Supplementary materials

Construction of a tightly controlled expression system for use in Pseudomonas

Liang Yang1‡, Lin Chen2, Lixin Shen1 and Kangmin Duan1,2*

1Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi’an, Shaanxi 710069, China

2Department of Oral Biology; Medical Microbiology, University of Manitoba, 780 Bannatyne Ave., Winnipeg, MB R3E 0W2 Canada

Running title: Zn-controlled expression system for Pseudomonas

*Correspondence author: KangminDuan, Department of Oral Biology; Medical Microbiology, University of Manitoba, 780 Bannatyne Ave., Winnipeg, MB R3E 0W2 Canada

Email Adresses:

Tel: (204)272-3185; Fax: (204)789-3913

‡ Current address: Department of Chemistry and Biochemistry, University of Oklahoma,

101 Stephenson Parkway, Norman, OK, 73019-5251, USA

Supplementary Table 1Bacterial stains and plasmid used in this study

Strains and Plasmids / Relevant Properties / Source or Reference
E. coli
DH10B / F-mcrAΔ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara, leu)7697 galUgalK λ- rpsLnupG /pMON14272/ pMON7124 / Invitrogen
P. aeruginosa
PAO1 / Wild type / Lab Collection
PAF / Mutant with a lacZ-GmR cassette inserted in PA2522; GmR / This study
Plasmids
pEX18Tc / Broad-host-range gene replacement vector; sacB, TetR / (Schweizer 1992)
pRK2013 / Broad-host-range helper vector; Tra+KanR / (Ditta et al. 1980)
pZ1918-LacZ / Vector, containing promoterlesslacZ and GmR(lacZ-GmR) / (Schweizer 1993)
pMS402 / Reporter vector carrying promoterlessluxCDABE, KanR TmpR / (Duan et al. 2003)
pKD-czcC / pMS402 carrying the promoter region of czcCBA; KanRTmpR / (Yang et al. 2011)
pUCP26 / Pseudomonas-Escherichia shuttle vector, TetR / (Olsen et al. 1982)
pPROEX HT / Expression vectors containing HTa, HTb and HTc, ApR / Invitrogen
pRS / pUCP26 deleted Plac promoter and MCS and ligated with the terminator, Kan resistance gene and czcCBA promoter region, KanRTetR / This study
pUC-Not-lux / pUC19 carryingluxABCDE in NotI site, ApR / Lab collection
pLY / Controlled expression system including pLY-A, pLY-B and pLY-C, KanRApRTetR / This study
pLY-Not-luxCDABE / pLY-C expression vector containing luxCDABE with its own RBS in NotI site, KanRApRTetR / This study
pLY-Kpn-luxABCDE / pLY-C expression vector containing luxABCDE without its own RBS in KpnI site, KanRApRTetR / This study
pLY-C-sacB / pLY-C expression vector containing sacBwith its own RBS in BamHI/EcoRI sites, KanRApRTetR / This study

Supplementary Table 2Primers used in this study

Name / Sequences
Paf-f / 5'-AAACTCGAGATCCAACTATTGCCAGTCTC-3'
Paf-r / 5'-CGTAAGCTTCCACCAGGTTCTTCTTCACC-3'
czcC-f / 5'-TCCCGTGACAGGTCATTCAG-3'
czcC-r / 5'-CAGGATCCGAAGTATCGGCATG-3'
MCS-BamHI-for / 5'-AAACAGACCATGTCGTACTAC-3'
MCS-HindIII-rev / 5'-GAGTAAACTTGGTCTGACAG-3'
pZE.05 / 5'-CCAGCTGGCAATTCCGA-3'

References:

Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A 77:7347-7351

Duan K, Dammel C, Stein J, Rabin H, Surette MG (2003) Modulation of Pseudomonas aeruginosa gene expression by host microflora through interspecies communication. Mol Microbiol 50:1477-1491

Olsen RH, DeBusscher G, McCombie WR (1982) Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol 150:60-69

Schweizer HP (1992) Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol 6:1195-1204

Schweizer HP (1993) Two plasmids, X1918 and Z1918, for easy recovery of the xylE and lacZ reporter genes. Gene 134:89-91

Yang L, Chen L, Shen L, Surette M, Duan K (2011) Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence. J Microbiol 49:107-114

Supplementary Figure 1: Dose-dependent induction of czcCBA expression by Zn. Expression measured with PAF in LB. Zn was added at 500 μM l-1 (cross), 400 μM l-1 (squares), 300 μM l-1 (asterisks), 200 μM l-1 (diamonds), 100 μM l-1 (circle), and 0 μM l-1 Zn (triangles). LB was used. Bacterial growth was monitored by measuring the OD600value.

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