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Keywords:

  • N-deoxyribosyltransferase;
  • Lactobacillus fermentum;
  • nucleotide salvage;
  • cell surface;
  • immunolocalization

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

N-deoxyribosyltransferases are essential enzymes in the nucleotide salvage pathway of lactobacilli. They catalyze the exchange between the purine or pyrimidine bases of 2′-deoxyribonucleosides and free pyrimidine or purine bases. In general, N-deoxyribosyltransferases are referred to as cytoplasmic enzymes, although there is no experimental evidence for this subcellular localization. In this work, the subcellular localization of N-deoxyribosyltransferase II (NTD) from Lactobacillus fermentum was examined by subcellular fractionation, transmission electron microscopy, and fluorescence microscopy. Our results indicate that L. fermentum NTD are distributed not only in the cytoplasm but also on the cell wall surface, and further studies showed that surface-attached NTD can be released into the culture broth and conventional buffers.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

Lactobacilli can be divided into two groups depending on whether or not they require deoxyribonucleosides for growth (Kaminski, 2002). Most lactobacilli that utilize the salvage pathway degrade exogenous nucleosides to the nucleobase and pentose sugar via a nucleoside phosphorylase. Others possess a special salvage system based on a nucleoside deoxyribosyltransferase and require a deoxynucleoside in combination with purine and pyrimidine bases for their DNA synthesis (Kilstrup et al., 2005).

N-deoxyribosyltransferases (EC 2.4.2.6), also called trans-N-deoxyribosylases, catalyze the transfer of a 2′-deoxyribosyl group from a donor deoxynucleoside to an acceptor nucleobase (Anand et al., 2004). This enzyme was initially described for lactobacilli and has also been found in certain species of Streptococcus (Chawdhri et al., 1991) and in some protozoans such as Crithidia luciliae (Steenkamp, 1991). Two types of N-deoxyribosyltransferase have been described in lactobacilli: type I is purine deoxyribosyltransferase (PTD), specific for the transfer of deoxyribose between two purines; type II is nucleoside 2′-deoxyribosyltransferase (NTD), which catalyzes the transfer of deoxyribose between either purines or pyrimidines (Holguin & Cardinaud, 1975; Miyamoto et al., 2007). Several dozen reports on lactobacilli N-deoxyribosyltransferase have been published since the initial study by Macnutt (Macnutt, 1950). The three-dimensional structure of these enzymes has been solved, and their kinetic mechanisms as well as their catalytic and substrate binding sites have been well characterized (Armstrong et al., 1996; Anand et al., 2004). The transfer reactions, catalyzed by either PTD or NTD, proceed following a ping-pong bi-bi mechanism by formation of a covalent deoxyribosyl enzyme intermediate (Danzin & Cardinau, 1974; Danzin & Cardinaud, 1976). As NTD has broader substrate specificity than PTD, it has attracted more attention. NTD also has a hydrolase function such that, in the absence of an acceptor base, the nucleoside is converted to its base and deoxyribose (Smar et al., 1991). Most antiviral or anticancer drugs are analogues of naturally occurring nucleosides. The use of purified enzyme or intact bacterial cells containing NTD enables a one-pot transglycosylation reaction at high yields, providing an interesting alternative to traditional multistep chemical methods (Fernandez-Lucas et al., 2010). Stereospecific reactions and high tolerance for various modifications in the bases also make NTD ideally suited to serve as biocatalyst for the production of nucleosides and nucleoside analogues (Okuyama et al., 2003; Mikhailopulo, 2007).

The main function of the salvage pathway in lactic acid bacteria seems to be rescuing nucleobases or nucleosides for nucleotide synthesis. It is vital for some lactobacilli. The salvage pathway systems containing N-deoxyribosyltransferases (or nucleoside phosphorylases), nucleoside deaminases, phosphoribosyltransferases, and nucleoside kinases in lactobacilli have been described by Kilstrup (Kilstrup et al., 2005). The subcellular location of a protein is critical for its physiologic function, and the enzymes of nucleoside catabolism have long been considered to have a periplasmic location (Taketo & Kuno, 1972) in Escherichia coli. The group translocation hypothesis used to explain nucleic acid bases transport was prevalent in the 1970s (Rader & Hochstadt, 1976), and this hypothesis states that the essential salvage pathway enzymes such as phosphoribosyltransferase are situated in the plasma membrane and facilitate the transport of nucleotide bases (Hochstadt, 1978). As the group translocation hypothesis has since been excluded by accumulated evidence (Pandey, 1984), purine nucleoside phosphorylases have been shown to be associated with the internal surface of the plasma membrane, whereas phosphoribosyltransferases appear to be located in the cytoplasm (Page & Burton, 1978). These early studies and hypotheses are inspiring, but were limited by the lack of visualization techniques. The question regarding the localization of nucleoside-catabolic enzymes is far from settled.

The enzymology of N-deoxyribosyltransferase from lactobacilli has been well characterized. In comparision, studies concerning its physiologic role have been limited. As an essential enzyme of nucleotide salvage, N-deoxyribosyltransferase has been considered intuitively to be an intracellular enzyme, although there is no experimental evidence for this subcellular localization. Knowledge of the precise subcellular localization would enable a much better understanding of how these enzymes interact and influence other salvage pathway enzymes or nucleoside transport systems.

Herein, we report the cloning and expression of the LAF 0141 homolog gene encoding a putative N-deoxyribosyltransferase from Lactobacillus fermentum CGMCC 1.2133, and we show that LAF 0141 homolog is a type II nucleoside 2′-deoxyribosyltransferase (NTD). The polyclonal antibodies raised against the purified recombinant protein are used to determine the subcellular localization of NTD in L. fermentum CGMCC 1.2133.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

Bacterial strains and growth conditions

The strain L. fermentum CGMCC 1.2133 (China General Microbiological Culture Collection Center, Beijing) was grown in modified MRS medium (Holguin & Cardinaud, 1975) for 20 h (to stationary phase) at 37 °C. Escherichia coli BL21 (DE3) was used as a host for gene expression and cultured at 37 °C in Luria–Bertani (LB) medium.

Sequencing analysis

Homology searches in the databases were carried out using the blast program. Sequence alignments for homology analysis were achieved using dnaman v.6.0 (Lynnon Biosoft, Quebec, Canada). Protein subcellular localization prediction was carried out by psortb 3.0 (Yu et al., 2010).

Cloning and construction of expression vectors

Genomic DNA was prepared from L. fermentum CGMCC 1.2133 according to the method described by Martin-Platero et al. (2007). PCR was done with L. fermentum CGMCC 1.2133 genomic DNA serving as a template and two primers LAF1 (5′-CATGCCATGG CT ATG TAC CAA AAC AAA GTT TAC CTC G-3′) and LAF2 (5′-CGGGATCC CCG TTT TCT TTA AAA GAC CTT CAT G-3′), corresponding to the LAF 0141 sequence (NCBI gi|184154617) of L. fermentum IFO 3956 strain. The restriction sites BamHI and NcoI are underlined. PCR amplification was carried out under standard conditions with Ex Taq Polymerase (TaKaRa, Dalian, China). The amplified 0.5-kb products were purified and cloned into pET28a (+) (Novagen, Darmstadt, Germany), giving pET-LAF, which was used to transform competent E. coli BL21 (DE3) cells. The exact sequence of the insertion into the designed plasmid was verified by sequencing both strands.

Expression and purification of recombinant protein

The E. coli cells harboring pET-LAF were grown at 37 °C in LB medium with 50 μg mL−1 of kanamycin until OD600 nm reaches 0.8. After induction with 0.5 mM isopropyl-β-D-thiogalactoside for 5 h, E. coli cells were harvested by centrifugation and washed once in 10 mM phosphate buffer (pH 7.3). The pellet was resuspended in 20 mM phosphate buffer (pH 6.5, buffer A) and disrupted using ultrasonic treatment. The lysate was centrifuged at 10 000 g for 30 min and filtered through a 0.22-μm membrane, then applied to a 1-mL Resource Q column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The column was equilibrated in buffer A and then the protein was eluted with a linear gradient of 0–1 M NaCl in buffer A. The fractions possessing N-deoxyribosyltransferase activity were dialyzed in buffer A and concentrated, then treated according to the procedure described above with a 1-mL Mono Q column (GE healthcare). The protein was further purified using gel filtration on a Superdex 75 column (GE healthcare) previously equilibrated with buffer A. All purification steps were carried out at 4 °C using an ÄKTA FPLC (GE Healthcare) system. Each fraction was analyzed using SDS-PAGE. Protein concentrations were measured using the BCA™ Protein Assay Kit (Thermo Scientific, Rockford, IL).

N-deoxyribosyltransferase activity assays

The standard reaction mixture contained 500 μL of cell extract or 0.05 mg of pure enzyme, and 10 mM thymidine as deoxyribose donor and 10 mM adenine as base acceptor in 50 mM citrate buffer (pH 5.9). Reactions were carried out in a total volume of 1 mL at 40 °C for 30 min and stopped by heating at 95 °C for 5 min. One unit of enzyme was defined as the amount of enzyme required to produce 1 μmol of products per minute under standard conditions. The mixture was diluted with water and then filtered through a 0.45-μm membrane. The production of deoxyadenosine was analyzed using high performance liquid chromatography (Waters 600 HPLC series, Waters Corporation, Milford, MA) at an absorbance of 254 nm, using a Diamonsil C18 column, 5 μm, 250 × 4.6 mm (Dikma Technologies, Beijing, China).

Preparation of polyclonal antibodies and Western blotting analyses

Polyclonal antibodies against N-deoxyribosyltransferase were raised in New Zealand rabbits following standard immunization procedures and then purified by Protein A Sepharose Fast Flow (Pharmacia Biotech, Uppsala, Sweden). The specificity of the antibodies was tested on Western blotting against the purified recombinant protein and the whole cell extract (Bhaduri & Demchick, 1983) of L. fermentum. For immunoblot analyses, protein samples were separated using SDS-PAGE in 12.5% polyacrylamide gel and transferred to a polyvinylidene difluoride membrane using the Multiphor II Western blotting system (Amersham Biosciences, Uppsala, Sweden). Purified polyclonal antibodies were used at dilutions of 1 : 1000 and horseradish peroxidase-conjugated goat anti-rabbit antibody at 1 : 3000. The signals were visualized using an HRP-DAB development kit (Tiangen Biotech Co. Ltd, Beijing, China).

Preparation of cytoplasmic protein and cell wall extract

The overnight cultures of L. fermentum were inoculated into fresh modified MRS broth and incubated for 20 h at 40 °C with gentle stirring (Holguin & Cardinaud, 1975). Lactobacillus fermentum cells were collected by centrifugation at 8000 g and washed once in 0.1 M phosphate buffer (pH 6.0). Cell-free extracts were prepared by sonication. Unbroken cells were removed by centrifugation at 10 000 g for 10 min. After ultracentrifugation at 100 000 g for 30 min, the supernatant contained cytoplasmic protein fractions, and the debris contained cell membrane and cell-walls fractions. The debris was washed twice with washing buffer (0.1 M phosphate buffer, pH 6.0) to exclude possible contamination with cytoplasmic proteins. The extraction of surface proteins of L. fermentum cells from 200 mL of medium was carried out according to the method of Saad (Saad et al., 2009): L. fermentum cells were incubated in 100 mM Tris–HCl buffer at pH 8.0 for 40 min at room temperature. After centrifugation at 10 000 g for 10 min, the supernatant was filtered through a 0.45-μm membrane. All the samples were precipitated with trichloroacetic acid and analyzed using Western blotting.

Electron microscopic immunogold localization

Lactobacillus fermentum intact cells were fixed in 0.5% glutaraldehyde, 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) overnight at 4 °C, and washed three times with 0.1 M phosphate buffer (pH 7.4). Lactobacillus fermentum cells were treated for 30 min with 0.1 M glycine to neutralize free aldehyde groups, then rinsed with 0.1 M phosphate buffer and dehydrated in a graded series of ethanol solutions (Kang et al., 2003). Lactobacillus fermentum cells were embedded in Epon-812 resin and cut into ultra-thin sections (70 nm) using an ultramicrotome (Lecia EM UC6, Leica, Nussloch, Germany). Sections were placed on copper grids and incubated for 20 min with 1% hydrogen peroxide, rinsed in 0.1 M Tris–HCl-buffered saline (TBS, pH 7.4) three times, and then incubated for 60 min in TBS with 1% bovine serum albumin. After removing the blotting buffer, the sections were incubated with the specific polyclonal antibodies (1 : 1000 dilution in TBS) for 2 h at room temperature, rinsed in TBS buffer six times, followed by incubation for 1 h at room temperature with the gold-labeled (5-nm colloidal gold) anti-rabbit IgG (1 : 30 dilution in TBS; Sigma, St. Louis, MO). Finally, sections were rinsed in TBS buffer and refixed in 2.5% glutaraldehyde for 10 min, double stained in uranyl acetate and lead hydroxide, and observed under a transmission electron microscope (Hitachi H-7650, Tokyo, Japan).

Immunofluorescence microscopic localization

Lactobacillus fermentum cells were washed once with PBS-citrate buffer (pH 4.5), then used to coat glass slides, and fixed with 3.5% paraformaldehyde for 20 min (Antikainen et al., 2007b). Some of L. fermentum cells were suspended in 1 mL 100 mM Tris–HCl (pH 8.0) after washing, and incubated at room temperature for 40 min before fixation. The samples were washed with TBS and blocked in 10% bovine serum albumin for 30 min. Following this, the samples were then incubated with anti-NTD antibody (1 : 50 dilution in TBS) at 37 °C for 1 h. After washing with TBS three times, the secondary DyLight 594 Goat Anti-Rabbit IgG Antibody (1 : 100 dilution in TBS; Jackson ImmunoResearch Laboratories, Inc., Baltimore Pike West Grove, PA) was added to the samples at 37 °C, which were then incubated for 30 min. The samples were rinsed in Milli-Q water and examined using differential interference contrast microscopy and fluorescence microscopy (Leica DMIRB, Wetzlar, Germany).

N-deoxyribosyltransferase activity of intact L. fermentum cells

To determine whether NTD retains its biologic activity when localized on the L. fermentum surface, enzymatic studies were carried out using whole cells. The standard reaction mixture employed with the purified NTD was used with whole L. fermentum cells. Reactions were carried out in a total volume of 1 mL (containing 0.25 g wet weight of cells) at 40 °C for 1, 2, 3, or 5 min and stopped by heating at 95 °C for 5 min. The L. fermentum cells were removed by centrifugation (10 000 g for 10 min). The supernatants were diluted with water and analyzed by measuring absorbance at 254 nm as described above. The NTD activity can be expressed in terms of transformation ratio (transformation ratio = molar concentration of deoxyadenosine produced/molar concentration of thymidine added). In a parallel group, the whole L. fermentum cells were incubated in 100 mM PBS-citrate buffer (pH 6.0) for 40 min with the supernatant completely removed before assays.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

Sequencing analysis

Lactobacillus fermentum CGMCC 1.2133 strain has high homology with L. fermentum IFO 3956, of which the genome has already been completely sequenced. To confirm whether any putative NTD had already been reported in this strain, we used NCBI blast Protein and found two putative N-deoxyribosyltransferase homologs in L. fermentum IFO 3956: LAF 0141 (NCBI gi|184154617), which encodes a 158-amino acid hypothetical protein, and LAF 0655 (NCBI gi|184155131), which encodes a 148-amino acid hypothetical protein. Their functions have not yet been revealed experimentally. Alignment of five amino acid sequences including LAF 0141, LAF 0655 and other reported NTDs (Fig. 1) showed that, in addition to the three critical catalytic sites for 2′-deoxyribosyl transfer activity (Armstrong et al., 1996; Anand et al., 2004; Miyamoto et al., 2007), the LAF 0141 gene encodes a substrate binding site that interacts with both purine and pyrimidine bases of 2′-deoxyribonucleosides (Miyamoto et al., 2007). This made LAF 0141 a perfect candidate as an NTD despite the fact that protein sequence identity between LAF 0141 and known NTDs (Kaminski et al., 2008) was only 34%.

image

Figure 1. Multiple sequence alignment of NTDs from Lactobacillus helveticus (AY064167), Lactobacillus leichmanni (Q9R5V5.3), L. fermentum (Q6YNI5.1), hypothetical protein LAF 0141 (YP_001842957.1), and LAF 0655 (YP_001843471.1). Identical amino acid residues are marked black and gray. Bullet indicate the catalytic sites of the three known enzymes. Arrow show the binding sites to the hydroxyl group binding of C5′ of 2′-deoxyribose. Two asterisks indicate the substrate binding sites: the first is only present in NTDs, rendering LAF 0141 a better candidate than LAF 0655 to test for NTD activity.

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Cloning and expression of NTD from L. fermentum

The LAF 0141 homolog from L. fermentum CGMCC 1.2133 was amplified using PCR, cloned, and overexpressed in E. coli BL21. The recombinant plasmid was sequenced and there were no differences at the nucleotide level between LAF 0141 and the homolog. To identify the function of the LAF 0141 homolog gene product, the recombinant protein was purified by a combination of two ion-exchange chromatography steps and further via a gel filtration column (Fig. 2a). Purified recombinant LAF 0141 homolog gene product migrated as an 18-kDa protein on 12.5% SDS-PAGE, which was identical with the theoretic molecular mass of 18.28 kDa (a total of 160 amino acids, with two additional amino acids present at the N-terminus). The concentration of the purified protein was 2.9 mg mL−1.

image

Figure 2. Purification and characterization of NTD. (a) SDS-PAGE of different purification steps of LAF 0141 homolog. Lane M, molecular weight standards; lane 1, total protein after Resource Q column; lane 2, total protein after Mono Q column; lane 3, 58 μg purified protein after gel filtration on a Superdex 75 column. Arrows on the right sides of the gels mark the positions of the purified protein. (b) HPLC chromatograms showing the enzymatic activity of NTD. Thymidine served as the deoxyribosyl donor and adenine the deoxyribosyl acceptor; deoxyadenosine and thymine are the products of the reaction (bottom). A control consisting of the standard reaction mixture, but without enzyme was performed (top).

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The N-deoxyribosyltransferase activity of the purified recombinant protein was determined by reactions between adenine and thymidine under standard conditions. The amount of deoxyribose transferred after 30 min in citrate buffer was 73.3%. The control reaction, which did not contain the enzyme, showed no conversion of the substrate to a product (Fig. 2b). As PTDs can only catalyze deoxyribosyl transfer to and from purines, and the nucleoside phosphorylases require inorganic phosphates for their enzyme reactions, the LAF 0141 homolog gene product should be classified as an NTD.

Subcellular localization of N-deoxyribosyltransferase

Subcellular localization of the NTD was determined using the polyclonal antibodies raised against recombinant NTD. The specificity of the purified antibodies was confirmed using whole cell extract of L. fermentum in Western blotting (Fig. 3a). The bacterial cells were separated into their different compartments, and NTD was detected both in the cytoplasmic fraction and the cell wall/plasma membrane fractions (Fig. 3b). Washing the debris with buffer could exclude possible contamination with cytoplasmic proteins. However, after two washes, NTD signal remains detectable in the washing supernatant indicating that the cell wall/plasma associated NTD might be washed off by the buffer.

image

Figure 3. Western blot analysis. (a) The specificity of the purified antibodies. Lane 1, SDS-PAGE of the whole cell extract; lane 2, Western blotting of the whole cell extract with anti-NTD. (b) Detection of NTD in the cytoplasmic fraction and the cell wall/plasma membrane fractions. Lane 1, Western blotting of the purified NTD with anti-NTD, positive control; lane 2, cytoplasmic fraction; lane 3, cell wall/plasma membrane fractions after one wash with washing buffer; lane 4, cell wall/plasma membrane fractions after two washes with washing buffer; lane 5, first washing buffer; lane 6, second washing buffer. Lane M, molecular weight standards.

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Immunogold labeling of NTD on ultrathin sections of lactobacilli cells was clearly visualized under the electron microscope, whereas background labeling was relatively low (Fig. 4). The electron-transparent granules can be inferred to be PHB (polyhydroxybutyrate) granules (data not shown). The positive signal, visualized as black dots, was localized within or in close proximity to the cell membrane. Unexpectedly, there were a number of gold particles spread over the surface of the cell wall (Fig. 4). According to PSORTb 3.0 analysis of the amino acid sequence of NTD, we found that NTD contains neither established cell wall-anchoring motifs nor signal sequences that could target it into secretory pathways. The immunofluorescence (Fig. 5a) and Western blotting results (Fig. 5b) support the surface association of N-deoxyribosyltransferase. This phenomenon is reminiscent of recent studies of the surface association of anchorless proteins in probiotics. These ‘anchorless’ proteins, including GroEL (Bergonzelli et al., 2006), EF-TU (Granato et al., 2004), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and enolase (Antikainen et al., 2007b), have been identified on the surface of lactobacilli. These housekeeping proteins do not possess any exporting motifs or surface-anchoring domains. The mechanism by which they cross the cytoplasmic membrane is still unknown. Enolase and GAPDH are essential intracellular glycolytic enzymes. However, the major function of surface GAPDH and enolase is the immobilization of human plasminogen onto the bacterial surface, subsequently enhancing its activation (Hurmalainen et al., 2007). In addition, enolase was found to bind to the extracellular matrix proteins, such as laminin and Collagen I (Antikainen et al., 2007a). They are considered to be anchorless multifunctional proteins or moonlighting proteins (Sanchez et al., 2008).

image

Figure 4. Immunogold staining of NTD on Lactobacillus fermentum thin sections. NTD was detected both in the cytoplasm and in contact with the cell membranes and cell surface by anti-NTD antibodies and 5-nm colloidal gold-labeled secondary antibody (a–f ). Sections processed without primary antibody did not label (g). The arrows indicate the location of NTD in the cytoplasm, and the arrowheads indicate NTD in contact with cell membranes or around the cell surface. The electron-transparent intracellular PHB granules were indicated by asterisks (a). Scale bar: 100 nm.

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image

Figure 5. Association of NTD with the cell wall of Lactobacillus fermentum. (a) Phase-contrast (A, C, E) and fluorescence (B, D, F) images are shown for indirect immunofluorescence of the cells. Lactobacilli cells were directly fixed (A, B, C, D) or fixed after incubation in Tris–HCl buffer, pH 8.0 (E, F), incubated with anti-NTD immunoglobulin and subsequently with DyLight 594 Goat Anti-Rabbit IgG. Control slide was incubated only with the labeled secondary antibody (C, D). The exposure time was the same (10 s) for all immunofluorescence photographs. (b) Western blotting analysis of the release of NTD from the cell surface. Lane 1, supernatant after incubation of the cells in 100 mM Tris buffer (pH 8.0); lane 2, culture supernatant after 20 h growth. (c) Release of NTD into buffer at pH values from 3.5 to 8.0. Cells from 80 mL of culture medium were incubated for 40 min in 100 mM PBS-citrate buffer at the indicated pH. The resulting supernatant was filtered through a 0.45-μm membrane filter. All the samples were precipitated with trichloroacetic acid and analyzed using Western blotting. Lane M, molecular weight standards.

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A few reports have shown that incubation in neutral or alkaline buffer can release enolase and GADPH from the surface of Lactobacilli, so that these extracellular proteins can be detected in the culture medium (Hurmalainen et al., 2007). Our results demonstrated that the NTD could also be released from the L. fermentum surface in Tris–HCl buffer at pH 8.0. Surface-exposed NTD was verified using indirect immunofluorescence (Fig. 5a), showing that the NTD was bound to the cell surface under normal culture conditions, whereas it was released after incubation in 100 mM Tris–HCl buffer at pH 8.0. This result was supported by Western blotting analysis of the supernatant (Fig. 5b). Microscopic examination of the cell suspension did not reveal any obvious cell lysis after 1 h of incubation, neither did we detect DNA in the cell-free supernatant (data not shown). Previous studies have also demonstrated that incubation would not result in the autolysis of Lactobacillus cells (Antikainen et al., 2007b; Hurmalainen et al., 2007). We have also detected NTD in the culture medium (pH value is 5.6 after 20 h culture) of L. fermentum (Fig. 5b). The release of NTD from the cell surface remained detectable after the incubation buffer was changed to 100 mM PBS-citrate buffer with pH values from 3.5 to 8.0 (Fig. 5c). This means that NTD could dissociate from the cell wall at pH values below or above its isoelectric point (the theoretic PI of NTD is 4.6), and indicates that the release of NTD is not just a response to the Tris–HCl buffer environment. This is not consistent with the ‘anchorless’ proteins thus far identified, including enolase and GAPDH, whose dissociation from the outer surface of Lactobacillus crispatus was favored when the pH was above the isoelectric point of these enzymes (Antikainen et al., 2007b). It has been reported that treatment with buffers normally used for cell washing (Tris–HCl or PBS) at pH 7.3 allowed the extraction of 12-fold higher protein concentrations compared with buffers adjusted to pH 4 (Sanchez et al., 2009), suggesting that most of the surface-associated proteins that interact with the cell envelop may depend on electrostatic interactions and thus are sensitive to pH. However, this does not apply to NTD. Further research is necessary to explore in detail the mechanism involved.

To determine whether the NTD activity also presents on the lactobacillus surface, enzymatic assay was carried out using whole lactobacillus cells. However, it is conceivable that lactobacillus cells may take up the highly concentrated substrates efficiently as other bacteria, as there have been many reports concerning nucleoside synthesis using bacteria whole cells (Fernandez-Lucas et al., 2007; Zheng et al., 2008), which implies that a set of membrane transportation system exists to facilitate the substrate import and product export. This uptake occurs very fast due to Nucleoside-specific membrane transporters in lactic acid bacteria (Kilstrup et al., 2005; Martinussen et al., 2010), namely, the conversion of nucleoside may be attributed to cytoplasmic enzyme when a whole cell assay was performed. Thus, considering that the incubation in conventional buffer will strip most of the NTD from the cell surface (Fig. 5a), whole cells after incubation in PBS-citrate buffer for 40 min were used in the same assay as a control. If surface-located NTD retain its biologic activity, washed cells were supposed to exhibit lower catalysis rate at the start point, at which time the activity of intracellular enzymes is yet limited by transportation kinetics. Data presented in Fig. 6 reveal a significantly reduced catalysis activity of washed whole cells after 1 min reaction. However, as the reaction time increases, the activity difference between washed cells and original cells gradually diminishes. This is consistent with our assumption that the uptake kinetics of nucleoside by lactobacillus is fairly fast. In a short period of reaction time, the conversion was mainly catalyzed by surface enzyme, as time elapsed, intracellular enzyme activity became dominant due to the function of membrane-located substrate and product transporters.

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Figure 6. NTD activity of whole Lactobacillus fermentum cells. The NTD activity was observed at 254 nm of the lactobacillus whole cells by determining the amount of deoxyribose transferred from thymidine to adenine in the buffer system as described in 'Materials and methods'. After washing with buffer, transformation ratio of the whole cells is lower at first, but progressively increased to a level similar to the non-washed cells. Each column represents the average of three parallel experiments. Error bars indicate standard deviation.

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From a physiologic point of view, the presence of NTD at the outside is puzzling, as the role of the enzyme is to balance the deoxynucleotide pools inside the cell (Kilstrup et al., 2005). Although NTD retains its deoxyribose transferase activity when localized on the cell surface, whether NTD do ‘moonlighting’, i.e. acquire another function when surface-associated (Jeffery, 2009), remains unclear. Current research is ongoing in our lab to determine its precise role on the surface of lactobacilli.

In conclusion, the data presented here show that NTD from L. fermentum can be added to a growing list of enzymes that one would expect to see only in the cytoplasm, but which have been detected on the cell surface (Granato et al., 2004). It is not known how these anchorless proteins cross the cytoplasmic membrane. They are thought to bind to the cell surface through non-covalent interactions and, thus, can be extracted by buffers or released into the culture medium. To our knowledge, we are the first to confirm experimentally the localization of an essential deoxynucleoside catabolic enzyme that has dual location both in the cytoplasm and on the surface in L. fermentum. The results reported here may serve as the basis for further work to characterize the surface-associated NTD, identify the specific roles of surface-associated NTDs in nucleoside metabolism or the extracellular environment, and also determine the surface-association mechanisms of the anchorless proteins.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

We express our gratitude to Professor Jan Martinussen (Center for Systems Microbiology, Department of Systems Biology, Technical University of Denmark) and the anonymous reviewers for their insightful suggestions, and to Professor Li Ying (Center of Biomedical Analysis, Tsinghua University) for her excellent technical assistance. This work was supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant No. J1030622), National Natural Science Foundation of China (Grant No. 20876088), and the National High Technology Research and Development Program of China (2010AA09Z405).

Statement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

The nucleotide sequence reported in this paper has been submitted to the GenBank with accession number JF331655.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. Statement
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml2369-sup-0001-FigureS1.docxWord document723KFig. S1. Micrographs of L. fermentum containing PHB inclusions.
fml2369-sup-0002-FigureS2.docxWord document72KFig. S2. DNA content of lactobacillus cells incubation supernatant measured using fluorescence spectroscopy.

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