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1.
J Bacteriol ; 189(16): 5903-15, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17557815

ABSTRACT

We present a comprehensive analysis of carbohydrate uptake systems of the soil bacterium Mycobacterium smegmatis and the human pathogen Mycobacterium tuberculosis. Our results show that M. smegmatis has 28 putative carbohydrate transporters. The majority of sugar transport systems (19/28) in M. smegmatis belong to the ATP-binding cassette (ABC) transporter family. In contrast to previous reports, we identified genes encoding all components of the phosphotransferase system (PTS), including permeases for fructose, glucose, and dihydroxyacetone, in M. smegmatis. It is anticipated that the PTS of M. smegmatis plays an important role in the global control of carbon metabolism similar to those of other bacteria. M. smegmatis further possesses one putative glycerol facilitator of the major intrinsic protein family, four sugar permeases of the major facilitator superfamily, one of which was assigned as a glucose transporter, and one galactose permease of the sodium solute superfamily. Our predictions were validated by gene expression, growth, and sugar transport analyses. Strikingly, we detected only five sugar permeases in the slow-growing species M. tuberculosis, two of which occur in M. smegmatis. Genes for a PTS are missing in M. tuberculosis. Our analysis thus brings the diversity of carbohydrate uptake systems of fast- and a slow-growing mycobacteria to light, which reflects the lifestyles of M. smegmatis and M. tuberculosis in their natural habitats, the soil and the human body, respectively.


Subject(s)
Biological Transport , Carbohydrate Metabolism , Membrane Transport Proteins/metabolism , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism , Carbohydrates , Gene Expression Regulation, Bacterial , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/physiology , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/physiology
2.
Appl Environ Microbiol ; 72(8): 5637-42, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16885321

ABSTRACT

The insertion element InsTipalpha was constructed to generate protein expression data. It randomly fuses the TetR-inducing peptide Tip to the affected reading frame. Fusion protein expression is quantified by Tet-regulated reporter gene expression. The expression patterns of tagged Escherichia coli genes fully agree with published data from transcriptional fusions or microarrays, validating the Tip tag approach.


Subject(s)
DNA Transposable Elements/genetics , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Gene Expression Regulation, Bacterial , Genes, Reporter , Peptides/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Mutation , Peptides/pharmacology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Tetracycline Resistance/genetics , Transcription, Genetic
3.
Mol Microbiol ; 61(5): 1237-51, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16925557

ABSTRACT

Members of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N-acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N-acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR-family regulator DasR, which controls the N-acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N-acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6-phosphate, a central molecule in N-acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment.


Subject(s)
Acetylglucosamine/metabolism , Bacterial Proteins/metabolism , Phosphotransferases/metabolism , Streptomyces coelicolor/metabolism , Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Genetic Complementation Test , Glucosamine/analogs & derivatives , Glucosamine/pharmacology , Glucose-6-Phosphate/analogs & derivatives , Glucose-6-Phosphate/pharmacology , Microscopy, Electron, Scanning/methods , Models, Biological , Mutation/genetics , Phenotype , Phosphotransferases/genetics , Protein Binding/drug effects , Regulon/genetics , Streptomyces coelicolor/genetics , Streptomyces coelicolor/ultrastructure , Substrate Specificity
4.
Nucleic Acids Res ; 32(11): 3418-26, 2004.
Article in English | MEDLINE | ID: mdl-15247334

ABSTRACT

Transcription factors (TFs) of bacterial helix-turn-helix superfamilies exhibit different effector-binding domains (EBDs) fused to a DNA-binding domain with a common feature. In a previous study of the GntR superfamily, we demonstrated that classifying members into subfamilies according to the EBD heterogeneity highlighted unsuspected and accurate TF-binding site signatures. In this work, we present how such in silico analysis can provide prediction tools to discover new cis/trans relationships. The TF-binding site consensus of the HutC/GntR subfamily was used to (i) predict target sites within the Streptomyces coelicolor genome, (ii) discover a new HutC/GntR regulon and (iii) discover its specific TF. By scanning the S.coelicolor genome we identified a presumed new HutC regulon that comprises genes of the phosphotransferase system (PTS) specific for the uptake of N-acetylglucosamine (PTS(Nag)). A weight matrix was derived from the compilation of the predicted cis-acting elements upstream of each gene of the presumed regulon. Under the assumption that TFs are often subject to autoregulation, we used this matrix to scan the upstream region of the 24 HutC-like members of S.coelicolor. orf SCO5231 (dasR) was selected as the best candidate according to the high score of a 16 bp sequence identified in its upstream region. Our prediction that DasR regulates the PTS(Nag) regulon was confirmed by in vivo and in vitro experiments. In conclusion, our in silico approach permitted to highlight the specific TF of a regulon out of the 673 orfs annotated as 'regulatory proteins' within the genome of S.coelicolor.


Subject(s)
Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/classification , Computational Biology/methods , Transcription Factors/chemistry , Transcription Factors/classification , Acetylglucosamine/metabolism , Bacterial Proteins/metabolism , Binding Sites , Consensus Sequence , Genome, Bacterial , Helix-Turn-Helix Motifs , Phosphotransferases/metabolism , Regulon , Response Elements , Software , Streptomyces/genetics , Streptomyces/metabolism , Transcription Factors/metabolism
5.
J Bacteriol ; 186(5): 1362-73, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14973030

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.


Subject(s)
Carbohydrate Metabolism , Gene Expression Regulation, Bacterial , Membrane Transport Proteins/metabolism , Streptomyces/enzymology , Transcription, Genetic , ATP-Binding Cassette Transporters/metabolism , Base Sequence , Biological Transport , Chromosomes, Bacterial , Molecular Sequence Data , Phosphoenolpyruvate Sugar Phosphotransferase System , Reverse Transcriptase Polymerase Chain Reaction , Streptomyces/genetics , Streptomyces/growth & development , Substrate Specificity
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