Supplemental Information. for. Structural and functional characterization of Pseudomonas aeruginosa AlgX: Role of AlgX in alginate acetylation
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1 Supplemental Information for Structural and functional characterization of Pseudomonas aeruginosa AlgX: Role of AlgX in alginate acetylation Laura M. Riley 1,#, Joel T. Weadge 1,#,$, Perrin Baker 1, Howard Robinson 2, Jereon D.C. Codée 3, Peter A. Tipton 4, Dennis E. Ohman 5, and P. Lynne Howell 1,6 1
2 Table S1: Bacterial Strains, Plasmids and PCR primers used in this Study. Antibiotic resistance markers are abbreviated as follows: Kan R, kanamycin resistance; Cm R, chloramphenicol resistance; Gm R, gentamicin resistance; bla, ampicillin/carbenicillin resistance. Cloning primers have P. aeruginosa DNA sequences of PAO1 (obtained from Winsor et al., 2009 [1]) highlighted in bold; embedded NsiI and NdeI restriction sites are underlined. The forward primers used for mutagenesis are shown with the mutations underlined. The reverse primers used for mutagenesis had the reverse complement sequence to that shown, as prescribed in the QuickChange Lightning kit. Characteristics Source Strain E. coli E. coli TOP10 TM F - mcra Δ(mrr-hsdRMS-mcrBC) φ80laczδm15 ΔlacX74 reca1 arad139 Δ(ara-leu) 7697 galu galk rpsl (Str r ) enda1 nupg λ- Invitrogen E. coli BL21- CodonPlus (DE3)- RP F - ompt hsds(r B - m B - ) dcm + Tet r gal λ(de3) enda [argu prol Cam r ] Stratagene B834(DE3) F - ompt hsdsb(r - B m - B ) gal dcm met(de3) Novagen P. aeruginosa PAO1 Wild-type pathogenic strain This lab PDO1208 PAO1 ::Gm R (derived from pex205-:gm) This study PDO1245 PAO1 + from PDO1208 x pdonr199- This study PDO1450 PAO1 (R115A) from PDO1208 x pdonr#- This study PDO1395 PAO1 (D174A) from PDO1208 x pdonr#- This study PDO1396 PAO1 (H175A) from PDO1208 x pdonr#- This study PDO1397 PAO1 (H176A) from PDO1208 x pdonr#- This study PDO1398 PAO1 (S269A) from PDO1208 x pdonr#- This study PDO1399 PAO1 (N270A) from PDO1208 x pdonr#- This study PDO1453 PAO1 (Y275A) from PDO1208 x pdonr#- This study PDO1400 PAO1 (D300A) from PDO1208 x pdonr#- This study PDO1401 PAO1 (S301A) from PDO1208 x pdonr#- This study PDO1452 PAO1 (Y328A) from PDO1208 x pdonr#- This study PDO351 PAO1 muca::gm R (Alg+) Wood et al., 2006 PDO1486 PAO1 + muca::gm R (Alg+) This study PDO1479 PAO1 (R115A) muca::gm R (Alg+) This study PDO1495 PAO1 (D174A) muca::gm R (Alg+) This study PDO1478 PAO1 (H175A) muca::gm R (Alg+) This study PDO1481 PAO1 (H176A) muca::gm R (Alg+) This study PDO1482 PAO1 (S269A) muca::gm R (Alg+) This study PDO1483 PAO1 (N270A) muca::gm R (Alg+) This study PDO1487 PAO1 (Y275A) muca::gm R (Alg+) This study 2
3 PDO1484 PAO1 (D300A) muca::gm R (Alg+) This study PDO1485 PAO1 (S301A) muca::gm R (Alg+) This study PDO1480 PAO1 (Y328A) muca::gm R (Alg+) This study PDO300 PAO1 muca23 (Alg+) Mathee et al., 1999 PDO1512 PDO300 muca23 :Gm (from pex205) (Alg-) This study PDO1513 PDO1512 muca23 :Gm (pbad510-) (Alg+) This study Plasmid pdonr-p4-p1r Gateway [attp4 ccdb Cm R attp1r] Kan R Invitrogen pdonr-221 Gateway [attp1 ccdb Cm R attp2] Kan R Invitrogen pdonr-p2r-p3 Gateway [attp2 ccdb Cm R attp3] Kan R Invitrogen pdest-r4r3 Gateway [attr4 ccdb Cm R attr3] bla Invitrogen pbad-dest49 Gateway [attr2 ccdb Cm R attr1] P BAD arac bla Invitrogen prk2013 ColE1-Tra(RK2)+ Kan R Figurski and Helinski, 1979 pss124 puc1918 with msf/oriv cartridge Suh et al., 2004 pucgm puc1918, aacc1 (Gm R ) ~850 bp cassette Schweizer, 1993 pex100t bla sacb orit gene replacement vector Schweizer and Hoang, 1995 pexapgw pex100t with [attr2 ccdb Cm R attr1], bla sacb orit pex183-r4r3 pex100t with [attr4 ccdb Cm R attr3], bla sacb orit pdonr150-gmr pdonr-221 with Gm R fragment from pucgm This study pdonr148-5 pdonr-p4-p1r with 5 half of, bla This study pdonr152-3 pdonr-p2r-p3 with 3 half of, bla This study pex205-:gm pex183-r4r3 with ::Gm R This study pdonr199- pdonr-221 with +, Kan R This study pdonr258-* pdonr199- with (R115A) This study pdonr261-* pdonr199- with (D174A) This study pdonr263-* pdonr199- with (H175A) This study pdonr265-* pdonr199- with (H176A) This study pdonr267-* pdonr199- with (W177A) This study pdonr268-* pdonr199- with (S269A) This study pdonr271-* pdonr199- with (N270A) This study pdonr272-* pdonr199- with (Y275A) This study pdonr274-* pdonr199- with (D300A) This study pdonr276-* pdonr199- with (S301A) This study pdonr279-* pdonr199- with (Y328A) This study pdonr296-* pdonr199- with (W400A) This study pex237- pexapgw with + (from pdonr199-) This study pex280-* pexapgw with (R115A) This study pex281-* pexapgw with (D174A) This study pex282-* pexapgw with (H175A) This study pex283-* pexapgw with (H176A) This study pex284-* pexapgw with (W177A) This study pex305-* pexapgw with (S269A) This study pex286-* pexapgw with (N270A) This study pex287-* pexapgw with (Y275A) This study pex288-* pexapgw with (D300A) This study pex289-* pexapgw with (S301A) This study Schweizer and Hoang, 1995 This study 3
4 pex307-* pexapgw with (Y328A) This study pex#-* pexapgw with (W400A) This study plg339 Tc R Kan R oriv(psc101) Stoker et al., 1982 plg369-msf plg339 with msf from pss124 in SmaI site, Tc R Stoker et al., 1982 pbad506-msf pbad-dest49 with msf/oriv from pss124 This study pbad510- pbad506-sf with + from pdonr199- This study pplhjtwx pet24b(+) with codon-optimized, full-length, Weadge et al., 2010 N- and C- terminal 6xHis tags, Kan R, laci pplhjtwx-174 pplhjtwx (D174A) This study pplhjtwx-176 pplhjtwx (H176A) This study pplhjtwx-269 pplhjtwx (S269A) This study Primer name Sequence (5ʹ - 3ʹ ) Cloning attb4-5ʹ X-F attb1r-5ʹ X-R attb2r-3ʹ X-F GGGGACAACTTTGTATAGAAAAGTTGTGGTGGCCTACAACGAGGT GGGGACTGCTTTTTTGTACAAACTTGTTAGCTGTTGCCGCAGAGTT GGGGACAGCTTTCTTGTACAAAGTGGAGTACGTCGACCGCTTCGA attb3-3ʹ X-R GGGGACAACTTTGTATAATAAAGTTGCGCGAGCTGGAGAACTTCA attb1-gm-f GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGCTCGAATTGACATA AGC attb2-gm-r GGGGACCACTTTGTACAAGAAAGCTGGGTGAGCTCGAATTGGCCGC G attb1-sd-f GGGGACAAGTTTGTACAAAAAAGCAGGCTGAGGACCCAGACGCATG AAA attb2-sd-r GGGGACCACTTTGTACAAGAAAGCTGGGTATCGTTTACCTCCCGGCC AC NsiI-M13F CCACCATGCATGTTGTAAAACGACGGCCAG NsiI-M13R GGTGGATGCATCAGGAAACAGCTATGACCAT NdeI-pUC19MCS-F CATATGCAGGAAACAGCTATGACCAT NdeI-pUC19MCS-R TACAACGTCGTGACTGGGACATATG in vivo mutagenesis R115A-F CTACCAGCCGACCGCCGGCCTGGTGAAC D174A-F CTACTTCAAGGGCGCCCACCACTGGACTC H175A-F CTACTTCAAGGGCGACGCCCACTGGACTCCCCATG H176A-F CTTCAAGGGCGACCACGCCTGGACTCCCCATGGC S269A-F CCTGGTCGGCACCGCCAACAGCGGCCC N270A-F GGTCGGCACCTCCGCCAGCGGCCCGGCC Y275A-F CAGCGGCCCGGCCGCCAACTTCGCCGGC D300A-F GGCGGCGGCTTCGCCAGTTCGCTGCTG S301A-F GGCGGCGGCTTCGACGCCTCGCTGCTGGCGTAC Y328A-F GAATTCGCCACCCACGCCGACATGGCGCAGAAG in vitro mutagenesis D174AAlgXFor CTATTATTTCAAAGGTGCCCATCACTGGACCCCGC H176AAlgXFor GGTGACCATGCCTGGACCCCGCACGG S269AAlgXFor CTGGTTGGCACCGCGAATAGCGGCCCGGC 4
5 References 1. G. L. Winsor, T. Van Rossum, R. Lo, B. Khaira, M. D. Whiteside, R. E. W. Hancock and F. S. L. Brinkman. (2009) Pseudomonas Genome Database: facilitating user-friendly, comprehensive comparisons of microbial genomes. Nucleic Acids Res. 37, D L. F. Wood, A. J. Leech and D. E. Ohman. (2006) Cell wall-inhibitory antibiotics activate the alginate biosynthesis operon in Pseudomonas aeruginosa: Roles of sigma (AlgT) and the AlgW and Prc proteases. Mol. Microbiol. 62, K. Mathee, O. Ciofu, C. Sternberg, P. W. Lindum, J. I. Campbell, P. Jensen, A. H. Johnsen, M. Givskov, D. E. Ohman, S. Molin, N. Hoiby and A. Kharazmi. (1999) Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide: a mechanism for virulence activation in the cystic fibrosis lung. Microbiology D. H. Figurski and D. R. Helinski. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. U. S. A. 76, S. J. Suh, L. A. Silo-Suh and D. E. Ohman. (2004) Development of tools for the genetic manipulation of Pseudomonas aeruginosa. J. Microbiol. Methods 58, H. D. Schweizer. (1993) Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques 15, H. P. Schweizer and T. T. Hoang. (1995) An improved system for gene replacement and xyle fusion analysis in Pseudomonas aeruginosa. Gene 158, N. G. Stoker, N. F. Fairweather and B. G. Spratt. (1982) Versatile low-copy-number plasmid vectors for cloning in Escherichia coli. Gene 18, J. T. Weadge, P. P. Yip, H. Robinson, K. Arnett, P. A. Tipton and P. L. Howell. (2010) Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 66,
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