For Western blot analysis, samples were solubilized in SDS-PAGE loading buffer, heated to 100 C for 10 min, and subjected to SDS-PAGE. O-antigen production is usually cryptic in K-12, because of a mutation in the synthesis genes, it possesses a prophage glucosylation cluster, which modifies the GlcNAc residue in an -l-Rha-(13)-d-GlcNAc motif found in the original O16 antigen. ATCC 33257 produces an O-antigen possessing the same disaccharide motif, but its assembly uses an ABC transporter-dependent system. harboring the O-antigen biosynthesis genes produced an O-antigen displaying reduced reactivity toward antisera raised against the native repeat structure, indicative of an Arsonic acid altered chemical structure. Structural determination using NMR revealed Arsonic acid the addition of glucose side chains to the repeat units. O-antigen modification was dependent on a functional ABC transporter, consistent with modification in the periplasm, and was eliminated by deletion of the glucosylation genes from your chromosome, restoring native level antisera sensitivity and structure. There are therefore no intrinsic mechanistic barriers for bacteriophage-mediated O-antigen glucosylation in ABC transporter-dependent pathways. isolates can contain up to four glucosylation systems, modifying different parts of the O-antigen structures (8). Clearly, these modification processes have a significant impact on diversity. Open in a separate window Physique 1. Structures of the O-polysaccharide repeat models. The component sugars are glucose (Glc), 2-acetylamino-2-deoxyglucose (O serotypes. Even though activated initial donor of the glucose residue is usually UDP-glucose, the direct donor is usually undecaprenol phosphate-glucose (11,C14). Early studies established an association between glucosylation modification and lysogenic bacteriophages (13, 15), and the same is true in (5). It is now known that this modification reaction requires acquisition of only three genes. The GtrB enzyme synthesizes undecaprenol phosphate-Glc, and GtrA is the putative exporter Arsonic acid for the lipid-linked donor; the corresponding genes are conserved across glucosylation systems. A third gene (designated or K-12 glucosylation machinery encoded by prophage KplE1 (19) to modify target residues supplied by a heterologous ABC transporter-dependent O-antigen from ATCC 33257 (observe Fig. 2). This O-antigen is built up of repeat units made up of an -l-Rha-(13)-d-GlcNAc disaccharide, and this represents a motif that is modifiable by glucosylation systems in both K-12 and (observe Fig. 1). Open in a separate window Physique 2. Organization of the O-antigen biosynthesis genes in K-12 (GenBankTM accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_010473″,”term_id”:”170079663″,”term_text”:”NC_010473″NC_010473) and ATCC 33257 (GenBankTM accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AY376146″,”term_id”:”34863491″,”term_text”:”AY376146″AY376146), as well as the KplE1 prophage (also named CPS-53) in the K-12 genome, encoding the Arsonic acid three Gtr proteins required for glucosylation of O-antigens. Experimental Procedures Bacterial Strains and Growth Conditions Bacterial cultures were produced with aeration at 37 or 30 C in LB base (Invitrogen), supplemented with appropriate antibiotics (100 g ml?1 ampicillin, 34 g ml?1 chloramphenicol, 100 g ml?1 kanamycin). ATCC 33257 was a gift from U. Mamat (Research Centre Borstel, Leibnitz Centre for Medicine and Biosciences, Borstel, Germany). The strains used in this study are derivatives of strain TOP10 (F?, Top10 to produce CWG1217 (and genes (recognized by lowercase); the primer sequences were 5-acgaataaattatctttaagaagaaacgttatatatctggctgtcgttcaGTGTAGGCTGGAGCTGCTTC-3 and 5-atgttcttcagtaataaaattaactagttcatcaaacccaactaatacatCATATGAATATCCTCCTTAG-3. Electrocompetent cells of Top10 made up of pSIM6 (22) were transformed with the linear PCR product, and after recovery overnight with aeration at 30 C, mutants were selected on LB agar made up of 15 g ml?1 kanamycin grown for 2 days at 30 C. The correct Rabbit Polyclonal to CtBP1 deletion was confirmed by a series of diagnostic PCR amplification reactions using primers with sequences located outside and within the deleted regions. Sensitivity to ampicillin indicated loss of pSIM6. To construct CWG1218 (in DH10b (GenBankTM accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NC_010473.1″,”term_id”:”170079663″,”term_text”:”NC_010473.1″NC_010473.1). The process for mutagenesis, confirmation, and curing of pSIM6 was the same as that explained for the parent, CWG1217, except mutants were selected on LB agar made up of 10 g ml?1 chloramphenicol at 30 C. Potential downstream (polarity) effects of the cassettes replacing or experienced no influence around the experimental strategy, so they were left in place in strains used to isolate polysaccharides for NMR experiments. However, they were subsequently excised using the pCP20 helper plasmid (21) to construct CWG1219 (complementation experiments. DNA Methods Custom oligonucleotide primers were obtained from Sigma. PCR amplification was performed using Pwo DNA polymerase (Roche Applied Science). The PureLink PCR purification kit (Invitrogen) was used to clean the PCR product. Plasmid DNA was purified from overnight cultures using the PureLink quick plasmid miniprep kit (Invitrogen). Chromosomal DNA was obtained using the PureLink genomic DNA mini kit (Invitrogen). Restriction digest and ligation reactions were performed according to the manufacturer’s instructions. Arsonic acid DNA sequencing was performed in the Genomics Facility of the Advanced Analysis Center at the University or college of Guelph. Plasmid Constructs pKM114 is usually a pMBL19 derivative made up of the 10.3-kb gene was PCR-amplified from pKM114 using primers designed to include an N-terminal FLAG tag in the gene product. The primer sequences (forward primer: 5-gatcgaattcATGinto pMBL19. The genes were PCR-amplified from BL21 (DE3) (forward primer, 5-gtcgaagaattcatATGACTTTTCTCTATCACTGATAGGGAGTGG-3; reverse.