Abstract
We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.
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References
Allison GE, Verma NK (2000) Serotype-converting bacteriophages and O-antigen modification in Shigella flexneri. Trends Microbiol 8:17–23. doi:10.1016/S0966-842X(99)01646-7
Amann E, Ochs B, Abel KJ (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315. doi:10.1016/0378-1119(88)90440-4
Baker JL, Çelik E, DeLisa MP (2013) Expanding the glycoengineering toolbox: the rise of bacterial N-linked protein glycosylation. Trends Biotechnol 31:313–323. doi:10.1016/j.tibtech.2013.03.003
Brown PK, Romana LK, Reeves PR (1991) Cloning of the rfb gene cluster of a group C2 Salmonella strain: comparison with the rfb regions of groups B and D. Mol Microbiol 5:1873–1881. doi:10.1111/j.1365-2958.1991.tb00811.x
Burrows LL, Lam JS (1999) Effect of wzx (rfbX) mutations on A-band and B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa O5. J Bacteriol 181:973–980
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97:6640–6645. doi:10.1073/pnas.120163297
Davies MR, Broadbent SE, Harris SR, Thomson NR, van der Woude MW (2013) Horizontally acquired glycosyltransferase operons drive salmonellae lipopolysaccharide diversity. PLoS Genet 9:e1003568. doi:10.1371/journal.pgen.1003568
DebRoy C, Fratamico PM, Yan X, Baranzoni G, Liu Y, Needleman DS, Tebbs R, O’Connell CD, Allred A, Swimley M, Mwangi M, Kapur V, Raygoza Garay JA, Roberts EL, Katani R (2016) Comparison of O-antigen gene clusters of all O-serogroups of Escherichia coli and proposal for adopting a new nomenclature for O-typing. PLoS One 11:e0147434. doi:10.1371/journal.pone.0147434
Duerr CU, Zenk SF, Chassin C, Pott J, Gütle D, Hensel M, Hornef MW (2009) O-antigen delays lipopolysaccharide recognition and impairs antibacterial host defense in murine intestinal epithelial cells. PLoS Pathog 5:e1000567. doi:10.1371/journal.ppat.1000567
Feldman MF, Marolda CL, Monteiro MA, Perry MB, Parodi AJ, Valvano MA (1999) The activity of a putative polyisoprenol-linked sugar translocase (wzx) involved in Escherichia coli O antigen assembly is independent of the chemical structure of the O repeat. J Biol Chem 274:35129–35138. doi:10.1074/jbc.274.49.35129
Fratamico PM, Yan X, Liu Y, DebRoy C, Byrne B, Monaghan A, Fanning S, Bolton D (2010) Escherichia coli serogroup O2 and O28ac O-antigen gene cluster sequences and detection of pathogenic E. coli O2 and O28ac by PCR. Can J Microbiol 56:308–316. doi:10.1139/W10-010
Gupta DS, Shashkov AS, Jann B, Jann K (1992) Structures of the O1B and O1C lipopolysaccharide antigens of Escherichia coli. J Bacteriol 174:7963–7970. doi:10.1128/jb.174.24.7963-7970.1992
Hobbs M, Reeves PR (1994) The JUMPstart sequence: a 39 bp element common to several polysaccharide gene clusters. Mol Microbiol 12:855–856. doi:10.1111/j.1365-2958.1994.tb01071.x
Hong Y, Reeves PR (2014) Diversity of O-antigen repeat unit structures can account for the substantial sequence variation of Wzx translocases. J Bacteriol 196:1713–1722. doi:10.1128/jb.01323-13
Hong Y, Reeves PR (2015) Model for the controlled synthesis of O-antigen repeat units involving the WaaL ligase. mSphere 1:e00074–00015. doi:10.1128/mSphere.00074–15
Hong Y, Cunneen MM, Reeves PR (2012) The Wzx translocases for Salmonella enterica O-antigen processing have unexpected serotype specificity. Mol Microbiol 84:620–630. doi:10.1111/j.1365-2958.2012.08048.x
Iguchi A, Iyoda S, Kikuchi T, Ogura Y, Katsura K, Ohnishi M, Hayashi T, Thomson NR (2015) A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster. DNA Res 22:101–107. doi:10.1093/dnares/dsu043
Iguchi A, Iyoda S, Seto K, Nishii H, Ohnishi M, Mekata H, Ogura Y, Hayashi T (2016) Six novel O genotypes from Shiga toxin-producing Escherichia coli. Front Microbiol 7:765. doi:10.3389/fmicb.2016.00765
Islam ST, Lam JS (2014) Synthesis of bacterial polysaccharides via the Wzx/Wzy-dependent pathway. Can J Microbiol 60:697–716. doi:10.1139/cjm-2014-0595
Jann B, Shashkov AS, Gupta DS, Panasenko SM, Jann K (1992) The O1 antigen of Escherichia coli: structural characterization of the O1A1-specific polysaccharide. Carbohydr Polym 18:51–57. doi:10.1016/0144-8617(92)90187-U
Jansson P-E, Lennholm H, Lindberg B, Lindquist U, Svenson SB (1987) Structural studies of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide. Carbohydr Res 161:273–279. doi:10.1016/S0008-6215(00)90084-3
King JD, Berry S, Clarke BR, Morris RJ, Whitfield C (2014) Lipopolysaccharide O antigen size distribution is determined by a chain extension complex of variable stoichiometry in Escherichia coli O9a. Proc Natl Acad Sci U S A 111:6407–6412. doi:10.1073/pnas.1400814111
Lederberg EM, Cohen SN (1974) Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol 119:1072–1074
Li D, Liu B, Chen M, Guo D, Guo X, Liu F, Feng L, Wang L (2010) A multiplex PCR method to detect 14 Escherichia coli serogroups associated with urinary tract infections. J Microbiol Methods 82:71–77. doi:10.1016/j.mimet.2010.04.008
Liu D, Cole RA, Reeves PR (1996) An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J Bacteriol 178:2102–2107. doi:10.1128/jb.178.7.2102-2107.1996
Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Reeves PR, Wang L (2014) Structural diversity in Salmonella O antigens and its genetic basis. FEMS Microbiol Rev 38:56–89. doi:10.1111/1574-6976.12034
Liu MA, Stent TL, Hong Y, Reeves PR (2015) Inefficient translocation of a truncated O unit by a Salmonella Wzx affects both O-antigen production and cell growth. FEMS Microbiol Lett 362:fnv053. doi:10.1093/femsle/fnv053
Marolda CL, Vicarioli J, Valvano MA (2004) Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit. Microbiology 150:4095–4105. doi:10.1099/mic.0.27456-0
McGrath BC, Osborn MJ (1991) Localisation of the terminal steps of O-antigen synthesis in Salmonella typhimurium. J Bacteriol 173:649–654. doi:10.1128/jb.173.2.649-654.1991
Murray GL, Attridge SR, Morona R (2003) Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol 47:1395–1406. doi:10.1046/j.1365-2958.2003.03383.x
Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656. doi:10.1128/MMBR.67.4.593-656.2003
Oldenburg KR, Vo KT, Michaelis S, Paddon C (1997) Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res 25:451–452. doi:10.1093/nar/25.2.451
Orr-Weaver TL, Szostak JW (1985) Fungal recombination. Microbiol Rev 49:33–58
Perepelov AV, Wang Q, Senchenkova SN, Feng L, Shashkov AS, Wang L, Knirel YA (2013) Structure and gene cluster of the O-antigen of Escherichia coli O154. Carbohydr Res 379:51–54. doi:10.1016/j.carres.2013.06.004
Raetz CRH, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71:635–700. doi:10.1146/annurev.biochem.71.110601.135414
Raymond CK, Sims EH, Kas A, Spencer DH, Kutyavin TV, Ivey RG, Zhou Y, Kaul R, Clendenning JB, Olson MV (2002a) Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa. J Bacteriol 184:3614–3622. doi:10.1128/JB.184.13.3614-3622.2002
Raymond CK, Sims EH, Olson MV (2002b) Linker-mediated recombinational subcloning of large DNA fragments using yeast. Genome Res 12:190–197. doi:10.1101/gr.205201
Saeui CT, Urias E, Liu L, Mathew MP, Yarema KJ (2015) Metabolic glycoengineering bacteria for therapeutic, recombinant protein, and metabolite production applications. Glycoconj J 32:425–441. doi:10.1007/s10719-015-9583-9
Samuel G, Reeves PR (2003) Biosynthesis of O-antigens: genes and pathways involved in nucleotide sugar precursor synthesis and O-antigen assembly. Carbohydr Res 338:2503–2519. doi:10.1016/j.carres.2003.07.009
Shanks RM, Kadouri DE, MacEachran DP, O’Toole GA (2009) New yeast recombineering tools for bacteria. Plasmid 62:88–97. doi:10.1016/j.plasmid.2009.05.002
Stevenson G, Dieckelmann M, Reeves PR (2008) Determination of glycosyltransferase specificities for the Escherichia coli O111 O antigen by a generic approach. Appl Environ Microbiol 74:1294–1298. doi:10.1128/AEM.02660-07
Tian T, Salis HM (2015) A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons. Nucleic Acids Res 43:7137–7151. doi:10.1093/nar/gkv635
Wang Q, Perepelov AV, Beutin L, Senchenkova SyN XY, Shashkov AS, Ding P, Knirel YA, Feng L (2012) Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase. Glycobiology 22:1321–1331. doi:10.1093/glycob/cws098
Willis LM, Whitfield C (2013) KpsC and KpsS are retaining 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules. Proc Natl Acad Sci U S A 110:20753–20758. doi:10.1073/pnas.1312637110
Acknowledgements
The yeast-E. coli shuttle vector pCRG16 and S. cerevisiae strain CRY1-2 were kindly provided by Elizabeth Sims (formerly of the University of Washington). The S. enterica O42 wzx clone was constructed by University of Sydney undergraduate student Carolyn Samer. The alignments in Supplementary Fig. S1 were performed by University of Sydney PhD student Dalong Hu.
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This study was funded by Australian Research Council Grant DP140104068.
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Liu, M.A., Kenyon, J.J., Lee, J. et al. Rapid customised operon assembly by yeast recombinational cloning. Appl Microbiol Biotechnol 101, 4569–4580 (2017). https://doi.org/10.1007/s00253-017-8213-9
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DOI: https://doi.org/10.1007/s00253-017-8213-9