Article Figures & Data
- FIG 1
Larval development time and TAG content in monoassociated D. melanogaster flies. Traits for D. melanogaster that were monoassociated with each of 41 bacterial strains were measured. Phylogenetic trees were calculated using 16S sequences with unweighted branch lengths. Taxon abbreviations are defined in Table 1. Significant differences between treatments after Bonferroni correction (P < 0.05) are indicated by different letters next to bars. (A) Differences in bacterial effects on larval time to pupariation (development time) were observed between strains. Survival analysis using a Cox mixed model was used to identify significant differences between treatments, with experimental replicate and vial as random effects. To facilitate visualization, data are presented as the mean times to development ± standard errors of the means (SEM). (B) Differences in bacterial effects on TAG content were observed between strains. A linear mixed model was used to identify significant differences between treatments, with experimental replicate as a random effect. Data are presented as mean TAG content ± SEM (mean of experimental means). Red, Proteobacteria; blue, Firmicutes; gray, Bacteroidetes.
- FIG 2
Experimental validation of candidate microbiota genes associated with host TAG level. (A) TAG content is shown for gnotobiotic flies monoassociated with either A. pasteurianus 3P3 or A. tropicalis DmCS_006 bearing the plasmids indicated. (B) Glucose content of fly diet after gnotobiotic rearing from egg to adulthood with recombinant strains bearing the indicated plasmids. Values are means ± standard errors for 3 experiments with 7 to 9 technical replicates each. Significant difference from the results for the control were determined by Dunnet’s test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
- FIG 3
Microbiota effects on diet. (A) The glucose contents of fly diet after gnotobiotic rearing from egg to adulthood with a subset of strains from the 41-strain panel are shown. Microbiota treatments are indicated along the x axes. Values are means ± standard errors for 3 experiments with 7 to 9 technical replicates each. Significant differences from the results for the control were determined by Dunnet’s test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (B) Correlation between TAG contents of gnotobiotic flies and glucose contents of food remaining after rearing. Statistics are from Spearman’s rank order test.
- TABLE 1
Bacterial strains
Strain name Abbreviationa GenBank accession no. Preferred mediumb Oxygen conditionsb Acetobacter aceti NBRC 14818 aace BABW00000000 mMRS Oxic Acetobacter malorum DmCS_005 amac JOJU00000000c mMRS Oxic Acetobacter pasteurianus 3P3 apa3 CADQ00000000b,d mMRS Oxic Acetobacter pomorum DmCS_004 apoc JOKL00000000c mMRS Oxic Acetobacter pasteurianus NBRC 101655 apan BACF00000000 mMRS Oxic Acetobacter tropicalis DmCS_006 atrc JOKM00000000c mMRS Oxic Acetobacter tropicalis NBRC 101654 atrn BABS00000000 mMRS Oxic Bacillus subtilis subsp. subtilis strain168 bsub NC_000964.3 LB Oxic Dysgonomonas mossii DSM 22836 dmos ADLW00000000 BHI Microoxic Escherichia coli strain K-12 substrain MG1655 ecok NC_000913.3 LB Oxic Enterococcus faecalis V583 efav NC_004668.1 BHI Oxic Enterococcus faecalis OG1RF efog NC_017316.1 BHI Oxic Enterobacter hormaechei ATCC 49162 ehor AFHR00000000 LB Oxic Gluconacetobacter europaeus LMG 18494 geur CADR00000000 Potato Oxic Gluconacetobacter hansenii ATCC 23769 ghan ADTV01000000 Potato Oxic Gluconacetobacter oboediens 174Bp2 gobo CADT00000000 Potato Oxic Gluconacetobacter xylinus NBRC 3288 gxyl NC_016037.1 Potato Oxic Gluconobacter frateurii NBRC 101659 gfra BADZ00000000 Potato Oxic Lactobacillus animalis KCTC 3501 lani AEOF00000000 mMRS Microoxic Lactobacillus brevis DmCS_003 lbrc JOKA00000000c mMRS Microoxic Lactobacillus brevis subsp. gravesensis ATCC 27305 lbga ACGG00000000 mMRS Microoxic Lactobacillus buchneri NRRLB-30929 lbuc NC_015428.1 mMRS Microoxic Lactobacillus casei W56 lcas NC_018641.1 mMRS Microoxic Lactobacillus fermentum ATCC 14931 lfer ACGI00000000 mMRS Microoxic Lactobacillus fructivorans DmCS_002 lfrc JOJZ00000000c mMRS Microoxic Lactobacillus fructivorans KCTC 3543 lfrk AEQY00000000 mMRS Microoxic Lactobacillus gasseri ATCC 33323 lgas NC_008530.1 mMRS Microoxic Lactococcus lactis BPL1 llac JRFX00000000 mMRS Microoxic Lactobacillus malefermentans KCTC 3548 lmle BACN00000000 mMRS Microoxic Lactobacillus mali KCTC 3596 = DSM 20444 lmli BACP00000000 mMRS Microoxic Lactobacillus plantarum DmCS_001 lplc JOJT00000000c mMRS Microoxic Lactobacillus plantarum WCFS1 lplw NC_004567.2 mMRS Microoxic Lactobacillus rhamnosus GG lrha NC_013198.1 mMRS Microoxic Lactobacillus versmoldensis KCTC 3814 lver BACR00000000 mMRS Microoxic Leuconostoc fallax KCTC 3537 lfal AEIZ00000000 mMRS Microoxic Providencia burhodogranariea DSM 19968 pbur AKKL00000000 LB Oxic Pseudomonas putida F1 pput NC_009512.1 LB Oxic Serratia marcescens ATCC 13880 smar N. Perna, personal communication LB Oxic Streptococcus mutans UA 159 smut NC_004350.2 BHI Microoxic Streptococcus parasanguinis FW 213 spar NC_017905.1 BHI Microoxic Sphingomonas wittichii RW1 swit NC_009511.1 BHI Oxic - TABLE 2
Top significant associations between larval development rate and phylogenetic distribution groupa
P value No. of COGs Notable annotated genes PDG (no. of Acetobacter strains, no. of Gluconacetobacter strains, and other taxa containing COG[s]) 2.20E−16 1 YciL protein 7, 4, E. hormaechei, E. coli, P. putida, S. marcescens 1.10E−15 3 Coenzyme PQQ synthesis B, C 7, 4, G. frateurii, E. coli, S. marcescens, P. putida, E. hormaechei 2.70E−15 1 Hypothetical protein 7, 4, G. frateurii, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei, 2 Enterococcus strains 4.30E−14 1 Ankyrin-like protein 7, 4, G. frateurii, E. coli, S. marcescens, P. putida, P. burhodogranariea, E. hormaechei 7.80E−13 11 Malate:quinone oxidoreductase 7, 4, G. frateurii, S. marcescens, S. wittichii, P. putida 8.00E−13 3 Zinc uptake regulation protein ZUR 6, 4, G. frateurii, E. coli, S. marcescens, P. putida, P. burhodogranaria, E. hormaechei, 2 Enterococcus strains 8.80E−13 1 Deoxyribodipyrimidine photolyase 7, 4, G. frateurii, E. coli, S. marcescens, S. wittichii, P. putida, E. hormaechei 1.00E−12 6 Glucose dehydrogenase (PQQ dependent) 6, 4, S. marcescens, S. wittichii, P. putida, E. hormaechei 2.70E−12 1 Hypothetical protein 7, 2, E. coli, S. marcescens, P. putida, P. burhodogranariea, E. hormaechei 2.90E−12 1 Major facilitator superfamily transporter 7, 3, G. frateurii, E. coli, S. marcescens, P. putida, P. burhodogranariea, E. hormaechei 5.20E−12 1 Paraquat-inducible protein B 7, 4, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei 3.40E−11 4 Succinate dehydrogenase iron-sulfur protein 7, 3, G. frateurii, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei 5.00E−11 1 ATP-dependent helicase HrpB 7, 4, E. coli, S. marcescens, E. hormaechei 6.00E−11 1 Hypothetical protein 7, 3, E. coli, S. marcescens, B. subtilis, P. putida, P. burhodogranariea, E. hormaechei 6.10E−11 1 Aminodeoxychorismate lyase 7, 4, G. frateurii, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei 6.70E−11 19 Flavin mononucleotide reductase YcdH 7, 4, G. frateurii, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei 7.20E−11 3 Multidrug resistance transporter HlyD 7, 4, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei, 2 Enterococcus strains, Dysgonomonas 8.40E−11 2 NADH-ubiquinone oxidoreductase chain E, F B. subtilis, 2 Streptococcus strains, 16 Lactobacillus strains, 1 Enterococcus sp., Leuconostoc 1.00E−10 2 Phenylalanine-tRNA ligase subunit beta 7, 4, G. frateurii, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei, 1 Enterococcus sp., Dysgonomonas 1.00E−10 1 Deoxyguanosinetriphosphate triphosphohydrolase B. subtilis, 2 Streptococcus strains, 16 Lactobacillus strains, Leuconostoc 1.00E−10 1 Thiamine pyrophosphokinase 7, 4, G. frateurii, S. marcescens, P. putida 1.30E−10 5 Oxidoreductase; alcohol dehydroenase cytochrome c subunit 7, 3, E. coli, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei 1.60E−10 1 Succinate dehydrogenase cytochrome b subunit 7, 4, G. frateurii, S. marcescens, S. wittichii, P. putida, P. burhodogranariea, E. hormaechei, 2 Enterococcus, Dysgonomonas 1.70E−10 1 Putative metal (Zn) chaperone 7, 4, G. frateurii, E. coli, S. marcescens, B. subtilis, P. putida, P. burhodogranariea, E. hormaechei 1.80E−10 3 Cytochrome o ubiquinol oxidase subunit II, III 7, 4, G. frateurii, S. marcescens, B. subtilis, P. putida, P. burhodogranariea, E. hormaechei ↵a For a full list, see Table S2.
- TABLE 3
Significant associations between adult TAG content and phylogenetic distribution groupa
P value No. of COGs Notable annotated genes PDG (no. of Acetobacter strains, no. of Gluconacetobacter strains, other taxon[s] containing COG[s]) 1.93E−009 1 NADH-dependent oxidoreductase 4, 4, G. frateurii, B. subtilis 2.34E−007 1 Gamma-glutamyltranspeptidase 7, 4, G. frateurii, S. wittichii, B. subtilis, L. malefermentans 2.41E−007 1 Dihydrolipoamide dehydrogenase 7, 4, G. frateurii, S. wittichii, B. subtilis, L. casei, 2 Enterococcus strains 3.36E−007 4 Gluconate 2-dehydrogenase subunit 4, 4, G. frateurii 4.06E−007 3 Copper efflux ATPase; metallo-dependent_hydrolases 7, 4, G. frateurii, B. subtilis 4.06E−007 1 MreC All but Acetobacteraceae family and 1 Enterococcus sp. 5.43E−007 5 Cytochrome c oxidase biogenesis; CarD-like transcriptional regulator; nitrogen fixation 7, 4, G. frateurii, S. wittichii, B. subtilis 6.45E−007 5 Thioredoxin peroxidase (phytoene) 4, 4, G. frateurii, S. wittichii 1.02E−006 1 Phytoene biosynthesis 4, 4, G. frateurii, L. plantarum, B. subtilis 1.15E−006 3 MotA 3, 4, G. frateurii 1.30E−006 2 5-Aminolevulinate synthase 4, 4, G. frateurii, S. marcescens, S. wittichii, B. subtilis 1.71E−006 1 TonB-dependent outer membrane channel 4, 3, G. frateurii 1.73E−006 2 Multidrug efflux pump acriflavin resistance protein 3, 4, G. frateurii 2.01E−006 2 Transcriptional regulator 3, 4, G. frateurii, S. wittichii 2.09E−006 1 Putative hexosyltransferase 6, 4, G. frateurii, B. subtilis 2.38E−006 1 PEBP family protein 3, 4, G. frateurii, S. wittichii 2.94E−006 1 Hydroxyacylglutathione 2, 4, B. subtilis 2.94E−006 2 Cob(II)yrinic acid reductase 7, 4, G. frateurii, B. subtilis, P. putida 3.12E−006 1 Hypothetical 2, 4, G. frateurii 3.46E−006 1 Hypothetical 5, 4, B. subtilis 3.59E−006 2 TPR superfamily; ubiquinol oxidase 7, 4, G. frateurii, B. subtilis, S. wittichii, P. putida 7.27E−006 1 Putative tRNA modifying enzyme 7, 4, G. frateurii, B. subtilis, S. wittichii, D. mossii 7.98E−006 1 N-Acetyltransferase 11 Lactobacillus strains, 2 Enterococcus strains, 2 Streptococcus strains, S. marcescens, E. coli, E. hormaechei 8.35E−006 1 Transcriptional regulator 2, 4, G. frateurii 1.46E−005b 2 Gluconate 2-dehydrogenase subunits 4, 4, G. frateurii, S. marcescens, P. putida
Supplemental Material
Table S1
Bacterial loads in monoassociated D. melanogaster. Table S1, DOCX file, 0.1 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S1
Number of COGs per phylogenetic distribution group (PDG). We determined the number of de novo COGs that contained sequences from exactly the same set of bacterial taxa (a PDG). The number of COGs per PDG is shown on the x axis, with the number of PDGs containing that number of COGs on the y axis (truncated for visualization of with multiple COGs). These data demonstrate that a majority of COGs have unique or nearly unique (shared with just one or a few other COGs) PDGs. Download Figure S1, TIF file, 2.5 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Dataset S1
Representative FASTA sequences and annotations for each COG. Representative amino acid sequences for each COG were selected using HMMER. Annotations are copied from the representative amino acid sequence annotation. Download Dataset S1, TXT file, 4.7 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Statistical association of PDGs and larval development rate. Taxon abbreviations are from Table 1. Table S2, CSV file, 0.5 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
The extracellular glucose oxidation pathway. This metabolic pathway oxidizes glucose to gluconate via glucose dehydrogenase (GDH) and then to 2-ketogluconate via gluconate dehydrogenase (GnDH). In the process, electrons (e−) are passed first to ubiquinone (Q), then to ubiquinol oxidase (Ubq Ox), and then to cytochrome c oxidase (Cyt-c Ox), which transfers the electrons to oxygen (O2). The synthesis of cytochrome oxidase protein (Sco1) is required for biogenesis of Cyt-c Ox. All proteins reside in the inner membrane (IM) of a Gram-negative bacterium. Multiple pathway components were identified as significantly associated with microbiota-dependent traits. For host TAG level, these included GnDH, Sco1, and an Ubq Ox subunit and for host development time, they included pyrroloquinoline quinone (PQQ) biosynthesis proteins B and C (not in diagram), GDH, and two Cyt-c Ox subunits. Download Figure S2, TIF file, 5 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S3
Statistical association of PDGs and adult fly TAG contents. Taxon abbreviations are from Table 1. Table S3, CSV file, 0.5 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Growth of recombinant bacterial strains in vitro and in vivo. (A) Proliferation of plasmid-bearing strains of A. tropicalis DmCS_006 and A. pasteurianus 3P3 on the surface of sterile fly food, as assessed by plating a known quantity of cells and determining the number of CFU present after 7 days of incubation at 25°C. Values are medians ± ranges of the results from three experiments, plotted on a logarithmic scale. None of the treatments were significantly different from one another in pairwise Mann-Whitney tests. (B) The abundance of bacteria in monocolonized, gnotobiotic, 5-day-old female flies was determined by homogenization and plating. CFUs are shown on a linear scale, each box delineates the first and third quartiles, the dark line is the median, and the whiskers show the range minus outliers that are shown as open circles. None of the treatments were significantly different from one another in pairwise Mann-Whitney tests. Download Figure S3, TIF file, 4.5 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S4
Analysis of variance in the effect of microbiota composition on diet contents. Table S4, DOCX file, 0.05 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Gnotobiotic rearing impacts protein content of the diet. (A to C) The nutritional content of fly diet after gnotobiotic rearing from egg to adulthood is shown, with the microbiota treatments indicated along the x axis. Values are means ± standard errors of the results from 3 experiments with 7 to 9 technical replicates each. Significant differences from the results for the control were determined by Dunnet’s test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Download Figure S4, TIF file, 6.8 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S5
Representation of candidate overexpression genes in sequenced Acetobacter and Gluconacetobacter genomes. Table S5, DOCX file, 0.1 MB.
Copyright © 2014 Chaston et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
Files in this Data Supplement:
- Dataset sd1, TXT - Dataset sd1, TXT
- Figure sf1, TIF - Figure sf1, TIF
- Figure sf2, TIF - Figure sf2, TIF
- Figure sf3, TIF - Figure sf3, TIF
- Figure sf4, TIF - Figure sf4, TIF
- Table st1, DOCX - Table st1, DOCX
- Table st2, CSV - Table st2, CSV
- Table st3, CSV - Table st3, CSV
- Table st4, DOCX - Table st4, DOCX
- Table st5, DOCX - Table st5, DOCX
