An expression-driven approach to the prediction of carbohydrate transport and utilization regulons in the hyperthermophilic bacterium Thermotoga maritima

Abstract

Comprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixed-model analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T. maritima relies extensively on ABC transporters for carbohydrate uptake, many of which are likely controlled by local regulators responsive to either the transport substrate or a key metabolic degradation product. Roles in uptake of specific carbohydrates were suggested for members of the expanded Opp/Dpp family of ABC transporters. In this family, phylogenetic relationships among transport systems revealed patterns of possible duplication and divergence as a strategy for the evolution of new uptake capabilities. The presence of GC-rich hairpin sequences between substrate-binding proteins and other components of Opp/Dpp family transporters offers a possible explanation for differential regulation of transporter subunit genes. Numerous improvements to T. maritima genome annotations were proposed, including the identification of ABC transport systems originally annotated as oligopeptide transporters as candidate transporters for rhamnose, xylose, beta-xylan, and beta-glucans and identification of genes likely to encode proteins missing from current annotations of the pentose phosphate pathway. Beyond the information obtained for T. maritima, the present study illustrates how expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable.

FIG. 1.

Loop design used for the study of carbon source utilization of T. maritima in the present study. The arrowheads correspond to the Cy5 channel, and the dotted arrow ends correspond to the Cy3 channel. Abbreviations for sugar names used in subsequent expression histograms are shown in parentheses.

FIG. 2.

Circular representation of the T. maritima genome showing locations of known carbohydrate transport proteins and Opp/Dpp family ABC transporter components. Least-squares mean estimates (see Materials and Methods) of transcript levels corrected for systematic errors are shown for selected operons whose carbohydrate specificity is predicted in this study. In this context, red and green denote transcript levels above (red) and below (green) the mean expression across all genes, where “0” represents the mean rather than no expression. Oligopeptide transporter subunits are represented in black, CUT1 transporter subunits are represented in gray, and CUT2 transporter subunits are represented in white.

FIG. 3.

Pentose-responsive loci of T. maritima. Predicted σ^A promoters are represented by arrows. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) A T. maritima locus which responds to the pentose sugars ribose, arabinose, and xylose contains genes for the utilization of the simple sugar d-ribose, a likely ribose transport system, and other genes likely to participate in the pentose phosphate pathway. (B) An arabinose utilization locus contains genes for the conversion of arabinose into d-xylulose-5-P. (C) Predicted pathway for the hydrolysis, transport and utilization of xylan, xylose, ribose, and arabinose by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S2 in the supplemental material.

FIG. 3.

Pentose-responsive loci of T. maritima. Predicted σ^A promoters are represented by arrows. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) A T. maritima locus which responds to the pentose sugars ribose, arabinose, and xylose contains genes for the utilization of the simple sugar d-ribose, a likely ribose transport system, and other genes likely to participate in the pentose phosphate pathway. (B) An arabinose utilization locus contains genes for the conversion of arabinose into d-xylulose-5-P. (C) Predicted pathway for the hydrolysis, transport and utilization of xylan, xylose, ribose, and arabinose by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S2 in the supplemental material.

FIG. 3.

Pentose-responsive loci of T. maritima. Predicted σ^A promoters are represented by arrows. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) A T. maritima locus which responds to the pentose sugars ribose, arabinose, and xylose contains genes for the utilization of the simple sugar d-ribose, a likely ribose transport system, and other genes likely to participate in the pentose phosphate pathway. (B) An arabinose utilization locus contains genes for the conversion of arabinose into d-xylulose-5-P. (C) Predicted pathway for the hydrolysis, transport and utilization of xylan, xylose, ribose, and arabinose by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S2 in the supplemental material.

FIG. 4.

Representative phylogenetic tree of substrate-binding proteins of peptide family transporters from T. maritima. All operons are shown on one strand to more clearly represent the relative positions of subunits. Black arrows represent substrate-binding proteins of the DppA family (COG0747), white arrows with diagonal stripes represent substrate-binding proteins of the OppA family (COG0747), dark gray arrows represent permease subunits of the DppB/OppB family (COG0601), light gray arrows represent permease subunits of the DppC/OppC family (COG1173), white arrows represent ATP-binding subunits of the DppD/OppD family (COG0444), and dotted white arrows represent ATP-binding subunits of the DppF/OppF family (COG4608). Other non-transport-related genes located between transporter subunits are represented as dotted arrows. Asterisks represent apparent lineage-specific gene expansions which have taken place since the divergence of T. maritima and the next closest fully sequenced organism. Black hairpins represent locations of GC-rich inverted repeats. The tree topology represented here is consistent with trees constructed using protein sequences for ATP-binding and permease subunits and was not altered by using pairwise or complete deletion of missing data or by tree construction method (neighbor-joining, minimum evolution, or maximum parsimony).

FIG. 5.

Expression results for transcripts detected at higher levels on β-linked polysaccharides. Small hairpin symbols represent locations of GC-rich inverted repeats, while large hairpin symbols represent locations of predicted rho-independent terminators (http://www.tigr.org/software/TransTermResults/btm.html). Predicted σ^A promoters are represented by arrows, and an asterisk denotes the position of a putative cellobiose regulator operator. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) Genes within a putative cellobiose transport operon (proposed designation CbtABCDF), including a likely regulator of cellobiose uptake and utilization (proposed designation CelR) and a colocalized mannan-responsive locus (including TM1224 [proposed designation ManR] and TM1226 [proposed designation MbtA]). (B) Genes within a glucomannan and galactomannan responsive locus include the Opp transporter components TM1746 to TM1752 (proposed designation MtpABCDF), Cel5A, and Cel5B and the β-mannosidase TM1624. (C) Genes within a β-glucan responsive locus (proposed designation BgtpABCDF), including a putative regulator of β-glucan uptake (proposed designation BglcR). (D) Predicted pathway for the utilization of β-glucans and glucomannan by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S1 in the supplemental material.

FIG. 5.

Expression results for transcripts detected at higher levels on β-linked polysaccharides. Small hairpin symbols represent locations of GC-rich inverted repeats, while large hairpin symbols represent locations of predicted rho-independent terminators (http://www.tigr.org/software/TransTermResults/btm.html). Predicted σ^A promoters are represented by arrows, and an asterisk denotes the position of a putative cellobiose regulator operator. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) Genes within a putative cellobiose transport operon (proposed designation CbtABCDF), including a likely regulator of cellobiose uptake and utilization (proposed designation CelR) and a colocalized mannan-responsive locus (including TM1224 [proposed designation ManR] and TM1226 [proposed designation MbtA]). (B) Genes within a glucomannan and galactomannan responsive locus include the Opp transporter components TM1746 to TM1752 (proposed designation MtpABCDF), Cel5A, and Cel5B and the β-mannosidase TM1624. (C) Genes within a β-glucan responsive locus (proposed designation BgtpABCDF), including a putative regulator of β-glucan uptake (proposed designation BglcR). (D) Predicted pathway for the utilization of β-glucans and glucomannan by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S1 in the supplemental material.

FIG. 5.

Expression results for transcripts detected at higher levels on β-linked polysaccharides. Small hairpin symbols represent locations of GC-rich inverted repeats, while large hairpin symbols represent locations of predicted rho-independent terminators (http://www.tigr.org/software/TransTermResults/btm.html). Predicted σ^A promoters are represented by arrows, and an asterisk denotes the position of a putative cellobiose regulator operator. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) Genes within a putative cellobiose transport operon (proposed designation CbtABCDF), including a likely regulator of cellobiose uptake and utilization (proposed designation CelR) and a colocalized mannan-responsive locus (including TM1224 [proposed designation ManR] and TM1226 [proposed designation MbtA]). (B) Genes within a glucomannan and galactomannan responsive locus include the Opp transporter components TM1746 to TM1752 (proposed designation MtpABCDF), Cel5A, and Cel5B and the β-mannosidase TM1624. (C) Genes within a β-glucan responsive locus (proposed designation BgtpABCDF), including a putative regulator of β-glucan uptake (proposed designation BglcR). (D) Predicted pathway for the utilization of β-glucans and glucomannan by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S1 in the supplemental material.

FIG. 5.

Expression results for transcripts detected at higher levels on β-linked polysaccharides. Small hairpin symbols represent locations of GC-rich inverted repeats, while large hairpin symbols represent locations of predicted rho-independent terminators (http://www.tigr.org/software/TransTermResults/btm.html). Predicted σ^A promoters are represented by arrows, and an asterisk denotes the position of a putative cellobiose regulator operator. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) Genes within a putative cellobiose transport operon (proposed designation CbtABCDF), including a likely regulator of cellobiose uptake and utilization (proposed designation CelR) and a colocalized mannan-responsive locus (including TM1224 [proposed designation ManR] and TM1226 [proposed designation MbtA]). (B) Genes within a glucomannan and galactomannan responsive locus include the Opp transporter components TM1746 to TM1752 (proposed designation MtpABCDF), Cel5A, and Cel5B and the β-mannosidase TM1624. (C) Genes within a β-glucan responsive locus (proposed designation BgtpABCDF), including a putative regulator of β-glucan uptake (proposed designation BglcR). (D) Predicted pathway for the utilization of β-glucans and glucomannan by T. maritima. Extracellular enzymes responsible for polysaccharide hydrolysis are shown, as well as periplasmic binding proteins, membrane-embedded permeases, associated ATP-binding subunits, and intracellular hydrolases. References for hydrolases shown in the pathway are listed in Table S1 in the supplemental material.

FIG. 6.

β-Xylan and xylose-responsive operons from groups 2 and 3 of Opp/Dpp family transporters. Small hairpin symbols represent locations of GC-rich inverted repeats found between substrate-binding proteins and other transporter subunits, and large hairpin symbols represent locations of predicted rho-independent terminators (http://www.tigr.org/software/TransTermResults/btm.html). Predicted σ^A promoters are represented by arrows, and an asterisk denotes the positions of putative xylan/xylose regulator operator. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) XtpGHJLM, a predicted xylooligosaccharide transport system, is divergently transcribed from xylanase Xyl10A. (B) XtpABCDF, a predicted xylose/xyloside transport system, is divergently transcribed from xylanase Xyl10B.

FIG. 7.

Rhamnose responsive locus containing Opp/Dpp family transporter from group 3 of Opp/Dpp family transporters. Small hairpin symbols represent locations of GC-rich inverted repeats found between substrate-binding proteins and other transporter subunits. Predicted σ^A promoters are represented by arrows. Substrate-binding proteins are outlined in bold and boxed. Spacing between genes is less than 30 bases unless indicated otherwise. (A) A rhamnose-responsive locus contains candidate genes likely to encode enzymes responsible for the transport and hydrolysis of rhamnose-containing di- or oligosaccharides, and the complete catabolism of the simple sugar l-rhamnose. (B) A predicted pathway for the utilization of l-rhamnose by T. maritima. A periplasmic binding protein, membrane-embedded permeases, associated ATP-binding subunits, and rhamnose catabolic enzymes are shown.