Abstract

Glycosylation is a frequent and heterogeneous posttranslational protein modification occurring in all domains of life. While protein N-glycosylation at asparagine and O-glycosylation at serine, threonine or hydroxyproline residues have been studied in great detail, only few data are available on O-glycosidic attachment of glycans to the amino acid tyrosine. In this study, we describe the identification and characterization of a bacterial protein tyrosine O-glycosylation system. In the Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051T, a polysaccharide consisting of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)] →4)-β-d-ManpNAc-(1→] repeating units is O-glycosidically linked via an adaptor with the structure -[GroA-2→OPO2→4-β-d-ManpNAc-(1→4)] →3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein SpaA. A +AH4-24.3-kb S-layer glycosylation (slg) gene cluster encodes the information necessary for the biosynthesis of this glycan chain within 18 open reading frames (ORF). The corresponding translation products are involved in the biosynthesis of nucleotide-activated monosaccharides, assembly and export as well as in the transfer of the completed polysaccharide chain to the S-layer target protein. All ORFs of the cluster, except those encoding the nucleotide sugar biosynthesis enzymes and the ATP binding cassette (ABC) transporter integral transmembrane proteins, were disrupted by the insertion of the mobile group II intron Ll.LtrB, and S-layer glycoproteins produced in mutant backgrounds were analyzed by mass spectrometry. There is evidence that the glycan chain is synthesized in a process comparable to the ABC-transporter-dependent pathway of the lipopolysaccharide O-polysaccharide biosynthesis. Furthermore, with the protein WsfB, we have identified an O-oligosaccharyl:protein transferase required for the formation of the covalent β-d-Gal→Tyr linkage between the glycan chain and the S-layer protein.

Introduction

Covalent attachment of glycans to the protein backbone via the amide nitrogen of an asparagine residue (N-glycosylation) or via the hydroxyl group of serine, threonine or hydroxyproline (O-glycosylation) has been reported for many natural glycoproteins (Varki et al. 1999). In contrast, as a rare event, in insect larvae (Chen et al. 1978; Kramer et al. 1980) as well as in glycogenin of glycogen-containing eukaryotic cells (Aon and Curtino 1985; Rodriguez and Whelan 1985), an O-glycosidic linkage between a tyrosine residue and α-d-glucose has been observed. In prokaryotes, O-glycosidic linkages of glycans via β-d-galactose or β-d-glucose residues to tyrosine βwere discovered as completely new types of linkage in the S-layer glycoproteins of Paenibacillus alvei, Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacterium thermosaccharolyticum strains, respectively (Christian et al. 1988; Altman et al. 1991, 1995; Messner et al. 1992, 1995; Bock et al. 1994; Schäffer et al. 2000). These S-layer glycoproteins share the common feature of S-layer proteins to self-assemble into 2D crystalline arrays on the supporting cell envelope layer (Messner et al. 2009; Sleytr and Messner 2009), covering the bacterium completely. The glycan chains protrude from the cell surface, comparable to the lipopolysaccharide (LPS) coating of Gram-negative bacteria (Messner et al. 2008). For several S-layer glycoprotein-carrying bacteria, polycistronic S-layer glycosylation (slg) gene clusters with a size of +AH4-16 to +AH4-25 kb have been identified and sequenced (Novotny, Pföstl, et al. 2004). These gene clusters include nucleotide sugar pathway genes that are arranged consecutively, glycosyltransferase genes, glycan processing genes and transporter genes, all of them exhibiting high homology to components involved in the biosynthesis of different bacterial surface polysaccharides (Novotny, Schäffer, et al. 2004).

In the mesophilic, Gram-positive bacterium P.alvei CCM 2051T, the S-layer O-glycan is a polymeric branched polysaccharide of, on average, 23 [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units linked via an adaptor with the structure -[GroA-2→OPO2→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein backbone (Altman et al. 1991; Messner et al. 1995) (Figure 1). Two predominant glycosylation sites are predicted on the S-layer protein, most probably corresponding to mono- and di-glycosylated S-layer protein as derived from the periodic acid-Schiff staining behavior of the S-layer glycoprotein after separation on a sodium dodecyl sulfate polyacrylamide (SDS-PA) gel (Zarschler et al. 2009). Recently, the initiation enzyme of S-layer glycan biosynthesis, WsfP, has been identified as part of a slg gene cluster of P. alvei CCM 2051T, and a gene disruption system has been developed for this organism (Zarschler et al. 2009).

Fig. 1

Schematic drawing of the S-layer glycoprotein glycan structure of P. alvei CCM 2051T.

Fig. 1

Schematic drawing of the S-layer glycoprotein glycan structure of P. alvei CCM 2051T.

In the present study, we report on the determination of the nucleotide sequence of the complete slg gene cluster of P. alvei CCM 2051T and the genetic assignment of these open reading frames (ORFs). Insertional mutants for nine of the 18 ORFs located in the slg gene cluster were created, which allows proposing a specific transport mechanism for the glycan across the cytoplasmic membrane and identifying the O-oligosaccharyl:protein transferase (O-OTase) catalyzing the transfer of the glycan chain to specific tyrosine residues of the S-layer target protein SpaA. Furthermore, we introduce a working model for the S-layer glycan biosynthesis route in P. alvei CCM 2051T based on the observed similarity between the proteins encoded by ORFs of the slg gene cluster and database entries, as well as on the effects of the disruption of selected ORFs of the slg gene cluster on S-layer glycan composition as determined by mass spectrometry.

Results

Identification of the P. alvei CCM 2051T slg gene cluster

Since l-rhamnose represents the main constituent of the adaptor region of the S-layer glycan of P. alvei CCM 2051T, we have chosen the genes responsible for dTDP-l-rhamnose biosynthesis as suitable candidates for the design of degenerate primers to obtain entry into a putative slg gene cluster. Using a genomic DNA bank of P. alvei CCM 2051T as a template for the PCR amplification reaction and the primer proof_RmlD_for, a +AH4-1.4-kb DNA fragment was obtained. After confirmation of the presence of the rmlD gene coding for a dTDP-4-dehydrorhamnose reductase, sequencing of upstream and downstream regions by chromosome walking revealed the presence of 18 ORFs contained in a 24.3-kb slg gene cluster, encoding components of the putative S-layer protein glycosylation machinery (Figure 2). Most of the putative gene products encoded by the assigned ORFs showed high homology to proteins involved in the biosynthesis of bacterial surface polysaccharides. Based on these sequence similarities, putative biological functions have been assigned to almost all of the ORFs of the slg gene cluster (Table I).

Fig. 2

Genetic organization of the slg gene cluster of P. alvei CCM 2051T. Predicted open reading frames are indicated by horizontal arrows with the respective gene designations indicated above the arrow. ORFs encoding similar functions in S-layer glycan biosynthesis have a similar gray shading code. ORFs flanking the slg gene cluster are indicated in black, and ORFs coding for proteins with unknown function are indicated in white. ORFs indicated in light gray encode putative glycosyltransferases. Wzm and wzt (dark gray) encode the two components of the putative ABC transporter. Squares indicate ORFs encoding proteins involved in the biosynthesis of nucleotide-activated sugar precursors. The lightest gray indicates the wsfB ORF encoding the putative O-OTase. Putative promoters and terminators are represented as flags and hairpins, respectively. The location of the Ll.LtrB insertion is indicated by vertical black arrows and by the numbers below, while positions are given relative to the initial ATG codon. Experimentally identified transcription units are depicted (1). The reverse transcription analysis and subsequent cDNA amplification are shown, with primer positions indicated as vertical black arrows (2). The nucleotide sequence of the slg gene cluster has been submitted to the GenBank/EBI Data bank with accession number HM011508.

Fig. 2

Genetic organization of the slg gene cluster of P. alvei CCM 2051T. Predicted open reading frames are indicated by horizontal arrows with the respective gene designations indicated above the arrow. ORFs encoding similar functions in S-layer glycan biosynthesis have a similar gray shading code. ORFs flanking the slg gene cluster are indicated in black, and ORFs coding for proteins with unknown function are indicated in white. ORFs indicated in light gray encode putative glycosyltransferases. Wzm and wzt (dark gray) encode the two components of the putative ABC transporter. Squares indicate ORFs encoding proteins involved in the biosynthesis of nucleotide-activated sugar precursors. The lightest gray indicates the wsfB ORF encoding the putative O-OTase. Putative promoters and terminators are represented as flags and hairpins, respectively. The location of the Ll.LtrB insertion is indicated by vertical black arrows and by the numbers below, while positions are given relative to the initial ATG codon. Experimentally identified transcription units are depicted (1). The reverse transcription analysis and subsequent cDNA amplification are shown, with primer positions indicated as vertical black arrows (2). The nucleotide sequence of the slg gene cluster has been submitted to the GenBank/EBI Data bank with accession number HM011508.

Table I

Predicted gene products encoded by the slg gene cluster of P. alvei CCM 2051T together with database homologies.

ORF Length/Mol. mass Conserved motifs and region
 
Related proteins
 
Name/putative function Organism Identity/similarity (%) Accession No. 
wsfB 781/87.5 Wzy_C (PF04932) 347-411 O-antigen polymerase Geobacillus sp. Y412MC10 43/65 ZP_03037638 
TPR2 (PF07719) 690-723 O-antigen polymerase Caldicellulosiruptor saccharolyticus DSM 8903 23/45 YP_001181337 
galE 328/36.3 Epimerase (PF01370) 3-251 UDP-glucose 4-epimerase Paenibacillus larvae subsp. larvae 65/80 ZP_02328485 
UDP-glucose 4-epimerase BRL-230010 Geobacillus sp. Y412MC10 64/79 ZP_03040569 
galU 290/33.0 NTP_transferase (PF00483) 5-216 UTP-glucose-1-phosphate uridylyltransferase Geobacillus sp. Y412MC10 73/85 ZP_03037639 
UTP-glucose-1-phosphate uridylyltransferase Bacillus weihenstephanensis KBAB4 69/82 YP_001647518 
wzm 232/27.4 ABC2_membrane (PF01061) 1-191 Wzm Aneurinibacillus thermoaerophilus 58/77 AAS49124 
ABC transporter, permease protein DSM 10155/G+-Vibrio sp. MED222 52/74 ZP_01065570 
wzt 434/48.7 ABC_tran (PF00005) 53-222 Wzt Aneurinibacillus thermoaerophilus 42/62 AAS49125 
Wzt DSM 10155/G+-Geobacillus tepidamans GS5-97T 57/79 ABM68319 
wsfA 520/60.5 GATase_2 (PF00310) Asn_synthase (PF00733) 2-157 Asparagine synthase (glutamine-hydrolyzing) Francisella philomiragia subsp. philomiragia ATCC 25017 52/69 YP_001678197 
224-475 Asparagine synthase (glutamine-hydrolyzing) Thioalkalivibrio sp. HL-EbGR7 29/50 YP_002514426 
tagD 139/16.3 CTP_transf_2 (PF01467) 5-131 Putative glycerol-3-phosphate cytidyltransferase Francisella philomiragia subsp. philomiragia ATCC 25017 64/86 YP_001678198 
Glycerol-3-phosphate cytidyltransferase Clostridium perfringens str. 13 58/78 NP_561399 
wsfC 1260/147.3 Glyphos_transf (PF04464) Glycos_transf_2 (PF00535) Glycos_transf_2 (PF00535) 99-279 Glycosyl transferase family 2 Anaeromyxobacter sp. Fw109-5 38/54 YP_001378599 
558-729 Glycosyltransferase Rickettsia felis URRWXCal2 39/58 YP_246702 
947-1124 
wsfD 457/53.3 PMT (PF02366) 15-264 Transmembrane protein Bacillus cereus G9241 30/51 ZP_00240365 
Transmembrane protein Paenibacillus larvae subsp. larvae BRL-230010 25/48 ZP_02327204 
wsfE 364/42.3 Glycos_transf_1 (PF00534) 187-342 Glycosyltransferase Clostridium acetobutylicum ATCC 824 29/48 NP_349666 
Glycosyl transferase, group 2 family protein Pseudomonas fluorescens Pf-5 23/42 YP_259145 
rmlA 247/54.6 NTP_transferase (PF00483) 2-236 Glucose-1-phosphate thymidyltransferase Geobacillus stearothermophilus ATCC 12980 76/84 AAQ23685 
Glucose-1-phosphate thymidylyltransferase Bacillus anthracis str. Ames 70/84 NP_843700 
rmlC 183/20.8 dTDP_sugar_isom (PF00908) 3-177 dTDP-4-dehydrorhamnose 3,5-epimerase Geobacillus sp. Y412MC10 70/85 ZP_03037628 
dTDP-dehydrorhamnose 3,5-epimerase Aneurinibacillus thermoaerophilus DSM 10155/G+- 72/83 AAL18012 
rmlB 341/38.5 Epimerase (PF01370) 3-241 dTDP-glucose 4,6-dehydratase Geobacillus sp. Y412MC10 76/88 ZP_03037627 
dTDP-glucose 4,6-dehydratase Geobacillus stearothermophilus NRS 2004/3a 72/85 AAR99612 
rmlD 286/32.2 RmlD_sub_bind (PF04321) 1-281 dTDP-4-dehydrorhamnose reductase Geobacillus stearothermophilus NRS 2004/3a 60/78 AAR99613 
RmlD Geobacillus tepidamans GS5-97T 59/76 ABM68332 
wsfF 314/36.8 Glycos_transf_2 (PF00535) 9-119 WsdG Aneurinibacillus thermoaerophilus DSM 10155/G+- 49/66 AAL18015 
Rhamnosyltransferase Oenococcus oeni ATCC BAA-1163 35/56 ZP_01544702 
wsfG 299/34.1 Glycos_transf_2 (PF00535) 5-191 Glycosyl transferase family 2 Geobacillus sp. Y412MC10 64/79 ZP_03037625 
WsaD Geobacillus stearothermophilus NRS 2004/3a 56/75 AAR99614 
wsfP 468/54.6 Bac_transf (PF02397) 281-468 WsaP Geobacillus stearothermophilus NRS 2004/3a 60/75 AAR99615 
WsbP Geobacillus tepidamans GS5-97T 60/75 ABM68334 
wsfH 336/38.4 Glycos_transf_2 (PF00535) 12-176 Glycosyl transferase Microcystis aeruginosa NIES-843 58/81 YP_001655327 
Glycosyl transferase family 2 Arthrospira maxima CS-328 61/79 ZP_03275272 
ORF Length/Mol. mass Conserved motifs and region
 
Related proteins
 
Name/putative function Organism Identity/similarity (%) Accession No. 
wsfB 781/87.5 Wzy_C (PF04932) 347-411 O-antigen polymerase Geobacillus sp. Y412MC10 43/65 ZP_03037638 
TPR2 (PF07719) 690-723 O-antigen polymerase Caldicellulosiruptor saccharolyticus DSM 8903 23/45 YP_001181337 
galE 328/36.3 Epimerase (PF01370) 3-251 UDP-glucose 4-epimerase Paenibacillus larvae subsp. larvae 65/80 ZP_02328485 
UDP-glucose 4-epimerase BRL-230010 Geobacillus sp. Y412MC10 64/79 ZP_03040569 
galU 290/33.0 NTP_transferase (PF00483) 5-216 UTP-glucose-1-phosphate uridylyltransferase Geobacillus sp. Y412MC10 73/85 ZP_03037639 
UTP-glucose-1-phosphate uridylyltransferase Bacillus weihenstephanensis KBAB4 69/82 YP_001647518 
wzm 232/27.4 ABC2_membrane (PF01061) 1-191 Wzm Aneurinibacillus thermoaerophilus 58/77 AAS49124 
ABC transporter, permease protein DSM 10155/G+-Vibrio sp. MED222 52/74 ZP_01065570 
wzt 434/48.7 ABC_tran (PF00005) 53-222 Wzt Aneurinibacillus thermoaerophilus 42/62 AAS49125 
Wzt DSM 10155/G+-Geobacillus tepidamans GS5-97T 57/79 ABM68319 
wsfA 520/60.5 GATase_2 (PF00310) Asn_synthase (PF00733) 2-157 Asparagine synthase (glutamine-hydrolyzing) Francisella philomiragia subsp. philomiragia ATCC 25017 52/69 YP_001678197 
224-475 Asparagine synthase (glutamine-hydrolyzing) Thioalkalivibrio sp. HL-EbGR7 29/50 YP_002514426 
tagD 139/16.3 CTP_transf_2 (PF01467) 5-131 Putative glycerol-3-phosphate cytidyltransferase Francisella philomiragia subsp. philomiragia ATCC 25017 64/86 YP_001678198 
Glycerol-3-phosphate cytidyltransferase Clostridium perfringens str. 13 58/78 NP_561399 
wsfC 1260/147.3 Glyphos_transf (PF04464) Glycos_transf_2 (PF00535) Glycos_transf_2 (PF00535) 99-279 Glycosyl transferase family 2 Anaeromyxobacter sp. Fw109-5 38/54 YP_001378599 
558-729 Glycosyltransferase Rickettsia felis URRWXCal2 39/58 YP_246702 
947-1124 
wsfD 457/53.3 PMT (PF02366) 15-264 Transmembrane protein Bacillus cereus G9241 30/51 ZP_00240365 
Transmembrane protein Paenibacillus larvae subsp. larvae BRL-230010 25/48 ZP_02327204 
wsfE 364/42.3 Glycos_transf_1 (PF00534) 187-342 Glycosyltransferase Clostridium acetobutylicum ATCC 824 29/48 NP_349666 
Glycosyl transferase, group 2 family protein Pseudomonas fluorescens Pf-5 23/42 YP_259145 
rmlA 247/54.6 NTP_transferase (PF00483) 2-236 Glucose-1-phosphate thymidyltransferase Geobacillus stearothermophilus ATCC 12980 76/84 AAQ23685 
Glucose-1-phosphate thymidylyltransferase Bacillus anthracis str. Ames 70/84 NP_843700 
rmlC 183/20.8 dTDP_sugar_isom (PF00908) 3-177 dTDP-4-dehydrorhamnose 3,5-epimerase Geobacillus sp. Y412MC10 70/85 ZP_03037628 
dTDP-dehydrorhamnose 3,5-epimerase Aneurinibacillus thermoaerophilus DSM 10155/G+- 72/83 AAL18012 
rmlB 341/38.5 Epimerase (PF01370) 3-241 dTDP-glucose 4,6-dehydratase Geobacillus sp. Y412MC10 76/88 ZP_03037627 
dTDP-glucose 4,6-dehydratase Geobacillus stearothermophilus NRS 2004/3a 72/85 AAR99612 
rmlD 286/32.2 RmlD_sub_bind (PF04321) 1-281 dTDP-4-dehydrorhamnose reductase Geobacillus stearothermophilus NRS 2004/3a 60/78 AAR99613 
RmlD Geobacillus tepidamans GS5-97T 59/76 ABM68332 
wsfF 314/36.8 Glycos_transf_2 (PF00535) 9-119 WsdG Aneurinibacillus thermoaerophilus DSM 10155/G+- 49/66 AAL18015 
Rhamnosyltransferase Oenococcus oeni ATCC BAA-1163 35/56 ZP_01544702 
wsfG 299/34.1 Glycos_transf_2 (PF00535) 5-191 Glycosyl transferase family 2 Geobacillus sp. Y412MC10 64/79 ZP_03037625 
WsaD Geobacillus stearothermophilus NRS 2004/3a 56/75 AAR99614 
wsfP 468/54.6 Bac_transf (PF02397) 281-468 WsaP Geobacillus stearothermophilus NRS 2004/3a 60/75 AAR99615 
WsbP Geobacillus tepidamans GS5-97T 60/75 ABM68334 
wsfH 336/38.4 Glycos_transf_2 (PF00535) 12-176 Glycosyl transferase Microcystis aeruginosa NIES-843 58/81 YP_001655327 
Glycosyl transferase family 2 Arthrospira maxima CS-328 61/79 ZP_03275272 

Genetic characterization of the slg gene cluster

The slg gene cluster of P. alvei CCM 2051T is flanked by ORFs coding for enzymes involved in lantibiotic biosynthesis (Bierbaum and Sahl 2009) and lipid/lipoteichoic acid biosynthesis (Chen et al. 1993), respectively. The closed spacings from ORF wzm to wsfH and their identical transcriptional direction indicate that these ORFs are transcribed as a single operon (Figure 2). To identify specific mRNA(s) of the gene cluster, total RNA of P. alvei CCM 2051T was isolated and reverse transcribed into cDNA. The amplification of cDNA using primer combinations spanning the regions wsfB/galE, galU/wzm, wzt/wsfA, wsfA, wsfC/wsfD, wsfD/wsfE, rmlB/wsfF and wsfH/pcrB revealed that the slg gene cluster is transcribed as a polycistronic unit starting with galU and ending with wsfH (Figure 3). No PCR products were obtained when primers annealing to wsfB/galE (1f/1r) and cDNA reverse transcribed with primer 1f or when primers annealing to wsfH/pcrB (9f/9r) and cDNA reverse transcribed with primer 1f were used (data not shown). This observation indicates that wsfB and galE are transcribed separately, implying that they are not part of the polycistronic slg gene cluster but of the S-layer glycosylation locus. PcrB is most possibly not involved in S-layer glycosylation.

Fig. 3

RT-PCR analysis of total RNA of P. alvei CCM 2051T. Reverse transcription was performed with the specific primer 4r targeted to wsfA (lanes 2–7) or 8r annealing to wsfF (lanes 9–20). Subsequent cDNA amplification was carried out with primer pairs 2f/2r annealing to galU/wzm (lanes 2–4), with 3f/3r targeted to wzt/wsfA (lanes 5–7), with 4f/4r annealing to wsfA (lanes 9–11), with 5f/5r targeted to wsfC/wsfD (lanes 12–14), with 6f/6r annealing to wsfD/wsfE (lanes 15–17) and with 8f/8r amplifying rmlB/wsfF (lanes 18–20). Lanes (a) show the specific PCR amplification products, using reverse transcribed single-strand cDNA as template; lanes (b) show control reactions, using DNase I-treated RNA without the cDNA-generating step as PCR template; lanes (c) show positive controls using genomic DNA as template. The 1-kb DNA Plus marker (Invitrogen) was used as DNA size marker (lanes 1, 8 and 20).

Fig. 3

RT-PCR analysis of total RNA of P. alvei CCM 2051T. Reverse transcription was performed with the specific primer 4r targeted to wsfA (lanes 2–7) or 8r annealing to wsfF (lanes 9–20). Subsequent cDNA amplification was carried out with primer pairs 2f/2r annealing to galU/wzm (lanes 2–4), with 3f/3r targeted to wzt/wsfA (lanes 5–7), with 4f/4r annealing to wsfA (lanes 9–11), with 5f/5r targeted to wsfC/wsfD (lanes 12–14), with 6f/6r annealing to wsfD/wsfE (lanes 15–17) and with 8f/8r amplifying rmlB/wsfF (lanes 18–20). Lanes (a) show the specific PCR amplification products, using reverse transcribed single-strand cDNA as template; lanes (b) show control reactions, using DNase I-treated RNA without the cDNA-generating step as PCR template; lanes (c) show positive controls using genomic DNA as template. The 1-kb DNA Plus marker (Invitrogen) was used as DNA size marker (lanes 1, 8 and 20).

The putative gene products of the slg gene cluster have been analyzed by extensive database comparison and are discussed in the order of their appearance within the analyzed region.

wsfB

The gene product of wsfB (Figure S1) contains 12 potential transmembrane domains and a conserved Wzy_C motif characteristic of O-antigen ligases responsible for the transfer of undecaprenyl-pyrophosphate-linked sugars to a target protein (Power et al. 2006). OTases, such as PglL from Neisseria meningitidis and PilO from Pseudomonas aeruginosa, show only low levels of amino acid similarity but possess similar transmembrane topology and short regions of high homology (Abeyrathne et al. 2005). Both OTases exhibit relaxed glycan specificity but require the translocation of the corresponding undecaprenyl-pyrophosphate-linked oligosaccharide substrates into the periplasm (Faridmoayer et al. 2007). Like PglL, WsfB possesses in its carboxy-terminal part a tetratricopeptide repeat (TPR) described as mediator for protein–protein interactions and the assembly of multiprotein complexes (D'Andrea and Regan 2003). A similar membrane spanning topology and the presence of the Wzy_C motif in WsfB supports the assumption that this enzyme belongs to the family of O-OTases transferring the S-layer glycan chain from the lipid carrier to certain tyrosine residues in SpaA.

galE and galU

The translation products of these two ORFs are homologous to the uridine diphosphate (UDP)-glucose 4-epimerase (GalE) and the uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU), respectively. GalE catalyzes the interconversion between UDP-glucose and UDP-galactose, and GalU mediates the transfer of UTP to glucose-1-phosphate resulting in UDP-glucose. The presence of galactose and glucose in the S-layer O-glycan of P. alvei CCM 2051T suggests that GalE and GalU are involved in the biosynthesis of the sugar precursors UDP-galactose and UDP-glucose.

wzm and wzt

The deduced 232- and 434-amino acid proteins encoded by wzm and wzt, respectively, reveal high similarity to proteins of the ABC-2 transporter family, involved in the transport of bacterial surface polysaccharides to the cell surface (Bronner et al. 1994). The presence of six transmembrane domains in the putative translation product of wzm suggests that this protein is the integral membrane component of the transporter. An ATP-binding site and an ATP transporter signature motif identified in the putative translation product of wzt indicate the involvement of this protein in the transport of sugars across the cytoplasmic membrane (Walker et al. 1982; Rocchetta and Lam 1997). Its extended carboxy-terminal part obviously contains an O-polysaccharide binding domain determining the transporter’s substrate specificity as observed for the polymannan O-antigenic polysaccharides of Escherichiacoli O8 and O9a. Furthermore, several amino acids identified in wzt of E. coli O9a to be critical for binding and export of O-antigenic polysaccharide were also found in the homologous protein of P. alvei CCM 2051T (Cuthbertson et al. 2005, 2007). For instance, G333 and G387 located in the carbohydrate-binding pocket of the E. coli protein correspond to G334 and G387 of Wzt of P. alvei CCM 2051T (Figure S2).

wsfA

Throughout the whole ORF, wsfA is homologous to genes coding for asparagine synthetase B (AsnB). This enzyme acts as a homodimer with each monomer being composed of a glutaminase domain, hydrolyzing glutamine to glutamic acid and a combined ammonia and asparagine synthetase domain, catalyzing the ATP-dependent conversion of aspartate to asparagine (Milman and Cooney 1979; Scofield et al. 1990). An asnB mutant of Corynebacterium glutamicum was isolated as a lysozyme- and temperature-sensitive mutant (Hirasawa et al. 2000), and in Mycobacterium smegmatis, AsnB is involved in the natural resistance to rifampin, erythromycin and novobiocin (Ren and Liu 2006). Up to date, no specific function could be assigned to the wsfA gene product of P. alvei CCM 2051T.

tagD

The stop codon of wsfA overlaps with the putative start codon of tagD. Comparison of the translation product of tagD with proteins in the database showed a high degree of amino acid homology to the glycerol-3-phosphate cytidyltransferase involved in the formation of cytidine diphosphate (CDP)-glycerol and pyrophosphate from cytidine triphosphate (CTP) and glycerol-3-phosphate (Mauel et al. 1991; Park et al. 1993). The presence of a glyceric acid phosphate residue in the adapter saccharide of the S-layer O-glycan of P. alvei CCM 2051T suggests that TagD catalyzes the synthesis of the building block CDP-glycerol.

wsfC

The protein encoded by wsfC is the largest found in the slg gene cluster, coding for a tripartite transferase of 147.34 kDa. Two glycosyltransferase family 2 motifs were predicted in the central and carboxy-terminal part, while a single CDP-glycerol:poly(glycerophosphate) glycerophosphotransferase motif was found at the amino-terminal part of the protein. The amino-terminal region shows similarity to the TagB protein of Bacillus subtilis, catalyzing, there, the incorporation of a single glycerol phosphate residue from CDP-glycerol to the nonreducing end of membrane-bound undecaprenyl-phosphate-linked N-acetylmannosamine-β-(1,4)-N-acetylglucosamine-1-phosphate (Bhavsar et al. 2005). The central part of WsfC is homologous to the glycosyltransferase LgtD of different Rickettsia strains. In Haemophilus influenzae and Neisseria gonorrhoeae, LgtD is involved in LPS and lipooligosaccharide biosynthesis, respectively, possessing acetylgalactosaminyltransferase and galactosyltransferase activity (Gotschlich 1994; Shao et al. 2002; Randriantsoa et al. 2007). Sequence homology searches for the predicted amino acid sequences of the carboxy-terminal part of WsfC showed homology with cyanobacterial and archaeal glycosyltransferases, with AglG being involved in the N-glycosylation of the Haloferax volcanii S-layer glycoprotein and showing hexuronic acid transferase activity (Yurist-Doutsch et al. 2008).

wsfD

The wsfD gene product contains nine potential transmembrane domains and is similar to uncharacterized transmembrane proteins of various Gram-positive bacteria. In the amino-terminal part, a dolichyl-phosphate-mannose-protein mannosyltransferase domain spanning seven transmembrane domains was predicted. In fungi, dolichyl-phosphate-mannose-protein mannosyltransferases (PMTs) are integral endoplasmic reticulum (ER) membrane proteins responsible for the initiation of protein O-mannosylation. The PMT of Saccharomyces cerevisiae, ScPmt1p, is an integral membrane glycoprotein of 817 amino acids, located in the ER and catalyzing the transfer of mannose from the lipid carrier Dol-P-β-d-mannose to serine/threonine residues of specific protein acceptors (Strahl-Bolsinger and Tanner 1991; Strahl-Bolsinger et al. 1993; Gentzsch et al. 1995). As ScPmt1p, WsfD possesses an amino-terminal loop, a large hydrophilic loop and a carboxy-terminal region, all facing the ER or the external face of the cytoplasmic membrane, respectively (Strahl-Bolsinger and Scheinost 1999; Girrbach et al. 2000). We speculate that, although no mannose residue was found in the S-layer O-glycan of P. alvei CCM 2051T, WsfD could be involved in the transfer of a hexose residue from a lipid carrier to the glycan chain.

wsfE

The 364-amino acid translation product of wsfE shows high similarity to several glycosyltransferases of various Clostridium, Pseudomonas and Vibrio strains (Nölling et al. 2001; Chen et al. 2003; Gross et al. 2007), and it contains a potential glycosyltransferase group 1 motif (GT1_wbuB_like). In E. coli, WbuB is involved in the biosynthesis of the O26 O-antigen, thereby acting as an N-acetyl-l-fucosamine (l-FucNAc) transferase (D'Souza et al. 2002).

rmlA, rmlC, rmlB, and rmlD

The rmlACBD gene products show a high degree of amino acid homology to the RmlACBD proteins involved in the biosynthesis of dTDP-l-rhamnose in different Geobacillus strains (Novotny, Schäffer et al. 2004; Zayni et al. 2007) and in other bacteria (Graninger et al. 2002). Since the adaptor oligosaccharide of the S-layer O-glycan of P. alvei CCM 2051T contains three l-rhamnose residues, it is conceivable that RmlACBD are providing the nucleotide-activated building block dTDP-l-rhamnose. The putative start codon of rmlD overlaps with the stop codon of rmlB.

wsfF and wsfG

The stop codon of wsfF overlaps with the putative start codon of wsfG. The protein products of these two ORFs possess a potential glycosyltransferase family 2 motif in their amino-terminal parts. The deduced protein sequence of WsfF shows significant homology to the putative sugar transferase WsdG of Aneurinibacillus thermoaerophilus DSM 10155/G+- and to different rhamnosyltransferases (Novotny, Pföstl et al. 2004). The protein encoded by wsfG is highly similar to the β 1,2-rhamnosyltransferase WsaF of Geobacillus stearothermophilus NRS 2004/3a, transferring an l-rhamnose residue to the linkage sugar galactose during S-layer glycan biosynthesis in this strain (Steiner et al. 2008, 2010). For WsfG, a single transmembrane-spanning domain was predicted at the carboxy-terminal part of the protein.

wsfP

The UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WsfP has been recently identified as the initiation enzyme of S-layer glycan biosynthesis in P. alvei CCM 2051T (Zarschler et al. 2009).

wsfH

The predicted protein WsfH shows high similarity to several glycosyltransferases found in different cyanobacteria (Kaneko et al. 2007; Welsh et al. 2008). A cytoplasmic glycosyltransferase family 2 motif and a single transmembrane-spanning domain were predicted at the amino- and carboxy-terminal part of the protein, respectively. Since WsfH also shares amino acid similarity with several polyprenyl-phosphate β-d-glucosyltransferases, it is likely that it is responsible for the intracellular transfer of a glucose residue to the membrane-associated lipid carrier undecaprenyl-pyrophosphate.

pcrP

The region downstream of wsfH contains a putative ρ-independent bacterial terminator followed by a putative promoter site allowing the transcription of an ORF coding for PcrB, an enzyme involved in lipid/lipoteichoic acid biosynthesis (Chen et al. 1993). This protein is not part of the slg gene cluster.

S-layer protein glycosylation in slg gene mutant backgrounds

To investigate the role of individual ORFs of the P. alvei CCM 2051Tslg gene cluster, insertional knockout mutants in nine ORFs were constructed. All mutants still produced the S-layer protein SpaA as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining (Figure 4). However, for SpaA produced in mutants with an insertion of Ll.LtrB in the wsfA, wsfB, wsfC, wsfE, wsfF, wsfG or wzt ORF, only a single protein band corresponding to the non-glycosylated S-layer protein could be detected on SDS-PA gels. The absence of glycosylation was corroborated by mass spectrometry (MS) analysis as described below. These findings suggest that mutations of the wsfA, wsfB, wsfC, wsfE, wsfF, wsfG and wzt ORFs had significant effects on SpaA glycosylation in P. alvei CCM 2051T. In contrast, knockout mutants in the wsfD and wsfH ORFs resulted in the production of glycosylated SpaA protein of similar mobility as that produced from wild-type cells. As described previously, insertion in the wsfP gene resulted in a glycosylation-deficient phenotype (Zarschler et al. 2009).

Fig. 4

Effect of insertional inactivation of ORFs from the P. alvei CCM 2051Tslg gene cluster. An aliquot of biomass (200 µg) from various P. alvei CCM 2051T mutant strains was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue G 250 staining. The mutant strains carry the Ll.LtrB insertion as indicated. Tri-banded appearance corresponds to non-glycosylated (N), monoglycosylated (M) and di-glycosylated (D) chimeric SpaA.

Fig. 4

Effect of insertional inactivation of ORFs from the P. alvei CCM 2051Tslg gene cluster. An aliquot of biomass (200 µg) from various P. alvei CCM 2051T mutant strains was analyzed by SDS-PAGE followed by Coomassie Brilliant Blue G 250 staining. The mutant strains carry the Ll.LtrB insertion as indicated. Tri-banded appearance corresponds to non-glycosylated (N), monoglycosylated (M) and di-glycosylated (D) chimeric SpaA.

To ensure that no polar effects on downstream gene expression caused by the intron insertion as described by Rodriguez et al. (2008) had occurred, but rather the inactivation of the target gene itself is responsible for the lack of S-layer glycosylation in the wsfA, wsfB, wsfC, wsfE, wsfF, wsfG or wzt mutants, the mRNA of the wsfE::Ll.LtrB mutant was analyzed for such effects. Using the reverse transcriptase polymerase chain reaction (RT-PCR) approach described above, no differences between wild-type and wsfE::Ll.LtrB cells could be observed, indicating, that despite the intron insertion the polycistronic mRNA is not interrupted (data not shown).

Structural characterization of S-layer glycans produced in slg gene mutant backgrounds

The elucidation of the glycan structures linked to the S-layer protein SpaA was accomplished by mass spectrometry of peptides derived from pronase digestion of S-layer glycoproteins isolated from different slg gene mutants. While in the S-layer protein fraction of the wsfA, wsfB, wsfC, wsfE, wsfF, wsfG and wzt mutants no glycans could be detected, the glycopeptides of the SpaA protein produced in either wsfD or wsfH mutant strains yielded tandem mass spectroscopy (MS/MS) results identical to that of the S-layer glycopeptides from the wild-type strain (Figure 5). The online MS/MS spectrum of the SpaA glycopeptides of wild-type P. alvei CCM 2051T confirmed the already known branched structure of the repeating units (Figure 5A). The three peaks at 528, 1055 and 1582 Da represent one, two or three repeating units of two hexoses and one N-acetylhexosamine residue each. Due to the collision energy chosen for the MS experiment, small fragments consisting of only a few S-layer glycan repeating units were obtained, which differs from the native long-chain S-layer glycan. The corresponding online MS/MS spectra of the wsfD::Ll.LtrB (Figure 5B) and the wsfH::Ll.LtrB (not shown) mutants showed four peaks at 366, 731, 1097 and 1462 Da, which are consistent with one to four repeating units containing one hexose and one N-acetylhexosamine residue. This observation indicates the absence of the α1,6-linked glucose residues of the repeating units in the glycosylated peptides of SpaA protein produced in wsfD::Ll.LtrB and wsfH::Ll.LtrB mutant strains, thus indicating the involvement of WsfD and WsfH in the glucosylation of the N-acetylmannosamine residues of the repeating units.

Fig. 5

Mass spectrometry of glycopeptides. Glycopeptides derived from pronase digestion of SpaA protein produced in P. alvei CCM 2051T wild-type (A) and wsfD::Ll.LtrB mutant strain (B) were analyzed by online MS/MS, showing the constitution of the different repeating units of the wild-type and the mutant strains. The spectrum of wsfH::Ll.LtrB mutant strain is identical to that observed for the wsfD::Ll.LtrB mutant strain and hence not shown. Please note that, to keep a constant scale in either mass spectrum, for the mutant strain, one more repeating unit is shown to account for the fact that in each unit one hexose is missing.

Fig. 5

Mass spectrometry of glycopeptides. Glycopeptides derived from pronase digestion of SpaA protein produced in P. alvei CCM 2051T wild-type (A) and wsfD::Ll.LtrB mutant strain (B) were analyzed by online MS/MS, showing the constitution of the different repeating units of the wild-type and the mutant strains. The spectrum of wsfH::Ll.LtrB mutant strain is identical to that observed for the wsfD::Ll.LtrB mutant strain and hence not shown. Please note that, to keep a constant scale in either mass spectrum, for the mutant strain, one more repeating unit is shown to account for the fact that in each unit one hexose is missing.

Discussion

In this study, we identified the slg gene cluster of the mesophilic, Gram-positive bacterium P. alvei CCM 2051T encoding the O-glycosylation of tyrosine residues of the S-layer protein of this organism. The sequenced +AH4-24.3-kb region contains 18 ORFs, of which the derived Wsf protein sequences show homology to proteins involved in the biosynthesis of different bacterial surface polysaccharides, such as LPS, exopolysaccharides and capsule polysaccharides. Both the observed similarity of the Wsf proteins with database entries of enzymes involved in bacterial polysaccharide biosyntheses as well as the disruption of the corresponding ORFs gave first insights into the S-layer glycan biosynthesis pathway of P. alvei CCM 2051T (Figure 6).

Fig. 6

Working model of S-layer glycan biosynthesis in P. alvei CCM 2051T. The initial transfer of a Gal residue from UDP-α-d-Gal to a lipid carrier is catalyzed by WsfP (A). The adaptor saccharide is formed by the α1,3-linkage of an l-Rha residue from dTDP-β-l-Rha to the linkage sugar d-Gal possibly performed by WsfG, followed by the transfer of two additional α1,3-linked l-Rha residues possibly by the action of WsfF (B). The glycan chain would be elongated by the activity of the aminosugar transferase WsfE and the tripartite transferase WsfC. WsfE may form the β1,4-linkage of a ManNAc residue from UDP-ManNAc to the third rhamnose residue. WsfC putatively adds a single glycerol phosphate from CDP-glycerol to the ManNAc residue of the adaptor oligosaccharide and may form the β1,3-linkage of a ManNAc residue to the third rhamnose residue as well as the β1,4-linkage of a Gal to the ManNAc residues of the repeating units. The glycan chain would be recognized by the carboxy-terminal part of Wzt and exported by the ABC transporter system through the cytoplasmic membrane (C). The transfer of cytoplasmic Glc to the lipid carrier would be carried out by WsfH and, after reorientation, is used at the external face of the cytoplasmic membrane by WsfD for α1,6-linkage of the Glc residues to ManNAc residues of the repeating units (D). The final transfer of the completed S-layer glycan to certain tyrosine residues of the S-layer protein is predicted to occur co-secretionally upon catalysis of the O-OTase WsfB (E). Eventually, the mature S-layer glycoprotein would be self-assembled at the cell surface (F). Please note that so far only the WsfP protein has been experimentally verified to perform its predicted role.

Fig. 6

Working model of S-layer glycan biosynthesis in P. alvei CCM 2051T. The initial transfer of a Gal residue from UDP-α-d-Gal to a lipid carrier is catalyzed by WsfP (A). The adaptor saccharide is formed by the α1,3-linkage of an l-Rha residue from dTDP-β-l-Rha to the linkage sugar d-Gal possibly performed by WsfG, followed by the transfer of two additional α1,3-linked l-Rha residues possibly by the action of WsfF (B). The glycan chain would be elongated by the activity of the aminosugar transferase WsfE and the tripartite transferase WsfC. WsfE may form the β1,4-linkage of a ManNAc residue from UDP-ManNAc to the third rhamnose residue. WsfC putatively adds a single glycerol phosphate from CDP-glycerol to the ManNAc residue of the adaptor oligosaccharide and may form the β1,3-linkage of a ManNAc residue to the third rhamnose residue as well as the β1,4-linkage of a Gal to the ManNAc residues of the repeating units. The glycan chain would be recognized by the carboxy-terminal part of Wzt and exported by the ABC transporter system through the cytoplasmic membrane (C). The transfer of cytoplasmic Glc to the lipid carrier would be carried out by WsfH and, after reorientation, is used at the external face of the cytoplasmic membrane by WsfD for α1,6-linkage of the Glc residues to ManNAc residues of the repeating units (D). The final transfer of the completed S-layer glycan to certain tyrosine residues of the S-layer protein is predicted to occur co-secretionally upon catalysis of the O-OTase WsfB (E). Eventually, the mature S-layer glycoprotein would be self-assembled at the cell surface (F). Please note that so far only the WsfP protein has been experimentally verified to perform its predicted role.

Seven ORFs located in the slg gene cluster are involved in the biosynthesis of nucleotide-activated monosaccharides. Next to galE converting UDP-glucose to UDP-galactose, galU transferring UTP to glucose-1-phosphate, resulting in UDP-glucose, is present. While tagD is involved in the formation of CDP-glycerol, the four rml genes code for the biosynthesis of dTDP-l-rhamnose. Since the S-layer glycan contains glucose, galactose, phosphoglyceric acid and rhamnose, the presence of these ORFs in the slg gene cluster is not surprising. However, no ORFs for the biosynthesis of nucleotide-activated N-acetylmannosamine are located in the slg gene cluster. This observation confirms the assumption that housekeeping genes are additionally required for S-layer glycan biosynthesis (Novotny, Schäffer et al. 2004).

As recently described and depicted in Figure 6A, the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WsfP acts as the initiation enzyme of S-layer glycan biosynthesis in P. alvei CCM 2051T (Zarschler et al. 2009).

The slg gene cluster encodes two rhamnosyltransferases (WsfF and WsfG) which are obviously involved in the assembly of the l-rhamnose-containing adaptor saccharide of the S-layer O-glycan, with WsfG transferring an l-rhamnose residue onto the linkage sugar galactose and WsfF acting as an α-l-rhamnose-1,3-α-l-rhamnosyltransferase (Figure 6B). Since WsfE is related to the aminosugar transferase WbuB, it might be responsible for the addition of an N-acetylmannosamine residue to the growing glycan chain (Figure 6C). Based on the observed similarity with the database, the proposed function of the tripartite transferase WsfC might be the transfer of a single glycerol phosphate from CDP-glycerol to the N-acetylmannosamine residue of the adaptor oligosaccharide and of a galactose residue to the N-acetylmannosamine residues of the repeating units. Since this enzyme exhibits a third transferase domain, it may also catalyze the reaction from glycerol phosphate to 2-phospho glyceric acid (Figure 6C).

The S-layer glycoproteins of the mutant strains wsfD::Ll.LtrB and wsfH::Ll.LtrB show identical migration behavior in SDS-PAGE compared to wild-type cells. MS analysis of the S-layer O-glycan of both mutants showed the lack of glucose residues being part of each repeating unit of the mature glycan, suggesting that both enzymes, WsfD and WsfH, are involved in the process of glucose addition to the glycan chain. Due to the similarity of WsfD to fungal Pmts and of WsfH to several polyprenyl-phosphate β-d-glucosyltransferases, we assume that WsfH transfers a glucose residue to undecaprenyl-pyrophosphate at the inner face of the cytoplasmic membrane, the lipid carrier is then re-orientated to the external face of the cytoplasmic membrane, and WsfD adds the glucose residue to the exported glycan chain (Figure 6D).

The identification of an ABC transporter system (Wzm and Wzt) and the loss of S-layer glycosylation in the wzt::Ll.LtrB mutant corroborate the assumption of an ATP hydrolysis-driven export of the undecaprenyl-pyrophosphate-linked glycan chain to the external face of the cytoplasmic membrane comparable to the ABC-transporter-dependent pathway of the LPS O-polysaccharide biosynthesis. According to this pathway, glycan chain extension is achieved by processive addition of sugar residues to the nonreducing terminus of the undecaprenyl-pyrophosphate-linked growing chain. Although not yet detected in P. alvei CCM 2051T, nonreducing terminal modifications, such as 2-O-methyl groups, were described as chain length termination signal recognized by the carboxy-terminal domain of Wzt (Cuthbertson et al. 2007). The polymer is then exported through the cytoplasmic membrane by the ABC transporter for ligation, independent of the presence of a Wzx flippase or Wzy polymerase homolog (Whitfield 1995; Raetz and Whitfield 2002). After the addition of glucose to the N-acetylmannosamine residue of each repeating unit by WsfD, the completed glycan chain would be transferred from the lipid carrier to specific tyrosine residues of SpaA by the O-OTase WsfB, possibly upon export of the S-layer protein across the cytoplasmic membrane (Figure 6E). This conclusion is based on the presence of the conserved Wzy_C motif in WsfB and on the similar transmembrane topology of the well characterized OTases WaaL, PglL and PilO, as well as on the loss of S-layer glycosylation in the wsfB::Ll.LtrB mutant. Eventually, the mature S-layer glycoprotein would be self-assembled at the cell surface (Figure 6F).

The role of WsfA in S-layer glycosylation biosynthesis remains still unclear. Since the wsfA::Ll.LtrB mutant shows an altered S-layer migration in SDS-PAGE due to the loss of the SpaA-linked glycan chains, its involvement in the glycosylation process is evident but needs to be further investigated.

Although several putative bacterial promoters and terminators have been identified by different prediction programs, most of the ORFs, namely those coding for GalU, Wzm, Wzt, WsfA, TagD, WsfCDE, RmlACBD and WsfFGPH, are transcribed by a single polycistronic mRNA. However, the UDP-glucose 4-epimerase GalE and the OTase WsfB are transcribed independently. A common phenomenon of bacterial polysaccharide biosynthesis gene clusters is the low % G C content compared to the respective bacterial genome as a whole (Novotny, Pföstl et al. 2004; Messner et al. 2008). For individual ORFs of the described slg gene cluster, the % G C content ranges between 25% and 43%, whereas it is 44.6% or 46.2% for the rest of the genome of P. alvei CCM 2051T, depending on the method of its determination (Claus and Berkeley 1986).

In conclusion, the current report describes the identification, annotation and characterization of the slg gene cluster of P. alvei CCM 2051T involved in tyrosine O-glycosylation of the S-layer protein of this organism. Considering the documented chemical stability of this rare O-glycosidic linkage type (Kolbe 1993), these data mark a starting point for further studies and applications in conjunction with the unique self-assembly feature of an S-layer protein matrix. This may lead to the future design of functional glycans and their controlled surface display for exerting biological activity in various settings.

Materials and methods

Bacterial strains and growth conditions

Bacterial strains, plasmids and primers are listed in Tables SI and SII. P. alvei CCM 2051T was obtained from the Czech Collection of Microorganisms (CCM, Brno, Czech Republic) and was cultivated at 37°C and 200 rpm in Luria–Bertani (LB) broth or on LB agar plates supplemented with 10 μg/ml chloramphenicol (Cm), when appropriate. E.coli DH5α (Invitrogen, Lofer, Austria) was grown in LB broth at 37°C supplemented with 30 μg/ml Cm, when appropriate.

Analytical and general methods

Genomic DNA of P. alvei CCM 2051T was isolated as described recently (Zarschler et al. 2009). Restriction and cloning enzymes were purchased from Invitrogen. The MinElute gel extraction kit (Qiagen, Vienna, Austria) was used to purify DNA fragments from agarose gels, and the MinElute reaction cleanup kit (Qiagen) was used to purify digested oligonucleotides and plasmids. Plasmid DNA from transformed cells was isolated with the Plasmid Miniprep kit (Qiagen). Agarose gel electrophoresis was performed as described elsewhere (Sambrook et al. 1989). Transformation of E. coli DH5 α was done according to the manufacturer’s protocol (Invitrogen). Transformants were screened by in situ PCR using RedTaq ReadyMix PCR mix (Sigma-Aldrich, Vienna, Austria), and recombinant clones were analyzed by restriction mapping and confirmed by sequencing (Agowa, Berlin, Germany). Transformation of P. alvei CCM 2051T was performed as described recently (Zarschler et al. 2009). SDS-PAGE was carried out according to a standard protocol (Laemmli 1970) using a Protean II electrophoresis apparatus (Bio-Rad, Vienna, Austria). Protein bands were visualized with Coomassie Brilliant Blue G 250 staining reagent. The isolation and purification of S-layer glycoprotein essentially followed published methods (Messner and Sleytr 1988).

PCR and DNA sequencing

Primers for PCR and DNA sequencing were purchased from Invitrogen, and PCR conditions were optimized for each primer pair (Table SII). PCR was performed using the Herculase ® II Fusion DNA Polymerase (Stratagene, La Jolla, CA) and the thermal cycler My CyclerTM (Bio-Rad). Amplification products were purified using the MinElute PCR purification kit (Qiagen). For the identification of the rml genes responsible for dTDP-l-rhamnose biosynthesis, the highly conserved seven amino acid stretch TDYVFDG of RmlD (dTDP-4-dehydrorhamnose reductase) was used for the design of the degenerate oligonucleotide primer proof_RmlD_for. For sequence determination of the slg gene cluster, chromosome walking was applied as previously described (Kneidinger et al. 2001; Pilhofer et al. 2007).

RT-PCR

Total RNA was extracted from P. alvei CCM 2051T using the RNeasy Protect Bacteria Mini Kit (Qiagen) and subsequently treated with RNase-free DNase I (Fermentas, St. Leon-Rot, Germany) to remove DNA contamination. First strand cDNA was synthesized utilizing the Revert AidTM Premium Reverse Transcriptase (Fermentas) according to the manufacturer’s instructions using a reverse primer specific for wsfB (1f), wsfA (4r), wsfF (8r) or pcrB (9r) (Table SII). After termination of the reaction by heating at 85°C for 5 min, one tenth of each cDNA reaction mixture was used as template for PCR using the Phusion™ High-Fidelity DNA Polymerase (New England Biolabs, Frankfurt/Main, Germany). PCR reactions were carried out with primer pairs annealing to wsfB/galE (1f/1r), galU/wzm (2f/2r), wzt/wsfA (3f/3r), wsfA (4f/4r), wsfC/wsfD (5f/5r), wsfD/wsfE (6f/6r), rmlB/wsfF (8f/8r) and wsfH/pcrB (9f/9r) (Table SII). As a positive control, genomic DNA was used, whereas DNase I-treated RNA without the cDNA-generating step served as a control for contamination of total RNA with chromosomal DNA. PCR products were analyzed by agarose gel electrophoresis.

Sequence analysis

Nucleotide and protein sequences were analyzed using the BLASTN and BLASTP sequence homology analysis tools (National Center for Biotechnology Information, Bethesda, MD). Open reading frames in the DNA sequence were identified by using the Clone Manager Professional Suite (SECentral, Cary, NC) and the ORF Finder analysis tool (National Center for Biotechnology Information). The TMHMM Server v. 2.0 transmembrane prediction program and the SignalP 3.0 Server (Center for Biological Sequence Analysis, Technical University of Denmark, Lyngby, Denmark) were used to identify putative protein transmembrane-spanning domains and the presence and location of signal peptide cleavage sites, respectively. The guanine-cytosine (G+-C) content of the entire slg gene cluster was determined using the GC Content and GC Skew program (Nano+Bio-Center, University of Kaiserslautern, Germany). For in silico reverse translation, the Sequence Manipulation Suite was used (Stothard 2000). Bacterial promoters, transcriptional terminators, operons and ORFs were predicted by the BProm and FindTerm modules of the FGenesB gene prediction program in Molquest software (SoftBerry Inc., Mount Kisco, NY). The presence of conserved motifs in a given protein sequence was analyzed by the Pfam protein families database (Finn et al. 2008). Physical and chemical parameters for a given protein were calculated using the ProtParam tool (Gasteiger et al. 2005).

Gene knockout

Specific disruption of nine ORFs located in the slg gene cluster of P. alvei CCM 2051T was performed as described recently (Zarschler et al. 2009). The Ll.LtrB targetron of pTT_wsfP1176 was retargeted prior to transformation into P. alvei CCM 2051T. For this purpose, identification of potential insertion sites and design of PCR primers for the modification of the intron RNA was accomplished by a computer algorithm (www.Sigma-Aldrich.com/Targetronaccess). For each ORF, insertion sites were chosen based on their location and intron insertion efficiency, and modifications of the intron RNA sequences were introduced via PCR by primer-mediated mutation (Table SIII). The retargeted Ll.LtrB targetron was subsequently digested with HindIII and BsrGI and ligated into pTT_wsfP1176 digested with the same restriction enzymes, thereby replacing the wsfP targetron. Creation of P. alvei CCM 2051T gene knockout mutants and confirmation of intron insertion was achieved as described (Zarschler et al. 2009). All mutant strains were analyzed for the migration behavior of the SpaA (glyco)protein on SDS-PA gels (Altman et al. 1995). Glycan structures linked to the S-layer protein SpaA of P. alvei CCM 2051T wild-type and mutant cells were analyzed by mass spectrometry.

Protein elution from SDS-PAGE and pronase digest

Aliquots of biomass were run on a 10% PA gel and stained with Coomassie Brilliant Blue G 250. Relevant SDS-PA gel bands were excised from the gel and destained (Stadlmann et al. 2008). Destained gel pieces were minced into small particles and loaded onto a BioRad Model 422 electro eluter. After elution from the gel, the protein was dialyzed against 10 mM ammonia formate buffer and precipitated using five volumes of acetone (−20°C, 1 h). Two hundred microliters of 0.15 M Tris-HCl buffer, pH 7.8 (containing 1 mM CaCl2 and 0.02% NaN3) was added to the dried protein, followed by proteolytic digestion using 1 μg pronase for 24 h at 37°C. Subsequently, an additional amount of 1 µg pronase was added, and incubation was continued for 24 h.

Glycopeptides (+AH4-10 μg of gel-eluted material) were enriched using a PGC-SPE cartridge (10 mg; Fisher Scientific, Vienna, Austria) according to a published protocol (Packer et al. 1998), except for the use of 150 mM ammonia formate buffer, pH 9.0, in 60% acetonitrile for elution. Prior to liquid chromatography (LC)-MS analysis, the samples were dried under vacuum and dissolved in 20 μl of distilled water.

Glycopeptide LC-electrospray ionization-MS/MS

Analysis of glycopeptides was performed by positive-ion LC-electrospray ionization (ESI)-MS/MS using a 50 × 0.32 mm porous graphitic carbon column (Thermo) (Pabst and Altmann 2008). A flow rate of 5 μl was maintained with a Dionex Ultimate 3000 cap flow system using 150 mM ammonia formate buffer (pH 9) as solvent A and acetonitrile as solvent B.

Glycopeptides were eluted from the separation column using a gradient from 5% to 45% of solvent B over 25 min. Analysis was carried out with a Waters Q-TOF Ultima Global mass spectrometer with standard ESI-source and a MassLynx V4.0 SP4 software for evaluation of obtained peaks. For online MS/MS experiments, the CE was set to 72 with a LM Res of 8 and a HM Res of 11. Capillary voltage, MS profile, cone voltage, RF LENS 1 setting and ESI probe adjustment were optimized to gain maximum signal intensity.

Supplementary Data

Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.

Financial support came from the Austrian Science Fund FWF, project P20745-B11 (to P.M.) and projects P19047-B12 and P21945-B20 (to C.S.), and the Hochschuljubiläumsstiftung der Stadt Wien, project H-02229-2007 (to K.Z.).

Conflict of interest statement

None declared.

Abbreviations

  • ABC

    ATP binding casstte

  • AsnB

    asparagine synthetase B

  • CDP

    cytidine diphosphate

  • Cm

    chloramphenicol

  • CTP

    cytidine triphosphate

  • ER

    endoplasmic reticulum

  • ESI

    electrospray ionization

  • G+-C

    guanine-cytosine

  • LB

    Luria–Bertani

  • LPS

    lipopolysaccharide

  • MS

    mass spectrometry

  • MS/MS

    tandem mass spectrometry

  • O-OTase

    O-oligosaccharyl:protein transferase

  • ORF

    open reading frame

  • PMTs

    dolichyl-phosphate-mannose-protein mannosyltransferases

  • SDS-PAGE

    sodium dodecyl sulfate polyacrylamide gel electrophoresis

  • TPR

    tetratricopeptide repeat

  • UDP

    uridine diphosphate

  • UTP

    uridine triphosphate

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Author notes

4
Present address: Institute of Genetics, General Genetics,Technische Universität Dresden, Zellescher Weg 20b, D-01217 Dresden, Germany; e-mail: christina.schaeffer@boku.ac.at