6

Cellvibrio japonicusCellvibrio sp BRCellvibrio sp OA-2007Cellvibrio sp PSBB023

Cellvibrio mixtusCellulomonas gilvusCellvibrio sp PR1

(a) Cellulose hydrolysis

01234

05

10152025

훼-GlucuronidaseXylanase

훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4

Acetyl xylan esteraseFerulic acid esterase

Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC



(b) Xylan hydrolysis

012345

0

1

2

-1 3-L-Neoagarooligosaccharide hydrolase -Agarase

-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1



(c) Agarose hydrolysis

Figure 3 Enzymes involving in cellulose (a) xylan (b) and agarose (c) hydrolysis and their encoding genes distributed in the genomes ofCellvibrio sp PR1Cellvibrio japonicus andCellvibrio sp BRCellvibrio sp OA-2007Cellvibrio sp PSBB023Cellulomonas gilvus andCellvibriomixtus


D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)

L-Ribulose-5-PRK (1)

RPE (1)

TKLTAL

3-P-Glyceraldehyde + fructose-6-P

L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose

D-Galactose-PGalK (2)

UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)

-xylosidase -glucuronidase xylanase acetyl xylan esterase ferulic acid esterase arabinofuranosidase

-agarase -neoagarooligosaccharide

hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1

Figure 4 Xylan and agarose metabolic pathways in strain PR1 proposed on the genome-wide analysis Yellow color marked pathway is thepentose phosphate pathway the number in parenthesis is the gene copy found in the genome GalK galactokinase (EC 2716) GalE UDP-glucose 4-epimerase (EC 5132) GalT UDP-glucose-hexose-1-phosphate uridylyltransferase (EC 27712) GalU UTP-glucose-1-phosphateuridylyltransferase (EC 2779) PGM phosphoglucomutase (EC 5422) XI xylose isomerase (EC 5315) XK xylulokinase (EC 27117)AI arabinose isomerase (EC 5314) RK ribulokinase (EC 27116) RI ribose 5-phosphate isomerase (EC 5316) RPE ribulose-phosphate3-epimerase (EC 5131) TKL transketolase (EC 2211) TAL transaldolase (EC 2212)

that were isolated from wastewater treatment plants [24] andsediments [25]

Strain PR1 has a higher xylanolytic activity than manyother microorganisms [10] Wu and He [10] concluded thatthe reason for the high xylanolytic activity of strain C mixtusJ3-8 was the abundance of GH11 xylanases the xylanaseslocated in GH11 are considerably more active than GH10xylanase for its high substrate specificity great stability andplasticity and small protein sizes In strain PR1 2 GH10 and2 GH11 xylanases were identified However the RNA-Seqresults suggested the GH10 xylanase-encoding genes (FPKM1265 for PRGL001867 and 671 for PRGL003904) had higherexpressions than GH11 xylanase-encoding genes (FPKM 569for PRGL000191 and 433 for PRGL003376) when xylan wasused as the primary substrate [2]The roles ofGH10 andGH11xylanases on xylan hydrolysis need to be further investigated

by the kinetics of enzyme-catalyzed reactions and specificprotein quantification

CBMs are usually considered to promote hydrolysisefficiency by binding an enzyme to the substrate In xylanasefamilies GH10 and GH11 the catalytic modules are appendedto a range of different CBMs that target crystalline cellulose(CBM 2 and CBM10) and xylan (CBM15 and CBM35) [9]The C japonicusUeda107 was found to have CBM2ndashCBM10-GH10 CBM2ndashCBM35-GH10 CBM15-GH10 and CBM60-GH11 xylanases to release soluble polysaccharides andoligosaccharides [9] Wu and He [10] detected 3 xylanase-binding CBMs (CBM2 CBM10 and CBM15) in strain Cmixtus J3-8However threeCBMbinding xylanases (CBM15-GH10 CBM57-GH11 and CBM60-GH11) were identified forstrain PR1 andneitherCBM2norCBM10was detected (TableS1) It seems that the CBM15 is a specific module that is only


detected in Cellvibrio strains such as C japonicus Cellvibriosp BR and C mixtus [9 10 26]

In addition six types of CBM (CBM5 CBM6 CBM12CBM2ndashCBM6 CBM5ndashCBM12 and CBM6ndashCBM12) struc-tures were detected from the binding domain of chitinases(GH18 and GH19) in strain PR1 GH18 chitinases are foundin various organisms and are ordinarily composed of onecatalytic domain and one or more domains that are involvedin chitin binding whereas GH19 chitinases are more likely tooriginate from plants and consisted of one or two domains[27] Many chitinolytic bacteria produce only GH18 chiti-nases and the production of bothGH18 andGH19wasmostlyreported in Streptomyces strains [27 28] The CBM5 sharessimilarities with some cysteine-rich chitin-binding domain(ChBDChiC ChBDChiA1 etc) and were usually found in thebinding domain of GH18 chitinases [27 29] However veryfew chitin-binding CBM6 and CBM12 have been reportedin bacteria The comparative genome analysis suggested thatthere are three CBM5-GH18 and one CBM6-GH19 chitinasesdetected in C japonicus Ueda107 and two CBM5-GH18chitinases were detected in Cellvibrio sp BR

Strain PR1 was a syntrophic bacterium to cyanobacteriawhen enriching and isolating cyanobacterium under pho-toautotrophic condition This strain was believed to bebeneficial in cyanobacteria growth by the bacterial-algalinteractions Based on this hypothesis we constructed aCellvibrio-microalgae cocultivation model for the promotionof microalgae growth The biomass production of ChlorellaChlamydomonas and Dunaliella was significantly enhancedby using this model the comparative transcriptome analysisindicated that the xylan hydrolysates or xylose was catalyzedinto some active substrates and responsible for the promo-tions [2] The utilization of monosaccharides is importantbecause the pentoses derived fromhemicelluloses are difficultto ferment by microorganisms Besides the organic carbonsources vitamins or other growth factors of strain PR1 arepossible substrates for enhancing microalgae growth StrainPR1 has complete enzyme systems for vitamin B1 (thiamine)and vitamin B

7(biotin) syntheses (Figures S1 and S2) The

thiamine-synthesizing genes includes thiC (ThiC phos-phomethylpyrimidine synthase EC419917) thiD (ThiDphosphomethylpyrimidine kinase EC27149) thiE (ThiEthiamine-phosphate pyrophosphorylase EC2513) thiF(ThiF adenylyltransferase EC277-) thiG (ThiG thiazolesynthase EC28110) thiI (ThiI thiamine biosynthesis ATPpyrophosphatase) thiL (ThiL thiamine-monophosphatekinase EC27416) thiO (ThiO glycine oxidase EC14319)thiS (ThiS sulfur carrier protein) and dxs (1-deoxy-D-xylulose-5-phosphate synthase EC2217) The biotin-synthesizing genes include bioA (adenosylmethionine-8-amino-7-oxononanoate aminotransferase EC26162) bioB(biotin synthetase EC2816) bioC (methyltransferases)bioD (dethiobiotin synthetase EC6333) and bioF (8-amino-7-oxononanoate synthase EC23147) It is knownthat over half of microalgal species are auxotrophic forcobalamin (vitamin B

12) and 20 require thiamine and 5

require biotin The important role of vitamins in controllingalgal growth is increasingly recognized [30 31]

This study found that strain PR1 shows distinctive dif-ferences in phenotype and genome from known Cellvibriospecies The genomic analysis provides some insights intothe functions of strain PR1 in polysaccharide hydrolysis andmonosaccharidemetabolism and possible syntrophic patternwith microalgae

Conflicts of Interest

The authors declare that there are no conflicts of interestsregarding the publication of this paper

Authorsrsquo Contributions

Zhangzhang Xie and Weitie Lin contributed equally to thiswork

Acknowledgments

This work was supported by the National Natural Sci-ence Foundation of China (no 21276099 no 41301318 no41473072) the Specialized Research Found for the DoctoralProgramofHigher Education of China (no 20120172120045)and the Fundamental Research Funds for the Central Univer-sities (no 2015ZM171)

References

[1] B S Harish J R Mekala and K Babu Uppuluri ldquoBioengi-neering strategies on catalysis for the effective production ofrenewable and sustainable energyrdquo Renewable and SustainableEnergy Reviews vol 51 article no 4568 pp 533ndash547 2015

[2] Z Xie W Lin and J Luo ldquoPromotion of microalgal growth byco-culturing with Cellvibrio pealriver using xylan as feedstockrdquoBioresource Technology vol 200 pp 1050ndash1054 2016

[3] X T Fu and S M Kim ldquoAgarase Review of major sourcescategories purification method enzyme characteristics andapplicationsrdquoMarine Drugs vol 8 no 1 pp 200ndash218 2010

[4] S-C Wu T-N Wen and C-L Pan ldquoAlgal-oligosaccharide-lysates prepared by two bacterial agarases stepwise hydrolyzedand their anti-oxidative propertiesrdquo Fisheries Science vol 71 no5 pp 1149ndash1159 2005

[5] A Grigorian L Araujo N N Naidu D J Place B Choudhuryand M Demetriou ldquoN-acetylglucosamine inhibits T-helper 1(Th1)T-helper 17 (Th17) cell responses and treats experimentalautoimmune encephalomyelitisrdquo Journal of Biological Chem-istry vol 286 no 46 pp 40133ndash40141 2011

[6] A A Klyosov ldquoTrends in biochemistry and enzymology ofcellulose degradationrdquo Biochemistry vol 29 no 47 pp 10577ndash10585 1990

[7] J AThomson ldquoMolecular biology of xylan degradationrdquo FEMSMicrobiology Letters vol 104 no 1-2 pp 65ndash82 1993

[8] Blackall et al ldquoValidation of the Publication of NewNames andNew Combinations Previously Effectively Published Outsidethe IJSB List No 22rdquo International Journal of SystematicBacteriology vol 36 no 4 pp 573ndash576 1986

[9] R T DeBoy E F Mongodin D E Fouts et al ldquoInsights intoplant cell wall degradation from the genome sequence of thesoil bacterium Cellvibrio japonicusrdquo Journal of Bacteriology vol190 no 15 pp 5455ndash5463 2008


[10] Y-R Wu and J He ldquoCharacterization of a xylanase-producingCellvibrio mixtus strain J3-8 and its genome analysisrdquo ScientificReports vol 5 Article ID 10521 2015

[11] G I Barrow andRK FelthamCowan and SteelrsquosManual for theidentification of medical bacteria Cambridge University PressCambridge UK 3rd edition 1993

[12] D Han H-L Cui and Z-R Li ldquo Halopenitus salinus spnov isolated from the brine of salted brown alga LaminariardquoAntonie van Leeuwenhoek International Journal of General andMolecular Microbiology vol 106 no 4 pp 743ndash749 2014

[13] J J McDade and R H Weaver ldquoRapid methods for thedetection of gelatin hydrolysisrdquo Journal of Bacteriology vol 77no 1 pp 60ndash64 1959

[14] M Mesbah U Premachandran and W B Whitman ldquoPrecisemeasurement of the g+c content of deoxyribonucleic acid byhigh-performance liquid chromatographyrdquo International Jour-nal of Systematic Bacteriology vol 39 no 2 pp 159ndash167 1989

[15] J R M Delamuta R A Ribeiro E Ormeno-Orrillo I S MeloE Martınez-Romero and M Hungria ldquoPolyphasic evidencesupporting the reclassification of Bradyrhizobium japonicumgroup Ia strains as bradyrhizobiumdiazoefficiens sp novrdquo Inter-national Journal of Systematic and Evolutionary Microbiologyvol 63 no 9 pp 3342ndash3351 2013

[16] J D Thompson T J Gibson F Plewniak F Jeanmougin andD G Higgins ldquoThe CLUSTAL X windows interface flexiblestrategies for multiple sequence alignment aided by qualityanalysis toolsrdquoNucleic Acids Research vol 25 no 24 pp 4876ndash4882 1997

[17] K Tamura D Peterson N Peterson G Stecher M Nei andS Kumar ldquoMEGA5 molecular evolutionary genetics analysisusing maximum likelihood evolutionary distance and max-imum parsimony methodsrdquo Molecular Biology and Evolutionvol 28 no 10 pp 2731ndash2739 2011

[18] Z Xie W Lin and J Luo ldquoGenome sequence of Cellvibriopealriver PR1 a xylanolytic and agarolytic bacterium isolatedfrom freshwaterrdquo Journal of Biotechnology vol 214 pp 57-582015

[19] B I Cantarel P M Coutinho C Rancurel T Bernard V Lom-bard and B Henrissat ldquoThe Carbohydrate-Active EnZymesdatabase (CAZy) an expert resource for glycogenomicsrdquoNucleic Acids Research vol 37 no 1 pp D233ndashD238 2009

[20] Y Yin X Mao J Yang X Chen F Mao and Y Xu ldquoDbCANA web resource for automated carbohydrate-active enzymeannotationrdquo Nucleic Acids Research vol 40 no 1 pp W445ndashW451 2012

[21] G L Miller ldquoUse of dinitrosalicylic acid reagent for determina-tion of reducing sugarrdquo Analytical Chemistry vol 31 no 3 pp426ndash428 1959

[22] D R Humphry G W Black and S P Cummings ldquoReclassi-fication of lsquoPseudomonas fluorescens subsp cellulosarsquo NCIMB10462 (Ueda et al 1952) as Cellvibrio japonicus sp nov andrevival of Cellvibrio vulgaris sp nov nom rev and Cellvibriofulvus sp nov nom revrdquo International Journal of Systematic andEvolutionary Microbiology vol 53 no 2 pp 393ndash400 2003

[23] J Mergaert D Lednicka J Goris M C Cnockaert P DeVos and J Swings ldquoTaxonomic study of Cellvibrio strains anddescriptin of Cellvibrio ostraviensis sp nov Cellvibrio fibrivo-rans sp nov and Cellvibrio gandavensis sp novrdquo InternationalJournal of Systematic and Evolutionary Microbiology vol 53 no2 pp 465ndash471 2003

[24] G Paes J-G Berrin and J Beaugrand ldquoGH11 xylanasesstructurefunctionproperties relationships and applicationsrdquoBiotechnology Advances vol 30 no 3 pp 564ndash592 2012

[25] Y J Rhee C R Han W C Kim D Y Jun I K Rhee and Y HKim ldquoIsolation of a novel freshwater agarolytic cellvibrio sp KY-YJ-3 and characterization of its extracellular 120573-agaraserdquo Journalof Microbiology and Biotechnology vol 20 no 10 pp 1378ndash13852010

[26] S J Millward-Sadler K Davidson G P Hazlewood G WBlack H J Gilbert and J H Clarke ldquoNovel cellulose-bindingdomains NodB homologues and conserved modular architec-ture in xylanases from the aerobic soil bacteria Pseudomonasfluorescens subsp cellulosa and Cellvibrio mixtusrdquo BiochemicalJournal vol 312 no 1 pp 39ndash48 1995

[27] Y Kezuka M Ohishi Y Itoh et al ldquoStructural Studies of aTwo-domain Chitinase from Streptomyces griseus HUT6037rdquoJournal of Molecular Biology vol 358 no 2 pp 472ndash484 2006

[28] T Watanabe R Kanai T Kawase et al ldquoFamily 19 chitinasesof Streptomyces species Characterization and distributionrdquoMicrobiology vol 145 no 12 pp 3353ndash3363 1999

[29] MHardt andRA Laine ldquoMutation of active site residues in thechitin-binding domain ChBDChiA1 from chitinase A1 of Bacilluscirculans alters substrate specificity use of a green fluorescentprotein binding assayrdquo Archives of Biochemistry and Biophysicsvol 426 no 2 pp 286ndash297 2004

[30] M T Croft M J Warren and A G Smith ldquoAlgae need theirvitaminsrdquo Eukaryotic Cell vol 5 no 8 pp 1175ndash1183 2006

[31] M A A Grant E Kazamia P Cicuta and A G Smith ldquoDirectexchange of vitamin B

12is demonstrated by modelling the

growth dynamics of algal-bacterial coculturesrdquo ISME Journalvol 8 no 7 pp 1418ndash1427 2014

Submit your manuscripts athttpswwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 201


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


with microalgae we comparatively studied the phenotypesand genome of strain PR1

2 Materials and Methods

21 Bacterial Strains and Culture Medium Strain Cellvibriosp PR1 was isolated from a freshwater sample collectedfrom the Pearl River (23∘81015840N and 113∘171015840E) The strain wasdeposited in the China General Microbiological CultureCollection Center (Beijing China) and the NITE BiologicalResource Center (Tokyo Japan) under the accession numbersCGMCC 114955 and NBRC 110968 respectively

The medium used for strain PR1 cultivation containsthe following 15 g Lminus1 NaNO

3 004 g Lminus1 K

2HPO4

0075 g Lminus1 MgSO4sdot7H2O 0036 g Lminus1 CaCl

2sdot2H2O 1mg

of glucose and 1ml of trace element solution The traceelement solution contains the following 286 g Lminus1H

3BO3

186 g Lminus1 MnCl2sdot4H2O 022 g Lminus1 ZnSO


Na2MoO4sdot2H2O 008 g Lminus1 CuSO


Co(NO3)2sdot6H2O 6 g Lminus1 citric acid 6 g Lminus1 ferric ammonium

citrate and 1 g Lminus1 EDTANa2 pH was adjusted to 72 Xylan

(Sigma-Aldrich USA) cellulose (Avicel PH-101 and CMCSigma-Aldrich USA) starch (Sigma-Aldrich USA) andagarose (Sigma USA) were used as the primary carbonsource for testing xylanase cellulase amylase and agaraseactivities respectively

22 Morphology and Physiological and Biochemical AnalysesThe cells of strain PR1 growing in exponential phase werecollected and used for the examination of cell morphology bytransmission electron microscopy (H-7650 Hitachi Japan)at 80 kV NaCl tolerance was tested in culture medium withadditional salt concentration ranging from 0 to 5 (wv)Growth was tested at different temperatures (4 10 15 2025 30 37 45 and 55∘C) and pH levels (4ndash10) The pHswere adjusted by using disodium hydrogen phosphate-citratebuffer solution (for pH 4ndash8) and borax-sodium hydrox-ide buffer solution (for pH 9-10) Anaerobic growth wasdetermined at 30∘C in an anaerobic chamber for 7 daysGram staining was performed following the standard Gramprocedure Motility was examined on a semisolid culturemedium supplemented with 05 (wv) of agar Catalase andoxidase activities were investigated following the proceduresby Barrow and Feltham [11] H

2S production was studied by

growing the strain in a tube that contains culture mediumsupplemented with 5 gL sodium thiosulfate and detectedby using a filter-paper strip saturated with lead acetate [12]Starch hydrolysis was determined on an agar plate supple-mented with 2 gL soluble starch and detected by floodingthe plate with Lugolrsquos iodine solution and gelatin hydrolysiswas performed by growing colonies on agar plates with5 gL gelatin and detected by filling the plates with Frazierrsquosreagent [13]The substrate-utilization profile was determinedin triplicate in a nonglucose medium that contains 1 gL ofeach substrate Nitrate reduction was tested by API 20NE(bioMerieux) according to the manufacturerrsquos instructionsThe enzyme activities were examined by using API ZYM(bioMerieux) The strips were incubated at 28∘C for 48 h

23 Genomic DNA GC Content and Fatty Acid Analysis ThegenomicDNAof strain PR1was isolated and purified by usinga TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit(TaKaRa Dalian China)The genomic DNAGC content wasdetermined by HPLC according to the method of Mesbahet al [14] with Escherichia coli K-12 as a reference Cellularfatty acids were extracted according to theMIDI protocol [15]and identified by using the standardMIDI SherlockMicrobialIdentification System (version 60)

24 16S rRNA Gene Sequence Analysis The complete 16SrDNA sequence of strain PR1 was obtained from its genomicsequence by using theDNAMANprogramandwas depositedin the GenBank under the accession number KT149658 The16S rRNA gene sequence of strain PR1 and related sequenceswere aligned by using CLUSTAL X [16] The phylogenetictreeswere reconstructed by theMEGAprogramversion 5 [17]using the neighbor-joining algorithms Bootstrap values werecalculated based on 1000 replicates

25 Genome Annotation and CAZyme Family IdentificationThe genome of strain PR1 was sequenced by using Illu-mina HiSeq 2000 sequencer the methods of data process-ing and assembly were presented in our previous work[18] The genomic sequence was deposited in the Gen-Bank database under the accession number JZSC00000000The predicted genes were annotated by comparing pro-tein sequences against public databases including KyotoEncyclopedia of Genes and Genomes (KEGG) Cluster ofOrthologous Groups of proteins (COG) Gene Ontology(GO) and Swiss-Prot by using BLASTp with an 119890-valuelt 000001 Two genomes from the closely related speciesthat include Cellvibrio sp BR (GenBank accession num-ber AICM000000001) and C japonicus Ueda107 (GenBankaccession number CP000934) were selected for the compar-ative analysis of functional genes that involve carbohydratemetabolism Gene-encoding glycoside hydrolases (GHs)carbohydrate esterases (CEs) glycosyltransferases (GTs)polysaccharide lyases (PLs) carbohydrate-binding modules(CBMs) and auxiliary activities (AAs) were identified byusing theCarbohydrate-Active EnZyme (CAZy) database [19]and dbCAN database [20] In brief CAZymes were analyzedbased on HMMer searches against the dbCAN database andBLASTp searches against the CAZy database using defaultparameters and an 119864-value cutoff of 1119890 minus 20 the annotationwas confirmed only when the two database searches yieldedpositive results the positive CAZymeswere assigned to an ECnumber

26 Enzyme Activity Assays The activity assays of xylanaseand agarase were performed by using the fermentation broththat was cultured in the medium with xylan and agarose asthe primary carbon source The fermentation was performedin a 1000mL Erlenmeyer flask containing 300mL of culturemedia and supplied with 1 g Lminus1 of each carbon source Thecultivation was maintained at 30∘C on a rotary shaker witha speed of 150 rpm Fermented broths were collected after24 hoursrsquo cultivation The supernatant from culture medium


(a)









9999

96

57

81

72

100

100 5474

9598

62

81

001


(b)



3 Results




2S production



150iso 2-OH


1811205967c orand


















30 35 40 45 5002468

1012141618

3 4 5 6 7 8 9 10 110

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

Enzy

me a

ctiv

ity (U

mL)

pH0 05 1 15 2 3 4 5

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)


(a)

Enzy

me a

ctiv

ity (U

mL)

3 4 5 6 7 8 9 10 11pH

0 05 1 15 2 3 4 5Extra NaCl ()

35 40 45 500

5

10

15

20

25

30

Temperature (∘C)

0

20

40

60

80

100

120En

zym

e act

ivity

( m

ax)

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

(b)

















4 Discussion




048

12


048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6




01234

05

10152025


훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4


Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC




012345

0

1

2


-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1






D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)









9999

96

57

81

72

100

100 5474

9598

62

81

001


(b)



3 Results




2S production



150iso 2-OH


1811205967c orand


















30 35 40 45 5002468

1012141618

3 4 5 6 7 8 9 10 110

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

Enzy

me a

ctiv

ity (U

mL)

pH0 05 1 15 2 3 4 5

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)


(a)

Enzy

me a

ctiv

ity (U

mL)

3 4 5 6 7 8 9 10 11pH

0 05 1 15 2 3 4 5Extra NaCl ()

35 40 45 500

5

10

15

20

25

30

Temperature (∘C)

0

20

40

60

80

100

120En

zym

e act

ivity

( m

ax)

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

(b)

















4 Discussion




048

12


048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6




01234

05

10152025


훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4


Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC




012345

0

1

2


-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1






D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















30 35 40 45 5002468

1012141618

3 4 5 6 7 8 9 10 110

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

Enzy

me a

ctiv

ity (U

mL)

pH0 05 1 15 2 3 4 5

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)


(a)

Enzy

me a

ctiv

ity (U

mL)

3 4 5 6 7 8 9 10 11pH

0 05 1 15 2 3 4 5Extra NaCl ()

35 40 45 500

5

10

15

20

25

30

Temperature (∘C)

0

20

40

60

80

100

120En

zym

e act

ivity

( m

ax)

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

(b)

















4 Discussion




048

12


048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6




01234

05

10152025


훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4


Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC




012345

0

1

2


-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1






D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


30 35 40 45 5002468

1012141618

3 4 5 6 7 8 9 10 110

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

Enzy

me a

ctiv

ity (U

mL)

pH0 05 1 15 2 3 4 5

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)


(a)

Enzy

me a

ctiv

ity (U

mL)

3 4 5 6 7 8 9 10 11pH

0 05 1 15 2 3 4 5Extra NaCl ()

35 40 45 500

5

10

15

20

25

30

Temperature (∘C)

0

20

40

60

80

100

120En

zym

e act

ivity

( m

ax)

0

20

40

60

80

100

120

Enzy

me a

ctiv

ity (

max

)

(b)

















4 Discussion




048

12


048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6




01234

05

10152025


훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4


Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC




012345

0

1

2


-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1






D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










4 Discussion




048

12


048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6




01234

05

10152025


훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4


Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC




012345

0

1

2


-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1






D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


048

12


048

121620

OHOH

OHOH

OH

OHOH

OH

OO

OO

OO

OO

n

HO

HOHO

HO

HO

Endoglucanase

0

2

4

6




01234

05

10152025


훽-Xylosidase

05

10152025

048

12

0

4

8

0

2

4


Arabinofuranosidase

OH

HO

OH

OH

HO

HO

HOHO

HO HOO

OO

O

OH O

O

O

O

OO

O

O

O

O

O

O

n

H3CO

H3CO

H3C

minusOOC




012345

0

1

2


-agarase

OH

OH OH

OH

OH

OH

OH

OHOH

HOO

O

OO

O

O

O

O

n

O1

1

1

1






D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


D-Xylose

L-Arabinose

D-Xylulose-5-P

D-XyluloseXI (5)

XK (1)

L-RibuloseAI (1)


RPE (1)

TKLTAL


L-Ribose-5-PRI (1)

glucosePyruvate

D-Galactose


UDP-Galactose

GalE (2)

Glucose-1-P

GalT (2)

UDP-Glucose

GalU (1)

Glucose-6-PPGM (2)



hydrolase

OOH

OH

OHOH

HO

OHOH

OH

OH

OH

OHOH

OO

OO

OO

O O

OOH

OHHO

n

OH

OH

OHHO

HO HOHO

HO

HOOO

O

O

O

O

O

O

O

O

OO

O

O

n

OHO

OHOH

OH

OH

O

OHHO

HOOH

O

HO

O

（3

（3

（3

minus

1

1



















Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology














Acknowledgments


References










































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Comparative Phenotype and Genome Analysis of Cellvibrio sp ...downloads.hindawi.com/journals/bmri/2017/6304248.pdf · sp. PR1 was isolated from a freshwater sample collected fromthePearlRiver(23∘8Nand113∘17E).Thestrainwas