Sporulation conditions influence the surface and adhesion properties of Bacillus subtilis spores.

Spore-forming bacteria of the Bacillus subtilis group are responsible for recurrent contamination of processing lines in the food industry which can lead to food spoilage. The persistence of B. subtilis would be due to the high resistance of spores to extreme environmental condition and their propensity to contaminate surfaces. While it is well known that sporulation conditions modulate spore resistance properties, little is known about their effect on surface and adhesion properties. Here, we studied the impact of 13 sporulation conditions on the surface and adhesion properties of B. subtilis 168 spores. We showed that Ca2+ or Mg2+ depletion, lower oxygen availability, acidic pH as well as oxidative stresses during sporulation lead to the release of more hydrophobic and adherent spores. The consequences of these sporulation conditions on crust composition in carbohydrates and proteins were also evaluated. The crust glycans of spores produced in a sporulation medium depleted in Ca2+ or Mg2+ or oxygen-limited conditions were impaired and contained lower amounts of rhamnose and legionaminic acid. In addition, we showed that lower oxygen availability or addition of hydrogen peroxide during sporulation decreases the relative amount of two crust proteins (CgeA and CotY) and the changes observed in these conditions could be due to transcriptional repression of genes involved in crust synthesis in late stationary phase. The fact that sporulation conditions affect the ease with which spores can contaminate surfaces could explain the frequent and recurrent presence of B. subtilis spores in food processing lines.

Parietal composition of Lichtheimia corymbifera: Differences between spore and germ tube stages and host-pathogen interactions.

The molecular composition and structural organization of the cell wall of filamentous fungi underlie the ability of the host to identify them as pathogens. Although the organization of the fungal cell wall, composed of 90% polysaccharides, is similar from one fungus to another, small variations condition their ability to trigger pattern recognition receptors. Because the incidence of mucormycosis, an emerging life-threatening infection caused by the species of the order Mucorales is increasing worldwide, the precise composition of the cell wall of two strains of Lichtheimia corymbifera was investigated in the early growth stages of germination (spores and germ-tubes) using trimethylsilylation and confocal microscopy. This study also characterizes the response of THP-1 cells to Mucorales. The study identified the presence of uncommon monosaccharides (fucose, galactose, and glucuronic acid) whose respective proportions vary according to the germination stage, revealing early parietal reorganization. Immunofluorescence studies confirmed the exposure of ß-glucan on the surface of swollen spores and germ-tubes. Both spores and germ-tubes of L. corymbifera promoted an early and strong pro-inflammatory response, through TLR-2. Our results show the singularity of the cell wall of the order Mucorales, opening perspectives for the development of specific diagnostic biomarkers.

Lichtheimia corymbifera is a causative agent of mucormycosis, an emerging invasive fungal infection. Deciphering cell wall composition can lead to the identification of a polysaccharide epitope, which could be used as a biomarker, useful for the diagnosis of mucormycosis.

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCEBacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

PII1: a protein involved in starch initiation that determines granule number and size in Arabidopsis chloroplast.

The initiation of starch granule formation is still poorly understood. However, the soluble starch synthase 4 (SS4) appears to be a major component of this process since it is required to synthesize the correct number of starch granules in the chloroplasts of Arabidopsis thaliana plants. A yeast two-hybrid screen allowed the identification of several putative SS4 interacting partners. We identified the product of At4g32190 locus as a chloroplast-targeted PROTEIN INVOLVED IN STARCH INITIATION (named PII1). Arabidopsis mutants devoid of PII1 display an alteration of the starch initiation process and accumulate, on average, one starch granule per plastid instead of the five to seven granules found in plastids of wild-type plants. These granules are larger than in wild-type, and they remain flat and lenticular. pii1 mutants display wild-type growth rates and accumulate standard starch amounts. Moreover, starch characteristics, such as amylopectin chain length distribution, remain unchanged. Our results reveal the involvement of PII1 in the starch priming process in Arabidopsis leaves through interaction with SS4.

Apart From Rhoptries, Identification of Toxoplasma gondii's O-GlcNAcylated Proteins Reinforces the Universality of the O-GlcNAcome.

O-linked ß-N-acetylglucosaminylation or O-GlcNAcylation is a widespread post-translational modification that belongs to the large and heterogeneous group of glycosylations. The functions managed by O-GlcNAcylation are diverse and include regulation of transcription, replication, protein's fate, trafficking, and signaling. More and more evidences tend to show that deregulations in the homeostasis of O-GlcNAcylation are involved in the etiology of metabolic diseases, cancers and neuropathologies. O-GlcNAc transferase or OGT is the enzyme that transfers the N-acetylglucosamine residue onto target proteins confined within the cytosolic and nuclear compartments. A form of OGT was predicted for Toxoplasma and recently we were the first to show evidence of O-GlcNAcylation in the apicomplexans Toxoplasma gondii and Plasmodium falciparum. Numerous studies have explored the O-GlcNAcome in a wide variety of biological models but very few focus on protists. In the present work, we used enrichment on sWGA-beads and immunopurification to identify putative O-GlcNAcylated proteins in Toxoplasma gondii. Many of the proteins found to be O-GlcNAcylated were originally described in higher eukaryotes and participate in cell shape organization, response to stress, protein synthesis and metabolism. In a more original way, our proteomic analyses, confirmed by sWGA-enrichment and click-chemistry, revealed that rhoptries, proteins necessary for invasion, are glycosylated. Together, these data show that regardless of proteins strictly specific to organisms, O-GlcNAcylated proteins are rather similar among living beings.

Identification of O-GlcNAcylated proteins in Plasmodium falciparum.

BACKGROUND: Post-translational modifications (PTMs) constitute a huge group of chemical modifications increasing the complexity of the proteomes of living beings. PTMs have been discussed as potential anti-malarial drug targets due to their involvement in many cell processes. O-GlcNAcylation is a widespread PTM found in different organisms including Plasmodium falciparum. The aim of this study was to identify O-GlcNAcylated proteins of P. falciparum, to learn more about the modification process and to understand its eventual functions in the Apicomplexans. METHODS: The P. falciparum strain 3D7 was amplified in erythrocytes and purified. The proteome was checked for O-GlcNAcylation using different methods. The level of UDP-GlcNAc, the donor of the sugar moiety for O-GlcNAcylation processes, was measured using high-pH anion exchange chromatography. O-GlcNAcylated proteins were enriched and purified utilizing either click chemistry labelling or adsorption on succinyl-wheat germ agglutinin beads. Proteins were then identified by mass-spectrometry (nano-LC MS/MS). RESULTS: While low when compared to MRC5 control cells, P. falciparum disposes of its own pool of UDP-GlcNAc. By using proteomics methods, 13 O-GlcNAcylated proteins were unambiguously identified (11 by click-chemistry and 6 by sWGA-beads enrichment; 4 being identified by the 2 approaches) in late trophozoites. These proteins are all part of pathways, functions and structures important for the parasite survival. By probing clicked-proteins with specific antibodies, Hsp70 and α-tubulin were identified as P. falciparum O-GlcNAc-bearing proteins. CONCLUSIONS: This study is the first report on the identity of P. falciparum O-GlcNAcylated proteins. While the parasite O-GlcNAcome seems close to those of other species, the structural differences exhibited by the proteomes provides a glimpse of innovative therapeutic paths to fight malaria. Blocking biosynthesis of UDP-GlcNAc in the parasites is another promising option to reduce Plasmodium life cycle.

O-GlcNAcylation is a key modulator of skeletal muscle sarcomeric morphometry associated to modulation of protein-protein interactions.

BACKGROUND: The sarcomere structure of skeletal muscle is determined through multiple protein-protein interactions within an intricate sarcomeric cytoskeleton network. The molecular mechanisms involved in the regulation of this sarcomeric organization, essential to muscle function, remain unclear. O-GlcNAcylation, a post-translational modification modifying several key structural proteins and previously described as a modulator of the contractile activity, was never considered to date in the sarcomeric organization. METHODS: C2C12 skeletal myotubes were treated with Thiamet-G (OGA inhibitor) in order to increase the global O-GlcNAcylation level. RESULTS: Our data clearly showed a modulation of the O-GlcNAc level more sensitive and dynamic in the myofilament-enriched fraction than total proteome. This fine O-GlcNAc level modulation was closely related to changes of the sarcomeric morphometry. Indeed, the dark-band and M-line widths increased, while the I-band width and the sarcomere length decreased according to the myofilament O-GlcNAc level. Some structural proteins of the sarcomere such as desmin, αB-crystallin, α-actinin, moesin and filamin-C have been identified within modulated protein complexes through O-GlcNAc level variations. Their interactions seemed to be changed, especially for desmin and αB-crystallin. CONCLUSIONS: For the first time, our findings clearly demonstrate that O-GlcNAcylation, through dynamic regulations of the structural interactome, could be an important modulator of the sarcomeric structure and may provide new insights in the understanding of molecular mechanisms of neuromuscular diseases characterized by a disorganization of the sarcomeric structure. GENERAL SIGNIFICANCE: In the present study, we demonstrated a role of O-GlcNAcylation in the sarcomeric structure modulation.

Glycosylation of BclA Glycoprotein from Bacillus cereus and Bacillus anthracis Exosporium Is Domain-specific.

The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracis is known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets.

Development of an SPME-GC-MS method for the specific quantification of dimethylamine and trimethylamine: use of a new ratio for the freshness monitoring of cod fillets.

BACKGROUND: Fish is a highly perishable food, so it is important to be able to estimate its freshness to ensure optimum quality for consumers. The present study describes the development of an SPME-GC-MS technique capable of quantifying both trimethylamine (TMA) and dimethylamine (DMA), components of what has been defined as partial volatile basic nitrogen (PVB-N). This method was used, together with other reference methods, to monitor the storage of cod fillets (Gadus morhua) conserved under melting ice. RESULTS: Careful optimisation enabled definition of the best parameters for extracting and separating targeted amines and an internal standard. The study of cod spoilage by sensory analysis and TVB-N assay led to the conclusion that the shelf-life of cod fillet was between 6 and 7 days. Throughout the study, TMA and DMA were specifically quantified by SPME-GC-MS; the first was found to be highly correlated with the values returned by steam distillation assays. Neither TMA-N nor DMA-N were able to successfully characterise the decrease in early freshness, unlike dimethylamine/trimethylamine ratio (DTR), whose evolution is closely related to the results of sensory analysis until the stage where fillets need to be rejected. CONCLUSION: DTR was proposed as a reliable indicator for the early decrease of freshness until fish rejection. © 2015 Society of Chemical Industry.

Monitoring the freshness of fish: development of a qPCR method applied to MAP chilled whiting.

BACKGROUND: Monitoring of early stages of freshness decay is a major issue for the fishery industry to guarantee the best quality for this highly perishable food matrix. Numerous techniques have been developed, but most of them have the disadvantage of being reliable only either in the last stages of fish freshness or for the analysis of whole fish. This study describes the development of a qPCR method targeting the torA gene harboured by fish spoilage microorganisms. torA encodes an enzyme that leads to the production of trimethylamine responsible for the characteristic spoiled-fish odour. RESULTS: A degenerate primer pair was designed. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies on 7-log DNA dilutions. The primer pair was used during a shelf-life monitoring study achieved on modified atmosphere packed, chilled, whiting (Merlangius merlangus) fillets. The qPCR approach allows the detection of an increase of torA copies throughout the storage of fillets in correlation with the evolution of both total volatile basic nitrogen (-0.86) and trimethylamine concentrations (-0.81), known as spoilage markers. CONCLUSION: This study described a very promising, sensitive, reliable, time-effective, technique in the field of freshness characterisation of processed fish.

Presence and function of a thick mucous layer rich in polysaccharides around Bacillus subtilis spores.

This study was designed to establish the presence and function of the mucous layer surrounding spores of Bacillus subtilis. First, an external layer of variable thickness and regularity was often observed on B. subtilis spores. Further analyses were performed on B. subtilis 98/7 spores surrounded by a thick layer. The mechanical removal of the layer did not affect their resistance to heat or their ability to germinate but rendered the spore less hydrophilic, more adherent to stainless steel, and more resistant to cleaning. This layer was mainly composed of 6-deoxyhexoses, ie rhamnose, 3-O-methyl-rhamnose and quinovose, but also of glucosamine and muramic lactam, known also to be a part of the bacterial peptidoglycan. The specific hydrolysis of the peptidoglycan using lysozyme altered the structure of the required mucous layer and affected the physico-chemical properties of the spores. Such an outermost mucous layer has also been seen on spores of B. licheniformis and B. clausii isolated from food environments.

Evolution of volatile compounds and biogenic amines throughout the shelf life of marinated and salted anchovies (Engraulis encrasicolus).

Producers of processed anchovies have developed hazard analysis and critical control points (HACCP) to guarantee the quality of their products. Nonetheless there is a lack of objective data to determine products' shelf life. The quality of a product is usually established on the basis of its safety and organoleptic properties. These parameters were assessed by monitoring the profiles of volatile compounds and quantitating six biogenic amines in samples of two types of processed anchovies during their shelf life. With regard to biogenic amines, quantities were below the regulatory limits throughout shelf life, except when a temperature abuse was applied for marinated samples. Moreover, this work highlights an optimum volatile profile at 5 and 6 months of storage for salted and marinated anchovies, respectively. This is the result of a higher content of six aldehyde and nine ketone compounds, mainly from lipid oxidation.

Characterization of the recombinant Candida albicans ß-1,2-mannosyltransferase that initiates the ß-mannosylation of cell wall phosphopeptidomannan.

The presence of ß-mannosides in their cell walls confers specific features on the pathogenic yeasts Candida albicans and Candida glabrata compared with non-pathogenic yeasts. In the present study, we investigated the enzymatic properties of Bmt1 (ß-mannosyltransferase 1), a member of the recently identified ß-mannosyltransferase family, from C. albicans. A recombinant soluble enzyme lacking the N-terminal region was expressed as a secreted protein from the methylotrophic yeast Pichia pastoris. In parallel, functionalized natural oligosaccharides isolated from Saccharomyces cerevisiae and a C. albicans mutant strain, as well as synthetic α-oligomannosides, were prepared and used as potential acceptor substrates. Bmt1p preferentially utilizes substrates containing linear chains of α-1,2-linked mannotriose or mannotetraose. The recombinant enzyme consecuti-vely transfers two mannosyl units on to these acceptors, leading to the production of α-mannosidase-resistant oligomannosides. NMR experiments further confirmed the presence of a terminal ßMan (ß-1,2-linked mannose) unit in the first enzyme product. In the future, a better understanding of specific ß-1,2-mannosyltransferase molecular requirements will help the design of new potential antifungal drugs.

Differentiation of fresh and frozen/thawed fish, European sea bass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), cod (Gadus morhua) and salmon (Salmo salar), using volatile compounds by SPME/GC/MS.

BACKGROUND: A simple method based on solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) was applied for studying the volatile profiles of whole fish samples of European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) and fillets of cod (Gadus morhua) and salmon (Salmo salar) during frozen storage in order to be able to differentiate a fresh product from one that has been frozen. Analysis of volatile compounds was performed on these two product types, fresh and after freezing/thawing following storage at - 20 °C for 30 and 90 days. RESULTS: More than a hundred volatile compounds were found by SPME/GC/MS. Statistical processing by principal component analysis and ascending hierarchical classification was used to classify the samples into categories and verify the possibility of separating fresh samples from those that had been frozen and thawed. The compounds to be used as differentiators were identified. Four compounds were common to all species: dimethyl sulfide, 3-methylbutanal, ethyl acetate and 2-methylbutanal. Not only were they found in larger quantities after thawing but they also increased with the duration of storage at - 20 °C. CONCLUSION: These four compounds can therefore be considered as potential markers of differentiation between a fresh product and one that has been frozen.

Environmental and biofilm-dependent changes in a Bacillus cereus secondary cell wall polysaccharide.

Bacterial species from the Bacillus genus, including Bacillus cereus and Bacillus anthracis, synthesize secondary cell wall polymers (SCWP) covalently associated to the peptidoglycan through a phospho-diester linkage. Although such components were observed in a wide panel of B. cereus and B. anthracis strains, the effect of culture conditions or of bacterial growth state on their synthesis has never been addressed. Herein we show that B. cereus ATCC 14579 can synthesize not only one, as previously reported, but two structurally unrelated secondary cell wall polymers (SCWP) polysaccharides. The first of these SCWP, →4)[GlcNAc(ß1-3)]GlcNAc(ß1-6)[Glc(ß1-3)][ManNAc(α1-4)]GalNAc(α1-4)ManNAc(ß1→, although presenting an original sequence, fits to the already described the canonical sequence motif of SCWP. In contrast, the second polysaccharide was made up by a totally original sequence, →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(ß1→, which no equivalent has ever been identified in the Bacillus genus. In addition, we established that the syntheses of these two polysaccharides were differently regulated. The first one is constantly expressed at the surface of the bacteria, whereas the expression of the second is tightly regulated by culture conditions and growth states, planktonic, or biofilm.

Overexpression of Man2C1 leads to protein underglycosylation and upregulation of endoplasmic reticulum-associated degradation pathway.

Unfolded glycoproteins retained in the endoplasmic reticulum (ER) are degraded via the ER-associated degradation (ERAD) pathway. These proteins are subsequently transported to the cytosol and degraded by the proteasomal complex. Although the sequential events of ERAD are well described, its regulation remains poorly understood. The cytosolic mannosidase, Man2C1, plays an essential role in the catabolism of cytosolic free oligomannosides, which are released from the degraded proteins. We have investigated the impact of Man2C1 overexpression on protein glycosylation and the ERAD process. We demonstrated that overexpression of Man2C1 led to modifications of the cytosolic pool of free oligomannosides and resulted in accumulation of small Man(2-4)GlcNAc(1) glycans in the cytosol. We further correlated this accumulation with incomplete protein glycosylation and truncated lipid-linked glycosylation precursors, which yields an increase in N-glycoprotein en route to the ERAD. We propose a model in which high mannose levels in the cytosol interfere with glucose metabolism and compromise N-glycan synthesis in the ER. Our results show a clear link between the intracellular mannose-6-phosphate level and synthesis of the lipid-linked precursors for protein glycosylation. Disturbance in these pathways interferes with protein glycosylation and upregulated ERAD. Our findings support a new concept that regulation of Man2C1 expression is essential for maintaining efficient protein N-glycosylation.

Freshness characterisation of whiting (Merlangius merlangus) using an SPME/GC/MS method and a statistical multivariate approach.

BACKGROUND: The freshness of whiting was studied at five stages of ice storage by comparing the analysis of volatile compounds obtained through solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) with two sensory methods. RESULTS: Of the volatile compounds identified, 38 were analysed using a statistical multivariate approach and classified according to their role in the estimation of freshness during storage as markers of freshness or spoilage. Regarding the evolution of the presence or absence of individual compounds, three categories were defined. For example, the volatile compounds propanal, hexanal, 1-penten-3-ol, pentanal, 2,3-pentanedione, 1-penten-3-one, heptanal, (E)-2-pentenal, 2,3-octanedione, (Z)-2-penten-1-ol, 1-pentanol, butanal, octanal, 3,5,5-trimethyl-2-hexene, 1-hexanol and 4,4-dimethyl-1,3-dioxane appeared highly relevant, because they are found throughout storage and can be divided into several categories that are directly related to the quality of fish. CONCLUSION: SPME/GC/MS combined with a statistical multivariate approach may be a useful method to identify volatile compounds and characterise fish freshness during storage.

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and N-acetylglucosamine metabolism in Bifidobacterium.

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial D: -fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the D: -fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism.

Bifidobacterium longum requires a fructokinase (Frk; ATP:D-fructose 6-phosphotransferase, EC 2.7.1.4) for fructose catabolism.

Although the ability of Bifidobacterium spp. to grow on fructose as a unique carbon source has been demonstrated, the enzyme(s) needed to incorporate fructose into a catabolic pathway has hitherto not been defined. This work demonstrates that intracellular fructose is metabolized via the fructose-6-P phosphoketolase pathway and suggests that a fructokinase (Frk; EC 2.7.1.4) is the enzyme that is necessary and sufficient for the assimilation of fructose into this catabolic route in Bifidobacterium longum. The B. longum A10C fructokinase-encoding gene (frk) was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on a Co(2+)-based column, and the pH and temperature optima were determined. A biochemical analysis revealed that Frk displays the same affinity for fructose and ATP (Km(fructose) = 0.739 +/- 0.18 mM and Km(ATP) = 0.756 +/- 0.08 mM), is highly specific for D-fructose, and is inhibited by an excess of ATP (>12 mM). It was also found that frk is inducible by fructose and is subject to glucose-mediated repression. Consequently, this work presents the first characterization at the molecular and biochemical level of a fructokinase from a gram-positive bacterium that is highly specific for D-fructose.