Glycobiology

Strains

H.halobium (DSM 670) was grown in 'complex medium" as described by Sumper and Herrmann, 1978. Escherichia coli strain DH5[alpha] was used for transformation experiments.

Chemicals and enzymes

TPCK-Trypsin was from Sigma (Deisenhofen, Germany).Mevinolin was extracted from mevinacor^® pellets (MSD Sharp & Dohme GmbH, München) containing 20 mg Lovastatin. Restriction enzymes and enzymes necessary for subcloning and sequencing were purchased from Boehringer Mannheim, Pharmacia (Uppsala), and MBI Fermentas (Vilnius). All other chemicals of analytical grade were obtained from Merck (Darmstadt).

Plasmids

pUC8-15 is a vector previously isolated from a halobacterial genomic library in our laboratory (Lechner et al., 1987) containing a 15 kb insert with the complete csg gene and EcoRI flanking sites. pWL-102 is a shuttle vector developed by Lam and Doolittle (1989) that was kindly provided by Dr. F. Pfeifer (TH Darmstadt).

Synthesis of oligonucleotides

Following oligonucleotides were synthesized with a Oligo 1000 DNA-synthesizer (Beckmann Instruments, Fullerton, CA). The number of the nucleotide position is related to the first nucleotide of the respective open reading frame.

Oligos from the csg gene for the csgX4 mutants

ONG4: 5[prime]GCG GCG AAT GCA gGa GAt CTG AAC GAT TAT CAG (100-132) replacing A112 for G, C114 for A and C117 for T and thereby introducing a new cutting site for BglII; ONV4: 5[prime]GCG GCG AAT GCA gtC GAC CT (100-119) replacing A112 for G and G113 for T and thereby introducing a new cutting site for SalI; ONL4: 5[prime]GCG GCG AAT GCA ttg GAC CTG AAC GAT TAT CAG (100-132) replacing A112 for T, G113 for T and C114 for G and thereby introducing a new cutting site for AvaIII; ONN4: 5[prime]GCG GCG AAT GCA AaC GAt CTG AAC GAT TAT CAG (100-132) replacing G113 for A and C117 for T and thereby introducing a new cutting site for MboI; ONI4: 5[prime]GCG GCG AAT GCA AtC GAt CTG AAC (100-123) replacing G113 for T and C117 for T and thereby introducing a new cutting site for ClaI; ON2: 5[prime]C CAG CTT CCG GAA GGT AAC GT (anti-sense, 236-256).

Oligonucleotides from the csg gene for making the csgV481 mutant

ON61: 5[prime]T GAG AAA TCG ATC GAA GTC GA (1395-1415); ON62: 5[prime]ACC Gac CGA GTT aAC GGC GTC GCT CTT (anti-sense, 1522-1548) replacing G1536 for A, A1543 for C and G1544 for A; ON63: 5[prime]GCC GTt AAC TCG gtC GGT GGC GTG (1531-1554) replacing C1536 for T, T1543 for G and C1544 for T and thereby introducing a new cutting site for HpaI; ON64: 5[prime]CTG TCG GAG CTC CGA GAT GTC (anti-sense, 1810-1830).

ON15U2: 5[prime]TTC GAG TCC GGG AGG CGG TTT A; ON3: 5[prime]CCG CAC CTG TGG GTC GCG T (1328-1346) of the HMG-CoA reductase gene responsible for the mevinolin resistance carried by the pWL-102 plasmid as described by Lam and Doolittle (1992).

Polymerase chain reaction for generation of mutagenized DNA fragments

Polymerase chain reaction was done in 100 µl containing 1.5 mM MgCl₂, 50 mM KCl, and 10 mM Tris-HCl, pH 8.3, 200 µM of dNTPs, 100 pmol of each oligonucleotide, varying amounts of template and 2.5 U of Taq-Polymerase with a Perkin-Elmer Cetus Thermal Cycler. Not more than 20 cycles were performed with denaturation at 94°C for 30 s, annealing at 66°C for 30 s, and extension at 72°C for 1 min. Plasmids were used as templates for generating fragments for the mutagenesis vectors.

Sequencing of PCR-products

For sequencing of PCR-products, fragments were separated on a preparative agarose gel, electroeluted, treated with T4-DNA-polymerase, phosphorylated and finally ligated into a SmaI and calf intestine phosphatase treated pUC18 vector. Sequencing of the inserts was performed by the dideoxy chain termination method (Sanger et al., 1977) using ³⁵S-labelled [alpha]-thio-dATP (Amersham).

Plasmid construction

DNA was cut according to the procedure given by the manufacturer and fragments purified by electroelution. Ligation, transformation, and preparation of plasmid from E.coli strains by the alkaline method was done according to Sambrook et al. (1989). Correct construction of the plasmids was confirmed by restriction analysis.

Construction of mutagenized csg genes with an exchange for Ser-4

Respective PCR reactions were performed with the oligonucleotide ONG4, ONV4, ONL4, ONN4, and ONI4, respectively as sense primer together with the antisense primer ON2 covering the Kpn2I cutting site. pUC8-15 containing the complete csg gene (Lechner and Sumper, 1987) served as template. The resulting 156 bp DNA-fragments were cloned into pUC18 vector and sequenced. A 5.0 kb SmaI-fragment derived from pUC8-15 containing the complete csg gene was cloned into pUC18 (pUC18-csg). From pUC18-csg a EcoRI-ApaI fragment was subcloned into pBluescript SK vector (pB-csg). Finally the 156 bp BsmI-Kpn2I fragment was replaced by a mutated fragment resulting in pB-csgG4, pB-csgV4 etc. (commonly denoted pB-csgX4). Substitution of the EcoRI-ApaI fragment of pUC18-csg with that of pB-csgX4 resulted in pUC18-csgX4 containing a 5 kb fragment with the complete csg gene carrying the desired mutation and about 1 kb 5[prime] and 3[prime] flanking regions.

Construction of the mutagenesis vector pRZ-csgX4

The shuttle vector pWL-102 (Lam and Doolittle, 1989) carrying the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene mediating mevinolin resistance as well as the pHV2 region for autonomous replication in halobacteria and the ampicillin resistance gene for cloning in E.coli cells was used. The 5 kb BamHI-KpnI fragment of pUC18-csgX4 was inserted into the BamHI-KpnI cloning site of pWL-102 resulting in pRZ-csgX4. Correct arrangement of the vector was analyzed by PCR.

Construction of the mutagenesis vector pRZ-csgV481

Two recombinant PCR-fragments, generated with ON61/ON62 and ON63/ON64 from pUC8-15, were used as template for amplifying a recombinant DNA-fragment with ON61 and ON64 carrying a codon which replaces Ser-481 for Val-481. This fragment was exchanged for the ClaI (1401)-SacI (1819) fragment of pUC18-csg which was finally used for construction of pRZ-csgV481 using the same procedures as for pRZ-csgX4.

Transformation of H.halobium

Transformation of H.halobium was done as described by Cline and Doolittle (1987). Two milliliters of H.halobium cells (absorbance at 578 nm: 0.300 ) were pelleted and resuspended in 180 µl of spheroblasting solution (2 M NaCl, 27 mM KCl, 50 mM Tris-HCl, pH 8.75, 15% sucrose). Spheroblasts were prepared by addition of 20 µl EDTA (pH 8.75) and incubation for at least 15 min at room temperature. Then 2 µg of mutagenesis vector was added. After 5 min, the transformation sample was gently mixed with an equal volume of 60% PEG 600 and incubated for a further 20 min at room temperature. Transformed bacteria were diluted with 1.6 ml basal salt solution (4.3 M NaCl, 80 mM magnesium sulfate, 10 mM trisodium citrate, 27 mM KCl, 1.4 mM calcium chloride, 50 mM Tris-HCl, pH 7.2, 15% sucrose) pelleted and resuspended in 2 ml regeneration medium (basal salt solution plus 0.3% yeast extract and 0.5% peptone from Difco). After incubation at 37°C with shaking over night aliquots of the transformed bacteria were plated onto agar plates containing 20 µM mevinolin. Growth of transformants was usually detected after 8 days. At this stage cells were streaked out onto new mevinolin plates and screened for correct vector integration by Southern blot analysis or PCR with subsequent analysis of additional restriction sites.

Isolation of recombinants from transformants

Mevinolin resistant transformants were grown at 37°C on plates without mevinolin. Single colonies from these plates were streaked out onto plates with mevinolin as well as onto plates without mevinolin and examined after 3 days. Bacteria unable to grow on mevinolin containing plates were finally subjected to Southern blot analysis or diagnostic restriction analysis to confirm the mutated genotype.

Southern blot analysis

Isolation of total halobacterial DNA from 1 ml cultured cells was performed as described previously (Lechner and Sumper, 1987; Sambrook et al., 1989). After electrophoresis on an 1% or 0.6% agarose gel, the DNA was denatured, neutralized, and transferred to a nylon membrane (Hybond N from Amersham Life Sciences, Braunschweig) by vacuum blotting using a VacuBlot apparatus (Pharmacia, Uppsala). Transferred DNA was immobilized by UV and hybridized with ³⁵S-labeled DNA fragments. Labeling of the DNA fragments was performed with random hexanucleotide primers and Klenow polymerase (Feinberg and Vogelstein, 1983, 1984). Hybridization conditions were as follows: 0.1% SDS, 5× Denhardts solution, 5× SSPE, 50% formamide, 10 mM dithiothreitol, 100 µg of sonified salmon sperm DNA per ml hybridization solution. Prehybridization was done without dithiothreitol for 4 h at 42°C. Subsequently 1 × 10⁵ c.p.m. of the radioactive probe per cm² membrane were added and hybridization performed at 42°C for 16 h. The filters were washed two times with 0.2× SSC containing 0.1% SDS and incubated with shaking in the same solution for 1 h at 65°C.

Analysis of the genotypes by diagnostic restriction analysis

For screening of transformants and mutants with a PCR-based method ON15U2 was used as sense primer. ON15U2 corresponds to a sequence region closely upstream to the SmaI site which is located upstream to the csg gene sequence. As anti-sense primer ON2 was used for csgX4 mutants and ON64 was used for csgV481. PCR (30 cycles) was performed with the respective total genomic DNA. The resulting fragments were precipitated and subjected to digestion with the respective restriction endonucleases. Fragments were finally analyzed by agarose gel electrophoresis.

Purification of the N-terminal tryptic peptide

A tryptic glycopeptide fraction of high molecular weight was isolated from a halobacterial cell envelope fraction as described previously (Paul et al. 1986). The cell envelope fraction was incubated twice for 10 h with 2.5% (w/w) N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin (Sigma) in 100 mM Tris-HCl buffer, pH 7.5 containing 5 mM CaCl₂. After digestion the sample was passed through a column of AG 50W-X8 H⁺ ion exchange resin (Bio-Rad) in water. The eluate was concentrated and chromatographed on a Bio-Gel P-10 column. Fractions from the void volume and positive in the phenol-sulfuric acid assay (Dubois et al., 1956 ) were further purified on a reversed phase column (Lichrosorb RP 18, Merck) under the following conditions: a gradient of acetonitrile in 20 mM ammonium acetate was applied from 0 to 100% in 60 min at a flow rate of 1 ml/min. Peaks detected at 226 nm were collected and again tested with an uronic acid detection assay (Blumenkrantz et al., 1973).

Determination of total sugar composition

Total sugar composition was determined according to Lechner and Wieland (1992) after methanolysis and pentafluoropropionylation. Derivatized sugars were analyzed on a Finnigan MAT gc-ms apparatus equipped with a DB-1701 capillary column at 1 ml/min He gas flow.

Sequencing of peptides

Sequence analysis of peptides was performed by Edman degra-dation using an automated gas-phase peptide sequencer (Applied Biosystems Inc.)

LC-MS of tryptic peptides

Wild type CSG and CSGV481 were purified to homogeneity from the respective cell envelope preparations by the phenol extraction method. An aliquot was subjected to trypsin digestion at 37°C for 16 h and separated on a Vydac C18-column (250 mm, 0.8 mm) directly connected to an ES-MS (Finnigan MAT SSQ7000). Simultaneously absorbance at 220 nm was monitored. Eluent A was 0.05% TFA in water and Eluent B was 70% acetonitrile containing 0.05% TFA. After elution of unbound material with 10% B a linear gradient was applied for elution of peptides reaching 100% B in 1 h.