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.The growth and nutritional requirements of mycobacteria have been intensely studied since the discovery of Mycobacterium tuberculosis (32). This resulted in an overwhelming body of literature on the physiology of mycobacterial metabolism in the years before the dawn of molecular biology (20,53,54). Carbon metabolism of mycobacteria has attracted renewed interest since the discovery that M. tuberculosis relies on the glyoxylate cycle for survival in mice (36,41). This observation indicates that M. tuberculosis uses lipids as the main carbon source during infection. On the other side, genes that encode a putative disaccharide transporter were essential for M. tuberculosis during the first week of infection, indicating that M. tuberculosis may switch its main carbon source from carbohydrates to lipids with the onset of the adaptive immune response (61). However, the nutrients and the corresponding uptake proteins are unknown for M. tuberculosis inside the human host. Surprisingly, this is also true for M. tuberculosis growing in vitro and for Mycobacterium smegmatis, which is often used as a fast-growing, nonpathogenic model organism to learn more about basic mycobacterial physiology. There is no doubt that the uptake pathways have been adapted to the habitats of M. tuberculosis and M. smegmatis, the human body and soil, respectively. Thus, much can be learned about the lifestyles of both organisms by a comparison of the complements of specific nutrient uptake proteins. Previously, 38 ATP-binding cassette (ABC) transport proteins...

Here we present the complement of the carbohydrate uptake systems of the strictly anaerobic probiotic Bifidobacterium longum NCC2705. The genome analysis of this bacterium predicts that it has 19 permeases for the uptake of diverse carbohydrates. The majority belongs to the ATP-binding cassette transporter family with 13 systems identified. Among them are permeases for lactose, maltose, raffinose, and fructooligosaccharides, a commonly used prebiotic additive. We found genes that encode a complete phosphotransferase system (PTS) and genes for three permeases of the major facilitator superfamily. These systems could serve for the import of glucose, galactose, lactose, and sucrose. Growth analysis of NCC2705 cells combined with biochemical characterization and microarray data showed that the predicted substrates are consumed and that the corresponding transport and catabolic genes are expressed. Biochemical analysis of the PTS, in which proteins are central in regulation of carbon metabolism in many bacteria, revealed that B. longum has a glucose-specific PTS, while two other species (Bifidobacterium lactis and Bifidobacterium bifidum) have a fructose-6-phosphate-forming fructose-PTS instead. It became obvious that most carbohydrate systems are closely related to those from other actinomycetes, with a few exceptions. We hope that this report on B. longum carbohydrate transporter systems will serve as a guide for further in-depth analyses on the nutritional lifestyle of this beneficial bacterium.

Analysis of culture supernatants obtained from Bifidobacterium longum NCC2705 grown on glucose and lactose revealed that glucose utilization is impaired until depletion of lactose. Thus, unlike many other bacteria, B. longum preferentially uses lactose rather than glucose as the primary carbon source. Glucose uptake experiments with B. longum cells showed that glucose transport was repressed in the presence of lactose. A comparative analysis of global gene expression profiling using DNA arrays led to the identification of only one gene repressed by lactose, the putative glucose transporter gene glcP. The functionality of GlcP as glucose transporter was demonstrated by heterologous complementation of a glucose transport-deficient Escherichia coli strain. Additionally, GlcP exhibited the highest substrate specificity for glucose. Primer extension and real-time PCR analyses confirmed that expression of glcP was mediated by lactose. Hence, our data demonstrate that the presence of lactose in culture medium leads to the repression of glucose transport and transcriptional down-regulation of the glucose transporter gene glcP. This may reflect the highly adapted life-style of B. longum in the gastrointestinal tract of mammals.Bifidobacteria are strictly anaerobic microorganisms that are found as commensals in the mammalian gastrointestinal tract (2). They predominate in infants' intestines and can represent up to 3% of the gut microbiota in adult humans (2). Together with lactobacilli, bifidobacteria are considered health-promoting bacteria and thus are used as food additives in the dairy industry (1, 9).Bifidobacteria can utilize a wide range of carbon sources. Some of them, such as oligofructose, inulin, and raffinose, have been identified as growth-promoting, bifidogenic compounds (8,10). This is further substantiated by sequence information from Bifidobacterium longum NCC2705, whose chromosome encodes a large variety of carbohydrate utilization genes (22). Nevertheless, little is known about the mechanisms of simple sugar transport, utilization, and regulation in bifidobacteria. Biochemical analyses of glucose transport revealed that a glucose-specific phosphotransferase system (PTS) is present in Bifidobacterium breve, and a potassium-dependent glucose permease, a facilitator for galactose, and a proton-driven symporter for lactose were described in Bifidobacterium bifidum (5,12,13). A sucrose permease gene from Bifidobacterium lactis was found as part of an operon that is induced by sucrose and raffinose and is subject to glucose repression (23). The isolation of a fructose kinase gene, frk, which is also repressed by glucose, has been related to fructose utilization in B. longum (3).In this report, we demonstrate that B. longum NCC2705 preferentially uses lactose over glucose when grown in the presence of both sugars. We further show that glucose transport is down-regulated by lactose, and we identify a glucose transporter gene that undergoes lactose repression. MATERIALS AND METHODSBacterial strains and culture ...

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS) controls sugar uptake and carbon utilization in low-GC Gram-positive bacteria and in Gram-negative bacteria. We have purified HPr from Streptomyces coelicolor cell extracts. The N-terminal sequence matched the product of an S. coelicolor orf, designated ptsH, sequenced as part of the S. coelicolor genome sequencing project. The ptsH gene appears to form a monocistronic operon. Determination of the evolutionary relationship revealed that S. coelicolor HPr is equally distant to all known HPr and HPr-like proteins. The presumptive phosphorylation site around histidine 15 is perfectly conserved while a second possible phosphorylation site at serine 47 is not wellconserved. HPr was overproduced in Escherichia coli in its native form and as a histidine-tagged fusion protein.Histidine-tagged HPr was purified to homogeneity. HPr was phosphorylated by its own enzyme I (EI) and heterologously phosphorylated by EI of Bacillus subtilis and Staphylococcus aureus, respectively. This phosphoenolpyruvate-dependent phosphorylation was absent in an HPr mutant in which histidine 15 was replaced by alanine. Reconstitution of the fructose-specific PTS demonstrated that HPr could efficiently phosphorylate enzyme II Fructose . HPr-P could also phosphorylate enzyme II Glucose of B. subtilis, enzyme II Lactose of S. aureus, and IIAMannitol of E. coli. ATP-dependent phosphorylation was detected with HPr kinase/phosphatase of B. subtilis. These results present the first identification of a gene of the PTS complement of S. coelicolor, providing the basis to elucidate the role(s) of HPr and the PTS in this class of bacteria.Keywords: phosphotransferase system; protein phosphorylation; HPr; sugar transport; Streptomyces.Streptomyces are high-GC, Gram-positive soil bacteria which grow vegetatively as a branching mycelial mass using a wide spectrum of carbon sources such as cellulose, chitin, xylan, and many mono-and disaccharides. Upon nutrient depletion this primary metabolic phase may switch to a secondary metabolic phase resulting, for example, in antibiotic production and morphogenesis [1]. Despite the interesting life cycle and the commercial importance, there is limited knowledge on nutrient sensing, carbohydrate transport, and regulation of carbohydrate utilization, and how these may influence secondary metabolic processes.So far, the mechanism of sugar uptake has been described for only a few carbohydrates. It was demonstrated that cellobiose, cellotriose, maltose, and xylobiose are taken up via ABC transport systems, and that fructose is apparently transported via a phosphoenolpyruvate:fructose phosphotransferase system (PTS) [2±5]. The utilization of carbohydrates in many cases is governed by carbon catabolite repression (CCR) [6]. CCR can be exerted by various carbohydrates such as glucose, fructose, mannitol, or gluconate [7]. The signal transduction pathway leading to CCR is not known. Mutations in glkA, encoding glucose kinase, ...

HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G؉C-content grampositive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G؉C-content grampositive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible highaffinity fructose transport with an apparent K m of 2 M. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor.