In silico and transcriptional analysis of carbohydrate uptake systems of Streptomyces coelicolor A3(2)

Abstract

Streptomyces coelicolor is the prototype for the investigation of antibiotic-producing and differentiating actinomycetes. As soil bacteria, streptomycetes can metabolize a wide variety of carbon sources and are hence vested with various specific permeases. Their activity and regulation substantially determine the nutritional state of the cell and, therefore, influence morphogenesis and antibiotic production. We have surveyed the genome of S. coelicolor A3(2) to provide a thorough description of the carbohydrate uptake systems. Among 81 ATP-binding cassette (ABC) permeases that are present in the genome, we found 45 to encode a putative solute binding protein, an essential feature for carbohydrate permease function. Similarity analysis allowed the prediction of putative ABC systems for transport of cellobiose and cellotriose, alpha-glucosides, lactose, maltose, maltodextrins, ribose, sugar alcohols, xylose, and beta-xylosides. A novel putative bifunctional protein composed of a substrate binding and a membrane-spanning moiety is likely to account for ribose or ribonucleoside uptake. Glucose may be incorporated by a proton-driven symporter of the major facilitator superfamily while a putative sodium-dependent permease of the solute-sodium symporter family may mediate uptake of galactose and a facilitator protein of the major intrinsic protein family may internalize glycerol. Of the predicted gene clusters, reverse transcriptase PCRs showed active gene expression in 8 of 11 systems. Together with the previously surveyed permeases of the phosphotransferase system that accounts for the uptake of fructose and N-acetylglucosamine, the genome of S. coelicolor encodes at least 53 potential carbohydrate uptake systems.

FIG. 1.

Schematic overview of carbohydrate uptake systems of S. coelicolor. Representative sugars for the depicted transport systems are fructose (Fru) for PTS, maltose (Mal) for ABC, glucose (Glc) for symport, and glycerol (Gly) for facilitated diffusion. Abbreviations: E, MalE; F, MalF; G, MalG; EIIA, enzyme IIA; EIIB, enzyme IIB; EIIC, enzyme IIC; PEP, phosphoenolpyruvate.

FIG. 2.

(A) ceb1 system. The depicted region includes six orfs, cebE1, cebF1, cebG1, bglC1, and shl1, comprising the ABC transport system and the first step of cellobiose utilization flanked by cebR1 and the probable secreted sugar hydrolase-encoding shl1. A palindromic region of 18 bp upstream of cebE1, shown to be part of the ceb operator (O_CebR) in S. reticuli, was detected (the half-sites are separated by asterisks). ORF numbers denote the lengths of the gene products. Genes that encode membrane proteins (generally with the suffix F or G) are shown in black (membrane-spanning proteins); genes that encode substrate-binding proteins are shown in grey (with the suffix E). White arrows represent metabolic genes, probably functionally correlated to the respective system. Vertically striped arrows denote genes for ATP binding and/or hydrolyzing protein. Cross-hatched arrows indicate regulatory genes. Genes of unknown function and/or presumably not involved in the respective system are represented by dotted arrows. Putative regulatory elements are shown in uppercase type and boldface type where in consensus with comparable sequences or to emphasize direct or inverted repeats therein. Dyad repeats are drawn as a stem-loop sign. Dotted lines represent cosmid borders. The numbers of intergenic base pairs are given in brackets, the location of an ORF on the cosmid is designated by SCXX.nn, with “c” denoting complementarity. (B) ceb2 locus. (C and D) bxl1 and bxl2 loci. (E and F) agl1 and agl2 loci. RIP, right imperfect palindrome; PBS, potential binding sequence (60). Abbreviations and designations for panels B to F are the same as for panel A. For further explanations, see the text.

FIG. 2.

(A) ceb1 system. The depicted region includes six orfs, cebE1, cebF1, cebG1, bglC1, and shl1, comprising the ABC transport system and the first step of cellobiose utilization flanked by cebR1 and the probable secreted sugar hydrolase-encoding shl1. A palindromic region of 18 bp upstream of cebE1, shown to be part of the ceb operator (O_CebR) in S. reticuli, was detected (the half-sites are separated by asterisks). ORF numbers denote the lengths of the gene products. Genes that encode membrane proteins (generally with the suffix F or G) are shown in black (membrane-spanning proteins); genes that encode substrate-binding proteins are shown in grey (with the suffix E). White arrows represent metabolic genes, probably functionally correlated to the respective system. Vertically striped arrows denote genes for ATP binding and/or hydrolyzing protein. Cross-hatched arrows indicate regulatory genes. Genes of unknown function and/or presumably not involved in the respective system are represented by dotted arrows. Putative regulatory elements are shown in uppercase type and boldface type where in consensus with comparable sequences or to emphasize direct or inverted repeats therein. Dyad repeats are drawn as a stem-loop sign. Dotted lines represent cosmid borders. The numbers of intergenic base pairs are given in brackets, the location of an ORF on the cosmid is designated by SCXX.nn, with “c” denoting complementarity. (B) ceb2 locus. (C and D) bxl1 and bxl2 loci. (E and F) agl1 and agl2 loci. RIP, right imperfect palindrome; PBS, potential binding sequence (60). Abbreviations and designations for panels B to F are the same as for panel A. For further explanations, see the text.

FIG. 3.

Two EFG-like gene arrangements are shown that represent the ngc/rok7B7/xyl gene locus. The conventions of presentation and designations are described in the legend to Fig. 2A.

FIG. 4.

The lac, smo, rbs1, rbs2, and rbs3 loci are shown. The conventions of presentation and designations are described in the legend to Fig. 2A.

FIG. 5.

The gal, glcP1, glcP, and gyl loci are shown. The conventions of presentation and designations are described in the legend to Fig. 2A.

FIG. 6.

Expression profiling of proposed carbohydrate uptake systems. For each system, RNA from S. coelicolor A3(2) grown without (left column, −) or in the presence of (right column, +) the indicated carbon source was used for RT-PCR. As an example of undoubted substrate induction, transcriptional analysis of fruA (encoding the specific fructose-PTS enzyme IIABC) is depicted (30).

FIG. 7.

Genetic map of S. coelicolor. A depiction of the positions of the gene clusters of newly identified transport systems (bxl2, rbs3, smo, rbs1, ceb1, agl1, gal, glcP1, gyl, ngc/xyl, rbs2, agl2, bxl1, glcP2, lac, and ceb2) is shown. The constant region of the chromosome is shown in black; the variable ends are in grey. The direction of the chromosome is counterclockwise.