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. Author manuscript; available in PMC: 2015 Apr 13.
Published in final edited form as: Microbiology (Reading). 2014 May 24;160(0 8):1807–1819. doi: 10.1099/mic.0.078162-0

Biochemical characterization of the major N-acetylmuramidase from Lactobacillus buchneri

Julia Anzengruber 1, Pascal Courtin 2,3, Ingmar J J Claes 4,†, Monika Debreczeny 5, Stefan Hofbauer 6, Christian Obinger 6, Marie-Pierre Chapot-Chartier 2,3, Jos Vanderleyden 4, Paul Messner 1, Christina Schäffer 1
PMCID: PMC4395873  EMSID: EMS62959  PMID: 24858286

Abstract

Bacterial cell wall hydrolases are essential for peptidoglycan remodelling in regard to bacterial cell growth and division. In this study, peptidoglycan hydrolases (PGHs) of different Lactobacillus buchneri strains were investigated. First, the genome sequence of L. buchneri CD034 and L. buchneri NRRL B-30929 was analysed in silico for the presence of PGHs. Of 23 putative PGHs with different predicted hydrolytic specificities, the glycosyl hydrolase family 25 domain-containing homologues LbGH25B and LbGH25N from L. buchneri CD034 and NRRL B-30929, respectively, were selected and characterized in detail. Zymogram analysis confirmed hydrolysing activity on bacterial cell walls for both enzymes. Subsequent reversed-phase HPLC and MALDI-TOF MS analysis of the peptidoglycan breakdown products from L. buchneri strains CD034 and NRRL B-30929, and from Lactobacillus rhamnosus GG, which served as a reference, revealed that LbGH25B and LbGH25N have N-acetylmuramidase activity. Both enzymes were identified as cell wall-associated proteins by means of immunofluorescence microscopy and cellular fractionation, as well as by the ability of purified recombinant LbGH25B and LbGH25N to bind to L. buchneri cell walls in vitro. Moreover, similar secondary structures mainly composed of β-sheets and nearly identical thermal stabilities with Tm values around 49 6C were found for the two N-acetylmuramidases by far-UV circular dichroism spectroscopy. The functional and structural data obtained are discussed and compared to related PGHs. In this study, a major N-acetylmuramidase from L. buchneri was characterized in detail for the first time.

INTRODUCTION

Bacterial peptidoglycan (PG) comprises a glycan backbone containing alternating β(1,4)-linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues that are cross-linked by peptide chains of varying composition (Schleifer & Kandler, 1972; Vollmer et al., 2008a).

Multilayered PG is the major cell wall component in Gram-positive bacteria; it is responsible for maintaining cell shape and morphology and confers resistance to internal turgor pressure (Delcour et al., 1999).

The bacterial cell wall is continuously expanded and renewed (Vollmer et al., 2008b). Bacterial PG hydrolases (PGHs) are produced by bacteria throughout their life cycle, and target either the PG sugar backbone or the peptide chains; they are involved in the regulation of cell wall growth, recycling and maturation of PG, and the separation of daughter cells (Vollmer et al., 2008b). According to their cleavage specificities, PGHs can be divided into four distinct classes: (i) N-acetylmuramidases, (ii) N-acetylglucosaminidases, (iii) N-acetylmuramoyl-l-alanine amidases and (iv) peptidases (including carboxypeptidases and endopeptidases) (Layec et al., 2008). Additional lytic transglycosylases may break the same bonds as muramidases, but generate anhydromuropeptides (Scheurwater et al., 2008).

Lactobacilli are major components of both foods and the human and animal microbiota. Given their GRAS (generally regarded as safe) status, they are intensively exploited in applied research. Only a few lactobacillar PGHs have been investigated in detail. For the endopeptidases Msp1 from Lactobacillus rhamnosus GG (Claes et al., 2012) and Lc-p75 from Lactobacillus casei BL23 (Regulski et al., 2012) as well as for the glucosaminidase Acm2 from Lactobacillus plantarum WCFS1 (Rolain et al., 2012), hydrolytic specificities have been experimentally proven; PGHs from Lactobacillus buchneri have not been studied up to now. L. buchneri is a facultative anaerobe that is of interest because of its property of efficient preservation of animal feed silage against aerobic spoilage. The obligatory heterofermentative nature and acid resistance makes this bacterial species well suited for application as a silage starter culture (Oude Elferink et al., 2001; Danner et al., 2003; Holzer et al., 2003; Eikmeyer et al., 2013). In the present study, the PGHs of L. buchneri CD034 (Heinl et al., 2012), isolated from grass silage, and L. buchneri NRRL B-30929 (Liu et al., 2011), isolated from an ethanol production plant, have been studied. L. buchneri possesses a typical Gram-positive cell wall profile with a thick PG of the A4α chemotype with d-Ala4→(d-Asp/d-Asn)→l-Lys3 interpeptide bridges (Schleifer & Kandler, 1972). As the outermost cell envelope decoration, L. buchneri possesses a self-assembling, 2D crystalline surface (S-) layer with oblique lattice symmetry (Möschl et al., 1993; Anzengruber et al., 2014). The L. buchneri S-layer protein of both strains is O-glycosylated at four serine residues within the motif Ser–Ala–Ser–Ser–Ala–Ser with, on average, seven Glc(α1-6) residues each (Möschl et al., 1993; Anzengruber et al., 2014).

Motif search within the L. buchneri CD043 and NRRL B-30929 genomes revealed the presence of one extra protein each containing the above glycosylation motif. These are the glycosyl hydrolase family 25 (GH25) domain-containing proteins LbGH25B (LBUCD034_0240) and LbGH25N (Lbuc_0200) from L. buchneri CD034 and L. buchneri NRRL B-30929, respectively, for which a database sequence similarity search predicted PG-hydrolysing capacity. According to MS evidence, indications for glucosylation of the two predicted PGHs were found (Anzengruber et al., 2014). Similar to the S-layer glycoproteins SlpB and SlpN from L. buchneri CD034 and L. buchneri NRRL B-30929, respectively, the highly homologous enzymes LbGH25B and LbGH25N have a basic pI of 9.5.

In this study, we identified in silico putative PGHs of L. buchneri CD034 and their homologues in L. buchneri NRRL B-30929, and characterized in detail the GH25 domain-containing proteins LbGH25B and LbGH25N. This includes (i) biochemical analysis of cell wall hydrolysing activity and specificity, (ii) determination of the subcellular localization of the enzymes, including an in vitro binding study to lactobacillar cell walls, and (iii) experimental insight into structural properties and thermal stability of the PGHs.

METHODS

Bacterial strains and culture conditions

L. buchneri CD034 (Heinl et al., 2012), L. buchneri NRRL B-30929 (Liu et al., 2011), L. rhamnosus GG (Claes et al., 2012) and L. plantarum CD033 (Spath et al., 2012) were grown in De Man–Rogosa–Sharpe (MRS) broth (BD Difco) (De Man et al., 1960) at 37 °C without shaking. Escherichia coli DH5α cells (Life Technologies) and E. coli BL21 Star BL21(DE3) cells (Life Technologies) were cultivated at 37 °C and 200 r.p.m. in Luria–Bertani (LB) medium supplemented with 50 μg kanamycin ml−1.

Construction of expression plasmids

The coding sequences for LbGH25B (LBUCD034_0240) and LbGH25N (Lbuc_0200) devoid of the N-terminal signal peptide sequences (amino acids 1–29 of the pre-proteins) were PCR-amplified with primers (Life Technologies) LbGH25B/N_F (5′-AATCATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGCTTTGACGCCGTCCAGTACC-3′, XbaI site underlined) and LbGH25B/N_His_stop_R (5′-AATCAAAGCTTTTAATGATGATGATGATGATGATTCAAATAACCGCGCCAAATCC-3′, HindIII site underlined). Similarly, the coding sequences for the N-terminal GH25 domain-containing region (amino acids 30–235) and the C-terminal region (amino acids 236–380) of LbGH25B were PCR-amplified with primers LbGH25B_F and GH25_His_stop_R (5′-AATCAAAGCTTTTAATGATGATGATGATGATGGTAGAAGCTGTTGTTTAATTG-3′, HindIII site underlined), and with C_termius_ LbGH25B_F (5′-AATCATCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGTTGAACTCCGGTTCAGCGGC-3′, XbaI site underlined) and LbGH25B_His_stop_R, respectively. PCR products were digested with XbaI and HindIII and ligated into pET28a+ expression vector (Novagen). For expression of the S-layer protein SlpB, plasmid pET28a-SlpB_His6 was constructed as described by Anzengruber et al. (2014). The resulting recombinant plasmids were propagated in E. coli BL21 Star BL21(DE3) for production of hexahistidine-tagged proteins.

Production and purification of recombinant proteins

Transformed strains were grown in 400 ml LB broth supplemented with kanamycin (50 μg ml−1) at 37 °C and 200 r.p.m. At the mid-exponential growth phase (OD600 of ~0.6), protein expression was induced with 1 mM IPTG and cultivation was continued for 4 h. Cells were pelleted (10 000 g, 20 min), resuspended in lysis buffer (50 mM sodium citrate buffer, pH 6.2, 0.1 % Triton X-100) and, after addition of lysozyme (800 μg ml−1; Sigma-Aldrich) and benzonase (50 U ml−1; Sigma-Aldrich), incubated for 30 min at 37 °C. Bacteria were lysed by ultrasonication (Branson sonifier, duty cycle 50 %; output 6) applying 10 cycles of 10 pulses with 30 s breaks, each, and inclusion bodies containing the recombinant proteins were pelleted. The proteins were extracted from the pellets with binding buffer [50 mM sodium citrate buffer, pH 5.5, 5 M guanidinium hydrochloride (GdHCl), 20 mM imidazol, 0.5 M NaCl] for 1 h at 4 °C. The extracts were subjected to centrifugation and membrane-filtration (0.45 μm pore size). The resulting protein samples were applied to a 1 ml HisTrap HP column (GE Healthcare) and recombinant proteins were recovered with elution buffer (50 mM sodium citrate buffer, pH 5.5, 5 M GdHCl, 1 M imidazol, 0.5 M NaCl). Recombinant His-tagged proteins were dialysed against 50 mM sodium citrate buffer, pH 5.5.

SDS-PAGE

SDS-PAGE was carried out on 10, 12 or 20 % slab gels in a Mini Protean electrophoresis apparatus (Bio-Rad) (Laemmli, 1970). Protein bands were visualized with colloidal Coomassie brilliant blue R-250 (CBB) and gels were imaged at 700 nm using the Odyssey imaging system (LI-COR).

Western immunoblotting

Polyclonal mouse antiserum was raised against purified recombinant LbGH25N (EF-BIO). Due to high amino acid homology (99 %) between LbGH25N and LbGH25B, the antiserum reacted with both proteins.

Proteins were transferred from the SDS-PAGE gel to a PVDF membrane (Bio-Rad) using a Mini Trans-Blot Cell (Bio-Rad). Muramidase-specific antiserum was used in combination with IR Dye 800CW goat anti-mouse antibody (LI-COR), and detection was performed at 800 nm using the Odyssey Infrared Imaging System.

Electronic circular dichroism (ECD) spectroscopy

Far-UV ECD spectra and temperature-mediated unfolding were recorded by ECD spectroscopy (Chirascan; Applied Photophysics). Prior to measurements the instrument was flushed with nitrogen at a flow rate of 5 l min−1. An integrated Peltier element was used for temperature control. Far-UV ECD spectra using 1.9 μM LbGH25B or LbGH25N in 12.5 mM sodium citrate buffer, pH 5.5, were recorded between 195 and 260 nm at 25 °C. The pathlength was 1 mm, spectral bandwidth was 1 nm and scan time per point was set to 10 s. Five scans were recorded for each protein and mean values were calculated. Secondary structure prediction was computed via the PSIPRED protein structure prediction server. Thermal unfolding was monitored at 222 nm between 20 and 95 °C with stepwise increments of 1.0 °C min−1 using 7.5 μM LbGH25B or LbGH25N in 50 mM sodium citrate buffer, pH 5.5. The pathlength was 1 mm, spectral bandwidth was 1 nm and scan time per point was set to 10 s. The fraction α of unfolded protein was calculated according to α=(θN − θ)/(θN − θU), with θN being the ellipticity (10−3 degrees) of the protein in the native folded state at 222 nm, θ the ellipticity at a defined temperature (T) and θU being the ellipticity of the completely unfolded state at 222 nm. Experiments were performed in duplicate and mean values and sd of the melting temperature (Tm) and the van’t Hoff enthalpy (ΔHm) were calculated.

Zymogram analysis

Cell wall hydrolysing activity of the PGHs was investigated by zymogram analysis (Lepeuple et al., 1998). As putative substrates, cell walls of L. buchneri CD034, L. buchneri NRRL B-30929 and E. coli BL21 Star BL21(DE3) as well as whole cells from L. rhamnosus GG were used.

L. buchneri CD034 cell walls were isolated and purified according to published protocols (Schäffer et al., 1999, 2000) with slight modifications. The high-molecular mass, polysaccharide-containing material obtained after lysozyme digestion and chromatography on Sephadex G-50 (2.5 × 120 cm, GE Healthcare) was further fractionated on a Sephacryl S-1000 column (XK16/40, GE Healthcare) at a flow rate of 1 ml min−1 with 50 mM NH4HCO3 as eluent. Carbohydrate-containing fractions were pooled and lyophilized.

L. buchneri NRRL B-30929 and E. coli BL21 Star BL21(DE3) cells were pelleted (10 000 g, 20 min), washed with 50 mM Tris/HCl, pH 7.2 (buffer A), and, subsequently, lysed by ultrasonication (duty cycle 50 %; output 8) applying six cycles of 10 pulses, with 30 s breaks, each. Cell wall fragments were sedimented (30 000 g, 15 min) and resuspended in buffer A, containing 1 % (v/v) Triton X-100. After three washing steps with buffer A, the cell walls were incubated in buffer A containing 5 M GdHCl at 4 °C for 30 min with shaking to remove the S-layer. The remaining pellet was washed twice with buffer A, boiled for 30 min in 1 % (w/v) SDS, and washed five times with MilliQ H2O to remove SDS. Cell walls were further treated with 10 % (w/v) TCA at 4 °C for 24 h to remove exopolysaccharides and teichoic acids, washed twice with 0.25 M Tris/HCl, pH 8.0, and washed four times with MilliQ H2O. The final pellet was kept at 4 °C. L. rhamnosus GG cells were autoclaved and treated with 10 % (w/v) TCA as described above.

Cells and cell wall preparations were co-polymerized at a concentration of 0.4 % (w/v) with SDS polyacrylamide gels to serve as enzyme substrates and zymogram analysis was performed as described by Claes et al. (2012). Bacterial cells were resuspended in zymogram sample buffer (62.5 mM Tris/HCl, pH 6.8; 2 % SDS; 0.01 % Bromophenol blue; 10 % glycerol), insoluble material was removed by centrifugation and soluble proteins were applied to zymogram gels. Recombinant LbGH25B and LbGH25N were also applied in purified form, as described above.

Determination of the hydrolytic specificity of LbGH25B and LbGH25N on PG

PG from L. rhamnosus GG, L. buchneri CD034 and L. buchneri NRRL B-30929 was prepared as described elsewhere (Claes et al., 2012). Bacterial cells were incubated in 50 mM Tris/HCl, pH 7.2, containing 5 M GdHCl for 30 min at 20 °C with shaking to remove the S-layer. Nucleic acids were removed by treatment with benzonase (50 U ml−1) in 20 mM Tris/HCl, pH 7.0, containing 1 mM MgCl2, at 37 °C for 18 h. PG was then treated with 10 % TCA (w/v) at 4 °C for 24 h with shaking to remove exopolysaccharides and teichoic acids, washed twice with 50 mM Tris/HCl, pH 8.0, and washed twice with MilliQ H2O. Subsequently, the material was incubated with 48 % (v/v) hydrofluoric acid at 4 °C to further eliminate residual exopolysaccharides and teichoic acids and washed as described above. The final pellet was freeze-dried and stored at −20 °C until further analysis.

The hydrolytic specificity of the enzymes was determined using PG from L. buchneri CD034, L. buchneri NRRL B-30929 and L. rhamnosus GG, respectively, as substrate. Purified PG (1 mg dry weight) was digested with either mutanolysin from Streptomyces globisporus (2500 U ml−1, Sigma-Aldrich) and/or purified recombinant His6-tagged LbGH25B (34 μg), LbGH25N (41 μg) or SlpB (14 μg) in 50 mM sodium citrate buffer, pH 5.5, containing 0.05 % (w/v) sodium azide, for 24 h at 37 °C with shaking. The resulting soluble muropeptides were reduced with sodium borohydride (Atrih et al., 1999). Degradation products were separated by (reversed-phase) HPLC with an Agilent Infinity 1290 ultraHPLC system on a Nucleodur C18 Pyramid column (150 × 2 mm; particle size, 1.8 μm; Macherey-Nagel) at 50 °C using a methanol gradient in ammonium phosphate buffer (Courtin et al., 2006). Muropeptides were analysed by MALDI-TOF MS using a Voyager-DE STR mass spectrometer (Applied Biosystems) as reported previously (Courtin et al., 2006).

Subcellular fractionation of L. buchneri strains

Supernatant

L. buchneri CD034 and NRRL B-30929 were grown overnight in MRS broth at 37 °C without shaking. After centrifugation (10 000 g, 20 min), proteins were precipitated from the supernatant by incubation with 20 % (w/v) TCA at 4 °C for 30 min, sedimented proteins (9000 g, 20 min) were washed twice with ice-cold 100 % acetone and the final pellet was air-dried.

Cell wall

The cell pellet was washed with buffer A and, subsequently, lysed by ultrasonication (duty cycle 50 %; output 8) by applying six cycles of 10 pulses, with 30 s breaks, each. Cell wall fragments were precipitated at 30 000 g for 15 min, and the resulting pellet was resuspended in buffer A, containing 1 % (v/v) Triton X-100, and washed four times with buffer A.

Cytosol

Cytosolic proteins were obtained from the supernatant of the centrifugation step directly after ultrasonication during the cell wall isolation described above. Proteins were precipitated with TCA, washed twice with ice-cold acetone and air-dried.

The resulting pellets were resuspended in Laemmli buffer at a concentration of 0.1 mg ml−1 (Laemmli, 1970) and run on SDS-PAGE gels.

In vitro protein–cell wall interaction studies

LbGH25B and LbGH25N were tested for their ability to interact with PG following a recently published protocol (Janesch et al., 2013). Briefly, 1 μg of purified protein was incubated with 0.2 mg of lyophilized L. buchneri NRRL B-30929 cell walls in 25 mM sodium citrate buffer, pH 5.5, in a total volume of 125 μl, for 1 h at 37 °C under gentle rotation. The mixture was centrifuged (16 100 g, 20 min), yielding a supernatant containing unbound protein and a pellet of insoluble cell wall components including attached protein. The pellet was washed twice to remove unbound proteins. Samples were investigated by SDS-PAGE followed by Western immunoblotting using muramidase-specific polyclonal antiserum. Mixtures without cell walls were used as control. The pellet and the supernatant fraction of the reactions as well as the controls were quantified using the Odyssey Infrared Imaging System (LI-COR). For comparability of datasets, the sum of the signals in the pellet fraction and the supernatant fraction of each reaction was set to 100 %. Experiments were performed in triplicate and sds were calculated.

Immunofluorescence staining and microscopy

The cellular localization of LbGH25B and LbGH25N was determined by immunofluorescence. L. buchneri CD034, L. buchneri NRRL B-30929 and L. plantarum CD033 were grown until the late-exponential phase (OD600 of ~1.5). Cells were washed with PBS and adsorbed on a glass slide for 2 h. All procedures were performed at room temperature, if not stated otherwise. Cells were washed twice with PBS and fixed for 30 min at −20 °C in 70 % (v/v) ethanol in PBS. After two washing steps with PBS, cells were incubated in blocking buffer (10 %, w/v, BSA in PBS) for 1 h, followed by incubation in blocking buffer containing muramidase-specific polyclonal antiserum or pre-immune serum (1: 10 dilution) for 1 h, washed once with blocking buffer and twice with PBS, and subsequently incubated in blocking buffer containing goat anti-mouse IgG (1: 100 dilution) conjugated to FITC (Sigma-Aldrich) for 2 h. The cells were washed once with blocking buffer and twice with PBS to remove unbound antibodies. One drop of 50 % (v/v) glycerol in PBS was added onto the cells and a coverslip was mounted. Confocal laser scanning microscopy analyses were performed using a Leica TCS SP5 II system. Images were taken with a 63.0× 1.40 oil-immersion objective. Immunofluorescence-stained bacterial cells were excited at 488 nm using an argon laser and detected at an emission bandwidth of 500–595 nm. Images were acquired and processed with Leica LAS AF software.

RESULTS

In silico search for predicted PGHs of L. buchneri

Putative PGHs of L. buchneri CD034 were identified in silico in the genome (GenBank Assembly ID: GCA_000298115.2) (Heinl et al., 2012) by searching for catalytic domains specific for PGHs (Layec et al., 2008). Twenty-three putative enzymes could be affiliated to the known classes of PGHs (Layec et al., 2008) according to their predicted hydrolytic specificities (Table 1). PGHs of bacteria of the phylum Firmicutes typically show a modular structure consisting of a catalytic and a cell wall-binding domain (Layec et al., 2008). Only three of the putative L. buchneri PGHs were predicted to contain a cell wall-binding domain. Among these is an amidase and an endopeptidase containing the non-catalytic SH3 cell wall-binding domain (Whisstock & Lesk, 1999), and a carboxypeptidase containing the cell wall association domain PBP5_C (Layec et al., 2008). The amino acid sequences of all putative L. buchneri CD034 PGHs were aligned with the respective L. buchneri NRRL B-30929 homologues (Liu et al., 2011); the calculated sequence identity was between 97 and 100 % (Table 1).

Table 1.

PGHs of L. buchneri CD034 and their homologues in L. buchneri NRRL B-30929 predicted in silico on whole-genome sequences

Locus tag MM* (kDa) SP† Putative hydrolytic specificity Catalytic domain‡ CW-binding domain§ NRRL B-30929 homologue∥ Identity (%)∫
LBUCD034_0240 40.8 Yes Muramidase GH25 Lbuc_0200 99
LBUCD034_0976 30.8 No Muramidase GH25 Lbuc_1390 53
LBUCD034_1564 30.5 No Muramidase GH25 Lbuc_1506 99
LBUCD034_1723 29.5 No Muramidase GH25 Lbuc_1566 100
LBUCD034_1884 34.4 Yes Muramidase GH25 Lbuc_1800 99
LBUCD034_2218 33.5 Yes Muramidase GH25 Lbuc_2119 99
LBUCD034_0537 23.5 No Glucosaminidase Glucosaminidase Lbuc_0501 99
LBUCD034_1423 28.2 Yes Glucosaminidase Glucosaminidase Lbuc_1294 99
LBUCD034_1424 54.2 Yes Glucosaminidase Glucosaminidase Lbuc_1295 99
LBUCD034_1762 24.0 Yes Glucosaminidase Glucosaminidase Lbuc_1692 100
LBUCD034_0238 25.2 Yes Amidase Amidase_2 Lbuc_0198 99
LBUCD034_0862 67.0 No Amidase Amidase_2 Lbuc_0803 98
LBUCD034_1137 34.2 No Amidase Amidase_3 SH3_3 Lbuc_1003 100
LBUCD034_2087 36.2 Yes Amidase Amidase_2 Lbuc_1998 99
LBUCD034_0481 47.7 Yes d-Alanyl-d-alanine carboxypeptidase Peptidase_S11 Lbuc_0439 98
LBUCD034_0620 45.1 Yes d-Alanyl-d-alanine carboxypeptidase Peptidase_S11 PBP5_C Lbuc_0602 97
LBUCD034_0662 43.3 Yes d-Alanyl-d-alanine carboxypeptidase Peptidase_S11 Lbuc_642 99
LBUCD034_0534 17.7 Yes Endopeptidase NLPC_P60 Lbuc_0498 99
LBUCD034_0586 19.2 Yes Endopeptidase NLPC_P60 Lbuc_0549 100
LBUCD034_1373 32.9 No Endopeptidase NLPC_P60 SH3_3 Lbuc_1248 100
LBUCD034_1574 202.2 No Endopeptidase NLPC_P60 Lbuc_1516 99
LBUCD034_1996 30.9 No Endopeptidase NLPC_P60 Lbuc_1908 96
LBUCD034_2219 17.8 Yes Endopeptidase NLPC_P60 Lbuc_2120 100
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*

Calculated molecular mass.

†

SP, signal peptide predicted with SignalP 4.1 server.

‡

Catalytic domains predicted with Pfam domain prediction. Muramidase (glyco_hydro_25; PF01183), glucosaminidase (PF01832), Amidase_2 (PF01510), Amidase_3 (PF01520), Peptidase_S11 (PF00768), endopeptidase (NLPC_P60; PF00877).

§

CW (cell wall)-binding domain; SH3_3, (PF08239); PBP5_C (PF07943).

∥

Locus tag of L. buchneri NRRL B-30929 protein, homology search by BLAST algorithm.

∫

Amino acid sequence identity by BLAST alignment.

LbGH25B and LbGH25N are 380-aa proteins with a calculated molecular mass of 40.8 kDa. A difference of 23 nt translates to a difference of only 2 aa, with Val182 and Ala260 in LbGH25B being replaced by Ile182 and Glu260, respectively, in LbGH25N. Either enzyme exhibits a conserved GH25 (Pfam01183) domain following a 29-aa signal peptide including a type I signal peptidase cleavage site at the N terminus, and a C-terminal part with unknown function (Fig. 1).

Fig. 1.

Fig. 1

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Schematic drawing of LbGH25B and LbGH25N variants. Native (n) LbGH25B and LbGH25N are both 380-aa proteins with a calculated molecular mass of 40.8 kDa. The amino acids Val182 and Ala260 in LbGH25B are replaced by Ile182 and Glu260, respectively, in LbGH25N. The putatively glycosylated sequence motif Ser–Ala–Ser–Ser–Ala–Ser is marked in dark grey. Recombinant (r) LbGH25B and LbGH25N are devoid of their 29-aa signal peptide (SP) but contain a C-terminal hexahistidine tag (black) for purification purposes. LbGH25B was dissected into an N-terminal protein portion containing the glycosyl hydrolase family 25 domain (GH25, Pf01183) and the C-terminal remainder of the protein.

Evaluation of secondary structure composition and thermal stability of LbGH25B and LbGH25N by ECD spectroscopy

ECD spectroscopy was used to investigate the secondary structure composition and compare the thermal stability of LbGH25B and LbGH25N. Fig. 2(a) depicts the ECD spectra in the far-UV region of both enzymes. The ellipticity of α-helices typically shows two minima at 208 and 222 nm, whereas the contribution of β-sheets gives rise to negative ellipticity around 212–214 nm, but with Δε values that are five times lower than those for α-helices (Kelly et al., 2005). The spectra of LbGH25B and LbGH25N show a similar overall fold of both proteins and the typical feature of proteins with a high β-sheet content, which correlates with computed secondary structure predictions. According to the PSIPRED secondary structure prediction server, LbGH25B and LbGH25N contain 28 % β-sheets, 16 % α-helices and 56 % random coils, and 28 % β-sheets, 17 % α-helices and 56 % random coils, respectively.

Fig. 2.

Fig. 2

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Comparison of secondary structure and thermal stability of LbGH25B and LbGH25N by ECD spectroscopy. Far-UV ECD spectra at 25 °C (a) and temperature-mediated unfolding followed at 222 nm (b) of LbGH25B (solid line) and LbGH25N (dotted line). Measurements were performed in 12.5 mM (a) and 50 mM (b) sodium citrate buffer, pH 5.5. The inset in (b) shows the van’t Hoff plots.

To gain more information about the thermal stability of the N-acetylmuramidases, temperature-mediated unfolding was followed by ECD spectroscopy in the far-UV region at 222 nm. The melting curves of both enzymes suggest a simple two-state transition with Tm values of 49.0±0.1 and 47.9±0.2 °C for LbGH25B and LbGH25N, respectively (Fig. 2b). A clear linear relationship between the equilibrium constants and the reciprocal temperature allowed us to calculate the van’t Hoff enthalpy for this transition to 413.4±9.2 and 364.4±4.2 kJ mol−1 for LbGH25B and LbGH25N, respectively.

Hydrolysing activity in zymograms

His-tagged LbGH25B and LbGH25N (Anzengruber et al., 2014) were purified from E. coli by nickel-affinity chromatography and the hydrolytic activity was investigated by zymogram assays. Additionally, enzyme activity of native LbGH25B and LbGH25N was assayed by applying a L. buchneri CD034 and L. buchneri NRRL B-30929 cell extract, respectively, to zymogram gels. To unravel a sequence-function relationship, LbGH25B was dissected into an N-terminal protein portion containing the GH25 domain and the C-terminal rest of the protein. The protein portions were produced in E. coli and assayed for their functionality. All protein variants used for zymography are schematically depicted in Fig. 1.

LbGH25B and LbGH25N were isolated at a final yield of 8 and 5 mg per gram of protein of E. coli cell pellet, respectively (Fig. 3a, lanes 1 and 2). Purity of the recombinant enzymes was analysed by SDS-PAGE following CBB staining (Fig. 3a), revealing both proteins migrating as a single band with a molecular mass of ~40 kDa (Fig. 3a, lanes 3 and 4). The N-terminal, GH25 domain-containing region and the C-terminal region of LbGH25B as present in extracts of E. coli cells carrying the respective expression plasmid (Fig. 3a, lanes 6 and 7) and purified (Fig. 3a, lanes 10 and 11) migrated at ~22 and ~18 kDa, respectively, which corresponds to the calculated masses based on the amino acid sequence of the truncated proteins. As a negative control, E. coli was transformed with empty pET28a vector (Fig. 3a, lane 5). Both L. buchneri CD034 and L. buchneri NRRL B-30929 cell extracts showed the characteristic protein pattern of L. buchneri cells, with the abundant protein band at ~60 kDa corresponding to the S-layer glycoproteins SlpB and SlpN, respectively (Anzengruber et al., 2014) (Fig. 3a, lanes 8 and 9).

Fig. 3.

Fig. 3

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SDS-PAGE and zymogram analysis. Purified recombinant LbGH25B (lane 1) and LbGH25N (lane 2); cell extracts of E. coli expressing LbGH25B (lane 3) and LbGH25N (lane 4); E. coli harbouring empty pET28a (lane 5); E. coli expressing the GH25 domain (lane 6) or the C-terminal domain (lane 7) of LbGH25B; cell extracts of L. buchneri CD034 (lane 8) and L. buchneri NRRL B-30929 (lane 9) as well as the purified recombinant GH25 domain (lane 10) and C-terminal domain (lane 11) of LbGH25B were run on SDS-PAGE (20 % polyacrylamide gel) and stained with CBB (a) or loaded on the zymogram gels (b–e) containing L. buchneri CD034 cell walls (20 % PA gel) (b), L. buchneri NRRL B-30929 cell walls (12 % PA gel) (c), L. rhamnosus GG cells (12 % polyacrylamide gel) (d) or E. coli BL21 Star BL21(DE3) cell walls (12 % PA gel) (e) as substrate. Zymogram gels were incubated overnight at 37 °C in potassium phosphate buffer containing 1 mM DTT and visualized by methylene blue staining. Arrows indicate hydrolytic bands of native LbGH25B and LbGH25N. Asterisks indicate hydrolytic bands of the LbGH25B N-terminal GH25 domain-containing region. Note the presence of a hydrolytic protein (~25 kDa) in the E. coli cell extracts (b). Mm1, Precision Plus protein kaleidoscope standard (Bio-Rad); Mm2, PageRuler Plus pre-stained protein ladder (Thermo Scientific).

Aliquots of the protein samples were applied to renaturing SDS-PAGE gels containing co-polymerized L. buchneri CD034 cell walls as a substrate (Fig. 3b). Translucent zones were observed for purified recombinant LbGH25B and LbGH25N as well as for LbGH25B and LbGH25N from E. coli cell extracts (Fig. 3b, lanes 1–4). Presumptive hydrolytic activity of an E. coli protein of ~25 kDa was detected in all of the E. coli cell extracts (Fig. 3b, lanes 3–7), including the negative control containing the empty plasmid (Fig. 3b, lane 5). Whereas the C-terminal region of LbGH25B did not show clearance in the zymogram gel, the N-terminal GH25 domain-containing region showed clearance (indicated by a star in Fig. 3b, lanes 6 and 10), confirming that the GH25 domain of the enzyme is crucial for its lytic activity. In regard to the PGH activity of L. buchneri CD034 and L. buchneri NRRL B-30929 cell extracts, a clearance zone was observed at ~40 kDa, which can probably be attributed to enzyme activity of native LbGH25B and LbGH25N, respectively (indicated by arrows in Fig. 3b, lanes 8 and 9). Cell wall hydrolysis by recombinant LbGH25B and LbGH25N was further confirmed on zymograms with L. buchneri NRRL B-30929 cell walls as a substrate (Fig. 3c). L. rhamnosus GG cells (Fig. 3d), which exhibit, like L. buchneri, an A4α PG chemotype with an interpeptide bridge (Schleifer & Kandler, 1972; Claes et al., 2012), were also shown to be a substrate for full-length LbGH25B and its N-terminal GH25 domain-containing region (indicated by the star in Fig. 3d), while the LbGH25B C-terminal region did not yield cleared zones. This is in agreement with the results obtained with L. buchneri CD034 cell walls. Interestingly, recombinant and native LbGH25B and LbGH25N were also able to hydrolyse the Gram-negative cell walls of E. coli, which are of the A1γ PG chemotype with direct cross-linking between d-Ala4 and meso-diaminopimelic acid3 (Schleifer & Kandler, 1972) (cleared band in Fig. 3e).

Hydrolytic specificity of LbGH25B and LbGH25N on PG

The hydrolytic specificity of LbGH25B and LbGH25N was determined by assaying the activity of the purified recombinant, His-tagged enzymes on L. buchneri CD034 and L. buchneri NRRL B-30929 PG, as well as on L. rhamnosus GG PG for which reference data were available (Claes et al., 2012). Isolated PGs were incubated with either LbGH25B, LbGH25N or the N-acetylmuramidase mutanolysin, a mixture of two major lytic enzymes from Streptomyces globisporus (Kawata et al., 1983). Soluble muropeptides were then separated by reversed-phase HPLC (Fig. 4), with mutanolysin-treated PG serving as a reference. Comparison of the muropeptide profiles obtained from L. buchneri CD034 PG digested with either mutanolysin, LbGH25B or LbGH25N revealed overall identity (Fig. 4a). An identical series of profiles was obtained for L. buchneri NRRL B-30929 PG digested with each of the three enzymes (Fig. 4b) as well as for L. rhamnosus GG PG (Fig. 4c). Furthermore, reversed-phase HPLC separation profiles of muropeptides obtained from PGs digested with a combination of LbGH25B or LbGH25N and mutanolysin did not differ from those treated with LbGH25B or LbGH25N alone (data not shown). This indicates that the muropeptides released by the two L. buchneri PGHs and by mutanolysin are identical, suggesting that LbGH25B and LbGH25N exhibit N-acetylmuramidase specificity like mutanolysin, cleaving the β-N-acetylmuramyl-(1→4)-N-acetylglucosamine bonds of bacterial PG. N-Acetylmuramidase specificity was further confirmed by MALDI-TOF MS analysis of main muropeptides (Table 2). Muropeptide structures were deduced from their determined m/z values and revealed acetylation of the GlcNAc–MurNAc disaccharide in both L. buchneri and L. rhamnosus PGs (Table 2). From this it can be concluded that L. buchneri muramidases can digest O-acetylated PG.

Fig. 4.

Fig. 4

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Reversed-phase HPLC separation profiles of muropeptides obtained from L. buchneri CD034 (a), L. buchneri NRRL B-30929 (b) and L. rhamnosus GG PG (c). PGs were digested by mutanolysin, LbGH25B, LbGH25N or SlpB (negative control). Main peaks (1–4, A–D) were analysed by MALDI-TOF MS (Table 2).

Table 2.

Main muropeptides from L. buchneri NRRL B-30929 and L. rhamnosus PG digested with LbGH25N

Peak* Proposed structure† Observed m/z Calculated‡ [M+Na]+
1 Tri-N 962.18 962.43
2 Tetra-D 1034.37 1034.45
3 Tetra-N 1033.35 1033.47
4 Tetra-N(Ac) 1075.52 1075.48
A Tri-N 962.33 962.43
B Tetra-N 1033.43 1033.47
C Tri-N(Ac) 1004.37 1004.44
D Tetra-N(Ac) 1075.38 1075.48
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*

Peak numbers refer to Fig. 4.

†

Tri, disaccharide tripeptide (l-Ala-d-iGln-l-Lys); Tetra, disaccharide tetrapeptide (l-Ala-d-iGln-l-Lys-d-Ala); Disaccharide, GlcNAc-MurNAc; Ac, acetylation; iGln, isoglutamine; N, d-Asn; D, d-Asp.

‡

Sodiated molecular ions were the most abundant on MALDI-TOF mass spectra for all muropeptides.

To exclude the possibility that the observed N-acetylmuramidase activity was caused by E. coli proteins that could not be completely removed after purification of LbGH25B and LbGH25N, or from lysozyme used for cell lysis, PGs were digested with recombinant SlpB as a negative control. SlpB was treated in the same way as LbGH25B and LbGH25N during expression and purification, but no PG-hydrolysing activity was observed (Fig. 4).

Of note, nearly identical muropeptide profiles were obtained from L. buchneri CD034 PG and L. buchneri NRRL B-30929 PG (Fig. 4a, b), supporting highly similar if not identical PG structures in these organisms.

Although L. buchneri and L. rhamnosus GG have a similar A4α PG chemotype with a d-Ala4→(d-Asp/d-Asn)→l-Lys3 inter-peptide bridge (Schleifer & Kandler, 1972; Claes et al., 2012), the muropeptide profiles differ due to PG modifications (acetylation of glycan chains, amidation of C-terminal d-Ala, cleavage of peptide chains) present to varying extents.

Cellular localization of LbGH25B and LbGH25N

To determine the cellular localization of the L. buchneri muramidases, cell fractionation followed by Western immunoblotting, an in vitro cell wall-binding assay with purified recombinant LbGH25B and LbGH25N, as well as indirect immunofluorescence microscopy of whole cells, was performed.

Proteins from the cell wall and TCA-precipitated proteins from the cytosol as well as the supernatant of L. buchneri CD034 and NRRL B-30929 cells, respectively, were subjected to Western immunoblotting for LbGH25B and LbGH25N detection. Distinct protein bands were observed in the cell wall fraction, while muramidase-specific antiserum could not detect the enzymes in either the cytosolic faction or the supernatant (Fig. 5a).

Fig. 5.

Fig. 5

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Cellular localization of LbGH25B and LbGH25N by (a) subcellular fractionation and (b) in vitro cell-wall binding. (a) L. buchneri CD034 and NRRL B-30929 proteins were isolated from cell walls (lanes 1 and 4), the cytosol (lanes 2 and 5), and the culture supernatant (lanes 3 and 6), run on SDS-PAGE (10 % gel) using equivalent sample amounts, Western blotted and detected with muramidase-specific antiserum. (b) Binding of recombinant LbGH25B and LbGH25N to cell walls of L. buchneri was tested. Proteins were incubated with (+) and without (−) cell walls. After incubation, the reactions were centrifuged to separate cell walls (with bound protein) from unbound protein. Analysis was done by SDS-PAGE (20 % gels) followed by Western immunoblotting using muramidase-specific antiserum. The figure represents one of three independent repeats of the experiment. S, supernatant; P, pellet; Mm, PageRuler prestained protein ladder (Thermo Scientific).

Furthermore, the ability of recombinant LbGH25B and LbGH25N to attach to isolated cell walls of L. buchneri was investigated in an in vitro cell wall-binding assay. Due to extensive treatment with denaturing agents, the cell wall preparation used for the assay was free of either native LbGH25B or native LbGH25N as confirmed by Western immunoblotting (data not shown). The results of the in vitro binding assay, as evidenced by densitometric quantification of the immunoblots, indicated that both recombinant proteins, despite the lack of a known cell wall recognition domain, are able to bind to the cell wall (Fig. 5b). Under the applied assay conditions, 77±4 % of LbGH25B and 77±2 % of LbGH25N used for the analysis were attached to L. buchneri cell walls as determined from three independent experimental repeats.

For localization of the PGHs in L. buchneri CD034 and L. buchneri NRRL B-30929 cells, muramidase-specific mouse antiserum in combination with FITC-conjugated goat anti-mouse, followed by immunofluorescence microscopy, was used (Fig. 6a, b), revealing a prevalence of the enzymes at the periphery of the bacterial cells. Closer inspection of the fluorescently labelled cells indicated accumulation of the enzymes at the cell poles and near emerging septa of dividing cells. Staining with pre-immune serum as a negative control revealed neither non-specific reactions nor fluorescence of L. buchneri cells (Fig. 6c, d). To exclude non-specific reactions of the muramidase-specific anti-serum with muramidases other than LbGH25B and LbGH25N, or with components of the Gram-positive cell surface, immunofluorescence staining was performed with L. plantarum cells. As shown in Fig. 6(e), no non-specific staining was detected.

Fig. 6.

Fig. 6

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Cellular localization of LbGH25B and LbGH25N by immunofluorescence microscopy. Muramidase-specific mouse antiserum in combination with FITC-conjugated goat anti-mouse IgG was used to visualize LbGH25B and LbGH25N on L. buchneri CD034 (a) and L. buchneri NRRL B-30929 (b) cells, respectively. Staining with pre-immune serum as a negative control revealed no fluorescence in L. buchneri CD034 (c) and L. buchneri NRRL B-30929 (d) cells. As an additional negative control, immunofluorescence staining with muramidase-specific mouse antiserum did not show a non-specific reaction in the Gram-positive bacterium L. plantarum CD033 (e).

DISCUSSION

Our aims were to investigate the PGH complement of L. buchneri and to biochemically characterize in detail the homologous cell-wall hydrolases LbGH25B and LbGH25N from L. buchneri CD034 and L. buchneri NRRL B-30929, respectively.

We provide clear evidence for cell wall-hydrolysing activity of the above enzymes using a combination of zymogram analysis (Leclerc & Asselin, 1989; Lepeuple et al., 1998) and determination of the PG cleavage site. Both LbGH25B and LbGH25N performed inter-species cell wall-hydrolysing activity by degrading L. buchneri CD034, L. buchneri NRRL B-30929 and L. rhamnosus GG cell walls. Relaxed substrate specificity was corroborated by hydrolysing activity on Gram-negative E. coli cell walls. Via muropeptide analysis with reversed-phase HPLC and MALDI-TOF MS, these enzymes were shown to possess N-acetylmuramidase specificity with cleavage of the β-(1→4) glycosidic bond between MurNAc and GlcNAc in the bacterial PG.

There are relatively few reports on bacterial muramidases with experimentally proven specificity in the literature, with none of these originating from lactobacilli (Vollmer et al., 2008b). Two autolytic muramidases, SF muramidase and pesticin, have been described in Enterococcus faecium (Kawamura & Shockman, 1983; Barrett et al., 1984) and in Yersinia pestis (Vollmer et al., 1997), respectively. Cellosyl produced by Streptomyces coelicolor degrades O-acetylated PG as present in Staphylococcus aureus and other pathogens (Rau et al., 2001). LytC and GHIP were identified in Streptococcus pneumoniae (García et al., 1999; Niu et al., 2013). Mutanolysin, consisting of two kinds of N-acetylmuramidases, M-1 and M-2, was isolated from culture broth of Streptomyces globisporus. While the hydrolysing activity of the M-2 enzyme was suppressed by the presence of O-acetylated MurNAc, M-1 was independent of the modification with O-acetyl groups (Kawata et al., 1983). In this study, LbGH25B and LbGH25N were shown to degrade the PG of various bacterial strains, including L. buchneri and L. rhamnosus PG, for which we could demonstrate MurNAc acetylation in this study, as well as E. coli PG, which does not contain O-acetylated MurNAc (Dezélée & Bricas, 1970). This indicates that the hydrolytic activity of the L. buchneri N-acetylmuramidases is not affected by O-acetylation of the PG glycan backbone.

Immunofluorescence microscopy clearly identified the N-acetylmuramidases as cell surface enzymes, indicating their possible role in cell wall growth and daughter cell separation, properties which are counted among the numerous functions of PGHs (Vollmer et al., 2008b). During bacterial cell wall growth, PG hydrolytic and synthetic reactions are coordinated temporarily and spatially to avoid cell lysis and guarantee a safe enlargement of the sacculus (Typas et al., 2012; Vollmer, 2012). A direct interaction between peptidoglycan synthases and hydrolases in multi-enzyme complexes has been observed in E. coli (Höltje, 1996a, b). In Gram-positive bacteria, synthases are involved in the synthesis of the inner, new PG layers, whereas hydrolases act, distant from the synthases, on the outer layers (Vollmer et al., 2008b). Further studies imply that bacteria have specific inhibitors to control their hydrolases, as was shown in Pseudomonas aeruginosa (Clarke et al., 2010). Interestingly, O-glycosylation was shown to control the activity of the glucosaminidase Acm2 from L. plantarum. Lack of O-glycosylation significantly increased enzyme activity, whereas removal of the PG-binding domains of Acm2 reduced this activity. A model was proposed in which access by Acm2 to its substrate may be hindered by O-glycosylation (Rolain et al., 2013). A similar control mechanism can be hypothesized for putatively O-glycosylated LbGH25B and LbGH25N.

Cell surface localization of LbGH25B and LbGH25N was further confirmed by subcellular fractionation, with no detectable enzyme in the culture supernatant. In contrast, lactobacillar endopeptidases Msp1 and Lc-p75 from L. rhamnosus GG and L. casei BL23, respectively, were found both attached to the cell wall and secreted in the culture supernatant (Claes et al., 2012; Regulski et al., 2012). Attachment of the N-acetylmuramidases to L. buchneri cell walls was additionally shown by in vitro cell wall-binding assays. Typically, PGHs exhibit a multi-domain structure including a catalytic domain and several copies of a cell wall-binding domain. Frequently found binding domains in GH25-containing proteins are LysM, SH3, PG_binding, CW_binding or Cpl_7 (Layec et al., 2008). Surprisingly, LbGH25B and LbGH25N do not contain any of these known domains, despite being, according to our experiments, cell wall-associated proteins. One might speculate that their basic pI value, which results in an overall basic net charge at neutral and low pH, may enhance their binding to negatively charged cell wall components (Vollmer et al., 2008b). The N-acetylmuramidase cellosyl, which also does not exhibit distinct cell-wall binding domains, possesses a basic pI of 9.3 and was shown to contain at its C terminus a prominent, long groove, very likely to be the PG-binding site, as predicted from its crystal structure (Rau et al., 2001).

Bacterial cell wall hydrolases, especially those originating from GRAS organisms, are interesting candidates as a new class of antimicrobials (Parisien et al., 2008). Structural stability is a relevant feature for the pharmaceutical and biotechnological use of proteins. In this regard, thermal denaturation studies can provide an evaluation of conditions that minimize unfolding and maintain biological activity. ECD demonstrated that LbGH25B and LbGH25N have similar overall protein folds, mainly composed of β-sheets. To date, the 3D structures of a few GH25 enzymes have been determined, including cellosyl [Protein Data Bank (PDB) code 1JFX] (Rau et al., 2001), BaGH25c from Bacillus anthracis (PDB code 2WAG) (Martinez-Fleites et al., 2009) and LytC and GHIP (PDB code 2WW5 and 4FF5, respectively) (Pérez-Dorado et al., 2010; Niu et al., 2013). Cellosyl, which shows, from all crystallized bacterial muramidases, the highest primary structure similarity to LbGH25B and LbGH25N, mainly at the N-terminal GH25 domain, comprises a single domain composed of an eight-stranded β-barrel flanked by seven α-helices (Rau et al., 2001).

To gain insight into protein stability, temperature-mediated unfolding was performed. The determined Tm values of around 49 °C for LbGH25B and LbGH25N reflect thermal stabilities of the enzymes as required for biotechnological and pharmacological exploitation. The values obtained are in accordance with that of the muramidase Cpl-7 of S. pneumoniae recently studied as an antibacterial agent (Bustamante et al., 2012). Identical thermal stability was observed for LbGH25B and LbGH25N with a simple two-state transition during thermal denaturation, suggesting that these enzymes are organized as one folding unit, which is in agreement with the single domain architecture of these enzymes based on their amino acid sequence. This has been observed for other bacterial muramidases, including cellosyl and GHIP (Rau et al., 2001; Pérez-Dorado et al., 2010; Niu et al., 2013).

To summarize, the data presented in this study characterize in detail, for the first time, the major N-acetylmuramidases from the genus Lactobacillus and contribute to the pool of bacterial cell wall hydrolases whose development as novel therapeutics pinpoints advantages over conventional antibiotics, such as low probability of developing resistance (Parisien et al., 2008). Food applications of bacterial cell wall hydrolases are being intensively studied (Callewaert et al., 2011), for instance, in the control of wine-spoiling lactic and acetic acid bacteria, which are due to specific PG structures mostly resistant to lysozyme (Blättel et al., 2009).

ACKNOWLEDGEMENTS

We thank Siqing Liu (US Department of Agriculture, Agricultural Research Service, Renewable Product Technology Research Unit, University of Illinois, Peoria, USA) for kindly providing L. buchneri NRRL B-30929. Financial support came from the Austrian Science Fund, FWF projects P21954-B20 (to C. S.) and P24305-B20 (to P. M.), the PhD programme ‘BioToP - Biomolecular Technology of Proteins’ (Austrian Science Fund, FWF project W1224) and the Hochschuljubiläumsstiftung der Stadt Wien, project H-2442/2012 (to J. A.). Work by the group of M.-P. C.-C. was supported by INRA (France) and Région Ile de France.

Abbreviations

CBB

Coomassie brilliant blue R-250

ECD

electronic circular dichroism

GdHCl

guanidinium hydrochloride

GH25

glycosyl hydrolase family 25

GlcNAc

N-acetylglucosamine

GRAS

generally regarded as safe

MurNAc

N-acetylmuramic acid

PG

peptidoglycan

PGH

peptidoglycan hydrolase

S-layer

surface layer

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