Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1
© Rolain et al.; licensee BioMed Central Ltd. 2012
Received: 14 June 2012
Accepted: 3 October 2012
Published: 15 October 2012
Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators.
Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH) complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the disappearance of specific cleavage products.
In this study, we showed that two PGHs of L. plantarum have a predominant physiological role in a range of growth conditions. We demonstrate that the N-acetylglucosaminidase Acm2 is the major autolysin whereas the D,L-endopeptidase LytA is a key morphogenic determinant. In addition, both PGHs have a direct impact on PG structure by generating a higher diversity of cleavage products that could be of importance for interaction with the innate immune system.
KeywordsLactobacillus plantarum Peptidoglycan Autolysin Peptidoglycan hydrolase Glucosaminidase Muropeptidase
The cell wall is an essential structure for the survival of bacteria, as it determines cell shape, and also preserves cell integrity from internal osmotic pressure. In Gram-positive bacteria, peptidoglycan (PG) is a major compound of the cell wall. This polymer consists of the repeating disaccharide N-acetylmuramic acid-(β-1,4)-N-acetylglucosamine (MurNAc-GlcNAc) connected to a peptidic stem which is linked to MurNAc. The composition of the peptidic stem varies between bacteria and, in Lactobacillus plantarum, is composed of L-Ala, D-Glu, meso-diaminopimelic acid (mDAP), D-Ala and D-lactate as last moiety[2, 3]. Neighboring glycan strands are cross-linked between the fourth amino-acid of the donor stem and the third amino-acid of the acceptor peptide, forming a three-dimensional network around the cell, termed sacculus. Bacteria produce a variety of enzymes that are able to degrade PG. These enzymes are collectively called peptidoglycan hydrolases (PGH) or autolysins in the case they cleave PG glycan strands or cross-links of the producer strain resulting in the destruction of the PG mesh and cell lysis. These enzymes have been shown to play a major role in different processes such as daughter cell separation, cell-wall turnover, autolysis, sporulation and germination, biofilm formation, resuscitation of dormant cells, and allolysis in genetic transformation[4, 5]. Most of the PGHs display a modular organization composed of different domains usually associated with functions related to cell wall binding (e.g. LysM, SH3 domains) or PGH enzyme activity. PGHs are divided into several major families depending on the activity of their catalytic domain; N-acetyl-glucosaminidases, -muramidases, and lytic transglycosylases hydrolyze β-1, 4-bonds of PG glycan strands, whereas N-acetyl-muramoyl-L-alanine amidases cleave the amide bond between the lactic acid side chain of MurNAc and L-Ala of the stem peptide and carboxy- and endo-peptidases cleave the peptidic stem. In the last family, the D,D- and L,D-carboxypeptidases form a distinct group of PGHs since they do not destroy the PG mesh and are generally considered as PG maturation enzymes.
Lactobacilli, including Lactobacillus plantarum, are among the most predominant bacterial species of Firmicutes that are present in the mammalian intestinal tract. Due to their ability to confer a health benefit on their host, specific strains of L. plantarum are marketed as probiotics[8, 9]. It is also known that species of the genus Lactobacillus are able to interact with receptors of the immune system in the gastrointestinal tract through PGH-dependent processes such as autolysis and/or cell wall turnover that release muramyl-peptides or PGH fragments themselves[10–15]. In L. plantarum WCFS1, the analysis of its genome sequence has revealed the presence of 16 putative PGHs (carboxypeptidases are excluded), including 12 candidates displaying similarity with well-characterized PGHs, 3 hypothetical (lytic) transglycosylases containing a WY domain, and a pseudogene (acm3, three fragments)[12, 16–18]. Among the well-characterized PGH homologs, Acm2, a putative N-acetylglucosaminidase composed of a catalytic domain associated to five C-terminal SH3 domains and an N-terminal O-glycosylated region rich in Ala, Ser, and Thr (AST domain), was previously shown to be responsible for the separation of daughter cells[19, 20]. In addition, we previously demonstrated that the PGH activity of Acm2 and LytH (putative N-acetylmuramoyl-L-alanine amidase) could be modulated through O-acetylation of glycan chains of L. plantarum PG. MurNAc O-acetylation triggers LytH activity, whereas GlcNAc O-acetylation inhibits Acm2 activity.
Besides some recent studies performed in L. rhamnosus GG and L. casei BL23 that focused essentially on the characterization of specific major PGHs, information concerning the functional role of the complete arsenal of PGHs in lactobacilli is lacking[11, 21, 22]. In this study, we investigate the functional role of the 12 more probable candidates of the PGH complement of L. plantarum WCFS1 by a systematic gene deletion strategy. Characterization of mutant cell morphology, growth, and autolytic behavior, in a range of conditions showed that 4 PGHs (Acm2, Lys2, LytA, and LytH) play important roles either in the cell cycle or in the autolysis process of L. plantarum. Notably, we demonstrated that the putative N-acetylglucosaminidase Acm2 is the major autolysin of L. plantarum and that the putative γ-D-glutaminyl-meso-diaminopimelate muropeptidase LytA is a major morphogenic determinant in L. plantarum, thereby establishing a novel role for D,L-endopeptidases of the NLPC/P60 family.
Bacterial strains, plasmids, and growth conditions
Bacterial strains and plasmids used in this study
Strain or plasmid
Source or reference
NZ7100 lp_2645 (acm2)::lox72
NZ7100 lp_3093 (lys2)::lox72
NZ7100 lp_1138 (acm1)::lox72
NZ7100 lp_1158 (lys1)::lox72
NZ7100 lp_1982 (lytH)::lox72
NZ7100 lp_3421 (lytA)::lox66-P32-cat-lox71
NZ7100 lp_2162 (lytB)::lox72
NZ7100 lp_1242 (lytD)::lox72
NZ7100 lp_0302 (mltA)::lox72
TR0011 lp_2645 (acm2)::lox72
Cloning host; F- ϕ80 lacZ∆ M15 ∆(lacZYA-argF) U169 endA1 recA1 hsdR17 (r k - m k + ) phoA supE44 thi-1 gyrA96 relA1 λ
MG1363 derivative, pepN::nisRK
Cmr Emr; pACYC184 derivative containing the cat gene under the control of the P32 constitutive promoter of Lactococcus lactis (lox66-P32-cat-lox71 cassette)
Emr; Cre expression vector
Cmr; shuttle vector containing P nisA promoter and start codon in NcoI site
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_2645 (acm2)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_3093 (lys2)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_1138 (acm1)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_1158 (lys1)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_1982 (lytH)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_3421 (lytA)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_2162 (lytB)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_1242 (lytD)
Cmr Emr; pNZ5319 containing both upstream and downstream homology fragments from lp_0302 (mltA)
Cmr; pNZ8048 derivative containing acm2 (lp_2645) gene in transcriptional fusion
Cmr; pNZ8048 derivative containing lys2 (lp_3093) gene in transcriptional fusion
DNA techniques and electrotransformation
General molecular biology techniques were performed according to the instructions given by Sambrook et al.. Electrotransformation of E. coli was performed as described by Dower et al.. Electrocompetent L. plantarum and L. lactis cells were prepared as previously described. L. plantarum chromosomal DNA was isolated as reported before. PCR were performed with Phusion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland) in a GeneAmp PCR system 2400 (Applied Biosystems, Foster City, CA). The primers used in this study were purchased from Eurogentec (Seraing, Belgium) and are listed in (Additional file1: Table S1).
Construction of deletion mutants
Construction of the deletion mutants of the L. plantarum PGH-encoding genes was performed as previously described[2, 29]. Briefly, a double cross-over gene replacement strategy was used to replace the target gene(s) by a chloramphenicol resistance cassette (lox66-P32-cat lox71). Subsequently, the lox66-P32-cat lox71 cassette was excised by temporal expression of the Cre recombinase using an unstable cre expression plasmid. This strategy was applied for the construction of strains TR0010 (Acm2-), TR0011 (Lys2-), TR0012 (Acm1-), TR0013 (Lys1-), TR0014 (LytH-), TR006 (LytA-), TR0015 (LytB-), TR0016 (LytD-) and TR0017 (MltA-) (Table1). In order to obtain the double mutant strain TR0018 (Acm2- Lys2-), strain TR0011 (Lys2-) was used for a next round of mutagenesis targeting for the deletion of acm2 following the same procedures as described above. Primers used to construct the deletion vectors and to validate the deletions events are listed in (Additional file1: Table S1).
Construction of the complementation vectors
The acm2 and lys2 ORFs were amplified by PCR using genomic DNA of L. plantarum NZ7100 and the primer pairs Acm2_NcoI/Acm2_XbaI or Lys2_NcoI/Lys2_XbaI, respectively (Additional file1: Table S1). The resulting amplicons were digested with NcoI and XbaI and cloned into similarly digested pNZ8048, yielding the expression plasmids pGITR0010 and pGITR0011, respectively. These plasmids contain either acm2 or lys2 under the control of P nisA that allows their induction in the presence of nisin. The integrity of the two plasmids was verified by DNA sequencing. These two expression vectors were electrotransformed into L. plantarum TR0010 for complementation studies. Primers used to construct the complementation vectors are listed in (Additional file1: Table S1).
Purification and structural analysis of peptidoglycan
PG from L. plantarum strains was prepared as previously described[2, 31]. PG was digested with mutanolysin and the resulting muropeptides were analyzed by reverse phase-high-pressure liquid chromatography (RP-HPLC) and MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization - Time Of Flight) mass spectrometry as previously reported.
Triton X-100-induced autolysis assays in buffer solution
L. plantarum strains were grown to mid-exponential phase (OD600=0.8). Cells were harvested by centrifugation at 5000 × g for 10 min at 4°C, washed once with 50 mM potassium phosphate buffer pH 7.0, and resuspended at an OD600 of 1.0 in 50 mM potassium phosphate buffer pH 7.0 supplemented with 0.05% Triton X-100[2, 33]. Cell suspensions were then transferred into 96-well sterile microplates with a transparent bottom (Greiner, Alphen a/d Rjin, the Netherlands) and incubated at 30°C. Autolysis was monitored by measuring the OD600 of the cell suspensions every 20 minutes with a Varioskan Flash multimode reader (Thermo Scientific). The extent of autolysis was expressed as the relative decrease in OD600 (given in percentages relative to the initial OD600).
SDS-PAGE and zymogram
The cell wall hydrolyzing activity was investigated by zymogram analysis. SDS-PAGE was performed with 8% (w/v) polyacrylamide separating gels. Renaturing SDS-PAGE was performed as previously described[2, 34]. The polyacrylamide gels contained L. plantarum NZ7100 autoclaved cells resuspended at OD600 of 0.8 as enzyme substrates. Disrupted cells used as samples were prepared as described before. Disrupted cells were boiled in denaturing sample buffer and centrifuged 1 min at 20.000 × g prior to loading. After sample migration, gels were washed for 30 min in deionized H2O and incubated overnight at room temperature in 50 mM Tris–HCl, pH 6.8, 1 mM DTT, containing 0.1% (v/v) Triton X-100. Subsequently, the gels were washed in deionized H2O, followed by staining with 0.1% Methylene Blue in 0.01% (w/v) KOH, and destained in deionized H2O.
Fluorescence microscopy and LIVE/DEAD staining
Microscopy analyses were performed using an Axio observer Z1 inverted microscope (Carl Zeiss). FM4-64 (Molecular Probes, Leiden, The Netherlands) and DAPI (4, 6-diamidino-2-phenylindole) (Sigma, Bornem, Belgium) staining were performed as previously reported. Bacterial membrane integrity was assessed by fluorescence microscopy using a LIVE/DEAD reduced biohazard viability/cytotoxicity kit (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. Analyses of micrographies were performed using the AxioVision 4.8. software (Carl Zeiss).
Transmission and scanning electron microscopy
L. plantarum cells were grown overnight at 28°C in MRS broth. Bacterial pellets were washed once in PBS and fixed overnight in a phosphate buffer (0.1 M and pH 7.4) containing 2.5% glutaraldehyde for transmission electron microscopy or 4% paraformaldehyde for scanning electron microscopy. After fixation, cells were washed, postfixed with 1% osmium tetroxide for 1 h, washed again and subjected to serial dehydration with ethanol. Samples for transmission electron microscopy were embedded in resin, thin-sectioned and stained with uranyl acetate and Reynold’s lead citrate. Samples for scanning electron microscopy were prepared by critical-point drying, mounted on an aluminum stub and covered with a thin layer of gold (20–30 nm). Finally, the samples were examined using a Transmission Electron Microscope (TEM) (LEO922; Zeiss) at the Institute of Condensed Matter and Nanosciences (Université catholique de Louvain, Belgium) or a Scanning Electron Microscope (SEM) (XL-20; Philips, Eindhoven, the Netherlands) at the Unité Interfacultaire de Microscopie Electronique (Facultés Universitaires Notre-Dame de la Paix, Belgium).
Nine of the twelve predicted peptidoglycan hydrolases could be inactivated in L. plantarum WCFS1
In order to investigate the functional role of each PGH, we aimed to construct a library of L. plantarum PGH mutant strains using the double cross-over gene replacement strategy based on the Cre-loxP system. During the first step of this strategy, we attempted to inactivate the targeted open reading frames through replacement by a chloramphenicol resistance cassette (lox66-P32-cat lox71). Using this approach, we obtained mutants for 9 of the 12 genes that were initially targeted (Figure1). Despite several attempts in which over 300 integrants were tested for their genotype, we were not able to obtain double cross over events for lytC, mltB, and mltC, suggesting that they might be essential for bacterial survival. Subsequently, the lox66-P32-cat lox71 cassette was successfully excised from 8 of the 9 mutants (all except the lytA that could no longer be transformed by electroporation) by temporal expression of the Cre recombinase.
Since in our tested conditions, four PGH-deficient strains (Acm2-, Lys2-, LytA-, and LytH-) were later shown to display phenotypic differences as compared to the wild type (see below), construction of complementation vectors for each of these PGH was attempted. The cloning of acm2 and lys2 genes under the transcriptional control of the nisin-inducible promoter[24, 30] was achieved and corresponding complemented strains were constructed. Despite numerous attempts, complementation of LytA- and LytH- mutant strains were not obtained since the cloning of lytA and lytH genes in various hosts (E. coli, L. lactis, and L. plantarum) was unsuccessful. We only obtained truncated gene copies of lytA, while for lytH, when full-length gene cloning was successful, all candidates contain point mutations resulting in inactive proteins (data not shown).
Acm2 and LytA are involved in cell separation and cell division
These morphological observations demonstrate that Acm2 and LytA are key actors of the cell cycle within the PGH complement of L. plantarum, since they play an important role during late cell separation and division process, respectively.
LytA inactivation has a strong negative impact on growth and viability
These results underpinned the prominent role of LytA in L. plantarum cell integrity that is apparent in a range of growth conditions, but also revealed that LytH is important under specific conditions (CDM).
Acm2 is the major autolysin of L. plantarum
Taken together, these data show that Acm2 is the dominant autolysin of L. plantarum but also that Lys2, LytH and LytA can contribute to autolysis under specific growth conditions. These results are in agreement with DNA microarray expression profiles that have shown that growth conditions and more particularly mild stress conditions can modulate the expression of PGH-encoding genes.
Lys2 and Acm2 are redundant peptidoglycan hydrolases involved in cell separation
Analysis of the peptidoglycan composition of Acm2 and LytA-deficient strains reveals their hydrolytic activity
These results pinpoint that the presence of Acm2 and LytA have a direct impact on PG structure with a more pronounced effect for LytA, which generated a higher diversity of cleavage products. These data also suggest that Acm2 cleaves between GlcNAc and MurNAc as expected from its predicted N-acetylglucosaminidase activity and its inhibition by O-acetylation of GlcNAc. Concerning LytA, our data indicate that it cleaves PG between D-Glu and mDAP, suggesting that LytA displays a γ-D-Glu-mDAP muropeptidase activity.
The PGH complement of L. plantarum WCFS1 contains at least 12 putative PGHs that were all predicted to be secreted. Moreover, most of them are anticipated to display a modular organization (8 PGHs), composed of catalytic domains linked to PG binding domains (SH3 or LysM). Furthermore, we highlighted that more than half of the PGHs of L. plantarum harbored a domain rich in Ala, Ser and Thr (named AST) which was recently shown to be glycosylated. The predominance of this domain among L. plantarum PGHs suggests that it may play a general role in the control of activity and/or stability of these enzymes, as was recently also hypothesized for a glycosylated PGH of L. rhamnosus GG. However, the functional role of the AST domain and of its glycosylated state in L. plantarum regarding to PGH activity/stability but also for spatial PGH localization remains to be investigated. In addition, it was recently shown that a similar domain rich in Ser and Thr (STp) from an extracellular protein of L. plantarum was resistant to intestinal proteolysis and was able to modulate in vitro cytokine production from intestinal dendritic cells. This reveals that such domain could be of importance in the dialogue between intestinal bacteria and the immune system.
In order to elucidate the physiological function(s) of L. plantarum PGHs, we underwent a systematic gene deletion strategy to obtain stable, marker-free mutants for each of them. As a result, 9 PGHs were identified as permissive for inactivation, whereas 3 PGHs remained refractory to deletion despite various attempts, suggesting that they may play an essential role in L. plantarum. Among these three PGHs, two belongs to the lytic transglycosylase family whose functional role is poorly understood in Gram-positive bacteria. We next investigated the role of the 9 PGHs with respect to cell morphology, growth, and autolysis in various conditions and identified four PGHs, namely Acm2, LytA, Lys2, and LytH, that could be associated to one or more phenotypic defect(s). Among these, Acm2 and LytA caught our attention since their deficiency resulted in the most severe morphological phenotypes. Acm2, is probably a N-acetylglucosaminidase, based on the analysis of the PG structure of its respective mutant strain. Preliminary results of PG degradation experiments with purified Acm2 support the proposed N-acetylglucosaminidase activity (T. Rolain, unpublished data). Phase-contrast, TEM and SEM microscopy show that Acm2 is strictly involved in the last step of cell separation during the septation process. This result is consistent with previous observations of an acm2 single cross-over mutant and the functional role of AcmA, the major N-acetylglucosaminidase of Lactococcus lactis, which shares 34% of identity with Acm2 and is also dedicated to cell separation in this species[20, 39]. For the second enzyme, renamed LytA, we provided strong evidence for its classification as a γ-D-Glu-mDAP muropeptidase based on the PG analysis of the mutant strain. The proposed LytA endopeptidase activity is consistent with the fact that all the previous known members of the NlpC/P60 family were characterized as γ-D-Glu-diaminoacid endopeptidases[11, 22, 40–42]. Unfortunately, all attempts to clone the corresponding gene or to purify its product in order to definitively prove its enzymatic activity remain so far unsuccessful. The absence of LytA results in a strongly hampered growth, loss of viability, and severe structural defects of the cell wall. However, we cannot exclude that polar effects on flanking genes might contribute to the observed phenotype, since we were unable to complement this mutant. Notably, the lytA gene is predicted to be monocistronic and flanked by genes which are completely unrelated to morphogenesis or cell wall assembly. Interestingly, suppressor mutants were obtained at low frequency without any genetic reorganization of the disrupted locus but displaying a wild-type cell morphology (data not shown), that may suggest that a gene encoding another D,L-endopeptidase of the NlpC/P60 family is activated to counteract the effects of lytA inactivation. Furthermore, numerous attempts to construct a double LytA/LytB-deficient strain remained so far unsuccessful, suggesting that both enzymes may play redundant but essential roles in L. plantarum. To our knowledge, this is the first time that the inactivation of a PGH containing an NlpC/P60 catalytic domain displays such a severe phenotype while the inactivation of members of this family, including members recently characterized in lactobacilli, leads to a cell-chaining phenotype similar to Acm2 inactivation[6, 11, 22]. Interestingly, deficiencies in the closely related PG endopeptidases belonging to the c ysteine, h istidine-dependent a mido-hydrolases/p eptidase (CHAP) family, such as PcsB from Streptococcus pneumoniae and LytN from Staphylococcus aureus, also strongly affect growth, cell morphology, and cell wall structure[43, 44]. Our data suggest that NlpC/P60 endopeptidases can also be implicated in other physiological roles than daughter cell separation and probably play an important role during the assembly of the cell wall by cooperating with the PG synthesis machinery during the division process, as recently shown for the CHAP endopeptidase PcsB from S. pneumoniae. Based on a recent transcriptome profiling study showing that the expression of a large set of PGH-encoding genes from L. plantarum are modulated by mild stress conditions, such as high salt concentration and elevated growth temperature, the contribution of each PGH to growth and autolysis was examined in different growth media as well as under stress conditions. These experiments revealed that Acm2 was responsible for the major part of the autolytic activity in all growth conditions tested and should be considered as the major autolysin of L. plantarum WCFS1. In addition, these experiments also showed that Lys2, LytH, and LytA contribute to the autolytic process but to a lesser extent than observed for Acm2 and only under specific growth conditions. We also show that the autolysis of Acm2- and LytA-deficient strains is increased under mild stress conditions showing that stress alters the composition of the PGH complement in L. plantarum, corroborating previously obtained transcriptome data. We hypothesized that Acm2 and Lys2, as well as LytA and other members of the NlpC/P60 family, displayed functional redundancy in specific growth conditions. This was further investigated for the potential redundancy between the N-acetylglucosaminidase Acm2 and the putative N-acetylmuramidase Lys2 where we show that Lys2 could fulfill the functional role of Acm2 regarding to autolysis and cell separation of daughter cells.
In conclusion, we show that the N-acetylglucosaminidase Acm2 and the γ-D-Glu-mDAP muropeptidase LytA are pivotal in the physiology of L. plantarum. Acm2 is the major autolysin under all conditions tested and is functionally involved in the final step of cell separation during division while LytA is playing a major morphogenic role since its absence not only resulted in delayed growth and loss of viability but also led to severe structural defects of the cell wall. For future applications, the identification of Acm2, in association with Lys2 under stress conditions, as key players in autolysis resistance offers the possibility to develop more robust starter strains or probiotics. In addition, the capacity to modulate L. plantarum PG composition by PGH inactivation could change its immunomodulatory properties by impacting on the repertoire of released muramyl peptides interacting with receptors of the innate immune system.
Domain rich in Ala, Ser, and Thr
Chemically define medium
Cysteine, histidine-dependent amido-hydrolases/peptidase
stress chemically define medium
SDS polyacrylamide gel electrophoresis
Scanning electron microscopy
Transmission electron microscopy
Work in the team of PH was supported by the National Foundation for Scientific Research (FNRS), the Université catholique de Louvain (Fonds Spéciaux de Recherche), and the Research Department of the Communauté française de Belgique (Concerted Research Action). Work in the team of MPCC was supported by INRA (Jeune Equipe grant). EB was the recipient of a Marie Curie fellowship for Early Stage Research Training (EST) of the FP6 LabHealth project (MEST-CT-2004-514428). TR held a doctoral fellowship from FRIA. PH is Research Associate of the FNRS. PAB is partially employed within the research programme of the Kluyver Centre for Genomics of Industrial Fermentation which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientific Research. We warmly thank Bernard Hallet for fruitful discussions, Wendy Glenisson for critically reading the manuscript, and Xavier Debolle for giving us access to the electron microscopy platform at FUNDP. We thank Pascale Lipnik and Christian Didembourg for helping us with electron microscopy.
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