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Microbial Ecology

SupplementaryMaterials

Spatial Patterns of bphA Gene Diversity Reveal Local Adaptation of Microbial Communities to PCB and PAH Contaminants

Juan L. Bouzat and Matthew J. Hoostal

Department of Biological Sciences

Bowling Green State University

Bowling Green OH, 43403

Table S1. Contaminant concentrations (mg contaminant/kg sediment) and physical attributes (% organic matter and grain size) of sedimentscollected from polluted (DC-14) and unpolluted (DC-15) locations of Duck Creek. Values in bold exceed the Ecological Reference Limit (ERL) whereas values in italics exceed the Human Health Reference Limits (HHRL) established by the EnvirontmenalProtection Agency (US EPA, 2007).

Contaminant / Concentration (mg/kg)
DC-14 / DC-15 / ERL / HHRL
Total PCBs / 0.34 / UN / 0.06 / 0.22
Aroclor 1254 / 0.20 / UN / NE / NE
Aroclor 1260 / 0.15 / UN / NE / NE
Total PAHs / 801.00 / 0.72 / 1.61 / NE
Anthracene / 32.40 / 0.52 / 0.06 / 22000.00
Benzo(a)anthracene / 87.20 / 0.07 / 0.11 / 0.62
Benzo(a)pyrene / 82.50 / 0.07 / 0.15 / 0.06
Benzo(b)fluoranthene / 10.70 / 0.11 / NE / 0.62
Benzo(g,h,i)perylene / 2.53 / 0.52 / NE / 2300.00
Benzo(k)fluoranthene / 38.60 / 0.52 / NE / 6.20
Chrysene / 80.90 / 0.09 / 0.17 / 62.00
Dibenz(a,h)anthracene / 9.74 / 0.52 / 0.03 / 0.06
Fluoranthene / 190.00 / 0.18 / NE / 0.42
Fluorene / 8.72 / 0.52 / 0.08 / 2700.00
Indeno(1,2,3-cd)pyrene / 32.90 / 0.52 / NE / 0.62
Naphthalene / 1.93 / 0.52 / 0.18 / 56.00
Phenanthrene / 68.40 / 0.06 / 0.20 / 22000.00
Pyrene / 150.00 / 0.14 / 0.20 / 2300.00
Heavy Metals
Arsenic / 86.00 / 52.10 / 9.79 / 0.39
Cadmium / 1.23 / 0.37 / 0.99 / 37.00
Lead / 226.00 / 78.20 / 35.80 / 400.00
Physical characterization / % total mass / ERL / HHRL
Total Organic Carbon (%) / 10.50 / 2.96 / NE / NE
Sediment Grain Size Analysis / % total mass / Classification
Sieve 4 / 12.5 / 9.5 / Sand
Sieve 10 / 15.5 / 9.1 / Sand
Sieve 35 / 13.7 / 8.0 / Sand
Sieve 50 / 10.4 / 11.0 / Sand
Sieve 100 / 17.8 / 14.7 / Sand
Sieve 200 / 3.4 / 13.3 / Silt/Clay

NE: not estimated; UN: undetectable.

Table S2. Rank abundances of bacterial taxa based upon characterizations of 298 16S rDNA sequences collected from DC-14 (polluted) and DC-15 (unpolluted) sample locations. Data indicates the number of sequences identified as particular bacterial taxonomic group based on a combination of phylogenetic analyses, RDP and BLAST queries (see Methods).

Polluted / Unpolluted
Bacterial Taxon / Number of Sequences / Bacterial Taxon / Number of Sequences
Betaproteobacteria / 45 / Bacteroidetes / 48
Bacteroidetes / 27 / Betaproteobacteria / 38
Firmicutes / 20 / Deltaproteobacteria / 9
Deltaproteobacteria / 9 / Chlorobi / 8
Gammaproteobacteria / 8 / Gammaproteobacteria / 7
Alphaproteobacteria / 7 / Alphaproteobacteria / 7
Chloroflexi / 7 / Firmicutes / 6
Chlorobi / 7 / Actinobacteria / 6
Actinobacteria / 6 / Chloroflexi / 5
Acidobacteria / 5 / Cyanobacteria / 3
Nitrospira / 4 / Candidate division BRCI / 3
Gemmatimonadetes / 2 / Acidobacteria / 2
Armatimonadetes / 1 / Gemmatimonadetes / 2
Planctomycetes / 1 / Planctomycetes / 2
Verrumicrobiota / 0 / Nitrospira / 2
Cyanobacteria / 0 / Verrumicrobiota / 1
Candidate division BRCI / 0 / Armatimonadetes / 0
Total = / 149 / Total = / 149

Table S3. Reference sequences used for the phylogenetic reconstruction of 16S rDNAsequences detected in Duck Creek (Figure S1). Reference sequences are listed with corresponding GenBank accession numbers, species name, and taxonomic classification.

Accession numberSpeciesTaxa

NR125648AltererythrobacteratlanticusAlphaproteobacteria

JX504811 MicrovirgavignaeAlphaproteobacteria

NR027547AnaeromyxobacterdehalogenansDeltaproteobacteria

M34407DesulfosarcinavariabilisDeltaproteobacteria

NR025358Thiobacillusdenitrificans Betaprotebacteria (order Hydrogenophilales)

FJ599672Acidovorax soli Betaproteobacteria (order Burkholderiales)

NR037091Clostridium bowmaniiFirmicutes (class Clostridia)

HM054473Bacillus hunanensisFirmicutes (class Bacillales)

JQ781598Pseudomonas putidaGammaproteobacteria

EF153292LamprobactermodestohalophilusGammaproteobacteria

NR024765RhodococcusjostiiActinobacteria

EF466110NocardioidessediminisActinobacteria

AJ585428Flavobacteriumpsychrolimna Bacteroidetes (class Flavobacteria)

CP002345PaludibacterpropionicigenesBacteroidetes (class Bacteroidia)

NR115472CytophagahutchinsoniiBacteroidetes (class Cytophagia)

KC430923ChitinophagapolysacchareaBacteroidetes (class Sphingobacteriia)

NR029287NitrospiramoscoviensisNitrospirae

NR041355LongilineaarvoryzaeChloroflexi

NR132330LitorilineaaerophilaChloroflexi

X62912Blastopirellula marinaPlanctomycetes

AJ290824ChlorobiumlimicolaChlorobi

Table S4. Twenty-six reference sequences used for the phylogenetic reconstruction of bphA sequences detected in Duck Creek (Figure 2). Reference sequences are listed with corresponding Genbank accession numbers, assigned gene name, species name, and taxonomic classification.

Accesion number Gene nameSpecies nameTaxa

AF546904 NidA Mycobacterium sp. S65 Actinobacteria

DQ537941 NidA Mycobacterium pallens Actinobacteria

5931576PhdANocardioides sp.Actinobacteria

166197617DfdANocardioidessp.Actinobacteria

AB302135 DfdA Rhodococcus sp. YK2 Actinobacteria

AJ401612NarARhodococcus sp. 1BNActinobacteria

33867057IpbARhodococcuserythropolisActinobacteria

X80041BphARhodococcusgloberulusActinobacteria

FJ183471ThnARhodococcusjostiiActinobacteria

CP000433EtbARhodococcusjostiiActinobacteria

226349831BnzARhodococcusopacusActinobacteria

AB110633DodARhodococcusopacusActinobacteria

313150331DfdATerrabacter sp.Actinobacteria

U51165DbdCXanthobacterpolyaromaticivoransAlphaproteobacteria

M86348BphABurkholderiasp. LB400Betaproteobacteria

AB70355BphAComomonastestosteroniBetaproteobacteria

EF600714 BphAPandoraeapnomenusa Betaproteobacteria

32562909BphA1Bacillus sp. JF8Firmicutes

U53507IpbAPseudomonas JR1Gammaproteobacteria

U15298TcbAPseudomonas sp.Gammaproteobacteria

1256702CumAPseudomonas fluorescensGammaproteobacteria

AF049851EdoAPseudomonas fluorescensGammaproteobacteria

M83673BphAPseudomonas pseudoalcaligenesGammaproteobacteria

AY831463CbzAPseudomonas putidaGammaproteobacteria

AF006691IpbAPseudomonas putidaGammaproteobacteria

J04996TodC1Pseudomonas putidaGammaproteobacteria

Figure S1. Bayesian phylogenetic reconstruction of 16S rDNA sequences characterized from Duck Creekand 21 reference sequences from major bacterial taxonomic groups.Reference sequencesare labeled in bold with the first letter of the genus followed by the first three letters of the species(GenBank accession numbers of reference sequences are reported in Table S3). Posterior probabilitiesgreater than 0.8 are denoted with black circles.Bacterial taxa from class Bacilli (phylum Firmicutes), class Alphabacteria (phylum Proteobacteria), and phylum Actinobacteria have thicker black branches, as these groups of bacteria represent potential members of the microbial community more likely to have bphA sequences with relatively broad substrate ranges.




Polluted Sediments




Unpolluted Sediments

Figure S2. Rarefaction curves of 16S rDNA sequences collected from polluted (DC-14) and unpolluted (DC-15) sediments at Duck Creek. The y-axis represents the number of new taxa discovered, as determined by a combination of RDP classifier, tBLASTx, and phylogenetic analyses, whereas the x-axis represents the number of clones analyzed. The plateau of the curves suggests that the number of clones collected from polluted and unpolluted locations was sufficient to determine predominance of major taxonomic groups at the level of bacterial phyla and classes.

Figure S3. Codon-specific analysis of positive selection based on dN/dS ratios from bphA sequences characterized from Duck Creek sediment samples. The ratio of dN/dS (y-axis) was calculated for each codon position (x-axis) using Hyphy software (Pond et al., 2005), implemented with the program MEGA. Thick bars along the x-axis denote substrate-binding regions of the BphA protein from Rhodococcusjostii. Asterisks denote codons critical to substrate specificity.

Figure S4. Multiple sequence alignment of previously characterized biphenyl dioxygenase homologs that recognize small, organic xenobiotics(CumAPflu, CumAfrom Pseudomonas fluorescens; and BphAPKF707, BphA from Pseudomonas pseudoalcaligenes KF707), a biphenyl dioxygenase that recognizes both PCBs and PAHs (BphARRHA1,BphA from Rhodococcusjostiisp. RHA1),as well as a translated consensus sequence of the bphAfrom Duck Creek sediments. Boxesrepresent the substrate-binding pocket of the BphA protein described in a study of R.jostii sp. RHA1 [73]. Asterisks indicate residues associated with substrate specificity in R. jostii. Alpha-helices (dashed lines), beta-sheet (solid lines), and critical residues are labeled according to their designation in the biphenyl dioxygenase of R. jostii.