Article Figures & Data
- FIG 1
B. thetaiotaomicron deprioritizes O-glycan metabolism in the presence of competing glycans and monosaccharides. (A) B. thetaiotaomicron growth curves in mixtures of homogalacturonan (HG) and PMOG. The dashed horizontal lines indicate the pause in exponential growth that occurs as a function of changing glycan mix concentrations. (B) Relative expression differences between early and late growth phases for B. thetaiotaomicron grown in a mixture of HG and PMOG (1.5 mg/ml and 7 mg/ml, respectively). Early and late samples were harvested before and after the pause, respectively. A total of seven previously identified O-glycan-responsive PULs (6) were monitored for changes in expression based on the criteria that they were induced more than 8.5-fold in pure PMOG and are not part of recombinational shufflons that might obfuscate temporal expression. PULs with statistically significant (P ≤ 0.05 by one-tailed Student's t test) increases in expression during early growth, PULs with statistically significant increases in later growth, and PULs with no significant changes in early versus late growth are shown. (C) A “spike-in” experiment in which B. thetaiotaomicron was grown to mid-exponential phase on 10 mg/ml PMOG alone and then HG (final concentration of 2.5 mg/ml) was abruptly introduced. Bacterial samples were harvested at 30-, 60-, 90-, and 120-min intervals after the introduction of HG. A corresponding negative control (minimal medium with no carbon) was used to monitor changes in O-glycan PUL expression (gray bars). Expression changes were determined relative to the culture grown in PMOG only (time zero [T0]). (D to H) Experiments similar to those described above, but with levan or chondroitin sulfate (CS) as the competing glycan. Additional experiments for pairwise combinations are shown in Fig. S1 in the supplemental material. (I) Aggregate expression differences of all PULs involved in metabolizing O-glycans and the non-O-glycans tested. Each symbol shows the value for one separate biological replicate of three conducted for each combination. (J) Exponential growth rates of B. thetaiotaomicron on all individual glycans tested. PG, pectic galactan; AP, amylopectin; AG, arabinogalactan; RG I, rhamnogalacturonan I. (Letters over each bar indicate data groupings in which the t test P values were <0.001). (K and L) Additional early/late growth phase experiments comparing the responses to a competing polysaccharide (levan and pectic galactan) and their corresponding monosaccharides (fructose and galactose). (M) HMO-responsive PUL (11) expression during early and late growth in a mixture of levan and human milk oligosaccharides (HMOs). A total of three biological replicates were conducted for each experimental condition, including bacterial growth curves. All values are means ± standard deviations (SD) (error bars), except in panels C and F, where they are means ± standard errors (SE) (error bars).
- FIG 2
(A) B. massiliensis growth curve in a potato amylopectin (starch)/PMOG mix. (B) Relative expression of B. massiliensis O-glycan-responsive PULs between early and late growth. Two later growth point transcripts were analyzed (absorbance at 600 nm values of 1.0 and 1.2) and compared separately back to the early growth point (A600 = 0.6). (C) B. fragilis growth curve in a potato amylopectin (starch)/PMOG mix. (D) Relative expression of B. fragilis O-glycan-responsive PULs between early and late growth in an APpot (potato amylopectin) (starch)/PMOG mix. (E) B. fragilis growth curve in an inulin/PMOG mix. (F) Relative expression of B. fragilis O-glycan-responsive PULs between early and late growth in an inulin/PMOG mix. Note the reciprocal amounts of starch and PMOG required for B. massiliensis analysis compared to similar experiments with B. thetaiotaomicron. All expression values are significant (P ≤ 0.05 by one-tailed Student's t test), except for those values labeled in black. All values are mean ± SD.
- FIG 3
(A) A schematic of the O-glycan-responsive PULs, BT4636-31 and BT4355-59, showing the presumed locations, based on previous microarray data (6), of noninducible promoters (bent arrows), ECF-σ-inducible promoters (colored bent arrows), predicted Ton boxes, and chimeric recombination sites. (B) Cellular organization of chimeric Sus-like systems encoded by the above PULs separating glycan sensing at outer membrane TonB-dependent transporters (yellow and red stars) and genetic regulation via inducible promoters and genes encoding ECF-σ and anti-σ factors. See Fig. S6E in the supplemental material for a detailed illustration of the genetic recombination scheme used. OM, outer membrane; IM, inner membrane. (C) Growth phase-specific expression of BT4634 and BT4357 in an undefined mixture containing O-glycans and glycosaminoglycans (GAGs), termed the “100 mM fraction” from PMOG purification (6). Previous microarray (black bars) and current qPCR (white bars and green striped bars) measurements are shown for comparison.
- FIG 4
(A) A schematic of the O-glycan-responsive BT4636-31 PUL showing the IGR deletion sequence. The full IGR sequence is shown in lowercase type with the deleted region shown in red and the retained ribosome binding site (RBS) shown in black. The respective stop (BT4635) and start (BT4634) codons are in uppercase type. (B) qPCR-based expression analysis of the wild-type and ΔBT4635-34 IGR B. thetaiotaomicron strains grown in PMOG as the only carbon source showing full PUL expression in the IGR deletion mutant. (C) Growth phase-specific expression profiles between wild-type and IGR deletion strains in a mixture of levan/PMOG. All other PMOG-responsive PULs previously tested were not altered during IGR deletion. All expression values are significant (P ≤ 0.05 by one-tailed Student's t test) except those labeled in black. (D) Expression analysis from an experiment in which levan was introduced into cells actively growing on PMOG with the same strains shown in panel C and normalized to a no-carbon control as shown in Fig. 1C and F. (E) Fusion of a Bacteroides-adapted super-glo gfp (sg-gfp) reporter gene downstream of the BT4635-34 IGR sequence and its 118-bp deletion variant and a constitutively expressed gene (BT0658) in the ΔBT4635-34 IGR strain as a genetic background. The micrograph images at the bottom validate expression of this reporter. The values below the micrographs are the 16S rRNA gene-normalized expression values by qPCR in MM-glucose and MM-PMOG. The sg-gfp gene was adapted for expression in Bacteroides by making a translational fusion to the first eight codons of a host glycan-responsive susC-like gene, BT1042. This gene is expressed in response to PMOG and is also derepressed in a mutant lacking expression of a gene encoding an associated anti-σ factor, BT1052. Note that combined loss of BT1052 and growth in PMOG result in increased gfp expression as measured by both qPCR and cell fluorescence. Cells were exposed to oxygen on ice for 30 min prior to imaging to allow the green fluorescent protein (GFP) fluorophore to fold. (F) Relative expression differences between early and late growth phases for the sg-gfp reporter strains (with or without the full 129-bp IGR) grown in a mixture of levan/PMOG. Parallel measurements of the levan-responsive BT1763 gene and the O-glycan-responsive genes, BT4134 and BT4634 (here deprioritized in the ΔIGR background) serve as controls.
- FIG 5
A model to explain the results reported in this study and based particularly on results of expression with the substrate at 100 mM. (Left) In the presence of preferred glycans, such as the CS and heparin glycosaminoglycans, PULs involved in catabolizing high-priority nutrients (red) are activated and liberate simple sugars that are ultimately transported into the cytoplasm, where they mediate repression of PULs that target lower-priority substrates. Since the PULs involved in degradation of some substrates that repress utilization of O-glycans can also be repressed in more-complex nutrient environments (26), it is possible that these gene clusters also harbor elements that mediate transcript repression via either the IGR-mediated mechanism described here or other unknown mechanisms. In the case of the nutrients present in the 100 mM fraction, the inducing substrate for the BT4355-59 PUL (green) is presumably present in either higher abundance and/or is more accessible, leading to deployment of this system early in growth. Alternatively, systems like BT4355-59 may be less susceptible to repression by monosaccharides present in certain high-priority substrates, which is consistent with the data shown in Fig. 1 and Fig. S1 in the supplemental material. (Right) After the preferred glycans are depleted and the corresponding repressive sugars are no longer present in the cytoplasm, expression of PULs that target low-priority glycans is released, but may require the action of other enzymes/Sus-like systems to uncover the glycan structure that is recognized by some systems such as that encoded by BT4636-31 (blue). (Left, inset) Thus, when the BT4636-31 IGR is absent during growth in the 100 mM substrate, repression is partially relieved (compare blue bars; P = 0.0007 by one-tailed Student's t test). Since the BT4636-31 target ligand is sensed later in growth (Fig. 3), possibly because it is only enzymatically uncovered after the action of other systems, this system is not fully deployed early in growth even when its associated IGR is missing.
Supplemental Material
Figure S1
(A to H) Growth- and phase-specific gene expression analysis on pairwise glycan combinations as described for levan, HG, and CS in Fig. 1 of the main text. (I) Gene schematics of the seven B. thetaiotaomicron PMOG PULs analyzed in this study. Functional annotations are indicated for genes predicted to encode hybrid two-component system (HTCS) regulators (pink), ECF-σ (green), anti-σ (red), susC-like (purple), susD-like (orange), glycoside hydrolase (GH) (blue) (with family membership indicated). Additional annotations are listed for some other cotranscribed genes shown in gray: carbohydrate binding module (CBM, with family indicated), M60-like protease, and sulfatase. Download Figure S1, PDF file, 1.1 MB.
Copyright © 2015 Pudlo 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
(A) Time course experiment in which a polysaccharide mixture, containing 2.5 mg/ml total of 11 different glycans (0.25 mg/ml each) that B. thetaiotaomicron can degrade, was added to actively growing PMOG cultures. A negative control in which no polysaccharides were added was performed as described in the legend to Fig. 1. (B to L) Spike in expression results from an experiment in which B. thetaiotaomicron was grown to mid-exponential phase on PMOG (T0) and then an equal amount of prereduced minimal medium containing one of the individual monosaccharides indicated was added to measure its ability to initiate PUL repression. Samples were taken 30 min after the addition for 2 h. Only one replicate was performed, and values represent the means of two quantitative reverse transcription-PCR (qRT-PCR) technical replicates. Download Figure S2, EPS file, 1.5 MB.
Copyright © 2015 Pudlo 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
Schematic representations of the various glycan structures presented to Bacteroides species in this study. The full names of the abbreviated polysaccharides are provided in the legends to Fig. 1 and Fig. S1 in the supplemental material. A bracketed end(s) of a glycan chain indicates that the glycan structure can be longer than that which is shown. Definitions of symbols used are provided in the key at the top right. Note that although the structures drawn here represent predominant linkages and monosaccharides in each of the glycans, there may be some differences based on extraction method and species or tissue source. Download Figure S3, EPS file, 1.4 MB.
Copyright © 2015 Pudlo 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
(A) Biphasic growth on different proportions of levan and human milk oligosaccharides (HMOs) showing that HMOs are given lower priority in this mixed growth condition. (B) Biphasic growth on different proportions of homogalacturonan and HMOs showing that HMOs are given lower priority in this mixed growth condition. (C) Biphasic growth of different B. thetaiotaomicron (Bt) strains on combinations of PMOG and one of two different fructans, levan or inulin, depending on which substrate the indicated strain showed optimal growth rate on. (D) Biphasic growth of different B. thetaiotaomicron strains on combinations of PMOG and homogalacturonan. For strains in both panels C and D, percent 16S rRNA gene identity to the type strain VPI 5482 is indicated for each strain along with the host source. Download Figure S4, PDF file, 0.3 MB.
Copyright © 2015 Pudlo 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 S5
(A) Phylogeny of several Bacteroidetes, including B. thetaiotaomicron, as well as Bacteroides fragilis and Bacteroides massiliensis, used in this study. (B) Cellular schematic of B. thetaiotaomicron's starch utilization system (sus) and homology with B. massiliensis's identically organized starch PUL. (C) B. massiliensis's putative starch-responsive susC is activated in the presence of starch compared to N-acetylglucosamine (GlcNAc) (B. massiliensis did not grow on glucose as the sole carbon source, despite growth on starch). (D) Growth curves of B. thetaiotaomicron when grown on PMOG (red), starch (green), and GlcNAc (gray). (E) Growth curves of B. massiliensis grown on PMOG (red), starch (green), and GlcNAc (gray) as the sole carbon source. (F) B. massiliensis grown on PMOG alone (red), starch alone (green), and an APpot/PMOG mix (3.5 mg/ml and 3 mg/ml, respectively; which is the same mix used in Fig. 2) to help determine possible metabolic shifts. (G) Growth profiles of B. fragilis grown on pairwise mixes of starch/PMOG. (H) Growth profiles of B. fragilis grown on pairwise mixes of inulin/PMOG. Download Figure S5, PDF file, 0.3 MB.
Copyright © 2015 Pudlo 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 S1
In vitro growth ability of Bacteroides thetaiotaomicron VPI 5482, Bacteroides massiliensis DSM17679, and Bacteroides fragilis NCTC 9343 on various glycans and simple sugars. Boxes in gray highlight substrates on which growth was not observed, while boxes in green represent substrates that support detectable growth. Table S1, PDF file, 0.1 MB.
Copyright © 2015 Pudlo 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
RNAseq-based gene expression changes in the whole-genome transcriptional profile of Bacteroides massiliensis DSM17679 grown on PMOG as the sole carbon source compared to an N-acetylglucosamine reference Table S2, PDF file, 0.8 MB.
Copyright © 2015 Pudlo 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 S6
(A) Analysis of 16S rRNA gene-normalized qPCR threshold cycle (Ct) values during the first growth phase in a mixture of PMOG and levan to determine whether 3′-5′ cyclic AMP (cAMP) is a signal (at 10 mM) for catabolite repression in B. thetaiotaomicron. There is no effect of cAMP under the conditions tested to suggest that this molecule causes derepression of O-glycan-responsive PULs assayed, as the only two significant changes result in increased repression. Values are the means ± SD of three replicates. Values that are significantly different by t test are indicated by asterisks as follows: *, P < 0.05; **, P < 0.001. Values that are not significantly different (n.s.) by t test are also indicated. (B) Expression profiles for cells grown on a mix of levan and PMOG using a strain lacking all eight capsular polysaccharide loci (B. thetaiotaomicron Δcps). We determined that the various capsular polysaccharides that B. thetaiotaomicron employs in an in vitro mixed cell population does not affect glycan prioritization. Values are the means ± SD of three replicates. Statistical significance by t test is indicated as follows: *, P < 0.05; **, P < 0.001; n.s., not significant. (C and D) Comparison of expression values between wild-type B. thetaiotaomicron and a mutant containing a disruption in the BT1754 gene, which encodes an essential regulator for levan utilization, when pregrown on PMOG and levan is introduced. Cells were harvested as described above with T0 and 30-min harvest for 2 h. Only one replicate was performed, and values represent the means of two qRT-PCR technical replicates. (E) Genetic manipulation scheme for generating the chimeric strain for BT4636-31 and 4355-59 PULs. Download Figure S6, EPS file, 2.3 MB.
Copyright © 2015 Pudlo 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 S7
We isolated the 100 mM NaCl-eluted fraction from porcine mucosal glycans (PMG, referred to herein as the “100 mM fraction”) similar to a previous study (6). Although most neutral porcine mucin O-glycans (PMOG) flow through an anion exchange column, the resulting fraction of 100 mM NaCl-eluted retained PGM contains residual MOG in addition to glycosaminoglycans (GAGs) and some N-linked glycans. The O-glycans contain more neutral sugars, while GAGs, including the chondroitin sulfate that is highest on the metabolic hierarchy of B. thetaiotaomicron (Bt), contain higher negative charge due to uronic acid, sulfate, and sialic acid content. Thus, growing B. thetaiotaomicron on this 100 mM fraction elicits a number of responses, including the chondroitin sulfate PUL, BT3328-3334, as well as O-glycan PULs. (A) Activation of B. thetaiotaomicron genes from the two O-glycan PULs used for chimera construction, BT4355-59 (red) and BT4636-31 (blue), when the bacteria were grown on PMOG as the sole carbon source. Values are the means ± SD of three replicates. (B) Growth on the 100 mM fraction exhibits an early (red) phase and a series of late (blue) growth phases. (C) Discordant transcript profiles from two B. thetaiotaomicron O-glycan-responsive PULs where one susC-like gene BT4357 is upregulated early in growth on the 100 mM fraction and another, BT4634, is repressed until later. This suggests that even individual PULs that target similar substrates can respond differently, possibly due to substrate structural/linkage diversity or sequential cleaving of substrates making other sugars or linkages available. Values are the means ± SD of two replicates determined from previous microarray data (6). (D) The two susC-like genes from the PULs described above with discordant expression grown on 100 mM NaCl fractionated PMG are both activated by the disaccharide, N-acetyllactosamine (LacNAc), that is a major component of the more-elaborate O-glycan chains contained in PMOG. (E) When the regulatory regions are swapped in the chimeric PUL B. thetaiotaomicron strain, activation still occurs, indicating that the PULs retain functionality. (F) Agarose gel image showing transcription of a region of the BT4635-34 PUL with an intergenic region (IGR) deleted. Notice the 118-bp difference between wild-type (wt) B. thetaiotaomicron and the ΔBT4635-34 IGR strain in reverse-transcribed cDNA and genomic DNA. Also, no amplifiable DNA for IGR-flanking genes was detected in only the extracted RNA. (G) Growth profiles for wild-type B. thetaiotaomicron and the ΔBT4635-34 IGR strain on PMOG alone (10/mg/ml total PMOG). (H) Growth phase-specific expression differences for the anti-σ (BT4635) and susC-like (BT4634) genes in either the 100 mM fraction or levan/PMOG, showing that these two adjacent genes, which flank the deleted IGR sequence, behave discordantly with the upstream anti-σ gene not showing similar repression. Download Figure S7, EPS file, 2.2 MB.
Copyright © 2015 Pudlo 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
RNAseq-based gene expression changes in the whole-genome transcriptional profile of Bacteroides fragilis NCTC 9343 grown on PMOG as a sole carbon source compared to glucose reference Table S3, PDF file, 0.9 MB.
Copyright © 2015 Pudlo 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
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- Table st3, PDF - Table st3, PDF
