burkholderia jiangsuensis sp. nov., a methyl parathion degrading bacterium, isolated from methyl...

Burkholderia jiangsuensis sp. nov., a methylparathion degrading bacterium, isolated frommethyl parathion contaminated soil

Xu-Yun Liu,13 Chun-Xiu Li,13 Xiao-Jing Luo,1 Qi-Liang Lai2

and Jian-He Xu1

Correspondence

Jian-He Xu

[email protected]

1Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of BioreactorEngineering, East China University of Science and Technology, Shanghai 200237, PR China

2State Key Laboratory Breeding Base of Marine Genetic Resources; Key Laboratory of MarineGenetic Resources, Third Institute of Oceanography, SOA; Key Laboratory of Marine GeneticResources of Fujian Province, Xiamen 361005, PR China

A methyl parathion (MP) degrading bacterial strain, designated MP-1T, was isolated from a waste

land where pesticides were formerly manufactured in Jiangsu province, China. Polyphasic

taxonomic studies showed that MP-1T is a Gram-stain-negative, non-spore-forming, rod-shaped

and motile bacterium. The bacterium could grow at salinities of 0–1 % (w/v) and temperatures of

15–40 6C. Strain MP-1T could reduce nitrate to nitrite, utilize D-glucose and L-arabinose, but not

produce indole, or hydrolyse gelatin. Phylogenetic analysis based on 16S rRNA gene sequences

demonstrated that MP-1T belongs to the genus Burkholderia, showing highest sequence

similarity to Burkholderia grimmiae DSM 25160T (98.5 %), and similar strains including

Burkholderia zhejiangensis OP-1T (98.2 %), Burkholderia choica LMG 22940T (97.5 %),

Burkholderia glathei DSM 50014T (97.4 %), Burkholderia terrestris LMG 22937T (97.2 %) and

Burkholderia telluris LMG 22936T (97.0 %). In addition, the gyrB and recA gene segments of

strain MP-1T exhibited less than 89.0 % and 95.1 % similarities with the most highly-related type

strains indicated above. The G+C content of strain MP-1T was 62.6 mol%. The major isoprenoid

quinone was ubiquinone Q-8. The predominant polar lipids comprised phosphatidyl ethanolamine,

phosphatidyl glycerol, aminolipid and phospholipid. The principal fatty acids in strain MP-1T were

C18 : 1v7c/C18 : 1v6c (23.3 %), C16 : 0 (16.8 %), cyclo-C17 : 0 (15.0 %), C16 : 1v7c/C16 : 1v6

(8.5 %), cyclo-C19 : 0v8c (8.1 %), C16 : 1 iso I/C14 : 0 3-OH (5.7 %), C16 : 0 3-OH (5.6 %) and

C16 : 02-OH (5.1 %). The DNA–DNA relatedness values between strain MP-1T and the three type

strains (B. grimmiae DSM 25160T, B. zhejiangensis OP-1T and B. glathei DSM 50014T) ranged

from 24.6 % to 37.4 %. In accordance with phenotypic and genotypic characteristics, strain

MP-1T represents a novel species of the genus Burkholderia, for which the name Burkholderia

jiangsuensis sp. nov. is proposed, the type strain is MP-1T (LMG 27927T5MCCC 1K00250T).

Methyl parathion (MP), a common organophosphoruspesticide, has been applied extensively for crop protection,especially in developing countries. However, it has toxiceffects on the water, air and soil (Barton et al., 2004). Avariety of MP strains of bacteria have been isolated fromsoil (Li et al., 2007; Liu et al., 2005).

In order to study the diversity of MP-degrading microbes,MP-degrading strain MP-1T was subjected to taxonomiccharacterization. On the basis of 16S rRNA gene sequenceanalysis, phylogenetic trees indicated that strain MP-1T

belongs to the genus Burkholderia, with high (.96.9 %)similarity to 16S rRNA sequences of six type strains:B. grimmiae DSM 25160T (Tian et al., 2013), Burkholderiazhejiangensis OP-1T (Lu et al., 2012), Burkholderia choica

3These authors contributed equally to this paper.

Abbreviations: LMG, Laboratorium voor Microbiologie; MCCC, MarineCulture Collection of China.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNAgene sequence of strain MP-1T is KJ400396. The gyrB genes of strainsMP-1T and Burkholderia grimmiae DSM 25160T are KJ535375 andKJ535376. The recA genes of strains MP-1T, DSM 25160T, Burkholderiazhejiangensis OP-1T, Burkholderia choica LMG 22940T, Burkholderiaterrestris LMG 22937T and Burkholderia telluris LMG 22936T areKJ535369, KJ535373, KJ535370, KJ535374, KJ535371 andKJ535372. The GenBank accession numbers for four draft genomesequences of strains MP-1T, DSM 25160T, OP-1T and Burkholderiaglathei DSM 50014T are JFHF00000000, JFHE00000000,JFHD00000000 and JFHC00000000.

Two supplementary figures and one supplementary table are availablewith the online version of this paper.

International Journal of Systematic and Evolutionary Microbiology (2014), 64, 3247–3253 DOI 10.1099/ijs.0.064444-0

064444 G 2014 IUMS Printed in Great Britain 3247

LMG 22940T (Vandamme et al., 2013), Burkholderia glatheiDSM 50014T (Zolg & Ottow, 1975), Burkholderia terrestrisLMG 22937T (Vandamme et al., 2013) and Burkholderiatelluris LMG 22936T (Vandamme et al., 2013). Thus,we investigated the characteristics of MP-1T further todetermine if it represents a new species of the genusBurkholderia.

The genus Burkholderia, a member of the class Betaproteo-bacteria, is a group of metabolically versatile Gram-stain-negative bacteria. To date, the genus Burkholderia com-prises 83 type strains with validly published names (http://www.bacterio.net/burkholderia.html), which are widelydistributed within diverse natural habitats, including water(Ali Khan & Ahmad, 2006; Zhang et al., 2010), fish (Gaoet al., 2013) and soil (Yoo et al., 2007). The genus was firstcreated to accommodate seven species from PseudomonasrRNA group II (Yabuuchi et al., 1992). Species of the genusBurkholderia are responsible for biological degradation,biological control or, as rhizosphere micro-organisms inagriculture, the promotion of plant growth. In the presentstudy, the exact taxonomic position of the newly isolatedstrain, MP-1T, was determined using phenotypic, geneticand chemotaxonomic analyses.

A conventional enrichment method was employed to isolateMP-degrading strains. About 1 g of sludge was sampledfrom a pesticide manufacturing company in Jiangsuprovince, PR China. The soil samples were inoculated intoa selective medium mineral salts medium (MSM), contain-ing: 1.0 g?l21 (NH4)2SO4, 0.8 g?l21 K2HPO4, 0.2 g?l21

KH2PO4, 0.2 g?l21 MgSO4, 0.08 g?l21 CaSO4, 0.005 g?l21

FeSO4.7H2O, 0.0033 g?l21 Na2MoO4?2H2O and 25 mg?l21

MP was supplemented as the sole carbon source. Theculture was incubated for about 3 days at 30 uC on a rotaryshaker at 180 r.p.m. The enrichment suspension was thenserially sub-cultured into fresh MSM containing a graduallyincreasing concentration of MP up to a maximum of200 mg?l21 every 3 days. After a few rounds of enrichmentcultivation, the suspension was serially diluted and spreadonto a peptone yeast (PY) agar plate (10.0 g?l21 peptone,5.0 g?l21 yeast extract, 5.0 g?l21 NaCl, 15.0 g?l21 agar,pH 7.0) supplemented with 200 mg?l21 MP. Among theisolates, a strain capable of degrading MP was isolated anddesignated strain MP-1T. It was maintained on PY agarat 4 uC and stored as glycerol suspensions (30 %, v/v) at280 uC. Biomass for chemotaxonomic studies was pre-pared by growing the strain in a shaker flask of PY brothat 30 uC for 2 days. Cells were harvested by centrifuga-tion (7690 g) and freeze-dried prior to physiological andbiochemical studies. One type strain of a species of thegenus Burkholderia (B. zhejiangensis OP-1T) was providedby Nanjing Agricultural University, while the other twostrains (B. grimmiae DSM 25160T and B. glathei DSM50014T) were purchased from the DSMZ culture collectioncentre (Braunschweig, Germany). These were used asreference type strains, and cultured under the same con-ditions in all analyses except for those of quinone andpolar lipids. Routine cultivation of the strains and most

phenotypic tests were performed on PY agar plates. Theother three strains (B. choica LMG 22940T, B. terrestris LMG22937T and B. telluris LMG 22936T) reported recently byVandamme et al. (2013), were purchased from Laboratoriumvoor Microbiologie (LMG).

Gram staining was conducted as described by Buck (1982).The cell size, morphology and flagellation pattern wereobserved by transmission electron microscopy (JEM-1230,JEOL) using colonies grown on PY agar plates for 2 days at30 uC. Spore morphology was examined in cultures grownon PY agar plates for 4 days. Cell motility was observedin vitro according to the hanging-drop method (Robbie,1945). The optimal temperature for growth was tested overa temperature range of 15–40 uC in PY broth, at 5 uC unitintervals. Growth over a pH range was determined inPY broth adjusted with HCl or NaOH to pH 3–10, at 1pH unit intervals. Cell growth was very poor at 15 or40 uC. NaCl tolerance was determined using PY brothsupplemented with 0, 0.5, 1, 2, 3, 4, or 5 % (w/v) NaCl.Physiological and biochemical properties were investigatedwith the API 20NE, API 20E and API ZYM systems(bioMerieux) according to the manufacturer’s instructions.B. grimmiae DSM 25160T, B. zhejiangensis OP-1T, B. glatheiDSM 50014T and strain MP-1T were all tested under thesame conditions.

Cultures were grown in PY broth for 2 days for totalDNA preparation using the extraction kit according to themanufacturer’s instructions (TIANamp Bacteria DNA Kit).The G+C content of strain MP-1T was calculated from itsdraft genome sequence (JFHF00000000, Liu et al., 2014). The16S rRNA gene sequence was amplified by PCR using a set ofuniversal bacterial primers (Collins et al., 1991). Theamplification of the DNA gyrase B subunit (gyrB) was alsoperformed using previously described primers (Tayeb et al.,2008). The recA gene sequence was amplified using the BUR1and BUR2 primers (Payne et al., 2005). The PCR productwas ligated into vector pMD18-T and sequenced (Majorbio).Sequence similarities between the MP-1T 16S rRNA genesequence and those of closely related species were determinedusing the EzTaxon-e server (Feng et al., 2012). Phylogenetictrees were reconstructed using the neighbour-joiningmethod (Saitou & Nei, 1987) in MEGA5.0 software (Tamuraet al., 2011). The topology of phylogenetic trees wasevaluated using bootstrap values based on 1000 replications.

Fatty acids from whole cells grown on PY plates at 30 uCfor 2 days were saponified, methylated and extractedusing the standard protocol of MIDI (Sherlock MicrobialIdentification System, version 6.0B). The fatty acids wereanalysed by gas chromatography (Agilent Technologies6850) and identified using the TSBA6.0 database of theMicrobial Identification System (Steele et al., 1997). Thefatty acid profile of strain MP-1T, B. grimmiae DSM25160T, B. zhejiangensis OP-1T and B. glathei DSM 50014T

were tested simultaneously. Isoprenoid quinones andpolar lipids were analysed by the Identification Service ofthe MCCC. The quinones were extracted according to a

X.-Y. Liu and others

3248 International Journal of Systematic and Evolutionary Microbiology 64

method based on one previously described (Tindall, 1990a,b). Isoprenoid quinones fell into different classes (e.g.menaquinones, ubiquinones) as determined by TLC onsilica gel, and were then analysed further by HPLC. Polarlipids were extracted from 200 mg freeze-dried cells using achloroform: methanol: 0.3 % (w/v) aqueous NaCl mixture1 : 2:0.8 (by vol.) (Bligh & Dyer, 1959). Polar lipids wereseparated by two-dimensional silica gel thin layer chro-matography and then identified according to the methoddescribed previously (Tindall et al., 2007).

DNA–DNA hybridization (DDH) estimated values werecalculated by the genome-to-genome distance calculator(GGDC2.0) (Auch et al., 2010a, b; Meier-Kolthoff et al.,2013).

Strain MP-1T is a Gram-stain-negative, non-spore-form-ing, rod-shaped, 0.6–0.9 mm61.3–1.6 mm bacterium withmotile subpolar flagella (Fig. S1, available with the onlineSupplementary Material). The diameter of the colonies was1.5–2.0 mm after incubation on PY agar plates at 30 uCfor 2 days. Colonies were circular, yellowish, moist and

translucent with a regular margin. The growth of strainMP-1T was observed at 0–1 % NaCl (w/v) (optimum0.5 %), at 15–45 uC (optimum 30 uC) and at pH 5–9(optimum pH 7). Other characteristics are described below.

A nearly full-length 16S rRNA gene sequence of strain MP-1T was obtained of 1498 bp. A BLAST search showed it hadhighest similarity with the sequences of B. grimmiae DSM25160T (98.5 %), B. zhejiangensis OP-1T (98.2 %), B. choicaLMG 22940T (97.5 %), B. glathei DSM 50014T (97.4 %), B.terrestris LMG 22937T (97.2 %) and B. telluris LMG 22936T

(97.0 %). A dendrogram based on the neighbour-joiningmethod was constructed, indicating the relationshipbetween strain MP-1T and related species of the genusBurkholderia (Fig. 1). Strain MP-1T with six close relativesconstituted a phylogenetic branch. However, it did notform a cluster with any type strain of species of the genusBurkholderia.

In order to analyse the affiliation of strain MP-1T withthe most closely related species of the genus Burkholderia,we performed a multilocus sequence analysis using two

Burkholderia graminis LMG 18924T (U96939)

0.01

99

89

100

8690

93

81

Burkholderia phenoliruptrix LMG 22037T (ASXI01000083)Burkholderia rhynchosiae WSM3937T (EU219865)

Burkholderia terricola LMG 20594T (AY040362)Burkholderia caledonica LMG 19076T (AE215704)

Burkholderia xenovorans LB400T (CP000270)Burkholderia bryophila LMG 23644T (AM489501)Burkholderia fungorum LMG 16225T (AF215705)

Burkholderia phenazinium LMG 2247T (U96936)Burkholderia phymatum LMG 21445T (CP001043)Burkholderia caribensis MWAP64T (Y17009)

Burkholderia hospita LMG 20598T (AY040365)Burkholderia sprentiae WSM5005T (HF549035)

Burkholderia tuberum LMG 21444T (AJ302311)Burkholderia kururiensis LMG 19447T (AB024310)

Burkholderia tropica LMG 22274T (AJ420332)Burkholderia ferrariae LMG 23612T (DQ514537)

Burkholderia silvatlantica LMG 23149T (AY965240)Burkholderia mimosarum LMG 23256T (AY752958)

Burkholderia sacchari LMG 19450T (AF263278)Burkholderia gladioli CIP 105410T (EU024168)

Burkholderia plantarii LMG 9035T (U96933)Burkholderia glumae LMG 2196T (AMRF01000003)

Burkholderia pyrrocinia LMG 14191T (U96930)Burkholderia multivorans ATCC BAA-247T (ALIW01000278)

Burkholderia oklahomenisis LMG 23618T (ABBG01000575)Burkholderia grimmiae DSM 25160T (JN256678)

Burkholderia zhejiangensis OP-1T (HM802212)

Burkholderia telluris LMG 22936T (HE981727)Burkholderia glathei DSM 50014T (Y17052)

Burkholderia choica LMG 22940T (AY949196)Burkholderia terrestris LMG 22937T (HE981726)

Achromobacter xylosoxidans DSM 10346T (Y14908)

Burkholderia jiangsuensis MP-1T (KJ400396)

Fig. 1. Neighbour-joining phylogenetic tree of strain MP-1T and representatives of other related taxa based on 16S rRNA genesequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. Bar, 0.01 ntsubstitution rate (Knuc) units.

Burkholderia jiangsuensis sp. nov.

http://ijs.sgmjournals.org 3249

housekeeping genes, gyrB and recA, which were obtainedfrom seven strains (Estrada-de los Santos et al., 2013). Thus,a gyrB functional gene segment (1342 bp) was obtainedfrom the genome sequence of strain MP-1T. Strain MP-1T

showed relatively low gyrB sequence similarity to thesequences of six closely related type strains (,89.0 %).The topology of the phylogenetic trees generated from gyrB(based on a 557 bp sequence) showed that strain MP-1T wasclosely related to B. zhejiangensis OP-1T, B. grimmiae DSM25160T and B. glathei DSM 50014T, but did not clusterwith any other species (Fig. 2). In addition, the recA genesequences of strain MP-1T and six closely related type strainsshowed less than 95.1 % similarities. Phylogenetic analysisdemonstrated that MP-1T did not constitute one clusterwith any strain (Fig. 3), indicating that strain MP-1T shouldnot be assigned to any recognized species.

The DNA G+C content of strain MP-1T was calculatedfrom draft genome sequences (62.6 mol%). There were tinydifferences between the values determined for strain MP-1T

and for the three other type strains (B. zhejiangensis OP-1T,B. grimmiae DSM 25160T and B. glathei DSM 50014T)(62.6–64.4 mol%). All DNA G+C contents were calculatedfrom draft genome sequences.

The principal fatty acids in strain MP-1T were identified asC18 : 1v7c/C18 : 1v6c (23.3 %), C16 : 0 (16.8 %), cyclo-C17 : 0

(15.0 %), C16 : 1v7c/C16 : 1v6 (8.5 %), cyclo-C19 : 0v8c(8.1 %), C16 : 1 iso I/C14 : 0 3-OH (5.7 %), C16 : 0 3-OH(5.6 %) and C16 : 0 2-OH (5.1 %). As shown in Table S1, themajor fatty acids of the seven type strains were all composedof C16 : 0, C16 : 0 3-OH, C16 : 1 iso I/C14 : 0 3-OH and C14 : 0,indicating that the fatty acid profile of strain MP-1T wasidentical to those of the six closely related type strains.However, there was a significant difference between C16 : 1

2-OH, cyclo-C17 : 0 and cyclo-C19 : 0v8c among seven strains.Although a few relevant data (C18 : 0, C18 : 1 2-OH, C19 : 0

10-methyl, C16 : 1v7c/C16 : 1v6c and C18 : 1v7c/C18 : 1v6c)about B. choica LMG 22940T, B. terrestris LMG 22937T

and B. telluris LMG 22936T are not indicated in the literature(Vandamme et al., 2013), the different proportions ofvarious fatty acids showed individual differences.

The major isoprenoid quinone of strain MP-1T wasdetermined to be ubiquinone Q-8. This is consistent withall other members of the genus Burkholderia (Lu et al.,2012; Tian, et al., 2013). The polar lipid profile of strainMP-1T contained phosphatidyl glycerol (PG), phosphatidylethanolamine (PE), aminolipid (AL) and phospholipid

Burkholderia phenazinium LMG 2247T (HQ849206)0.1

98

89

86

92

9498

Burkholderia bryophila LMG 23644T (HQ849188)Burkholderia xenovorans LMG 21463T (HQ849219)

Burkholderia fungorum LMG 16225T (GU144391)Burkholderia rhynchosiae WSM3937T (HE994045)

Burkholderia caledonica LMG 19076T (HQ849189)Burkholderia graminis LMG 18924T (GU144400)

Burkholderia terricola LMG 20594T (HQ849215)Burkholderia phenoliruptrix LMG 22037T (HQ849207)Burkholderia sprentiae WSM5005T (HE994059)Burkholderia tuberum LMG 21444T (HQ849217)

Burkholderia ferrariae LMG 23612T (HQ849193)Burkholderia tropica LMG 22274T (HQ849216)

Burkholderia sacchari LMG 19450T (HQ849212)Burkholderia mimosarum LMG 23256T (HQ849202)Burkholderia silvatlantica LMG 23149T (HQ849213)

Burkholderia hospita LMG 20598T (GU144377)Burkholderia phymatum LMG 21445T (HQ849208)Burkholderia caribensis MWAP64T (EU024190)

Burkholderia grimmiae DSM 25160T (KI535376)Burkholderia zhejiangensis OP-1T (HF547269)

Burkholderia glathei DSM 50014T (HF985172)Burkholderia terrestris LMG 22937T (HF985170)Burkholderia telluris LMG 22936T (HF985161)Burkholderia choica LMG 22940T (HF985163)

Burkholderia kururiensis LMG 19447T (HQ849201)Burkholderia oklahomensis LMG 23618T (HQ849205)Burkholderia pyrrocinia LMG 14191T (HQ849211)

Burkholderia multivorans ATCC BAA-247T (HQ849203)Burkholderia gladioli CIP 105410T (EU024237)

Burkholderia glumae LMG 2196T (HQ849198)Burkholderia plantarii LMG 9035T (HQ849210)

Achromobacter xylosoxidans DSM 10346T (JQ914275)


Fig. 2. Neighbour-joining phylogenetic tree of strain MP-1T and representatives of other related taxa based on gyrB genesequences. Bootstrap values (expressed as percentages of 1000 replicates) are shown at branch points. Bar, 0.1 ntsubstitution rate (Knuc) units.



(PL) (Fig. S2). However, two type strains of the genusBurkholderia (B. grimmiae DSM 25160T, B. zhejiangensisOP-1T) contained PG, PE, uncharacterized AL andunknown PL (Lu et al., 2012; Tian et al., 2013). The majorprofile of MP-1T was similar to that of closely relatedstrains, except for the absence of glycolipid (GL) comparedto B. grimmiae DSM 25160T.

The draft genome sequence of the novel strain MP-1T

(JFHF00000000, Liu et al., 2014), along with those of threetype strains [B. grimmiae DSM 25160T (JFHE00000000), B.zhejiangensis OP-1T (JFHD00000000) and B. glathei DSM50014T (JFHC00000000)], were deposited in GenBank.

Estimated values of DDH between the four typestrains were calculated using GGDC2.0 with the BLAST+

alignment method. The estimated DDH values betweenstrain MP-1T and the three type strains were 24.6 %––37.4 %, which are below the threshold of 70 % for thedelineation of a species (Wayne et al., 1987). This resultconfirms that strain MP-1T represents a novel speciesof the genus Burkholderia.

Phylogenetic, phenotypic and chemotaxonomic analysesassigned strain MP-1T to the genus Burkholderia. Strain

MP-1T could be set apart from closely related species dueto some differential phenotypic characteristics, which arelisted in Table 1. Owing to the low estimated DDH value(,38 %) with closely related species, the isolate MP-1T

should not be assigned to any previously reported species.In conclusion, strain MP-1T is proposed to represent anovel species of the genus Burkholderia, for which the nameBurkholderia jiangsuensis sp. nov. is suggested.

Description of Burkholderia jiangsuensis sp. nov.

Burkholderia jiangsuensis (jiang.su.en’sis. N.L. fem. adj.jiangsuensis of Jiangsu, a province of the People’s Republicof China, where the type strain was isolated).

Cells are Gram-stain-negative, non-spore-forming, rod-shaped, 0.6–0.9 mm61.3–1.6 mm, motile by a subpolarflagella. The diameter of colonies is 1.5–2.0 mm afterincubation on PY agar plates at 30 uC for 2 days; coloniesare circular, yellowish, moist and translucent with a regularmargin. Growth is observable at 0–1 % NaCl (w/v)(optimum 0.5 %), at 15–45 uC (optimum 30 uC) and atpH 5–9 (optimum pH 7). With the API 20NE kit nitrate isreduced to nitrite and D-glucose, L-arabinose, D-mannose,D-mannitol, N-acetylglucosamine, potassium gluconate,

Burkholderia phenazinium LMG 2247T (AY619668)

Burkholderia bryophila LMG 23644T (HQ849133)Burkholderia xenovorans LMG 21463T (HQ849164)

Burkholderia fungorum LMG 16225T (AJ549505)

0.02

97100

9591

90

95

Burkholderia rhynchosiae WSM3937T (HE994064)Burkholderia caledonica LMG 19076T (AY619669)

Burkholderia graminis LMG 18924T (AJ551267)Burkholderia terricola LMG 20594T (HQ398599)

Burkholderia phenoliruptrix LMG 22037T (HQ849150)

Burkholderia sprentiae WSM5005T (HE994077)Burkholderia tuberum LMG 21444T (AY644642)

Burkholderia ferrariae LMG 23612T (HQ849137)Burkholderia tropica LMG 22274T (HQ849161)

Burkholderia sacchari LMG 19450T (AY644641)Burkholderia mimosarum LMG 23256T (HQ849146)

Burkholderia silvatlantica LMG 23149T (HQ849157)

Burkholderia hospita LMG 20598T (FJ958192)

Burkholderia phymatum LMG 21445T (AY644640)Burkholderia caribensis MWAP64T (AY644639)

Burkholderia grimmiae DSM 25160T (KJ535373)

Burkholderia zhejiangensis OP-1T (KJ535370)Burkholderia glathei DSM 50014T (AY619666)

Burkholderia terrestris LMG 22937T (KJ535371)

Burkholderia telluris LMG 22936T (KI535372)Burkholderia choica LMG 22940T (KJ535374)

Burkholderia kururiensis LMG 19447T (AY619654)

Burkholderia oklahomensis LMG 23618T (HQ849148)

Burkholderia pyrrocinia LMG 14191T (HQ849155)Burkholderia multivorans ATCC BAA-247T (HE981730)

Burkholderia gladioli CIP 105410T (AY619665)

Burkholderia glumae LMG 2196T (AJ551324)Burkholderia plantarii LMG 9035T (AJ551323)

Achromobacter xylosoxidans DSM 10346T (NC023061)


Fig. 3. Neighbour-joining phylogenetic tree of strain MP-1T and representatives of other related taxa based on recA genesequences. Bootstrap values (expressed as percentages of 1000 replicates) are shown at branch points. Bar, 0.02 ntsubstitution rate (Knuc) units.



capric acid, malic acid, trisodium citrate, phenylacetic acidare used and adipic acid weakly used, but not argininedihydrolase, urease, b-glucosidase, b-galactosidase andmaltose. It cannot denitrify, produce indole and hydrolysegelatin. With the 20E kit, it is positive for citrate utilizationand weakly positive for acetoin production (Voges–Proskauer), glucose, rhamnose and arabinose, but negativefor lysine decarboxylase, ornithine decarboxylase, H2Sproduction, tryptophan deaminase, gelatinase, inositol,sorbitol, sucrose, melibiose and amygdalin. With APIZYM test strips it is positive for alkaline phosphatase,esterase (C4), lipase (C8), leucine aminopeptidase andacid phosphatase, and weakly positive for valine amino-peptidase and naphthol-AS-Bl-phosphoamidase, butnegative for lipase (C14), cystine aminopeptidase, trypsin,a-chymotrypsin, a-galactosidase, b-galactosidase, b-glucur-onidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosa-minidase, a-mannosidase and a-fucosidase. It has the

ability to degrade MP, a common organophosphoruspesticide, which can be utilized as the sole carbon sourcefor growth. The major fatty acids are C18 : 1v7c/C18 : 1v6c,C16 : 0, cyclo-C17 : 0, C16 : 1v7c/C16 : 1v6, cyclo-C19 : 0v8c,C16 : 1 iso I/C14 : 0 3-OH, C16 : 0 3-OH and C16 : 0 2-OH.The respiratory quinone is Q-8. The polar lipids arecomprised of PG, PE, AL and PL.

The type strain, MP-1T (LMG 27927T5MCCC 1K00250T),was isolated from MP-contaminated soil of a pesticidemanufacturing company in Jiangsu Province, PR China.The chromosomal DNA G+C content of the type strain is62.6 mol%

Acknowledgements

This work was supported financially by the Ministry of Science and

Technology (2012AA022206C and 2011CB710800). The authors are

Table 1. Differential physiological characteristics between strain MP-1T and related species of the genus Burkholderia

Strain numbers: 1, MP-1T; 2, B. grimmiae DSM 25160T; 3, B. zhejiangensis OP-1T; 4, B. choica LMG 22940T; 5, B. glathei DSM 50014T; 6, B. terrestris

LMG 22937T; 7, B. telluris LMG 22936T. Reactions using API 20NE, API 20E and API ZYM tests were tested in this study. Characteristics are scored

as: +, positive; 2, negative; W, weakly positive; ND, no data.

Characteristics Strain number

1 2 3 4D 5 6d 7§

API 20NE

Reduction of nitrate to nitrite + + + – + – +

D-Glucose fermentation W – – ND – ND ND

D-Glucose + + + ND + ND ND

L-Arabinose + + W – + – +

Capric acid + + + – + W +

Adipic acid W W W + W – –

Trisodium citrate + W W – + + +

Phenylacetic acid + + + W + + +

API 20E

b-Galactosidase – – – ND W ND ND

Citrate utilization + + W ND + ND ND

Acetoin production (Voges–Proskauer) W + + ND – ND ND

Fermentation of glucose W + + ND – ND ND

Fermentation of mannitol – + W ND + ND ND

API ZYM

Alkaline phosphatase + + + – + + +

Esterase (C4) + + + + + + W

Esterase/lipase (C8) + + + + + + W

Lipase (C14) – w – ND W ND ND

Leucine aminopeptidase + + + ND ND ND ND

Valine aminopeptidase W W W – W W –

Cystine aminopeptidase – W W – W – –

a-Chymotrypsin – – – ND W ND ND

Acid phosphatase + + + ND + ND ND

Naphthol-AS-Bl-phosphoamidase W W W ND W ND ND

DNA G+C content (mol%)* 62.6 63.0 62.7 63.0 64.4 62.0 64.0

*DNA G+C contents were calculated from draft genome sequences.

D, d, §, Data from Vandamme et al. (2013); the rest are data from this study.



grateful to Professor Qing Hong at Nanjing Agricultural Universityfor kindly providing type strains.

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