Comparative single-cell genomics reveals potential ecological niches for the freshwater acI

14 Pages·2014·1.69 MB·English

by  Trevor W Ghylin

TheISMEJournal(2014)8,2503–2516
&2014InternationalSocietyforMicrobialEcology Allrightsreserved 1751-7362/14
www.nature.com/ismej
ORIGINAL ARTICLE
Comparative single-cell genomics reveals potential
ecological niches for the freshwater acI
Actinobacteria lineage
Trevor W Ghylin1, Sarahi L Garcia2,3, Francisco Moya1, Ben O Oyserman1,
Patrick Schwientek4, Katrina T Forest2, James Mutschler2, Jeffrey Dwulit-Smith2,
Leong-KeatChan4,ManuelMartinez-Garcia5,10,AlexanderSczyrba4,6,RamunasStepanauskas5,
Hans-Peter Grossart7,8, Tanja Woyke4, Falk Warnecke3,{, Rex Malmstrom4, Stefan Bertilsson9
and Katherine D McMahon1,2
1Department of Civil and Environmental Engineering, University of Wisconsin-Madison, Madison, WI, USA;
2DepartmentofBacteriology,UniversityofWisconsin-Madison,Madison,WI,USA;3JenaSchoolforMicrobial
Communication (JSMC) and Microbial Ecology Group at Friedrich Schiller University Jena, Jena, Germany;
4DOE Joint Genome Institute, Walnut Creek, CA, USA; 5Bigelow Laboratory for Ocean Sciences, East
Boothbay, ME, USA; 6Center for Biotechnology (CeBiTec), Bielefeld University, Bielefeld, Germany;
7DepartmentofLimnologyofStratifiedLakes,Leibniz-Institute forFreshwaterEcologyandInland Fisheries,
Stechlin, Germany; 8Institute for Biochemistry and Biology, Potsdam University, Potsdam, Germany and
9Department of Ecology and Genetics, Limnology and Science for Life Laboratory, Uppsala University,
Uppsala, Sweden
Members of the acI lineage of Actinobacteria are the most abundant microorganisms in most
freshwaterlakes;however,ourunderstandingofthekeystotheirsuccessandtheirroleincarbon
and nutrient cycling in freshwater systems has been hampered by the lack of pure cultures and
genomes. We obtained draft genome assemblies from 11 single cells representing three acI tribes
(acI-A1, acI-A7, acI-B1) from four temperate lakes in the United States and Europe. Comparative
analysis of acI SAGs and other available freshwater bacterial genomes showed that acI has more
genecontentdirectedtowardcarbohydrateacquisitionascomparedtoPolynucleobacterandLD12
Alphaproteobacteria,whichseemtospecializemoreoncarboxylicacids.TheacIgenomescontain
actinorhodopsin as well as some genes involved in anaplerotic carbon fixation indicating the
capacity to supplement their known heterotrophic lifestyle. Genome-level differences between the
acI-A and acI-B clades suggest specialization at the clade level for carbon substrate acquisition.
Overall, the acI genomes appear to be highly streamlined versions of Actinobacteria that include
some genes allowing it to take advantage of sunlight and N-rich organic compounds such as
polyamines, di- and oligopeptides, branched-chain amino acids and cyanophycin. This work
significantlyexpandstheknownmetabolicpotentialofthecosmopolitanfreshwateracIlineageand
itsecological andgenetictraits.
TheISME Journal(2014)8, 2503–2516;doi:10.1038/ismej.2014.135; published online5August2014
Introduction nutrient eutrophic (Wu et al., 2007), low-nutrient
oligotrophic (Humbert et al., 2009) and dystrophic
Members of the acI lineage within the phylum
lakes (Newton et al., 2006). They are also abundant
Actinobacteria are an intriguing group of free-living
in some marine estuaries (Glockner et al., 2000).
ultramicrobacteria that dominate many freshwater
Moreover,asalineage,acIshowsasmallerseasonal
ecosystems (Newton et al., 2011), including high-
abundance variation when compared to other major
freshwater bacteria (Allgaier and Grossart, 2006;
Salcher et al., 2010; Newton et al., 2011; Eckert
Correspondence: KD McMahon, Department of Civil and
et al., 2012; Ro¨sel and Grossart, 2012). Due to their
Environmental Engineering, and Bacteriology, University of
Wisconsin1550,LindenDriveRoom5552,Madison,WI53706,USA. high abundance and high metabolic activity
E-mail:[email protected] (Warnecke et al., 2005; Allgaier and Grossart, 2006;
10Currentaddress:UniversityofAlicante,Alicante,Spain. Salcher et al., 2010), the acI bacteria likely have a
{Deceased.
critical role in carbon and other nutrient cycling in
Received8January2014;revised13June2014;accepted22June
2014;publishedonline5August2014 freshwater systems, yet their genomic features
EcogenomicsoftheacIlineage
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remain elusive. This is in part due to the historical and metabolizing pentoses such as xylose. The
lack of isolated representatives and limited authors also confirmed an actinorhodopsin gene in
genomic data. the genome.
Onthebasisof16SrRNAgenesequenceanalysis, Here, we greatly expand on the single-cell gen-
the acI lineage comprises three distinct clades (A, B ome-basedanalysis(Garciaetal.,2013)byanalyzing
andC;Newtonetal.,2011).Eachofthesecladeshas 10 additional acI single-amplified genomes (SAGs)
been further subdivided into ‘tribes,’ members of from three different acI tribes and four different
which share X97% 16S rRNA gene sequence lakes. The additional genomes allowed us to con-
identity(intotal13suchtribes).Themostabundant firm key gene functions and genomic features (for
and prevalent tribes are acI-A1, acI-A6, acI-A7 and example, GC content) that are conserved within the
acI-B1 (Newton et al., 2011). The tribes seem to lineage while also revealing clade-specific differen-
niche partition based on pH, with some clades (for tiation. We also compare the gene content of acI
example, acI-A1 and acI-B2) preferring slightly genomes to that of cultivated members of their
more acidic environments (Newton et al., 2007). parent order, Actinomycetales, and to that of other
Several single-cell targeted studies have abundant freshwater bacteria. This kind of analysis
attempted to elucidate acI’s capability to take up was not possible with only one draft genome (the
and consume specific carbon sources, using fluor- previously published acI-B1 SAG called SCGC
escent in situ hybridization (FISH) combined with AAA027-L06). Our data show that acI members
catalyzed reporter deposition and microautoradio- have unique traits that likely provide a competitive
graphy (MAR). These studies have shown that acI advantage when scavenging for energy, carbon,
members can take up glucose (Buck et al., 2009), nitrogen and phosphorus in freshwater habitats.
leucine(Bucketal.,2009;Perezetal.,2010;Salcher Thepresenceofseveralgenesinvolvedintheuptake
et al., 2010; Eckert et al., 2012), acetate (Buck et al., and metabolism of organic nitrogen compounds
2009), thymidine(Perez etal.,2010),N-acetylgluco- suggests that N-rich organic matter may be a
samine (NAG) (Beier and Bertilsson, 2011; Eckert significant source of carbon, nitrogen and energy
etal.,2012)anddi-NAG(BeierandBertilsson,2011; for acI biosynthesis.
Eckert et al., 2012; Tada and Grossart, 2014).
A recent MAR-FISH study performed on fall
samples from Lake Zu¨rich in Switzerland showed Materials and methods
thatacIbacteriaconsumedanaminoacidmixtureas
well (Salcher et al., 2013). The acI in this study did SAG generation and selection
not take up acetate, fructose, arginine, aspartate, Water samples (1ml) were collected from the
glutamate, glutamine, serine, glycine or alanine. upper 0.5 to 1m of each of four lakes (Mendota,
However, due to the inherent limit of phylogenetic Sparkling, Damariscotta, Stechlin) and cryopre-
resolution associated with rRNA-targeted FISH served (Supplementary Table S1), as previously
probes, many of these studies generally cannot described (Martinez-Garcia et al., 2012; Garcia
attribute the ability to uptake key substrates to et al., 2013) (Supplementary Online Material).
clades or tribes within the lineage. Only a few Bacterial SAGs were generated and identified at
studies have used FISH probes that differentiate theBigelowLaboratorySingleCellGenomicsCenter
between members of the acI-A and acI-B clades. (SCGC; http://www.bigelow.org/scgc),asdetailedin
One such study, performed by Buck et al., Martinez-Garcia et al. (2012) (Table 1).
showedevidenceofsubstrate-basednichepartition- Ten SAGs from lakes Mendota, Sparkling and
ing, where acI-B-positive cells could consume Damariscotta were selected during the SAG library
acetate but acI-A-positive cells could not (Buck screening step described in Martinez-Garcia et al.
et al., 2009). (2012).Partial16SrRNAgenesamplifiedpreviously
A few recent studies have used (meta)genomics- (Martinez-Garcia et al., 2012) were phylogenetically
based analysis to study acI Actinobacteria. Ghai classified to the freshwater ‘tribe’ level by insertion
et al. analyzed metagenomic data from lakes, into reference trees in the ARB software package
estuaries (Ghai et al., 2012), and rivers (Ghai et al., (Ludwig et al., 2004; Newton et al., 2011). SAGs
2011), and found that acI members have a lower were selected in order to compare among acI tribes
thanexpectedGCgenomiccontent.Martinez-Garcia and source lakes. The one SAG from Lake Stechlin
et al. (2012) used single-cell genomics and found was selected from a separate library constructed at
that the origins of more than 80% of rhodopsins theSCGCasdescribedinGarciaetal.(2013)andits
found among lake bacteria belong to the actinobac- 16S rRNA gene was 100% identical to the
teria. Another study reported the first nearly acI-B1 SAG previously analyzed (AAA027-L06).
complete acI genome from the acI-B1 tribe obtained Phylogenetic reconstruction was conducted by
using single-cell genomics (Garcia et al., 2013). In maximum likelihood (RAxML; Stamatakis et al.,
short, the genome was small (estimated to be just 2008) with 1000 bootstrap runs on the CIPRES web
over 1Mbp) and low GC (B42%). Metabolic recon- portal (www.phylo.org) using near full-length refer-
struction indicated that members of acI-B1 are ence 16S rRNA gene sequences from a manually
facultative aerobes with a capability for taking up curated alignment (Newton et al., 2011) and a 50%
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Table1 AcISAGmetadataandoverallfeatures
FullSAGID IMGtaxon Tribe Estimatedgenome Lake Date Mbp Estimatedcomplete %GC Scaffolds Genes
OID completeness(%)a collected genomesize(Mbp)
SCGCAAA027-M14 2236661003 acI-A1 41 MEN 5/19/09 0.8 2.0 47 22 863
SCGCAAA278-O22 2236661007 acI-A1 96 DAM 8/19/09 1.1 1.1 48 43 1238
SCGCAAA044-N04 2236661005 acI-A7 78 DAM 4/28/09 1.3 1.7 46 23 1341
SCGCAAA023-J06 2236661001 acI-A7 36 SPA 5/28/09 0.7 1.9 45 98 818
SCGCAAA024-D14 2264265190 acI-A7 71 SPA 5/28/09 0.8 1.1 45 82 892
SCGCAAA027-J17 2236661002 acI-B1 84 MEN 5/19/09 1.0 1.2 42 81 1094
SCGCAAA028-A23 2236661004 acI-B1 80 MEN 5/19/09 0.8 1.0 42 64 913
SCGCAAA278-I18 2236661006 acI-B1 74 DAM 8/19/09 0.9 1.2 41 54 1037
SCGCAAA023-D18 2236661009 acI-B1 51 SPA 5/28/09 0.8 1.6 40 67 827
SCGCAAA027-L06 2505679121 acI-B1 499 MEN 5/19/09 1.2 1.2 42 75 1282
SCGCAB141-P03 2236876028 acI-B1 34 STE 5/25/10 0.7 2.0 41 66 736
Abbreviations:DAM,DamariscottaLake(Mesotrophic);MEN,LakeMendota(Eutrophic);SAG,single-amplifiedgenome;SPA,SparklingLake
(Oligotrophic);STE,LakeStechlin(Oligotrophic).
aEstimatedbasedonthepresenceofsingle-copygenesfoundinotherActinomycetales,asdescribedinMaterialsandmethods.
basefrequencyfilter(total1402positions;Figure1). 2009). No putative contaminants were found in any
For four SAGs only short fragments (B400bp) were of the assemblies.
available. Bootstrap values are indicated above GeneswereidentifiedusingProdigal(Hyattetal.,
nodes with greater than 50% support and the scale 2010). ThepredictedCDSs (coding DNAsequences)
bar represents 10 base substitutions per 100 nt were translated and used to search the National
positions. Center for Biotechnology Information (NCBI) non-
redundant database, UniProt, TIGRFam, Pfam,
KEGG, Clusters of Orthologous Group (COG) and
Genome sequencing, assembly, contamination InterPro databases. The tRNAScan-SE tool (Hacker
detection and annotation and Kaper, 2000) was used to find tRNA genes,
Thedraftassembliesofthesingle-cellgenomeswere whereas ribosomal RNA genes were found by
generated at the DOE Joint genome Institute (JGI) searchesagainstmodelsoftheribosomalRNAgenes
usingtheIlluminatechnology.AnIlluminastandard built from SILVA (Pruesse et al., 2007). Other non-
shotgun library was constructed and sequenced coding RNAs such as the RNA components of the
using the Illumina HiSeq 2000 platform by pooling protein secretion complex and the RNase P were
libraries for approximately 10 SAGs per lane. All identifiedbysearchinggenomesforthecorrespond-
general aspects of library construction and sequen- ingRfamprofilesusingINFERNAL(Makarovaetal.,
cing performed at the JGI can be found at http:// 1999). Additional gene prediction analysis and
www.jgi.doe.gov/.RawIlluminasequencedatawere manual functional annotation was performed
filtered for known Illumina sequencing and library within the Integrated Microbial Genomes (IMG;
preparationartifactsandthenscreenedandtrimmed Markowitz et al., 2012) platform developed by the
accordingtothek-merspresentinthedataset.Reads Joint Genome Institute, Walnut Creek, CA, USA
representing highly abundant k-mers were removed (http://img.jgi.doe.gov).
such that no k-mers with a coverage of more than
(cid:2)30 were present after filtering. Contigs with an
average k-mer depthoflessthan (cid:2)2wereremoved. Genome completeness and size estimates
The following steps were then performed for Genome size and completeness were estimated
assembly:(1)filteredIlluminareadswereassembled using a conserved single-copy gene (CSCG) set that
using Velvet version 1.1.04 (Zerbino and Birney, has been determined from all finished actinobacter-
2008). The VelvetOptimiser script (version 2.1.7) ial genome sequences (n¼151) in the IMG database
was used with default optimization functions (n50 (Markowitz et al., 2012). The set consists of 158
for k-mer choice, total number of base pairs in large CSCGsthatwerefoundtooccuronlyonceinatleast
contigs for cov_cutoff optimization). (2) 1 to 3kbp 95% of all genomes by analysis of an abundance
simulatedpairedendreadswerecreatedfromVelvet matrix based on hits to the protein family (Pfam)
contigsusingthewgsimsoftware.(3)thenormalized database(Puntaetal.,2012).HiddenMarkovmodels
Illumina reads were assembled together with simu- of the identified Pfams (Supplementary Table S2)
lated read pairs using Allpaths-LG (version 41043) wereusedtosearchallSAGassembliesbymeansof
(Gnerre et al., 2011). the HMMER3 software (Eddy, 2011). Resulting best
Weemployedacombinationoftetramerprincipal hitsabovethetrustedcutoff(TCfieldasprovidedin
component analysis and blast searches against the HMM files from Pfam) were counted and the
reference databases to identify contigs that may completeness was estimated as the ratio of found
originate from DNA contaminants (Woyke et al., CSCGtototalCSCGsinthesetafternormalizationto
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SCGC AAA027−M14, HQ663196, 410 bp
SCGC AAA278−O22, JGI Gene OID=2236668838
59 Rio III Reservoir clone AL_9 , HQ008541 acI-A1
81 Trout Lake clone TR1C6, EU117945
Lake Grosse Fuchskuhle clone FukuN30, AJ289996
Lake Gossenkoellesee clone GKS2−103, AJ290024
Trout Lake clone LT1C1, EU117824
97 Lake Mendota clone ME7D10, EU117768
61 Chinese hypereutrophic lake clone ML−3−12.2, DQ520158
acI-Phila
Trout Lake clone LT1C9, EU117829
80 Mirror Lake clone MI1C8, EU117868
74
Candidatus Planktophila limnetica, FJ428831
Lake Mendota clone ME7B3, EU117764
99 TLroaukte L Satkeec hclloinn cel oLnTe1 B, D8,Q E3U11613768720 acI-A4 acI-A
51 Lake Schoehsee clone S7, AJ575508
Lake Niegocin clone N3, AJ575530
100 Lake Mendota clone ME001A11, FJ827873 acI-A5
100 Trout Lake clone LT1F11, EU117842
SCGC AAA023−J06, JGI Gene OID=2236662227
SCGC AAA044−N04 JGI Gene OID=2236666169
acI-A7
Lake Stechlin clone , DQ316356
100 Green Lake clone GR1E8, EU117702
Trout Lake clone TR1C7, EU117946
SCGC AAA024−D14, HQ663054, 431 bp
97 100 LLaakkee KMinennedroetta f oclsomneid MKE0074D,5 G, EQU381714797270 acI-A6
81 Dongping Lake clone DP3.2.21, FJ612142
Jimi Hendrix Bog Lake clone JB1E11, EU117749
Lake Mendota clone ME7C3, EU117767
Cox Hollow Lake clone CH1A5, EU117603
Trout Lake clone TR1B1, EU117936
Rio III Reservoir clone FL_45 , HQ008593
SCGC AAA023−D18, JGI Gene OID=2236670490
Rimov Reservoir clone R6, AJ575502
Yellowstone Lake clone YL195 , HM856556
acI-B1
Toolik Lake clone, AF534431
Dongping Lake clone DP10.2.14, FJ612345
SCGC AAA027−L06, JGI Gene OID=2506125100
SCGC AB141−P03
SCGC AAA028−A23, JGI Gene OID=2236664786 acI-B
SCGC AAA278−I18, HQ663560, 405 bp
100 SCGC AAA027−J17, HQ663173, 411 bp
Lake Stechlin clone ST11−16, DQ316361
67 100 LCarkyes tFalu Bchosgk Luahklee cclloonnee FCSBW02111F−92,, HDQQ533126139591 acI-B2
98 Ike Walton Lake clone IW1E8, EU117739
Adirondack Lake clone ADK−GRe02−15, EF520354
100 Crystal Bog Lake clone CB2F8, HQ532245 acI-B3
100 Lake Grosse Fuchskuhle clone SW10, AJ575554
95 Firefly Lake clone FF1D8, EU117669
Lake Schoehsee clone S8, AJ575509 acI-B4
97 Brandy Lake clone BR1E4, EU117574
MiYu Reservoir clone MYY33 , GU305723
Rio III Reservoir clone FL_17, HQ008573
100 Lake Zurich clone ZS−2−02., FN668289 acI-C2
100 Chinese hypereutrophic lake clone ML−9−97.2, DQ520197
100 Lake Delton clone DE1H8, FJ916092
Lake Gatun clone 5C231028 , EU803457
acI-C1
53 Trout Lake clone LT1E5, FJ916087
99 Yellowstone Lake clone YL206 , HM856567
Corynebacterium glutamicum, Z46753
95 Mycobacterium bovis, M20940
Rhodococcus opacus DSM 43205 (T), X80630
77 Acidothermus cellulolyticus 11B, AJ007290
99 Nocardioides sp. JS614, NC_008699
Propionibacterium freudenreichii subsp shermanii, Y10819
83 Thermobifida fusca, AF002264
89 Intrasporangium calvum DSM 43043, NC_014830
98 Cellulomonas fimi DSM 20113 (T), X79460
100 Arthrobacter chlorophenolicus, AF102267
Streptomyces flavogriseus ATCC 33331, NC_016114
0.1 changes per site
Figure1 PhylogeneticplacementoftheSAGswithintheacIlineageandrelativetoothersequencedactinobacterialgenomes,andtothe
previouslysequencedAAA027-L06SAGbasedonnearlyfull-length16SrRNAgenesequences.Whenonlyshort(B400bp)amplified
gene fragments were available (Martinez-Garcia et al., 2012), sequences were added after tree construction within the ARB Software
using the maximum parsimony criterion (Ludwig et al., 2004). These shorter sequences are noted along with their corresponding
accessionnumber.ShadedsequencesarefromSAGs.
95%.Theestimatedcompletegenomesizewasthen tribe were downloaded from the IMG website
calculated by dividing the estimated genome cover- (http://img.jgi.doe.gov/; Supplementary Table S3).
age by the total assembly size. A presence/absence COG list was generated and
used to determine COG prevalence (% of genomes
containing a certain COG). The data were used to
Comparative analysis determineCOGprevalence(%ofgenomescontaining
Annotations with the COGs of proteins for a certain COG) in acI as well as the other
each SAG and comparison genome from the organisms of interest. The COG list was then
Actinomycetales order, Polynucleobacter genus, sorted based on difference in prevalence
and freshwater Alphaproteobacteria LD12 (% prevalence in acI divided by % prevalence
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in other group) between acI and a group of acImetabolism:howdoestheacIlineagemakealiving
interest. COGs at the top of this sorted list had in freshwater?
the greatest difference in prevalence between acI Carbon and energy. The previously published
and the group of interest and were considered AAA027-L06 acI-B1 genome indicated a facultative
overrepresented in acI. aerobic lifestyle with mostly complete archetypal
Average protein identity between SAG pairs was central metabolism (glycolysis, pentose phosphate
calculatedusingall-versus-allblastpfrompredicted pathway and citrate cycle), oxidative phosphoryla-
codingsequencesinall11SAGstoidentifybesthits tion machinery, and the ability to ferment pyruvate
foreachSAGpair.Resultswereparsedusingcustom (Garcia et al., 2013). These and other features were
perl scripts to identify reciprocal blast best hits that largely conserved among our ten newly sequenced
had alignments over at least 50% of both the query acI SAGs (Figure 2), with exceptions attributable
and the subject sequence lengths. Averages were either to incomplete genome recovery or niche
calculatedforeachpairbasedonthereciprocalblast diversification among clades or tribes (discussed
best hit results. furtherbelow).AsnotedforAAA027-L06,theSAGs
seem capable of metabolizing glucose but lack an
obvious glucosetransport system.This iscuriousin
light of MAR-FISH-based studies clearly demon-
Codon bias strating the ability of acI to incorporate glucose
For each genome, we calculated the frequency of (Buck et al., 2009; Salcher et al., 2013). However,
each amino acid codon out of the total codons most SAGs did contain ABC-type sugar transport
encodingthecorrespondingaminoacid,onceinthe components (for example, COG1653) (Table 2 and
ribosomal protein genes and once in the rest of the Supplementary Table S5) and using the Transporter
genes. We then calculated the average difference Classification Database (Saier et al., 2009), a peri-
between codon frequencies in the ribosomal pro- plasmic component with closest match to the
teins and in the rest of the proteins in the genomes. glucose-binding protein in Thermus thermophilus
In order to calculate the genome, CAIave (Codon (TC#3.A.1.1.24) was found in eight SAGs but was
Adaptation Index) for each organism we computed missing in AAA027-L06. Thus, we cannot confirm
the average of CAI values overall genes in the nor refute the ability of all acI members to take up
genome (Rocha, 2004). The CAI of a gene was glucose based on genomic evidence alone. Other
computed as described by (Sharp and Li, 1987). transporters that the 11 acI SAGs have in common,
include ABC-type transporters for ribose/xylose/
arabinose/galactoside, polyamines, dipeptides and
Results and discussion branched-chain amino acids (Table 2). The poly-
amine transporters are likely being used for putres-
Genome statistics and phylogenetic affiliation cine uptake, as the SAGs harbor downstream
We sequenced ten acI SAGs and analyzed them pathways for its eventual conversion to succinate
along with the one previously sequenced SAG via the transamination pathway (Dasu et al., 2006;
(Garcia et al., 2013). These 11 SAGs belong to three Chouetal.,2008);(Figure2).Genesfortheuptakeof
different acI tribes and originate from four different carboxylic acids in acI-A1, acI-A7 and acI-B1 were
lakes. A phylogenetic tree of these SAGs is pre- not found in any of the sequenced genomes,
sented in Figure 1. Our genome completeness consistent with MAR-FISH-based studies (Buck
estimates ranged from 34% to a nearly complete et al., 2009; Salcher et al., 2013).
genome.Onlyfourofthe158Pfamsusedforgenome Most SAGs also contained a putative cyanophy-
completion estimates were absent in all 11 SAGs cinase(COG4242),allowingacItoaccessthisC-and
(Supplementary Table S2). Back calculating the N-storage compound synthesized by cyanobacteria.
estimated genome size of the SAGs, it could be Homologs of cyanophycinase are typically found in
observed that all 11 of them were small (1–2Mbp) cyanobacteriawheretheyareusedtobreakdownan
and had relatively low GC% (40 to 48%). This low intracellular granular C and N storage molecule,
GC% had been observed already in metagenomic cyanophycin (Richter et al., 1999). Cyanophycin
assemblies (Ghai et al., 2012). The previously synthetase and/or cyanophycinase homologs have
analyzed acI-B1 genome also determined low GC been detected in about 10% of heterotrophic
content (B42%) and small genome size (B1.16 genomes analyzed (Krehenbrink et al., 2002;
Mbp; Garcia et al., 2013). Our comparative genomic Fueser and Steinbuechel, 2007). The acI SAGs
analysis confirms that these features are broadly analyzed here have the genes required to break
representative of the acI lineage and further distin- down cyanophycin granules and take up the result-
guishesamongtheacItribes,withacI-B1havingthe ingdipeptidesandaminoacidsasasourceofenergy
lowest GC (Table 1 and Supplementary Table S4). and N. However, it is unclear whether this cyano-
The average genome completeness was 68% (based phycinase can be secreted, as we were unable to
on the fraction of CSCGs). Therefore, we would identify an obvious secretion signal peptide.
expect to encounter a core, single-copy gene in Secreted cyanophycinases have been reported in
about seven of the 11 SAGs. both Gram negative and Gram positive soil bacteria
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GLYCOLYSIS PENTOSE PHOSPHATE PATHWAY
Xylose
Glucose Glucono- 6-Phospho-
1,5-lactone-6P gluconate Xylulose
Glucose-6P Arabino-
Hexulose-6P Ribulose-5P Xylulose-5P
Fructose-6P Xylulose-5P
Ribose-5P Ribose
Fructose-1,6P2
PRPP
Phenylalanine Glyceraldehide-3P
Tryptophan
1,3-Biphosphoglycerate
Isoleucine
Valine 3-Biphosphoglycerate Spermidine
Leucine Extracellular
2-Biphosphoglycerate Polyamine
Homo-
CO2 spermidine Putrescine
(atmosphere) Phosphoenolpyruvate
?
Pyruvate 1,3-Diaminopropane 4-aminobutyraldehyde
?
Acetate S-Acetyldihydro- 3-Amino
lipoamide-E propanaldehyde 4-aminobutyrate
Glutamine
Acetyl-CoA NH
β-alanine Succinate semialdehyde 3
Acetaldehyde
Glutamate Proline
Ethanol Aspartate Ornithine
Citrate
cis- L-arginino Fumarate
Oxaloacetate Aconitate succinaseUREA
CYCLE
Malate Isocitrate
Arginine
Fumarate TCA CYCLE Oxalosuccinate Citruline
?
Succinate 2-Oxoglutarate n(Asp-4-Arg)dipeptide
Succinil-CoA 3-Carboxy-1-
Cyanophycin
hydroxypropyl-ThPP
S-Succinyl-
dihydrolipoamide-E
lipDoiahmydidroe--E LipoaEmide 75-10500-%752%5-500-%25% Percentage
acI-A (1 & 7)
acI-B (1)
Figure2 CentralcarbonmetabolismandotherrelevantmetabolicpathwaysidentifiedinacISAGs.Circlesdenotegenesencodingthe
necessary enzymes are present in that clade (blue for acI-A and green for acI-B), with the size of the circle being proportional to
thepercentageofSAGswithinthatcladethatwerefoundtocontainthatgene.Thepresenceandabsenceofgeneswasdeterminedwithin
theIMGenvironmentbasedonKEGGannotations.
(Obst et al., 2002, 2004; Sallam et al., 2011), but in polypeptides. We did not identify homologs of
these cases signal sequence cleavage was demon- genes involved cyanophycin synthesis. The poten-
strated or inferred. Although the SAG genomes tial ability of acI to break down cyanophycin hints
encode canonical Sec pathway components as well at a potential interaction between cyanobacteria
as twin arginine translocation genes, the predicted (or other microbes making cyanophycin) and acI,
cyanophycinase polypeptides do not have these whereby acI members acquire energy, C and N
signal translocation sequences. Similarly, sortases from this polymer synthesized by others. Curiously,
that would anchor secreted proteins to the cell wall we found two tandem copies of cyanophycin
were predicted in the acI genomes, but the sortase synthetase in each of the two Polynucleobacter
motif was not found in the cyanophycinase genomes but not in acI. This has implications for
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Table2 SelectDOCuptakeCOGsinacI,LD12,PnecCandActinomycetalesa
Abbreviations:Actm,non-acIActinomycetales;Carbs,carbohydrates;COG,ClustersofOrthologousGroup;DOC,dissolvedorganiccarbon;
PnecC,Polynucleobacternecessariusasymbioticus.
CellsareshadedaccordingtopercentofgenomesthatcontainthatCOG,toaidinvisualinterpretationofnumbers.AfulllistofDOCuptakeCOGs
ispresentedinSupplementaryTableS5.
aDOCCOGlistbasedon(Poretskyetal.2010).bLD12,acosmopolitanfreshwaterAlphaproteobacteriatribethatisasistergrouptoPelagibacter
ubiqueandSAR11.Numberofgenomesincludedintheanalysisisprovidedinparentheses.
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potentialinteractionsbetweenacIandPolynucleobacter acI lineage, though further experimental work is
when they co-occur (that is, Polynucleobacter needed to confirm whether such a chromophore is
synthesizing cyanophycin and acI breaking it actually synthesized by acI.
down). Co-occurrence of acI and Polynucleobacter Rhodopsin in Pelagibacter ubique and other
sp. has already been observed in laboratory enrich- marine bacteria promotes survival during nutrient
ments (Garcia et al., 2014). However, this kind of starvation periods (Gomez-Consarnau et al., 2007;
interaction is only speculative until experimental DeLong and Beja, 2010; Go´mez-Consarnau et al.,
confirmation can be made. 2010; Steindler et al., 2011); thus actinorhodopsin
Others have found that acI abundances change may serve a similar function in acI. In such an
through the seasons with maxima in spring and fall auxiliary system, it remains to be explored why
(AllgaierandGrossart,2006)andthatacIabundance there is not co-localization of the rhodopsin gene,
positively correlates with solar radiation (Warnecke the oxygenase gene and the core carotenoid synth-
et al.,2005), suggesting they gain some benefit from esis operon, in acI genomes. Interestingly, actinor-
higher light intensities either through phototrophy hodopsin expression in a German lake was not
orconsumptionofphotochemicallyproducedlabile linked directly to sunlight but rather to a circadian
dissolved organic carbon (DOC). The latter possibi- schedule with a maximum expression rate just
lityiscontradictedbytheapparentinabilityofacIto before dawn (Wurzbacher etal., 2012). Comparative
utilize carboxylic acids (see above), amajorproduct genomicanalysisalsorevealedthepresenceofgenes
of photochemical DOC reactions. Instead, previous involved in anaplerotic carbon fixation (carbonic
work suggested that actinorhodopsins are broadly anhydrase and phosphoenolpyruvate carboxylase).
distributed in freshwater Actinobacteria and The acI SAGs lack the RuBisCO enzyme and other
provide the potential for phototrophy (Sharma pathways for carbon fixation, but phosphoenolpyr-
et al., 2008, 2009; Martinez-Garcia et al., 2012; uvatecarboxylasemayprovidethemwiththeability
Garciaetal.,2013;Salkaetal.,2014).Weconfirmed to synthesize oxaloacetate, an intermediate in the
the presence of actinorhodopsin in all but three of tricarboxylic acid cycle cycle that can be used to
the eleven acI SAGs (AAA023-J06, AAA028-A23 replenish precursors needed for growth (Figure 2).
andAB141-P03)makingthisgenealikelypartofthe This may provide acI with the ability to grow
broadly shared genes of the acI lineage. Moreover, photoheterotrophically using actinorhodopsin,
enzymes required for synthesis of the presumed though we can only speculate in the absence of
actinorhodopsin chromophore, retinal or its precur- experimental evidence. Notably, a marine Bacteroi-
sors were identified in 10 of the 11 acI SAGs, detes (Polaribacter) lacking RuBisCO but harboring
suggesting the likely assembly of functional proteorhodopsin has been shown to increase CO
2
rhodopsininvivo.IntheacISAGs,thefourenzymes fixation rates in the light (Gonza´lez et al., 2008).
that lead from farnesyl pyrophosphate, the ubiqui-
tous sterol precursor, to b-carotene, the bicyclic Nitrogen, phosphorus and sulfur. As reported
conjugated chromophore, (Martinez et al., 2007) are previously for the acI-B1 SAG AAA027-L06, the
most commonly encoded in a cluster. The final lineage appears to lack identifiable genes involved
pathway enzyme, a b-carotene cleaving oxygenase, in sulfate, sulfite, nitrate and nitrite assimilation.
was somewhat surprisingly sequenced only in two Seven SAGs did harbor ammonia permease
of the 11 SAGs, and not co-located with the (COG0004), suggesting free ammonia is an N-source
rhodopsin gene as it is in many marine proteo- for some or all acI members. We found no evidence
bacteria (Martinez et al., 2007; Riedel et al., 2013; forureatransportorcatabolism.The11SAGsdonot
Vollmers et al., 2013). However, one of these SAGs harboracompleteureacycleamongthem.However,
places the oxygenase near a b-carotene pathway genome content across the lineage shows the
gene,likelyindicatingthatitoncecomposedpartof capability to consume N-rich carbon sources,
a full pathway operon. The genes necessary for including polyamines, cyanophycin, di- and oligo-
synthesis of a carotenoid glycoside ester sit just peptides and branched-chain amino acids such as
upstream of the cluster, suggesting a secondary leucine, isoleucine and valine. This has intriguing
chromophore for actinorhodopsin similar to the implications for acI’s success in freshwater, as it
salinixanthin discovered hydrophobically clinging seems to obtain both C and N from the same
toxantho-andGleobacterrhodopsins(Lueckeetal., substratecompounds.Thisobservationwouldbuild
2008; Imasheva et al., 2009). At the protein level, a onpreviousobservationsbasedonMAR-FISHusing
change of bacteriorhodopsin’s tryptophan 138 to a probes targeting the entire Actinobacteria phylum
glycineprovidesspaceforthefour-ketoringofsuch suggesting that acI contributes more than would
a putative secondary chromophore, and six of the have been expected based on its biomass to total
hydrophobic amino acid side chains that would amino acid turnover in lakes (Salcher et al., 2010).
interact with this putative rhodopsin-hugging Indeed, genes involved in acquisition of these
photon-funnel are shared between the actinorho- N-rich compounds also generally have high codon
dopsins and xanthorhodopsin (Balashov et al., biasintheSAGs(SupplementaryTableS6),indicat-
2010). This has exciting implications for the role of ing possibly high expression rates. Some prior
a chromophore other than retinal inmembers of the research has focused on organic-N concentrations
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or sources for freshwater bacterioplankton. Buck relative of the marine SAR11 (10 genomes) and
etal.(2009)usedMAR-FISHtotestglucose,leucine Polynucleobacter sp. (two genomes; Table 2 and
and acetate assimilation in freshwater. They found Supplementary Table S5). We found that several
that Actinobacteria had the highest percentage of COGs involved in polyamine uptake and meta-
cells incorporating leucine but not glucose or bolism were more common in acI than in the other
acetate, indicating a preference for nitrogen-rich analyzedgenomes.Infact,the10LD12genomesdid
organics. Salcher et al. (2013) also found that acI not contain any of these COGs. Other interesting
was active in leucine uptake. Some MAR-FISH COGs that were more common in acI were nucleo-
studies have suggested that another nitrogen-rich tide transporters, severalsugartransportersand two
compound, NAG may be an important acI substrate ribose/xylose transporters. Overall, acI had more
(Beier and Bertilsson, 2011; Eckert et al., 2012, COGs for general carbohydrate transport than LD12
2013).However,wecouldnotidentifymachineryto and Polynucleobacter.
support the ability to take up and incorporate this As described above, the acI genomes lacked
substance. This may well simply be due to the fact carboxylic acid and dicarboxylic acid transporters
that the biochemical pathways involved have not while they were present in Polynucleobacter and
been identified. It is probable that one of the many LD12. Compared with the other freshwater bacteria,
COGs involved in the uptake of carbohydrates general amino acid uptake COGs were under-
enables NAG uptake (Table 2). represented in acI SAGs and COGs for di- and
We found no evidence of genes for sulfite/sulfate oligopeptides were overrepresented. The di- and
assimilation or reduction in the acI SAGs, as was oligo-peptide transporters may have some connec-
previouslyreportedforAAA027-L06,suggestingthe tion with the cyanophycinase genes found in acI
proposed dependence on cysteine synthase for (and not in LD12 or Polynucleobacter). COGs
sulfur incorporation holds for the entire lineage involved in lipid transport were underrepresented
(Garciaetal.,2013).Cysteinesynthasewasfoundin inacI(ascomparedwithLD12andPolynucleobacter),
eightoftheelevenSAGs.AcI’sapparentrelianceon with only the glycerol transporter, COG1133 (long
reduced sulfurcompoundsis an interesting similar- chain fatty acid transport) and COG2867 (Oligoketide
ity to that of the ubiquitous marine SAR11 clade, cyclase/lipid transport protein) present.
whichwasthefirstfree-livingbacteriarecognizedto Taken altogether, these findings provide genome-
relyonreducedsulfurforgrowth(Groteetal.,2012; level confirmation of experimental observations
Carini et al., 2013). (Salcher et al., 2013): the differences in types of
We searched for evidence of phosphorus acquisi- DOC uptake genes found in each lineage indicate
tion strategies that may allow for high-affinity potentialspecializationinsubstratepreferencesand
transport (Pst) or low-affinity high rate of transport ecological niches. AcI genomes indicate specializa-
(Pit). Six of the acI genomes have a PstSCAB tion in polyamines, di- and oligopeptides and
transport system. Three other genomes had 75% of carbohydrates (including pentoses). LD12 genomes
these genes; the partial gene absence was likely due indicate specialization in lipids and carboxylic
to incomplete genome recovery. Only the two most acids, whereas Polynucleobacter specializes in gen-
incomplete SAGs were missing the majority of the eral carbohydrates and carboxylic acids.
Pst complex. The associated regulatory protein
PhoU and phosphorus starvation activated protein
PhoH were also found in most of the genomes. The Comparative genomics hints at the basis for ecological
presence of the Pst complex (in lieu of low-affinity differentiation within the acI lineage
Pit phosphorus transport complex) provides acI We sought to determine whether differences in
with the ability to acquire phosphorus in low- gene content might show potential niche separa-
nutrient settings, further indicating that these tion between members of the acI lineage (for
organisms are well adapted to phosphorus limita- example, separation of acI-A vs acI-B) (Figure 3,
tion, which is a common feature of these and most Supplementary Table S7). It should be noted that
freshwater lakes (Magnuson et al., 2006). We could this analysis is preliminary due to the incomplete-
not find any evidence for the ability to metabolize ness of the SAGs. Little is known about the ecology
phosphonates in the acI SAGs. of the acI tribes as most previous studies lacked
phylogenetic resolution to the tribe level. However,
Newton et al. (2007) found that acI tribes differ-
Shared and differential gene content involved in entiated based on pH with acI-B2 and acI-A1
heterotrophic growth among freshwater bacteria correlating with acidic lakes and acI-B1 and acI-A6
We asked whether the differential gene content correlating with alkaline lakes. The ratio of terrest-
among sequenced freshwater bacterial genomes rially sourced carbon to in-lake-derived carbon also
might also explain the ecological success of acI seemstopredicttribedistribution,withacI-B1,-A6,
during growth. First, we examined DOC uptake and -A7 generally preferring lakes with lower color-
Clusters of Orthologous Groups (COGs) for acI to-chlorophyll (CtCh) ratios as compared with other
versus other Actinomycetales (424 genomes) and tribes (Jones et al., 2009). This ratio is calculated
freshwaterbacteriasuchasLD12Alphaproteobacteria using measurements of chromophoric dissolved
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tobeanABCtransportsystemforribose(COG1172),
but the acI-B1 genomes uniquely carried an
acI-A7
auxillary component (ribose pyranase - COG1869)
106
that may give this tribe access to certain pentose
68
acI-A1 isomers(SupplementaryTableS8).TheacI-B1SAGs
58 did carry an aromatic ring-cleaving dioxygenase
723 88
(DOPA4,5-dioxygenase)thatwasnotfoundinacI-A
65 or any other Actinomycetales (COG3805) but is
acI-B1
commonly found in fungal genomes. Homologs of
85
thisproteinarekeyforthebiosynthesisofpigments
such as betalains (red and yellow pigments) in
plants, suggesting the role of a unique pigment in
Figure 3 Distribution of 1193 COGs found in the acI lineage acI-B1.
based on the analysis of 11 SAGs. Shared and differential COG
We note that based on the genome recoveries
content among acI tribes within the clades based on the
(Table 1), the probability of an acI ‘shared COG set’
comparisonoftwoacI-A1SAGs,threeacI-A7SAGsandsixacI-
B1SAGs.Markedly,moreCOGsaresharedamongacItribesthan gene missing from all the genomes due to incom-
areuniquetoanyparticulartribe.Wenotethattheprobabilityof plete recovery is 0.000003% in acI, 0.003% in acI-
anacI‘sharedCOGset’genebeingmissingfromallthegenomes B1, 2.7% in acI-A1 and 4.1% in acI-A7, for any
due to incomplete genome recovery is 0.000003% for all of acI,
single COG. These probabilities were calculated by
0.003% in acI-B1, 2.7% in acI-A1 and 4.1% in acI-A7, for any
singleCOG. multiplyingtheprobabilityofagenemissingineach
genome (that is, same as percent of genome
incompleteness). For example, the acI-A1 genome
organic matter (by light absorbance at 440nm) and SCGC AAA027-M14 is only 41% complete, there-
chlorophyll-a concentrations, making it a useful fore the probability of a ‘shared COG set’ gene
proxy for the extent of allochthony of a lake missing fromthegenomeis59%.The probabilityof
ecosystem. Interestingly, acI-A1 is found in lakes the same gene missing from the acI-A1 genome
withbothlowandhighCtCh.Asthetribesincluded SCGCAAA278-O22is4%andtheprobabilityofthe
in the current study are usually found in lakes with genemissingfrombothgenomesduetoincomplete-
low CtCh, it remains to be learned whether other ness is 2.7% (that is, 59 (cid:2) 4%). These probabilities
tribes (for example, acI-B2, acI-A4, acI-A5) have indicatethatthe‘sharedCOGset’basedontheSAGs
different gene content that relates to life at high for acI and acI-B1 are essentially complete, but that
CtCh. the ‘shared COG set’ for acI-A and corresponding
Members of the acI-A clade contained markedly tribes is likely to expand as much as 4.1% with the
more unique COGs than members of the acI-B sequencing of more genomes.
clade (compare Supplementary Table S7 with The average protein similarity between the acI
Supplementary Table S8), suggesting they are more SAGs indicated diversity within clades and a range
metabolically versatile. However, this might be invariationwithintribes,attheprotein-codinggene
explained by the fact that two acI-A tribes are level (Supplementary Table S9). The average amino
representedinourSAGcollectionascomparedwith acid identity across tribes (65–70% acI-A1 vs acI-
one acI-B tribe. Nevertheless, we found that the A7) indicate that the acI-A clade is similar to a
alpha and beta-galactosidases were common to traditional taxonomic genus, which has been esti-
acI-A1 and acI-A7, but were absent in all acI-B1 mated to have 65%–70% average protein identity
SAGs (Supplementary Table S7). The presence of (Konstantinidis and Tiedje, 2007). The comparison
these genes in acI-A SAGs suggests a specialization ofaminoacididentityacrossclades(60–65%amino
in breaking down poly- or oligo-saccharides as acid identity acI-B1 vs acI-A1 and acI-A7) indicates
compared with acI-B. Interestingly, an over- that the acI lineage is comparable to a traditional
representation of glycosidases such as alpha and taxonomic family.
beta-galactosidaseswasfoundinfreshwatersystems
compared with marine systems (Eiler et al., 2013).
Thiseffectmaybelargelyduetothepresenceofthe Shared gene content within acI distinguishes the
ubiquitous acI-A clade in freshwater. Zakharova lineage from other Actinomycetales
etal.(2013)studiedbacteriaassociatedwithdiatom Any genes that distinguish acI clades from their
degradation in the near bottom water layer of Lake relatives within the Actinomycetales are good
Baikal and found high beta-galactosidase activity. candidates for further inquiry into their role in acI
They also found that Actinobacteria were a signifi- ecophysiology, as they may explain why acI is such
cant portion of the bacterial community. The acI-A an omnipresent and successful resident of fresh-
clade also had two glycerol uptake COGs that were water systems (Newton et al., 2011). However, we
not found in acI-B1. The acI-A7 tribe had three identified only two COGs that were found in acI to
COGs involved in amino acid uptake as well as an the exclusion of all other sequenced Actinomyce-
NAG kinase and oligopeptide transporter not found tales: an aromatic ring-cleaving dioxygenase
in acI-A1 or acI-B1. Both clades share what appears (COG3805) and an uncharacterized conserved
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