LA BANCA ORTODOXA Y LOS FONDOS FINANCIEROS PRIVADOS

Materials: All chemicals were reagent grade and purchased from Sigma-Aldrich or VWR. Restriction enzymes, DNA polymerases, and T4 DNA ligase were purchased from New England BioLabs. The following compounds were used for the susceptibility testing and their classes/mode of action and sources are represented in parenthesis: chloramphenicol (Sigma-Aldrich), erythromycin (macrolide; Sigma-Aldrich), ciprofloxacin (fluoroquinolone; Sigma-Aldrich), norfloxacin (fluoroquinolone; Sigma- Aldrich), nalidixic acid (quinolone precursor; Sigma-Aldrich), sodium dodecyl sulfate (detergent; BioRad), acriflavine hydrochloride (intercalator; Acros Organics), ethidium bromide (intercalator; Amresco), cetyltrimethyl-ammonium bromide (quaternary ammonium compound; Amresco), benzalkonium chloride (quaternary amino compound; Alfa Aesar), methyl viologen (redox cycling drug, Acros Organics), Oxacillin (penicillin, Sigma), Cloxacillin (Penicillin, sigma), Piperacillin (Penicillin,TCI America).

Table 3.1: E. coli Strains and plasmids in this study:

Strain Genotype Source/reference

W3110 Wild Type, F-,_λ-,_{IN(rrnD-rrnE)1,rph-1 E. coli genetic stock}

center, Yale University

BC202 W3110, ΔyqjA::TetR, ΔyghB781::KanR (Thompkins,

Chattopadhyay et al. 2008)

BC202KS W3110, ΔyqjA::TetR_{, ΔyghB781<>FRT This work}

JW0826 Δ(araD-araB)567, ΔlacZ4787(::rrnB-

3), λ-, Δcmr-742::kan, rph-1, Δ(rhaD-

rhaB)568, hsdR514

(Baba, Ara et al. 2006)

JW0531 Δ(araD-araB)567, ΔlacZ4787(::rrnB-

3), ΔemrE750::kan, λ-, rph-1, Δ(rhaD-

rhaB)568, hsdR514

(Baba, Ara et al. 2006)

JW0451 Δ(araD-araB)567, ΔlacZ4787(::rrnB-

3), ΔacrB747::kan, λ-, rph-1, Δ(rhaD-

rhaB)568, hsdR514

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Strain Genotype Source/reference

W3110∆mdfA W3110, ∆cmr-742::kan This work

W3110∆emrE W3110,∆emrE-750::kan This work

W3110∆acrB W3110,∆acrB-747::kan This work

BC202∆mdfA BC202KS, ∆cmr-742::kan This work

BC202∆emrE BC202KS, ∆emrE-750::kan This work

BC202∆acrB BC202KS, ∆acrB-747::kan This work

XL1 Blue recA1 endA1 gyrA96 thi-1 hsdR17

supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (TetR₎

Stratagene

Plasmids Genotype Source/reference

pBADHisA Expression vector; araBAD promoter,

AmpR

Invitrogen. pCP20 FLP+, λ cI857+, λ ρR Repts, AmpR,

CamR

(Cherepanov and

Wackernagel 1995)

pBAD-yqjA H6-yqjA expression; araBAD

promoter,Ampr

(Sikdar, Simmons et al. 2013)

pBAD- yqjA(E39A)

H6-yqjA(E39A) expression; araBAD

promoter, Ampr

This work pBAD-

yqjA(E39Q)

H6-yqjA(E39Q) expression; araBAD

promoter, Ampr

This work pBAD-

yqjA(D51A)

H6-yqjA(D51A) expression; araBAD

promoter, Ampr

This work pBAD-

yqjA(D51N)

H6-yqjA(D51N) expression; araBAD

promoter, Ampr

This work

pBAD-YghB H6-yghB; araBAD promoter,AmpR (Sikdar, Simmons et al.

2013) pBAD-

yghB(E39A)

H6-yghB(E39A) expression; araBAD

promoter, Ampr

This work pBAD-

yghB(D51A)

H6-yghB(D51A) expression; araBAD

promoter, Ampr

This work

pBAD-MdfA H6-mdfA expression; araBAD

promoter,AmpR

(Sikdar, Simmons et al. 2013)

pACYC184-yqjA pACYC184 expressing E.coli yqjA. CamR

(Thompkins,

Chattopadhyay et al. 2008)

pACYC184-yghB pACYC184 expressing E.coli yghB. CamR

(Thompkins,

Chattopadhyay et al. 2008)

pBB0250-GFP BB0250-GFP expression. lac promoter.

AmpR

(Liang, Xu et al. 2010) pBB0250(E39A)-

GFP

BB0250(E39A)-GFP expression, lac promoter. AmpR

This work (Table 3.1 continued)

81 Table 3.2 Primers used in the study

Primer Name Sequence ( 5’-3’) YqjA E39A_FWD GTTCCTTGCAAACGGCTTGCTTCCGGCGGCCTTTTTACC YqjA E39A_REV GCCGTTTGCAAGGAACAAAATTACAAACAAGACAAAATAC YqjA D51A _FWD CCGGGCGCCAGTTTACTGGTATTGGTCGGCGTGTTGATTGC G YqjA D51A_REV GTAAACTGGCGCCCGGTAAAAAGGCCGCCGGAAGCAAGC CG YqjA E39Q_FWD GTTCCTTCAAAACGGCTTGCTTCCGGCGGCCTTTTTACC YqjA E39Q_REV GCCGTTTTGAAGGAACAAAATTACAAACAAGACAAAATAC AAC YqjA D51N_FWD CGGGCAACAGTTTACTGGTATTGGTCGGCGTGTTGATTGCG YqjA D51N_REV GTAAACTGTTGCCCGGTAAAAAGGCCGCCGGAAGCAAGCC G YghB E39A_FWD GTTTTTAGCAAACGGCCTGCTGCCCGCCTCATTTTTGCC YghB E39A_REV GCCGTTTGCTAAAAACAGCGTGGCAAACATGAC YghBD51A_F WD GCCAGGCGCCAGCTTGTTGATACTGGCAGGCGCATTGATT GCCCAGGGG YghB D51A_REV CAAGCTGGCGCCTGGCAAAAATGAGGCGGGCAGCAGGCC GTTTTC BB0250 E39A _FWD CTATTTCTGCGGATGCAATAGTACTAATGGGTGGAATTTTA TCTAG BB0250 E39A _REV GCATCCGCAGAAATAGGAACATTAAGTCCTGCTAAAATTA GTAACG BB0250 D40A _FWD CCTATTTCTGAGGCTGCAATAGTACTAATGGGTGGAATTTT ATC BB0250 D40A _REV CTATTGCAGCCTCAGAAATAGGAACATTAAGTCCTGCTAA AATTAG

Plasmids Genotype Source/reference

pBB0250(D40A)- GFP

BB0250(D40A)-GFP expression. lac promoter. AmpR

This work

pBB0 GFP expression vector. lac promoter.

AmpR

(Liang, Xu et al. 2010) (Table 3.1 continued)

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Bacterial growth conditions: Bacterial cultures were grown in Luria-Bertani broth (LB) (1% tryptone, 0.5% yeast extract and 1% NaCl) unless otherwise stated. LB media of pH 6.0 was buffered with 100 mM 2-(N-morpholino)-ethanesulfonic acid (MES) (Sikdar, Simmons et al. 2013). Solid media were obtained by addition of 1.5 % agar (w/v). When required, the media was supplemented with ampicillin (Amp) 100 µg/ml, kanamycin (Kan) 30 µg/ml, tetracycline (Tet) 12.5 µg/ml, chloramphenicol (Cam) 30 µg/ml), arabinose (0.002% or 0.1%, w/v) or isopropyl-1-thio-β-D-galactopyranoside (IPTG; 0.5 mM). All cultures were grown at 30 °C unless otherwise indicated.

Strain construction: The multiple-deletion mutants were generated in E. coli W3110 and correct configuration was verified through PCR with primers flanking the appropriate gene. In order to eliminate the kanamycin-resistance (kanR) gene from BC202, plasmid pCP20 expressing FLP recombinase was used as described previously (Cherepanov and Wackernagel 1995). Since this protocol requires overnight incubation at 42 C to cure pCP20 (nonpermissive for BC202), the yghB::kanR strain was first made markerless (W3110 background). YqjA::tetR _{(Thompkins, Chattopadhyay et al. 2008) was}

then introduced by P1 transduction. Additional mutations (i.e. ΔemrE::kanR, ΔmdfA::kanR, or ΔacrB::kanR) were introduced by P1 transduction with P1vir lysate prepared from the

indicated E. coli strains (Table 3.1) obtained from the Keio collection (Baba, Ara et al. 2006). P1 transductions were carried out as described (Silhavy, Berman et al. 1984). All strains and genotypes are listed in Table 3.1.

Microscopy: Overnight cultures of E. coli strains were diluted 1:100 in fresh LB media with suitable antibiotics and additives, and grown to OD600 ~ 0.6 at 30 °C in a

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applied to a 1% agarose coated glass slide for imaging. A Leica DM-RXA2 deconvolution microscope was used for all the Differential Interference Contrast (DIC) micrographs. Observations were made by a 100X, 1.30-numerical-aperture oil immersion objective lens. The images were captured through a DIC filter by a cooled Cooke SensiCamQE 12 bit, 1, 3-megapixel, charge-coupled device digital camera and recorded using Slidebook software (Intelligent Imaging Innovation, Denver, CO).

Susceptibility to toxic compounds: For testing the susceptibility on solid medium, overnight cultures of E. coli strains were freshly diluted 1:100 in LB media with appropriate antibiotics and additives, and grown to OD600 ~ 0.6 at 30°C in a shaking

incubator. 5 μl of serially log10-diluted cells were spotted on LB agar plates containing

various concentrations of corresponding biocides. Growth was analyzed after incubation for 20 to 24 hours at 30 °C. All experiments were repeated at least three times.

Minimal inhibitory concentration (MIC) determination: The MICs of biocides were carried out using a two-fold dilution technique in either 1.5 x 15 cm glass tube or in 96- well microtiter plates in liquid medium (Wiegand, Hilpert et al. 2008). Overnight cultures were freshly diluted 1:100 into LB medium with suitable antibiotics and additives. Exponentially growing cultures at OD600 ~1.0 were inoculated at a density of 105 cells per

ml into LB medium supplemented with a series of two-fold dilutions of indicated biocide. Cell growth was determined visually after incubation at 30°C for 24 hours. All experiments were repeated at least three times.

Site-directed mutagenesis: Site-specific mutants were created by using previously published protocol (Zheng, Baumann et al. 2004). The primers carrying the site-specific mutations (Table 3.2) were used in a PCR reaction to amplify a vector containing the

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specified wild type gene. The PCR product was then digested with Dpn1 and used to transform competent XL1-blue cells. Colonies obtained after transformation were screened by colony PCR using gene-specific primers. The mutations confirmed by DNA sequencing conducted at the LSU College of Science Genomics Facility.

Membrane preparation and Western blotting: Cell membranes was prepared from exponential phase cultures of BC202 containing appropriate plasmid DNA grown under inducing condition (arabinose or IPTG) using a previously published protocol (Doerrler and Raetz 2002). Equal amounts of protein were resolved by 12 % SDS-PAGE and transfer to PVDF. Western blotting was done using penta-His (Qiagen) or GFP (JL-8; Clontech) primary antibody at 1:5000 dilution and goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (Pierce) at 1:5000. Detection was carried out using the ImmunStar HRP kit (Bio-Rad).