ORIGINAL PAPER
Diversity of Crenarchaeota in terrestrial hot springs in Tengchong,China
Zhao-Qi Song •Jing-Quan Chen •Hong-Chen Jiang •
En-Min Zhou •Shu-Kun Tang •Xiao-Yang Zhi •Li-Xin Zhang •Chuan-Lun L.Zhang •Wen-Jun Li
Received:2November 2009/Accepted:12March 2010ÓSpringer 2010
Abstract Diversity of Crenarchaeota was investigated in eight terrestrial hot springs (pH 2.8–7.7;temperature 44–96°C)located in Tengchong,China,using 16S rRNA gene phylogenetic analysis.A total of 826crenarchaeotal clones were quenced and a total of 47operational taxonomic units (OTUs)were identified.Most (93%)of the identified OTUs were cloly related (89–99%)to tho retrieved from hot springs and other thermal environments.Our data showed that temperature may predominate over pH in affecting crenarchaeotal diversity in Tengchong hot springs.Crenarchaeotal diversity in moderate-temperature (59–77°C)hot springs was the highest,indicating that the
moderately hot-temperature springs may provide optimal conditions for speciation of Crenarchaeota.Keywords Crenarchaeota ÁDiversity ÁTerrestrial hot springs ÁTengchong
Introduction
Archaea are divided into Euryarchaeota,Crenarchaeota,Korarchaeota and Nanoarchaeota (Dawson et al.2006).Numerous studies have been performed to investigate the diversity of Euryarchaeota and Crenarchaeota in different environments (e review by Schleper et al.2005;Zhang et al.2010).The cultivated euryarchaeotes consist of diver mesophilic and thermophilic anaerobes and halo-philes,whereas the cultivated crenarchaeotal members mainly consist of hyperthermophilic sulfur-dependent thermophilic species (Huber et al.2006)and recently
retrieved ammonia-oxidizing archaeal strains (Ko
¨nneke et al.2005;de la Torre et al.2008).The phylum Cre-narchaeota reprents one of the deep-branching phyloge-netic lineages within prokaryotes (Woe et al.1990;Winker and Woe 1991).Since 1990s,multiple studies using culture-independent approaches have shown the ubiquity of the crenarchaeotes in oceans (DeLong 1992;Fuhrman et al.1992),lakes (Schleper et al.1997),soils (Ochnreiter et al.2003),and other low temperature environments.Recently,mesophilic Crenarchaeota are named Thaumarchaeota bad on the genome quence of Cenarchaeum symbiosum which was discovered in marine environment (Broc
hier-Armanet et al.2008).The majority of known species of hyperthermophilic Crenarchaeota grow optimally at temperatures C 80°C (Stetter 1996;Boone and Castenholz 2001).Most of them are able to
Communicated by A.Oren.
Z.-Q.Song ÁJ.-Q.Chen ÁE.-M.Zhou ÁS.-K.Tang ÁX.-Y.Zhi ÁW.-J.Li (&)
The Key Laboratory for Microbial Resources of the Ministry of Education,Yunnan University,Kunming,China e-mail:wjli@ynu.edu
Z.-Q.Song ÁJ.-Q.Chen ÁE.-M.Zhou ÁS.-K.Tang ÁX.-Y.Zhi ÁW.-J.Li
Laboratory for Conrvation and Utilization of Bio-Resources,Yunnan Institute of Microbiology,Yunnan University,Kunming 650011,China
H.-C.Jiang
Geomicrobiology Laboratory,State Key Laboratory of Geological Process and Mineral Resources,
China University of Geosciences,Beijing 100083,China L.-X.Zhang ÁW.-J.Li
快速违章查询
Chine Academy of Sciences,Institute of Microbiology,Beijing 100101,China
C.-L.L.Zhang
Department of Marine Sciences,University of Georgia,Athens,GA 30602,USA
Extremophiles
DOI 10.1007/s00792-010-0307-6"创业项目网"
grow chemolithoautotrophically on H2,S,or H2S,using O2,NO3-,S,or Fe3?as electron acceptors(Stetter1996; Spear et al.2005;Dawson et al.2006),the compounds of which are widely prent in hot springs.
Hot springs are a type of extreme environments widely distributed all over the world,and they are unique with respect to physical,chemical and geographical character-istics(Hugenholtz et al.1999;Marteinsson et al.2001; Kvist et al.2005;Spear et al.2005).Multiple studies have shown that hot springs harbor very diver creanarchaeotal populations(Takai and Sako1999;Jackson et al.2001; Kanokratana et al.2004;Kvist et al.2005;Meyer-Dombard et al.2005;Skirnisdottir et al.2000;Spear et al. 2005;Huang et al.2007;Costa et al.2009;Vick et al. 2010).Recent molecular studi
es showed that hot springs of moderate temperature(55–70or65–70°C)posss the highest bacterial and archaeal diversity(Lau et al.2006). This obrvation is in contrary with the general ecological principle that more extreme environments decrea diver-sity(Frontier1985;Hacine et al.2004).Is thefinding of Lau and colleagues applied to other geothermal features in the world?If yes,could the temperature range be wider for hot springs having the highest crenarchaeotal diversity?
To answer the above questions,we investigated the crenarchaeotal diversity in hot springs in Tengchong,China with the u of crenarchaeotal16S rRNA gene phylogenetic analysis.The results showed that temperature predominated over pH in affecting crenarchaeotal diversity in the investi-gated hot springs,and the crenarchaeotal diversity in mod-erate-temperature(59–77°C)hot springs was the highest. Materials and methods
Site description and sample collection
Tengchong(in western Yunnan Province,China)is located on the northeastern edge of Tibet–Yunnan geothermal zone between the Indian and Eurasian plates,one of the most active geothermal areas in the world.Tengchong hot springs posss a variety of hydrothermal phenomena,such as hydrothermal explosions,geyrs,fumaroles,boiling springs and hot springs(Kearey and Wei1993).
The investigated hot springs in this study are located in an area of one square kilometer or so in Tengchong National Geological Park(N24°570,E98°260).Surface diment or mat material was collected into sterile50-mL Falcon conical tubes at each hot spring.A total of eight samples were collected from different hot springs with different characteristic of pH and temperature.All samples were stored in dry ice in thefield and during transportation, and then at-80°C in the laboratory until further analysis. At each sampling location,pH and temperature were measured using a HachÒpH-meter equipped with a pH/temperature probe.
DNA extraction
Total DNA was extracted from5to10g of each sample using an UltraClean Mega DNA soil kit(Mo Bio Labora-tories,Inc.,Carlsbad,CA,USA)according to the manu-facturer’s instructions.The precipitated DNA was gel-purified using Agaro Gel DNA Fragment Recovery Kit Ver.2.0(TaKaRa,Dalian,China).
Construction of crenarchaeotal16S rRNA gene clone libraries
Purified DNA was ud as the template for the amplifica-tion of crenarchaeotal16S rRNA genes by specific primers Cren7F:50-TTC CGG TTG ATC CYG CCG GAC C-30and Cren518R:50-GCT GGT WTT ACC GCG GCG GCT GA-30 (Perevalova et al.2008).PCR amplification consisted of an initial d
enaturation at95°C for10min,followed by denaturation at95°C for45s,annealing at72°C for2min; 30cycles,with the last cycle being followed by afinal extension at72°C for20min.Individual reagents and their concentrations were as follows:19PCR buffer with 1.5mM Mg2?,dNTPs(100l M each),0.25l M each pri-mer, 2.5U of DNA polymera(Ex-Taq)(TaKaRa, Dalian,China),and*50ng of total DNA.PCR products were purified using an Agaro Gel DNA Fragment Recovery Kit Ver.2.0(TaKaRa,Dalian,China)according to the manufacturer’s instructions.
Purified PCR products were ligated into pMD19-T Vector system(TaKaRa,Dalian,China)and transformed into competent Escherichia coli JM109cells according to the manufacturer’s instructions.Eight clone libraries were constructed,one for each of the eight hot spring samples in Tengchong.70to160positive clones per library were randomly lected and plasmid DNA was purified using DNA Fragment Purification Kit Ver.2.0(TaKaRa,Dalian, China).Restriction fragment length polymorphism(RFLP) analysis was performed on the randomly lected clones in each clone library according to procedures described elwhere(Song et al.2009).Unique RFLP patterns were identified visually and reprentatives of each RFLP were lected for16S rRNA gene phylogenetic analysis. Phylogenetic analysis
Clones reprenting all identified RFLP types were quenced using primer M13?on an ABI3100aut
omated quencer.The quences were edited using the DNASTAR program v.5.0.The potential prence of chimeric quen-ces was examined with Bellerophon(Huber et al.2004).
Extremophiles
The condary-structures of all obtained quences were analyzed using the Vienna RNA Package(Hofacker2003). Potential chimeric quences were removed.Operational taxonomic units(OTUs)were identified using DOTUR1.53 (Schloss and Handelsman2005).The quences of C98% identity were clustered into one OTU.One quence was lected from each OTU for phylogenetic analys.The reprentative quences of each OTU and lected clost references(bi.v)were pooled and aligned using CLUSTALX1.83.Phylogenetic analysis was performed using distance-bad Neighbor-Joining method with MEGA version4.0(Tamura et al. 2007).Bootstrap analysis was performed using1000 pudoreplications.
Statistical analys
Species richness was calculated for each of the hot springs using the nonparametric estimators ACE(abundance-bad coverage estimator).Coverage was calculated with the equation C=1-n/N,wh
ere n is the number of unique OTU quences obrved and N is the number of total OTUs.In addition,a rarefaction analysis of the number of unique OTUs versus the estimated OTUs was performed with the u of Rarefaction version 1.3(www. uga.edu/strata/software/Software.html).The F statistical analysis was calculated for asssing the degree of differ-entiation between microbial communities and comparing the phylogenetic diversity of each community with the total phylogenetic diversity of the combined communities.F is defined by the equation F ST=(h T-h W)/h T,where h T is the phylogenetic diversity of the two communities combined and h W is the phylogenetic diversity of each community (Martin2002;Whitaker et al.2003;Mathur et al.2007). Linear regression analys were performed to evaluate the correlation between the F st and temperature/pH distances using SYSTAT SigmaPlot.v10.0package(Version10.0).
Nucleotide quence accession numbers
The quences obtained in this study were deposited in the GenBank databa under accession numbers:GU075731–GU075793.
Results
Characteristics of sampling sites
The eight investigated hot springs in Tengchong posss temperature ranging from44to96°C with a pH gradient of 2.8–7.7(Table1).
Table1Description of hot spring samples investigated in this study
ND not determined Sample Sample type Temp(°C)pH Total dissolved
solids(TOD)(mg/L)
Peal spring(PLS)Brown sandy diment96 4.3245
Great boiling pot A(GBA)Ashen geyrite84 6.62070
Wuming spring(WMS)Gray diment777.7ND Huangguanqing spring(HGS)Sandy diment74 2.8ND
Bridge spring A(BSA)Black mat597.5656 Shuirebaozhaqu(SRQ)Green diment457.5ND
Great boiling pot F(GBF)Red diment44 3.4ND
Bridge spring B(BSB)Green mat447.5650
Table2Diversity indices of crenarchaeotal16S rRNA gene clone libraries retrieved from eight hot springs in Tengchong,Yuanan,China
Community No.
clones No.
OTUs
Richness
(ACE)
C coverage
(%)
Avg.BLAST identity
for all phylotypes(%)a
Shannons index
(Chao and Shen)
Simpson
index(MLE)
PLS754 4.0(4.0,4.0)10094 1.03(0.86,1.19)0.433(0.208,0.658) GBA676 6.0(6.0,6.0)10095.5 1.44(1.27,1.61)0.287(0.146,0.428) WMS1481515.5(15.0,20.5)99.393.9 2.41(2.23,2.58)0.116(0.085,0.148) HGQ1541010.0(10.0,10.0)10094.6 2.11(2.02,2.20)0.137(0.107,0.166) BSA1491111.5(11.0,17.5)99.394.1 2.09(1.87,2.31)0.149(0.101,0.197) SRQ615 5.0(5.0,5.0)10096.2 1.34(1.18,1.50)0.306(0.180,0.431) BSB846 6.0(6.0,6.0)10095.3 1.51(1.36,1.66)0.266(0.188,0.345) GBF8877.0(7.0,7.0)10097 1.64(1.49,1.80)0.241(0.170,0.311) a Average percentage similarity among all phylotypes from a given location with clost matches in the NCBI GenBank databa Extremophiles
T a b l e 3C l o n e s f r o m i n v e s t i g a t e d h o t s p r i n g s a n d c l o s e s t r e l a t i v e s d e t e r m i n e d b y D O T U R p r o g r a m a n d B L A S T
O T U I D P L S G B A
W M S
H G S B S A S R Q G B F B S B C l o s e s t r e l a t i v e (%16S r R N A g e n e i d e n t i t y )G e n B a n k a c c e s s i o n n o .
O T U 1P L S 9(4)
H o t s p r i n g c l o n e H v e r d 026A (93)
D Q 441483
O T U 2P L S 21(9)
H o t s p r i n g c l o n e H v e r d 026A (93)
D Q 441483
O T U 3P L S 51(28)
H o t s p r i n g c l o n e H v e r d 026A (93)
D Q 441483
O T U 4G B A 6(3)
夏天到了H o t s p r i n g H L 1e n v .11(93)
E U 239999
O T U 5
W M S 1(4)
H o t s p r i n g c l o n e S S W _L 4_E 05(96)
E U 635928
O T U 6
W M S 5(1)
H o t s p r i n g c l o n e S S W _L 5_H 08(94)
E U 635922
O T U 7W M S 51(2)H o t s p r i n g c l o n e Y N P _B P _A 49(91)D Q 243731O T U 8
W M S 15(7)
H o t s p r i n g c l o n e Y N P _O b P _A 5(97)
D Q 243757
O T U 9
W M S 60(1)
E p i t h e r m a l g o l d m i n e c l o n e H A u D -L A 30(98)A B 113632
O T U 10
W M S 38(5)
H o t s p r i n g c l o n e L H C 4_L 2_G 04(92)
E U 635907
O T U 11
W M S 11(18)
H o t s p r i n g c l o n e Y N P _B P _A 60(91)
D Q 243732
O T U 12
W M S 27(5)
H y d r o t h e r m a l flu i d s c l o n e p Y K 04-13(91)
A B 235330
O T U 13
W M S 10(20)
H y d r o t h e r m a l A r c h a e a c l o n e a 87Y 32(91)
D Q 417485
O T U 14
taffetaW M S 64(1)
H o t s p r i n g c l o n e Y N P _B P _A 22(89)
D Q 228585
O T U 15道德小故事
W M S 3(5)
H o t s p r i n g c l o n e H v e r d 014N (94)
D Q 441506
O T U 16
H G S 14(2)
H o t s p r i n g c l o n e H v e r d 050N (94)
D Q 441516
O T U 17
H G S 17(14)
H o t s p r i n g c l o n e p J P 41(96)
L 25301
O T U 18
H G S 29(8)
H y d r o t h e r m a l a r c h a e o t a c l o n e a 87Y 32(92)
D Q 417485
O T U 19
H G S 61(6)
H y d r o t h e r m a l a r c h a e o t a c l o n e a 87Y 32(90)
D Q 417485
O T U 20
H G S 5(3)
S u l fid e v e n t c l o n e 4136-1-92(93)
E U 427996
O T U 21
B S A 3(2)
G e o t h e r m a l w a t e r c l o n e 10-H -08(95)
A B 201309
O T U 22B S A 8(11)G e o t h e r m a l w a t e r c l o n e 10-H -08(96)A B 201309O T U 23
B S A 56(22)
E p i t h e r m a l g o l d m i n e c l o n e H A u D -l a 42(97)
A B 113634
O T U 24
B S A 29(19)
M a n g r o v e c l o n e M K C S B -A 3(90)
D Q 363754
O T U 25
B S A 65(9)
H y d r o t h e r m a l flu i d c l o n e (94)
A B 301869
O T U 26
B S A 63(1)
S u l f u r o u s f r e s h w a t e r c l o n e 2C 3(89)
A J 937875
O T U 27
B S A 76(6)
R h i z o s p h e r e a r c h a e o t a c l o n e (92)
E F 021134
O T U 28
B S A 30(18)
R h i z o s p h e r e a r c h a e o t a c l o n e (94)
E F 021161
O T U 29
S R Q 68(3)
L a k e s e d i m e n t c l o n e L C D A R C H 142(98)
E U 247289
O T U 30
S R Q 46(39)
H o t s p r i n g D G G E b a n d B L 1017(90)
E U 586818
O T U 31
S R Q 62(16)
S o i l c l o n e A r c C -s _c B 07(97)
E U 307056
O T U 32
G B F 17(26)
T h e r m a l s o i l c l o n e Y N P F F A 108(91)
A B 072728
O T U 33
G B F 42(7)
M e t a l l o s p h a e r a s p .J 1(90)
须知和需知
A F 167083
O T U 34
G B F 2(9)
E p i t h e r m a l g o l d m i n e c l o n e (97)
A B 072728
O T U 35
G B F 9(13)
T h e r m a l s o i l c l o n e Y N P F F A 108(97)
A F 391993
Extremophiles
Diversity and phylogenetic analysis of Crenarchaeota
A total of eight crenarchaeotal 16S rRNA gene clone libraries (one for each hot spring sample)were constructed,and a total of 826clones were randomly picked and sub-jected to RFLP analysis (Table 2).The coverage calcula-tion (Table 2)and rarefaction analysis (data not shown)indicated that the analyzed clones almost covered the diversity of crenarchaeotal populations in every hot spring.One clone was lected from each RFLP type in each clone library,and a total of 64clones were quenced.All the obtained quences could be grouped into 47OTUs with the cutoff of 98%similarity (Table 2).
Most if not all retrieved OTUs in this study were cloly related (90–99%)to tho from thermal environments (Table 3).Fourteen OTUs obtained in this study were grouped into the class Thermoprotei ,and were cloly related (90–99%identity)to hyperthermophilic or ther-mophilic strains,and other OTUs were affiliated with clone quences retrieved from thermal environments.
Among the investigated hot springs,the crenarchaeotal communities in the three moderate-temperature (59–77°C)hot springs (WMS,HGS and BSA)were most diver,containing 15,10and 11OTUs,respectively.In contrast,the numbers of OTUs in other investigated hot springs were apparently lower than in the hot springs (Table 2).
Statistical analysis
Most F st values were higher than 0.2,indicating that community structures were significantly different (Lau et al.2009).It also was found that there were lower F st values between hot springs having similar temperatures.Furthermore,to show the relationships between tempera-ture/pH and crenarchaeotal community structures,linear regression analysis was performed bad on the F st and temperature/pH distances (Fig.2).It is clear that the F st values were linearly correlated with the temperature dis-tances (R 2=0.55)among Tengchong hot springs (Fig.2);pH values,however,do not have an obvious linear corre-lation with F st .
Discussion
Crenarchaeotal diversity in eight hot springs (temperature 44–96°C;pH 2.8–7.7)was investigated in this study.So far,most of the cultured Crenarchaeota are hyperthermo-philic and acidophilic microorganisms with an optimal growth temperature of 80–90and 65–70°C,respectively (Huber et al.2006).To our best knowledge,with the
T a b l e 3c o n t i n u e d
O T U I D P L S
G B A
W M S
H G S
B S A
S R Q G B F华为找手机
B S B
C l o s e s t r e l a t i v e (%16S r R N A g e n e i d e n t i t y )
G e n B a n k a c c e s s i o n n o .
O T U 36
B S B 127(11)P e t r o l e u m c o n t a m i n a t e d s o i l (99)
A B 161339
O T U 37
B S B 143(32)V u l c a n i s a e t a d i s t r i b u t a (98)
A B 063641
O T U 38P L S 4(59)
G B A 2(30)
S u l f o l o b u s s p .D G G 22(97t o 98)
F J 489516
O T U 39
G B A 34(39)
H G S 56(7)
T h e r m o p h i l i c r e a c t o r c l o n e (96)
D Q 383349
O T U 40
W M S 54(1)
H G S 15(20)
H o t s p r i n g c l o n e Y N P _O b P _A 5(97t o 98)
D Q 2437572
O T U 41
W M S 16(9)H G S 27(16)
H o t s p r i n g c l o n e H v e r d 014N (97t o 98)
D Q 441506
O T U 42
B S A 1(3)
国家放假安排
B S B 44(8)
S o i l c l o n e A r c C -u _c E 05(94)E U 307069
O T U 43
S R Q 14(7)
G B F 25(3)
R h i z o s p h e r e a r c h a e o t a c l o n e (98t o 99)
E F 020745
O T U 44G B A 8(3)
W M S 12(9)
H G S 13(11)
S u l fid e v e n t c l o n e 4136-1-92(93)E U 427996O T U 45
B S A 36(2)
G B F 55(38)B S B 125(37)E p i t h e r m a l g o l d m i n e c l o n e (97t o 98)
A B 072728
O T U 46
G B A 7(4)
S R Q 20(34)G B F 60(5)
B S B 6(4)
H o t s p r i n g c l o n e p J P 41(97t o 98)
L 25301
O T U 47
G B A 1(21)
W M S 13(9)H G S 23(17)B S A 51(7)
B S B 139(8)H o t s p r i n g c l o n e S S E _L 4_H 05(97)
E u 635920
T h e n u m b e r i n t h e p a r e n t h e s e s m e a n s t h e n u m b e r o f p e r c e n t a g e o f t h e t o t a l c l o n e l i b r a r y
Extremophiles
