Comparative genomic and physiological analysis provides insights into the role of Acidobacteria in

15 Pages·2012·0.95 MB·English

RESEARCH ARTICLE
Comparative genomic and physiological analysis provides
Acidobacteria
insights into the role of in organic carbon
utilization in Arctic tundra soils
Suman R. Rawat1, Minna K. Ma¨nnisto¨2, Yana Bromberg1 & Max M. Ha¨ggblom1
1DepartmentofBiochemistryandMicrobiology,SchoolofEnvironmentalandBiologicalScience,Rutgers,TheStateUniversityofNewJersey,
NewBrunswick,NJ,USA;and2FinnishForestResearchInstitute,Rovaniemi,Finland
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CDoeprraerstmpoenntdoefncBeio:cMheamxiMstr.yHaa¨ngdgblom, Abstract d fro
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Acidobacteria are among the most abundant bacterial phyla found in terrestrial
Microbiology,Rutgers,TheStateUniversityof h
NewJersey,NewBrunswick,NJ08901,USA. ecosystems, but relatively little is known about their diversity, distribution and ttp
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Tel.:[phone];fax:[phone] most critically, their function. Understanding the functional activities encoded ://a
8965;e-mail:[email protected] in their genomes will provide insights into their ecological roles. Here we ca
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describe the genomes of three novel cold-adapted strains of subdivision 1 e
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Received19December2011;revised20 Acidobacteria. The genomes consist of a circular chromosome of 6.2 Mbp for ic
February2012;accepted6March2012. .o
Finalversionpublishedonline30April2012. GMrPa5nAulCicTelXla9, manadllen5s.i0sMMbpP5AfoCrTTXe8r,rig4lo.3buMs bspaanfeonrsisGSrPa1nPuRlic4e.llaIn tuadnddritiicoonla, up.co
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DOI:10.1111/j.1574-6941.2012.01381.x G. tundricola has five mega plasmids for a total genome size of 5.5 Mbp. The /fe
three genomes showed an abundance of genes assigned to metabolism and m
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Editor:DirkWagner transport of carbohydrates. In comparison to three mesophilic Acidobacteria, c
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namely Acidobacterium capsulatum ATCC 51196, ‘Candidatus Koribacter versa- rtic
Keywords tilis’ Ellin345, and ‘Candidatus Solibacter usitatus’ Ellin6076, the genomes of le
Y Granulicella;Terriglobus;tundra;soilorganic the three tundra soil strains contained an abundance of conserved genes/gene -ab
G matter;carbohydrate-activeenzymes. clusters encoding for modules of the carbohydrate-active enzyme (CAZyme) stra
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O family. Furthermore, a large number of glycoside hydrolases and glycosyl trans- t/8
2
L ferases were prevalent. We infer that gene content and biochemical mechanisms /2/3
O encoded in the genomes of three Arctic tundra soil Acidobacteria strains are 41
shaped to allow for breakdown, utilization, and biosynthesis of diverse struc- /4
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C 7
tural and storage polysaccharides and resilience to fluctuating temperatures 4
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E and nutrient-deficient conditions in Arctic tundra soils. 9 b
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Introduction habitats, only a few have been cultivated and described. t o
O Acidobacteria have been divided in up to 26 phylogenetic n 0
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L Acidobacteria represent one of the most abundant and subdivisions based on 16S rRNA gene phylogeny (Barns A
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O umbeinqtusito(Buasrnbsacteetriaall.,ph1y9l9a9;foJuanndsseinn,g2lo0b0a6l; sFoiielreernveitroanl.-, eatbuanl.,da2n0t0l7y)doeftewchteicdhisnubsdoiivliseionnvsiro1,nm3,e4n,tsan(dJo6neasreemt aols.t, ril 20
I 1
B 2007; Jones et al., 2009; Kielak et al., 2009; Lauber et al., 2009). The phylogenetic diversity, ubiquity, and abun- 9
O 2009;Lee&Cho,2009;Eichorstet al.,2011),andtheyare dance of this group suggest that they play important eco-
widely distributed in Arctic and boreal soils (Goulden logical roles in soils. Acidobacteria are assumed to be
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et al., 1998; Neufeld & Mohn, 2005; Dedysh et al., 2006; genetically and metabolically diverse, as they inhabit a
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Ma¨nnisto¨ et al., 2007, 2009; Lee et al., 2008; Pankratov wide variety of natural environments over a range of tem-
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M et al., 2008, 2011; Campbell et al., 2010; Chu et al., 2010). perature, salinity, organic matter, and pH (Jones et al.,
Nevertheless, relatively little is still known about their 2009; Faoro et al., 2010; Ganzert et al., 2011). The abun-
functional and ecological roles in these soils. Despite a dance of Acidobacteria correlates with soil pH, with sub-
large collection of Acidobacteria 16S rRNA gene sequences group 1 Acidobacteria being the most abundant in slightly
in databases representing diverse species from various acidic soils (Kishimoto et al., 1991; Ma¨nnisto¨ et al., 2007;
FEMSMicrobiolEcol82(2012)341–355 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
342 S.Rawatetal.
Kleinsteuber et al., 2008; Jones et al., 2009; Lauber et al., 2003; Davis et al., 2005). Our study provides genomic
2009; Chu et al., 2010). An increasing number of Acido- insights into the ecology of these Acidobacteria communi-
bacteria have recently been cultivated and described (for ties in turnover of soil organic carbon in Arctic and bor-
references see Ma¨nnisto¨ et al., 2012). Nonetheless, the eal environments.
paucity of well-characterized Acidobacteria hampers our
understanding of the physiology and ecological function
Materials and methods
of these organisms, as well as how they will respond and
adapttoenvironmentalchangeinthesesoilenvironments.
Habitat and strains of Acidobacteria
Arctic and boreal environments cover over 20% of the
terrestrial surface and harbor about one-third of the total Granulicella mallensis MP5ACTX8T (= DSM 23137 = ATCC
global soil carbon pool (Loya & Grogan, 2004). However, BAA-1857), G. tundricola MP5ACTX9T (= DSM 23138 =
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little is known about the microbial communities that uti- ATCC BAA-1859), and T. saanensis SP1PR4T (= DSM ow
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lize this large carbon pool, their activity, and community 23119 = ATCC BAA-1853) were isolated from Arctic tun- lo
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dynamics, despite their critical role in carbon mineraliza- dra heaths located in northern Finland (Männistö et al., d
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tion and potential impact on atmospheric CO2 and future 2011,2012).Allstrainsoriginatedfromtheorganiclayerof fro
climate change. Acidobacteria have been reported to dom- soil samples collected from oligotrophic wind-swept hills m
inate soils rich in soil organic matter and are involved in that experience large annual temperature variation and fre- http
m(Eiiccrhoobrisatletdeagl.r,a2d0a1ti1o;nPaonfkrlaitgonvoceetllaul.l,os2i0c11p)l.aUntsinbgiocmomas-s qtiuoenntinfretehzees–ethsaitwescyiscledsomininthaeteadutbuymndwanadrfspshrirnugb.sVoefgetthae- s://ac
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bined molecular- and cultivation-based approaches, we Ericaceaefamily,whichproduceacidicorganicmatterwith de
have demonstrated that members of subdivision 1 Acido- a high C/N ratio (Eskelinen et al., 2009). The soil organic mic
bacteria are a dominant bacterial group that are active at matter content is high, c. 30–50% and acidic (pH 4.8–5.2). .ou
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low temperatures and resilient to multiple freeze–thaw Strains were cultivated from soil samples using R2A agar .c
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cycles in acidic tundra soils of northern Fennoscandia. In (SP1PR4) or a mixture of carboxymethyl cellulose (CMC), m
addition, Acidobacteria comprise up to > 50% of starch, and xylan as carbon sources (MP5ACTX8 and /fem
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sequences in clone libraries (Ma¨nnisto¨ et al., 2007, 2009). MP5ACTX9), and once obtained as pure cultures, the e
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A concerted effort led to the cultivation of several new strains were maintained and grown on R2 agar or broth /a
slow-growing and fastidious cold-adapted Acidobacteria (adjusted to pH 5.5, Difco) and stored at (cid:1)70 °C in 20% rticle
belonging to the genera Terriglobus and Granulicella glycerol. -a
b
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(Ma¨nnisto¨ et al., 2011, 2012). It appears that soils natu- tra
rally exposed to harsh and changing environmental con- Growth of strains and DNA extraction ct/8
ditions may harbor frost-tolerant and resilient bacterial 2/2
species. We hypothesize that these conditions have The tundra soil strains were grown aerobically on half- /3
4
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selected a stable bacterial community dominated by strength R2A medium, pH 5.5 at 20 °C. Genomic DNA /4
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Acidobacteria that is only minimally affected by tempera- of high sequencing quality was isolated using a hexade- 7
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ture fluctuation and freeze–thaw cycles. cyltrimethylammonium (CTAB) method (Doyle & Doyle, 99
b
Here, we report on the analysis of the genomes of three 1990) modified for genomic DNA extraction from bacte- y
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novel cold-adapted strains of subdivision 1 Acidobacteria, rial cells. Cells (OD of not more than 1.2) were u
600 nm e
Granulicella mallensis strain MP5ACTX8, Granulicella treated with 2% SDS and 250 lg mL(cid:1)1 proteinase K to st o
tundricola strain MP5ACTX9, and Terriglobus saanensis lyse the cells and incubated at 37 °C for 1 h. Then, CTAB n 0
6
strain SP1PR4, isolated from Arctic tundra soils (Fig. 1). buffer (1% CTAB, 0.75 M NaCl, 50 mM Tris pH 8, A
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Osuuprpogretneodmbiyc panhaylsyiosilsogoifcatlhcehtahrarecetertiuznatdiroanstooilasstsreasisnsthies 1100 mmiMn. ETDheTAsu)spweanssioadndwedasaenxdtrainctceudbaotnedceawtit6h5c°hClorfoo-r ril 20
1
mechanisms promoting their activity, dominance, and form/isoamyl alcohol (24 : 1) and then with phenol/chlo- 9
survival in these soil environments. These strains are roform/isoamyl alcohol (24 : 1). Finally, the DNA was
compared with three other Acidobacteria strains, for precipitated from the supernatant with 0.6 vol isopropa-
which finished genomes are available (Ward et al., 2009; nol ((cid:1)20 °C) at room temperature for 30 min. Genomic
Challacombe et al., 2011), namely Acidobacterium capsul- DNA was pelleted, washed with 70% ethanol, and dried.
atum ATCC 51196 isolated from acid mine drainage in The pellet was resuspended in TE (10 mM Tris, 1 mM
Japan (Kishimoto et al., 1991) and two other soil strains, EDTA) buffer containing 1 lL RNAse (10 mg mL(cid:1)1) and
‘Candidatus Koribacter versatilis’ Ellin345 and ‘Candida- incubated at 37 °C for 20 min. The DNA was evaluated
tus Solibacter usitatus Ellin6076’, both isolated from soils according to the quality control guidelines provided by
of rye grass/clover pasture in Australia (Joseph et al., the DOE Joint Genome Institute (DOE-JGI).
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)341–355
PublishedbyBlackwellPublishingLtd.Allrightsreserved
GenomicanalysisofthreetundrasoilAcidobacteriaspecies 343
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Subdivision 1 d
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Fig.1. Phylogenetictreebasedon16SrRNA m
ic
genesequencesshowingtherelationshipsof .o
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thethreetundrasoilstrainsandothercultured p
strainsofthephylumAcidobacteria.Bootstrap .co
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values(expressedaspercentagesof1000 /fe
replicates)of>50%areshownatbranch m
s
points.Theevolutionaryhistorywasinferred e
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uosnintghethTeammuarxaim–Nuemimlikoedliehloforodmmaetthootadlboafsed Subdivision 3 /artic
le
1167unambiguouslyalignednucleotide -a
poofstihtieonpshyinlutmhePfilannatlodmaytcaesteest.,TSwinogumlisepmhbaeerras Subdivision 4 bstra
c
acidiphilaATCCBAA-1392T(AM850678),and t/8
2
Isosphaerapallidastrain563(AJ231193)were Subdivision 6 /2
usedasoutgroup(notshown).Strainswith /3
4
finishedgenomesareindicatedinbold. 1/4
Accessionnumbersareinparentheses.Bar: 9
Subdivision 8 7
0.05substitutionspernucleotideposition. 49
9
b
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Genome sequencing and assembly Ivlelursmioinna0.7se.6q3ue(nZceirnbginoda&taBwirenreey,a2s0se0m8)b,leadndwtihtehcVoEnLsVeEnT-, uest o
Finished genomes for strains G. mallensis MP5ACTX8 sus sequences were computationally shredded into 1.5 kb n 0
(JGI ID 4088692), G. tundricola MP5ACTX9 (JGI ID overlapping fake reads (shreds). The 454 Newbler consen- 6 A
4w0e8r8e6g9e3n),eraatnedd aTt. sDaOanEenJsoisintSPG1ePnRo4me(JGInIstiItDute40u8s8in6g90a) rsueasdshpraeidrss,wtheereIlilnutmeginraateVdELiVnETthcoen4s5en4supsaisrhedre-dens,danlidbrtahrye pril 20
combination of Illumina (Bennett, 2004) and 454 tech- using parallel phrap, version SPS – 4.24 (High Perfor- 19
nologies (Margulies et al., 2005). Three libraries, an Illu- mance Software, LLC). The software CONSED (Ewing et al.,
mina GAii shotgun library, a 454 Titanium standard 1998; Gordon et al., 1998) was used in the following fin-
library, and a paired-end 454 library were constructed. ishing process. Illumina data were used to correct poten-
All general aspects of library construction and sequencing tial base errors and increase consensus quality using the
performed at JGI can be found at http://www.jgi.doe.gov/. software POLISHER developed at JGI (Alla Lapidus, unpub-
The 454 Titanium standard data and the 454 paired-end lished data). Possible misassemblies were corrected using
data were assembled together with NEWBLER, version 2.3. gapResolution (Cliff Han, unpublished data), Dupfinisher
The Newbler consensus sequences were computationally (Han & Chain, 2006), or sequencing cloned bridging
shredded into 2 kb overlapping fake reads (shreds). PCR fragments with subcloning. Gaps between contigs
FEMSMicrobiolEcol82(2012)341–355 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
344 S.Rawatetal.
were closed by editing in Consed, by PCR, and by Bubble number of genes in A (at a given HSSP cutoff). At each
PCR (J-F Cheng, unpublished data) primer walks. A total HSSP cutoff (range (cid:1)5 to 45), we computed the numbers
of 291, 153, and 28 additional PCRs, and 5, 6, and 0 of genes overlapping between all different combinations
shatter libraries were necessary to close gaps and to raise of genomes. This measure represents the distance of a
the quality of the finished genomes of G. mallensis particular sequence alignment from a homology threshold
MP5ACTX8, G. tundricola MP5ACTX9, and T. saanensis curve, a function of alignment length, and percent
SP1PR4, respectively. A combined depth of coverage of sequence identity. In other words, alignments are mapped
2319, 2949, and 2199 was achieved for the three ge- to a two-dimensional space where points above the HSSP
nomes of G. mallensis MP5ACTX8, G. tundricola curve represent pairs of functionally similar proteins. The
MP5ACTX9, and T. saanensis SP1PR4, respectively. distance of one such point to the curve is correlated with
the reliability of function transfer; that is, the percentage
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of functionally similar sequence pairs at HSSP distance ow
Sequence analysis,annotation, and n
above 30 is higher than at HSSP distance above 0. We lo
bioinformatics a
mapped the percentage of genes in each genome common d
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Gene prediction and/or functional annotation of genomes to (1) only the three tundra genomes vs. (2) all six fro
were retrieved through the Integrated Microbial Genome genomes. To compute the ‘random’ baseline for this m
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(IMG) system supported by DOE-JGI Microbial Annota- curve, we randomly selected from HAMAP (Lima et al., ttp
tuisoinngPPirpoedliingeal (aDsOpaEr-tJGofIthMeAOPa)k. RGidengeesNwateiroenaildLeanbtiofiread- 2si0m09il)arsgiexnogmeneosmizees(4o–f5 Kfulglyenesse)q:uAeznocteodbacbtearctveirniaelanwditihi, s://ac
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tory genome annotation pipeline, followed by a round of Escherichia coli (strain K12), Methylacidiphilum inferno- de
m
manual curation using the JGI GenePRIMP pipeline (Pati rum (isolate V4), Novosphingobium aromaticivorans ic
et al., 2010). The coding sequences (CDS) were translated (strain DSM 12444), Rhodopseudomonas palustris (strain .ou
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and used to search the National Center for Biotechnology ATCC BAA-98/CGA009), and Vibrio fischeri (strain .c
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Information (NCBI) nonredundant database, UniProt, MJ11). Of these six, we randomly chose E. coli, A. vine- m
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TIGRFam, Pfam, PRIAM, KEGG, COGs, and InterPro landii, and V. fischeri to be the reference outliers. m
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databases. These data sources were combined to assert a e
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product description for each predicted protein. Noncod- /a
ing genes and miscellaneous features were predicted using Assays for substrate and enzyme activities rticle
tRNAscan-SE, RNAMMer, Rfam, TMHMM, and signalP. Carbon source utilization assays and enzymatic activities -a
b
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Comparative genome analysis was carried out in part were tested and reported earlier (Ma¨nnisto¨ et al., 2011, tra
using the Integrated Microbial Genome (IMG) portal. 2012), where utilization of sugars was detected by ct/8
The DNA and protein sequences were retrieved by web- growth on 96-well plates, and hydrolysis of various poly- 2/2
based databases (e.g., NCBI Query and BLAST searches). saccharides was assayed as CO2 production (Ma¨nnisto¨ /34
1
To facilitate the predictive analysis of the open reading et al., 2011, 2012). Here, we assayed the utilization of /4
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frames of the sequenced genomes, relevant information soluble polysaccharides alginate, CMC, laminarin, liche- 7
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was extracted from databases such as clusters of ortholo- nan, pectin, pullulan, and starch on 96-well plates with 9
b
gous genes (COGs) (http://www.ncbi.nlm.nih.gov/COG) VL55 mineral medium (pH 5.5; Sait et al., 2002) supple- y
(Tatusov et al., 1997) and CAZy (http://www.cazy.org) mented with yeast extract (100 mg L(cid:1)1) and 1–2 g L(cid:1)1 gu
e
s
(Cantarel et al., 2009). Phylogenetic and molecular evolu- of the polysaccharide. CMC hydrolysis was tested for t o
tionary analyses were conducted for DNA- or protein- colonies spotted on replicate diagnostic plates with 0.5 n 0
6
based sequences subjected to alignment using CLUSTALW and 1% (w/v) of CMC sodium salt as sole source of A
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andHoMmUSoClLoEgyu-sdinegrivMeEdGAsveecrosinodnar5y(Tsatrmuuctruareet aol.f, 2p0r1o1t)e.ins c(1ar0b0omngiLn(cid:1)1)VLan55d gmroewdniumfor c2onwteaeinkisn.gForymeaasttionextorfacat ril 20
1
(HSSP) distances (Sander & Schneider, 1991; Rost, 2002) zone of clearance around colonies after staining with 9
between protein sequences were computed as follows: (1) iodine was used as a preliminary indication of enzyme
PSI-BLAST (Altschul et al., 1997) all-against-all the activity (Kasana et al., 2008). Hydrolysis of CMC and
sequences in the six genomes (three iterations, inclusion xylan was further assayed on plates containing
e-value (h and e) parameters = 10–10); (2) extract 0.25 g L(cid:1)1 of peptone and yeast extract, 20 g L(cid:1)1 of
sequence identity and alignment length without gaps for agar, and 2 or 5 g L(cid:1)1 of each polysaccharide in VL55
each alignment; (3) apply the HSSP formula described in buffer (pH 5.5). The plates were incubated at 20 °C for
Rost (2002) to compute the distance. In short, the simi- up to 4 weeks, and the polysaccharide hydrolysis was
larity of genome A to genome B is computed as the num- detected by flooding the plates with 0.1% Congo red for
ber of genes in A that have homologues in B to the total 15 min (Teather & Wood, 1982).
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)341–355
PublishedbyBlackwellPublishingLtd.Allrightsreserved
GenomicanalysisofthreetundrasoilAcidobacteriaspecies 345
Utilization of chitin by the strains was assayed using (Fig. 2). In addition, G. tundricola has five mega plasmids
chitin azure (chitin from crab shells covalently linked ranging in size from 1.1 9 105 to 4.7 9 105 bp (Fig. S1)
with Remazol Brilliant Violet 5R (RBV) dye; Sigma) and foratotalgenomesizeof5.5 Mbp.Amongthefivestrains
by a chitobiase assay as described by O’Brien & Colwell of subdivision 1 Acidobacteria, G. mallensis has the largest
(1987). For the chitin azure assay, 0.5 mL of substrate genome size of 6.2 Mbp. The general genome and physio-
(0.5 g L(cid:1)1 in VL55 buffer) was mixed with 0.5 mL of logical features of the three tundra soil strains are com-
culture grown for 5 days on glucose (1 g L(cid:1)1) and yeast pared with those of A. capsulatum, ‘K. versatilis’, and
extract (0.5 g L(cid:1)1). Chitinase activity was detected after ‘S. usitatus’ and shown in Table 1. The genome GC con-
5 h, 20 h, 42 h, and 10 days incubation at room temper- tent for the three tundra strains ranges from 57% to 60%.
ature by measuring the absorbance at 550 nm. Chitobiase The genomes of G. mallensis, G. tundricola, and T. saan-
activity was assayed by the filter paper spot test using ensis are estimated to encode for 4907, 4706, and 4279
D
4-methylumbelliferyl-N-acetyl-b-D-glucosaminide. Biomass protein CDSs, respectively, with ~70% of the genes with ow
for the assay was grown on R2A (pH 5.5) for 5 days and predicted functions (68–72% COGs) (Table 1). The nlo
a
ca. 0.2 mL of loop-full of the colonies rubbed on antibi- genomesofG. mallensisandT. saanensisconsistofalarge d
e
ointoiccudluismks.wCeroenitnroclluddiesdk,sawndith2o0ultLsuobfstthraetesuabnstdrawteitshooluu-t ptheartceanthoigfhCerDSnsumwibthersiogfnaglenpeesptairdeesin(4vo3–lv4e4d%i)n,sturagngsepstoirntg/ d from
h
tion pipetted to each disk. Chitobiase activity was translocation processes in these two tundra soil strains as ttp
tduerteecbteydexapftoersu2r0e–t3o0UmVinligohfti.ncubation at room tempera- cthormeepatruenddtroa tshoeilostthrearinssubcodnivtiasiinoend1aAlcairdgoebanctuemriab.erThoef s://ac
a
sequences in paralogous clusters (50–54%). In compari- de
m
Genome submissions sboenlo,ngthineg ltaorgseubgdeivniosmione 3sizAecid(o9b.9acMterbipa)haosfan‘S.inucsriteaatsuesd’ ic.ou
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ThefinishedgenomesaresubmittedtoNCBIwiththefollow- numberofparalogs,whichhasbeenaccountedforbygene .c
o
ingaccessionnumbers/TaxonIDs:G. mallensisMP5ACTX8 acquisition and horizontal gene transfer events (Challa- m
/fe
(CP003130/682795), G. tundricola MP5ACTX9 (CP002480/ combeet al.,2011).Weidentifiedmobilegeneticelements m
s
696844), G. tundricola MP5ACTX9 plasmids (CP002481, in all three tundra soil strains, with a large number in the e
c
CP002482, CP002483, CP002484 and CP002485), and genome of G. tundricola (n = 154), encoding for phage /a
T. saanensisSP1PR4(CP002467/401053). integrases,transposases,andISelements(TableS1).Gran- rticle
ulicella tundricola also contained five mega plasmids -a
b
s
Results and discussion (> 100 kb in size). We did not find any CDSs for clus- tra
tered, regularly interspaced, short palindromic repeats ct/8
General genome features and metadata (CRISPRs) in the genome of any of the three tundra soils 2/2
strains; however, a CDS for a CRISPR-associated protein /3
4
1
The genomes of three tundra soil strains, G. mallensis, (AciX8_2932) of the Cas5 family was present in the gen- /4
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G. tundricola, and T. saanensis, consist of one circular omeofG. mallensis.Thissuggests thatthegenomes ofthe 7
4
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chromosome of 6.2, 4.3, and 5.0 Mbp, respectively tundrasoilstrainsaresubjectedtogenetransferevents. 9
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t o
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6
A
p
ril 2
0
1
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Fig.2. Circular representation of the genomes of Granulicella mallensis MP5ACTX8, Granulicella tundricola MP5ACTX9, and Terriglobus
saanensis SP1PR4 displaying relevant genome features. The outermost circles (circles 1 and 2) show the forward and reverse strand of protein
codinggenes(CDSs)coloredbyCOGsfunctionalcategories;circle3showsRNAgenes(tRNAsingreen,rRNAsinred,otherRNAsinblack);circle
4showsCDSsencodingforCAZymefamilies;circle5showsGCplot(G+Cdistribution);andcircle6showsGCskew.
FEMSMicrobiolEcol82(2012)341–355 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
346 S.Rawatetal.
a
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‘S.usitatus’Ellin6076 9965640 901723461.9 6352 65 8003114 7940(99.2)4898(61.2) 3042(38.0)4997(62.4) 1882(23.5)5963(74.5) 2858(35.7)1779(22.2) Ryegrasssoil pasture,Austr 3Solibacter¶Mesophile¶Acidophile
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‘K.versatilis’Ellin345 5650368 505659358.4 5847 38 48372 4779(98.8)3080(63.7) 1699(35.1)3167(65.5) 1271(26.3)2995(61.9) 1425(29.5)1284(26.6) Ryegrasssoil pasture,Austr 1Koribacter¶Mesophile¶Acidophile Mbp. Download
05,0.12 ed from
A.capsulatumATCC51196 4127356 355203160.5 4845 3– 3425– 3377(98.6)2248(65.6) 1129(33)2294(67) 1036(30.2)1579(46.1) 831(24.3)842(24.6) Acidicmine drainage,Japan 1Acidobacterium–§2037–§36.0 0.12Mbp;pACIX9 https://academic
X904, .oup.c
T.saanensisSP1PR4 5095226 457820657.3 5448 33 433399 4279(98.8)2890(66.7) 1389(32.0)3152(72.7) 1176(27.1)2197(50.7) 1865(43.0)1082(24.9) Tundrasoil, Finland 1Terriglobus‡–430‡–4.57.5 19Mbp;pACI om/femsec/a
pecies 903,0. rticle-a
s X b
ofsixAcidobacteria G.tundricolaMP5ACTX9 5503984* 476204560.0 5246 33 4757163 4706(98.9)3313(69.6) 1393(29.3)3276(68.8) 1154(24.2)2412(50.7) 1307(27.5)1106(23.3) Tundrasoil, Finland 1Granulicella†–428†–3.56.5 902,0.3Mbp;pACI stract/82/2/341/49
andphysiologicalfeatures G.mallensisMP5ACTX8 6237577 545209357.9 5347 33 496090 4907(98.9)3511(70.8) 1396(28.2)3496(70.5) 1210(24.4)2679(54.0) 2203(44.4)%)1291(26) Tundrasoil, Finland 1Granulicella†–428†–3.56.5 CIX901,0.48Mbp;pACIX 7499 by guest on 06 Ap
ComparisonofgeneralgenomeTable1. GenomedataGenomesizebasepairs(bp) DNAcodingregion(bp)G+Ccontent(mol%) TotalRNAgenestRNAgenes rRNAgenesOtherRNAgenes TotalnumberofgenesPseudogenes TotalproteinCDSs(%)Withfunctionprediction(%) Withoutfunctionprediction(%)WithCOGs(%) WithTIGRfam(%)Inparalogousclusters(%) Codingforsignalpeptides(%)Codingfortransmembraneproteins( MetadataIsolationsource(Habitat) Acidobacteriasubdivision(SD)Genus Temperaturerange(°C)pHrange *PlasmidsinG.tundricolaMP5ACTX9,pA†¨¨DatafromMannistoetal.(2012).‡¨¨DatafromMannistoetal.(2011).§DatafromKishimotoetal.(1991).¶DatafromWardetal.(2009). ril 2019
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)341–355
PublishedbyBlackwellPublishingLtd.Allrightsreserved
GenomicanalysisofthreetundrasoilAcidobacteriaspecies 347
The physiology of the three tundra soil strains, (a)
G. mallensis, G. tundricola, and T. saanensis, has been mes 0.1
o
Ais+cdsooe4’.,imslccaarptsnipotabdrsraeuadi2‘ltnaSi8svt.uie°nuamCsrdie,)tdaate‘ctKatcuoao.slwid’mlv-ieatap(rhrdMsaeaarsteap¨ptidnrltriaeensid’sin,wessnt(aio¨tgtnAherddo.ewtct‘iaShnap.eal.stTu,usamtli2aebtm0atelues1tuopm11spe,’,.rhaT2‘(itKlK0uhic.1ires2evhs)tesiu,rtmfsrnraaaoodtinnitmrldoa-s ologues in only 3 tundra genmber of CDS per genome000000......000000456789 HSSP 34 HSSP 22 TGGAA.v... sevmtuairnaanaegldnlleeareinncnsodsiliisais
mu
esiAsttocriaaldailno.t,ebsda1ac9rtf9eer1roi;amabWlsettareratmdoinpsegetrraoaarwtele.,aaec2tni0dva0oin9rp;ohanCcimlihedsaei.lcnlTatpschoHe(mTtrwbaabeonlegeGet1ra)(a.lpn.,HuA2llil0c3e1.sl51il–ax) o of CDS with hovs. the total n000...000123 Dow
6.5), while T. saanensis grows at a pH range of 4.5–7.5. Rati 00 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 nlo
Ratio of CDS with homologues in all 6 Acidobacteria genomes a
vs. the total number of CDS per genome de
d
General genome comparisons (b) G. mallensis MP5ACTX8 G. tundricola MP5ACTX9 from
4907 CDSs (6.2 Mb) 4706 CDSs (5.5 Mb) h
Organisms occupying the same environmental niche are ttp
itnhteuoituivtseildyemrs.orHeefruen,cwtieoansaslelysseredlaotergdantoismeacfhunoctthioernathlasinmtio- s://ac
a
larity using whole-genome homology. Comparisons of the 520-535 de
m
six Acidobacteria genomes were made by all-against-all 1679 1640 ic
sequence comparisons at the level of protein CDS, using .ou
p
HSSP distance as a measure of similarity (Rost, 2002). In .c
o
short, the similarity of genome A to genome B is com- 1900-1966 m
/fe
puted as the number of genes in A that have homologues 382-398 m
s
in B to the total number of genes in A (at a given HSSP 458-466 e
c
cutoff). At each cutoff (range (cid:1)5 to 45), we computed /a
the similarity of all six genomes in our study. For our threeG teunnedsra c Aomcidmoobna octnelyri ato the 1249 rticle
analysis, we assume that a higher number of functionally -a
G. mallensis 380 b
s
sitiimveillayr, hpirgohteeirnsstriinndgiecnatceyoinveraasslligonrignagnihsmomsoimloiglyaridteyc.rIenatsue-s GT.. s tauanndericnosilsa 334790 T4.2 s7a9a CnDenSssis ( 5S.0P 1MPbR) 4 tract/8
the number of homologous genes; that is, with increasing Fig.3. Comparison of protein CDSs encoded in the genomes of six 2/2
HSSP cutoffs, there is a decrease in the percentage of strains of Acidobacteria. Functional similarity was assessed using /34
1
genes common to all six genomes (Fig. 3a, X-axis). How- whole-genome homology comparing the three tundra soil strains, /4
9
ever, highly similar genes are still assigned homology even Granulicella mallensis, Granulicella tundricola, and Terriglobus 7
4
at high stringency thresholds, as illustrated by the increase saanensis, to three other strains of Acidobacteria, Acidobacterium 99
capsulatum, ‘K.versatilis’, and ‘S.usitatus’. (a) At each cutoff (range b
in the percentage of genes common to only the three y
(similar) tundra genomes (Fig. 3a, Y-axis; HSSP (cid:1)5 to (cid:1)5 to 45), we computed the similarity of all six genomes. For each gu
HSSP cutoff, the percentage of genes in each tundra genome e
s
HSSP 34). Thus, the increase in HSSP cutoffs stringency commononlytothethreetundragenomesvs.toallsixgenomeswas t o
from (cid:1)5 to 22 (Fig. 3a; beginning of the curve plateau) mapped.The‘random’baselineforthiscurve(grayline)isrepresented n 0
6
progressively better groups the tundra genomes closer to byAzotobactervinelandii(seeMaterialsandmethodsandFig.S2for A
ethaechthorteheerot(hienrcrsetarsaiinngs oalfoAngcidtohbeacYt-earixais)(daencrdeaaswinagyaflroonmg fnuullm‘rbaenrdoofmg’egnreasphc)o.mWmitohnprtoogarellssgiveenloymsterisctdeerccruetaosfefssi(n(cid:1)5fatvoor22o)f,tthhee pril 20
the X-axis). Cutoffs stricter than 22 remove homologues overlap set of only the tundra genomes, that is, for a given tundra 19
geneX,homologuesinnontundragenomesarelesssimilartoXthan
from both sets, with the tundra overlap set being replen-
itstundrahomologues,thus‘droppingout’oftheoverlapsetearlier.
ished by the nontundra ‘drop-outs’ until HSSP = 34.
(b) Venn diagram showing the number of genes in all possible
Beyond this threshold, fewer and fewer genes find homo- genomeoverlapsetsatHSSP=22.Eachgenomeisrepresentedwith
logues in any of the genomes. the corresponding total number of CDSs. Numbers in intersections
The gene pool shared by the genomes of three tundra indicate the number of shared homologues between two or three
soil strains G. mallensis, G. tundricola, and T. saanensis at genomes.Homologuessharedonlybythreetundrasoilstrainsandnot
found in the genomes of the other three Acidobacteria strains are
HSSP = 22 (beginning of curve plateau, Fig 3a) is
shown inthetriangle.Numbers outsidethe intersections within each
depicted by a Venn diagram (Fig. 3b; numbers in each
circlerepresentnumberofgenesspecifictoeachgenome.
intersection indicate shared CDSs). Some genes were
FEMSMicrobiolEcol82(2012)341–355 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
348 S.Rawatetal.
specific to one strain only: 1679 CDSs in G. mallensis, hydrate transport and metabolism [G] (9–10%), with the
1640 CDSs in G. tundricola, and 1249 CDSs in T. saanen- highest abundance in genomes of G. mallensis and T. sa-
sis. 1900–1966 CDSs were shared by all tundra genomes anensis among all six Acidobacteria strains. This was fol-
with more than half (1551–1586 CDSs; data not shown) lowed by amino acid transport and metabolism [E] (7–
of these genes shared by all six Acidobacteria strains (the 8%), energy production and conversion [C] (5–6%), and
three tundra soil strains and A. capsulatum, ‘K. versatilis’, lipid transport and metabolism [I] (3–4%). For cellular
and ‘S. usitatus’). Further analysis identified a gene pool processes (Cp), the majority of genes were assigned to cell
shared only by the three tundra Acidobacteria (but not wall/membrane/envelope biogenesis [M] (8–9%) followed
identified in three other species) consisting of 380 CDSs by signal transduction mechanisms [T] (4–5%) and for
in G. mallensis, 370 CDSs in G. tundricola (including information storage and processing (Isp) to transcription
plasmids: 21 genes in pACIX902, 20 genes in pACIX901, [K] (7–9%). We infer that the genomes of the three tun-
D
11 genes in pACIX903, four genes in pACIX904, and two dra soil strains encode for functions involved in transport ow
n
genes in pACIX905), and 340 CDSs in T. saanensis and utilization of nutrients, mainly carbohydrates for lo
a
(Fig 3b, box). This gene pool was assigned to 261 COG energy production and cell biogenesis to maintain cell d
e
d
and 273 pfam functions (Table S2), while 47 genes had integrity in cold tundra soils. fro
no assigned function. Many of the CDSs in this gene pool m
h
were assigned via COG annotations to functions of Carbohydrate transport and metabolism ttp
mTheetaseboinlicsmludeadndglytrcaonsisdpeorhtydorfolcaasrebso(hGyHdrsa)teosf f(aTmabillyeGSH2)1. To explore the genetic potential of the three tundra soil s://ac
a
(pfam00232), GH2 (pfam00703, pfam00754, pfam02836), strains to metabolize organic carbon, we analyzed their de
m
GH20 (pfam00728), GH28 (pfam00295), GH31 genomes for CDSs predicted to code for modules that ic
(pfam01055), GH57 (pfam03065, pfam09210), GH88 catalyze the breakdown, biosynthesis, or modification of .ou
p
(pfam07470), and GH92 (pfam07971), alginate lyase of carbohydrates of the carbohydrate-active enzymes (CAZy) .c
o
polysaccharide family PL5 (pfam05426), glycosyl transfer- family (http://www.cazy.org; Cantarel et al., 2009). CDSs m
/fe
ases (GTs) of family GT1 (pfam00534), GT2 predicted to encode for CAZymes were more abundant in m
s
(pfam00535), and GT9 (pfam01075), and transporters of the genomes of the three tundra soil strains, G. mallensis e
c
Caenorhabditis elegans ORF (CEO) family (DUF1632)/ (n = 321), G. tundricola (n = 215), and T. saanensis /a
sugar transport protein (pfam06800) and major facilitator (n = 244), as compared to the genomes of two other rticle
superfamily (MFS) (pfam00083, pfam07690). strains of subdivision 1, A. capsulatum (161) and ‘K. ver- -a
b
s
satilis’ (135) (Fig. 5). The genomes of G. mallensis, tra
Functionaldiversity in Acidobacteria genomes G. tundricola, and T. saanensis contained gene modules ct/8
spanning four major CAZyme super families of glycoside 2/2
For all six strains of Acidobacteria, predicted genes were hydrolases (GHs) (n = 166, 103, 110, respectively), glyco- /3
4
assigned to four main functional categories – metabolism syl transferases (GTs) (n = 77, 74, 90), polysaccharide ly- 1/4
9
(Me), cellular processes (Cp), information storage and ases (PLs) (n = 9, 4, 4), carbohydrate esterases (CEs) 7
4
processing (Isp), and poorly characterized (Pc) within the (n = 16, 15, 16), and noncatalytic carbohydrate-binding 99
b
Cluster of Orthologous Groups (COG) database (Tatusov modules (CBMs) (n = 53, 19, 24). This indicates that the y
g
et al., 1997) as shown in Fig. 4. For metabolism (Me), tundra soil strains are abundant in genes encoding for u
e
s
the highest percent of genes could be assigned to carbo- functional activities required for rearrangement of oligo- t o
n
0
6
18 Metabolism(Me) A
s) otal CDSs 111624 "GTT"AGSK.... .. sc mtvuuaaesanapirldnstlsaereuaitncnlutasiossltiiliiuss"as " m [[[[[[[[QHCFGEFI]]]]]]]]LNNAECCSipnmuueaoiecccrdeibrollnneegtonozrooyhday(cid:415)(cid:415)apmynacdddrrsieeeoypdradotttmttrurrrreaaateacnnn(cid:415)ttnarasssnsoapppbpdnnooooosmrrralrpitttntteoeaaadatrsnnnnatcdddbdboaiommmnonmldsvieeeseyetmttmntraaaastbebbbihotooooeanllllsiiibisssisosmmmm,litsrmansportandcatabolism pril 2019
% of t% 10 C[[[DOOe]]]llCPPueoollssal(cid:425)(cid:425)rdrripaavrinnsossiollcaane(cid:415)(cid:415),soocsnnheaarsollmmm(Cooopdds)oiififimccaae(cid:415)(cid:415)pooannr(cid:415),pp(cid:415)rrooonttieeniignnttuurrnnoovveerr,cchhaappeerroonneess
ontent ( 68 [[[[PTNM]]]]SICnCigeoenlrlllgamealntnorvi(cid:415)acelniiltoosydnpuaetncrb(cid:415)adinoossngepecmornreeet(cid:415)csaohisnandannimsdmeotsuatbeorlimsmembrane
ene c 4 [[VU]]DInetfreancesellumlaercthraanffiiscmkisngandsecre(cid:415)on Fig.4. ComparisonofgenecontentbyCOG
G
Informa(cid:415)onStorageandprocessing(Isp) functionalcategoriesgroupedbyfourmajor
2 [J]Transla(cid:415)on,ribosomalstructureandbiogenesis
[K]Transcrip(cid:415)on categories:metabolism(Me),cellularprocesses
0 [L]DNAReplica(cid:415)on,recombina(cid:415)onandrepair
[C][G][E][F][H][I][Q] [D][O][M][N][P][T] [U][V] [J][K][L] [R][S] Poorlycharacterized(Pc) (Cp),informationstorageandprocessing(Isp),
MMee CCpp IIsspp PPcc [[RR]]GGeenneerraallffuunncc(cid:415)(cid:415)oonnpprreeddiicc(cid:415)(cid:415)oonnoonnllyy andpoorlycharacterized(Pc)inthegenomes
[S]Func(cid:415)onunknown
COG functional categories ofsixstrainsofAcidobacteria.
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)341–355
PublishedbyBlackwellPublishingLtd.Allrightsreserved
GenomicanalysisofthreetundrasoilAcidobacteriaspecies 349
7.0 grew on a number of plant- and microbe-based polysac-
Carbohydrate-binding module (CBM)
charides as single carbon sources (Table 2).
otal) 66..00 CPoalrybsoahcycdhraartied ee slytearsaesse (sP (LCsE) s) Hemicelluloses are highly complex heteropolysaccha-
of t Glycosyl transferases (GTs) ridesrequiring abattery ofenzymes, belongingto GHand
s (% 5.0 Glycoside hydrolases (GHs) CE families, required for hydrolysis of xylan-, mannan-,
e
ym 44.00 and arabinofuranosyl-containing hemicelluloses (Shallom
Z
A
C & Shoham, 2003). We identified predicted CDSs encoding
g for 3.0 for hemicellulolytic enzymes in the genomes of the three
n
odi 22.00 tundra soil strains (Table S2). These included endoxylan-
c
n ases represented by family GH10 and exoxylanases repre-
e
Ss 1.0 sented by family GH39 that successively hydrolyze xylan
D D
C intoshortxylooligomersandxylose.Inaddition,CDSsfor ow
00.00 G.mallensis G.tundricola T.saanensis A.capsulatum K.versa(cid:415)lis S.usitatus acetylxylanesterasesofcarbohydrateesterasefamiliesCE1 nlo
a
and CE4 that hydrolyze the acetyl substituents of xylose d
Fig.5. Distribution of gene content (% of total CDSs) encoding for e
d
four major CAZy families: glycoside hydrolases (GHs), GTs, moietieswereidentifiedinthegenomesofallthreetundra fro
polysaccharide lyases (PLs), carbohydrate esterases (CEs), and soil strains (Table S3). Predicted CDSs for family GH3, m
noncatalyticcarbohydrate-bindingmodules(CBMs)inthegenomesof GH43, and GH51, which represent a-L-arabinofuranosid- http
sixstrainsofAcidobacteria. afoseusndreqinuirseodftwtooodclexayvleansL-aarnadbinGoHfu1ra,nGosHe2,sidaendchGaHin5s s://ac
a
and polysaccharides. Predicted gene modules encom- requiredforhydrolysisofb-mannan-basedpolymers,were de
m
passed 59 different families of glycoside hydrolases (GHs, also present in the genomes of the three tundra soil ic
21 GTs, seven PLs, nine CEs, and 12 CBMs (Table S3), strains. In addition, a large number of CDSs of family .ou
p
emphasizing the elaborate set of enzymes needed for GH27, GH36, and GH57 that represent a-galactosidases .c
o
breakdown of different types of plant and/or microbial and GH1, GH3, GH30, and GH116, which represent m
polysaccharides as well as for the biosynthesis of various b-glucosidases, were identified in tundra soil strains /fem
s
polysaccharides. The three tundra Acidobacteria strains (Table S3). By cultivation assays, xylan (from birch wood) e
c
contained a large number of predicted CDSs encoding for degradation was not detected in any of the three tundra /a
sugar transporters of the major facilitator superfamily. strain as assayed by turbidity, CO2 production, or Congo rticle
red staining (Ma¨nnisto¨ et al., 2011, 2012; Table 2). Fur- -a
b
s
Biodegradation of structuraland storage ther studies are underway to assay the xylanase activities tra
polysaccharides againstdifferentsubstratesandunderdifferentconditions. ct/8
Degradation of cellulose requires three enzyme activi- 2/2
Cellulose and hemicelluloses are the most abundant plant ties, including endoglucanase, exoglucanase (or cellobio- /3
4
structural carbon polymers found in the biosphere, and hydrolase), and b-glucosidase. Recently, cellulases were 1/4
9
therefore, their degradation by microorganisms represents described within 13 GH families, of which GH5 and GH9 7
4
a significant part of the carbon cycle. The efficient degra- appear to have the largest number of biochemically char- 99
b
dation of polysaccharides requires the concerted action of acterized bacterial cellulases with both endo- and exocel- y
g
many catalytic enzymes and/or noncatalytic CBMs, which lulase activity, while no exocellulase activity is identified u
e
s
facilitate the targeting of enzymes to the insoluble poly- for GH8 in the CAZy database (Sukharnikov et al., 2011). t o
saccharides (Warren, 1996). The tundra soil Acidobacteria We identified CDSs belonging to five different glycoside n 0
6
are versatile heterotrophs isolated using selective plant- hydrolase families that represent cellulases: GH5, GH8, A
p
boardseedr tcoarebxopnlorseouthrceems e(tMaba¨onlnicispto¨oteenttaial.l,o2f0t1h1e, t2h0r1ee2)t.unIn- GidHen9t,ifiGeHd1in2,thanedgeGnHom51es(oTfabGle. mS3a)ll.enCsDisSasnfdorG.GtHun5dwriecore- ril 20
1
dra soil strains to hydrolyze biomass polysaccharides, we la, but not in T. saanensis. CDS for GH9 was only identi- 9
analyzed their genomes for CDSs predicted to code for fied in G. mallensis. GH9 cellulases were also present in
main-chain and side-chain cleaving enzymes of the CAZy the genomes of A. capsulatum, ‘K. versatilis’, and ‘S. usita-
family. The genomic data were validated by biochemical tus’ (Ward et al., 2009). However, no CDSs for exoglu-
assays to bridge genome predictions to biochemical activi- canases or cellobiohydrolase were identified in the
ties encoded in their genomes. We identified predicted genomes of any of the three tundra soil strains.
CDSs of CAZyme families involved in breakdown of plant Carbohydrate-binding modules that are likely involved in
structural polysaccharides such as hemicelluloses, cellu- cellulose degradation were identified in the three tundra
loses, pectin, and storage polysaccharides such as starch/ strains (Table S2), which included CBMs binding to GH16
glycogen (Table S3). The tundra Acidobacteria strains in both Granulicella strains and those binding to GH27 and
FEMSMicrobiolEcol82(2012)341–355 ª2012FederationofEuropeanMicrobiologicalSocieties
PublishedbyBlackwellPublishingLtd.Allrightsreserved
350 S.Rawatetal.
Table2. Comparisonofcarbonsubstrateutilization/hydrolysisbysixstrainsofAcidobacteria
G.mallensis G.tundricola T.saanensis A.capsulatum ‘K.versatilis’ ‘S.usitatus’
MP5ACTX8* MP5ACTX9* SP1PR4† ATCC51196‡ Ellin345§ Ellin6076§
Utilizationof
Mono-anddisaccharides
D-arabinose (cid:1) (cid:1) (cid:1) nd + +
Cellobiose + + + + + +
D-Fructose + + + nd + +
D-galactose + + + + + +
D-glucose + + + + + +
Lactose + + + + + +
Lactulose + + nd nd nd nd Do
D-lyxose (cid:1) (cid:1) (cid:1) nd nd nd wn
D-maltose + + + + nd nd loa
d
D-mannose + + + + + + e
d
D-ribose + (cid:1) + nd + nd fro
Sucrose + + + nd + + m
DD--txryelohsaelose ++ ++ ++ ++ nndd nndd https
D-melezitose + + + nd nd nd ://a
c
D-raffinose + + + nd nd nd ad
N-acetyl-D-glucosamine + + + nd nd nd em
Polysaccharides ic
.o
Laminarin + + + nd nd nd u
p
Pectin + + + nd nd + .c
Lichenan + (cid:1) (cid:1) nd nd nd om
Starch + + + + nd + /fe
m
Xylan (cid:1) (cid:1) (cid:1) + + + s
e
Pullulan (cid:1) + (cid:1) nd nd nd c
/a
ACleglliunlaotsee (cid:1)(cid:1) (cid:1)(cid:1) (cid:1)(cid:1) nd nd n+d rticle
Chitin (cid:1) (cid:1) (cid:1) nd nd nd -ab
Chitosan (cid:1) (cid:1) (cid:1) nd nd nd stra
Enzymaticandotherassays c
Cellulosehydrolysis(plateassay) + + (cid:1) nd nd nd t/82
Chitinase (cid:1) (cid:1) (cid:1) nd nd nd /2/3
Chitobiase + + + nd nd nd 4
1
/4
nd,nodataavailable. 97
4
*DatafromMa¨nnisto¨ etal.(2012)orthisstudy. 9
†DatafromMa¨nnisto¨ etal.(2011)orthisstudy. 9 b
‡DatafromKishimotoetal.(1991). y g
§DatafromWardetal.(2009). ue
s
t o
GH36 in G. mallensis and T. saanensis genomes, but notin Subdivision 1 Acidobacteria have been linked to cellulose n 0
6
G. tundricola.CBMsbindingtoGH64andGH55represent- degradation in sphagnum peat, but the rates of cellulose A
isntrgaibns-,1,w3-hgilluecCanBaMse6wbeinrediindgentotifiGeHd5i5naanlldthGrHee16tuwnadsraidseoni-l dTeogrdaedtaetrimoninearweheextthreermtehlye plorwese(nPcaenkorfaatonveeatsialyl.,de2g0r1a1d)-. pril 20
1
tified in genomes of G. mallensis and G. tundricola, respec- able substrate would trigger CMC hydrolysis, the strains 9
tively. CBM6 is reported to have both xylan and cellulose- were inoculated on plates containing CMC with peptone
bindingfunction(Borastonet al.,2004). and yeast extract and with cellobiose, peptone, and yeast
Although the three tundra soil Acidobacteria contained extract. After 3 weeks of incubation, G. mallensis strain
cellulases from several glycoside hydrolase families, none MP5ACTX8 scored positive for CMC hydrolysis in plates
of them effectively utilized cellulose when assayed by tur- with both amendments, while G. tundricola strain
bidity or CO production (Ma¨nnisto¨ et al., 2011, 2012; MP5ACTX9 produced clearing zones, indicative of CMC
2
Table 2). No increase in turbidity or CO production was hydrolysis, only in plates amended with cellobiose. Terri-
2
detected after 3 weeks of incubation in liquid culture with globus saanensis strain SP1PR4 did not produce a clearing
CMC and a small amount (100 mg L(cid:1)1) of yeast extract. zone on either of the plates. Further studies are needed to
ª2012FederationofEuropeanMicrobiologicalSocieties FEMSMicrobiolEcol82(2012)341–355
PublishedbyBlackwellPublishingLtd.Allrightsreserved