Article Figures & Data
Figures
- FIG. 1.
ydeI mRNA accumulation requires PhoP and PmrA. Real-time quantitative PCR was used to evaluate ydeI mRNA levels and samples were normalized to a gapA control. (A) ydeI mRNA levels after exposure to polymyxin B for 40 min in LB medium using strains with the indicated genes deleted. (B) ydeI mRNA levels in early stationary phase cells in LB medium. WT, wild type. The data are representative of three independent experiments.
- FIG. 2.
LPS and lipid A from ydeI mutant strains and the wild type are indistinguishable. (A) LPS was isolated from wild-type (WT) and strains with deletions of the genes indicated, separated by SDS-PAGE, and silver stained. (B) MALDI-TOF mass spectra of lipid A from wild-type and phoP and ydeI mutant strains. The mass/charge (m/z) ratios and related structures are as follows: 1,796 unmodified hexa-acyl lipid A; 1,813, hexa-acyl lipid A with 2-OH myristate; 1,937, hexa-acyl lipid A with Ara4N; 2,036, unmodified hepta-acyl lipid A; and 2,167, hepta-acyl lipid A with Ara4N.
- FIG. 3.
OmpD specifically copurifies with YdeI. (A) Bound proteins were eluted from YdeI-HA using an HA dipeptide, resolved by SDS-PAGE, and analyzed by silver staining. Eluted fractions (E1 to E3) from a pYdeI-HA strain (left) and from a wild-type (WT) strain (right). The 40-kDa band was isolated and submitted for mass spectrometric analysis. Lanes shown are from the same gel, but the image was cropped to remove unloaded lanes. (B) Bound proteins were eluted from YdeI-HA or GST-HA with low pH, resolved by SDS-PAGE, and analyzed by silver staining. Each strain harbored endogenous OmpD or chromosomal OmpD-3xFLAG, as marked. GST-HA (28 kDa) is marked with an asterisk. Lanes shown are from the same gel, but the image was cropped to remove unloaded lanes. (C) The flowthrough (FT) and eluate from the GST-HA sample (B) were subject to immunoblotting with anti-FLAG and anti-HA antibodies.
- FIG. 4.
OmpD-3xFLAG coimmunoprecipitates with chromosomally expressed YdeI-HA. Strains encoding chromosomally expressed, epitope-tagged YdeI-HA and OmpD-3xFLAG were grown overnight in LB medium, harvested, and lysed, and YdeI-HA was immunoprecipitated with mouse anti-HA antibody coupled to protein G-agarose beads. Supernatant (SN) or agarose bead immunoprecipitate (IP) from equivalent numbers of bacteria was resolved by SDS-PAGE and analyzed by immunoblotting with (A) an anti-FLAG antibody or (B) an anti-HA antibody. Molecular weight markers are indicated by bars to the left; the heavy and light chains of the immunoprecipitating antibody are indicated by arrows to the right of (B).
- FIG. 5.
ompD mutant strains are sensitive to polymyxin B and cathelicidin antimicrobial peptide. Strains harboring deletions of ydeI, ompD, or both genes were incubated in the presence of 4 μg of polymyxin B/ml (A) or 50 μM cathelicidin antimicrobial peptide (B) and plated for CFU. Controls included wild-type (WT) and a phoP insertion mutant. The data are reported as percent survival relative to the wild type. Asterisks represent P values on triplicate samples, as determined by a Student t test (P < 0.001).
- FIG. 6.
YdeI is in the periplasm. Immunoblotting of whole-cell (WC) lysates and fractions from an YdeI-HA strain (KDE686). The upper blot was probed with an anti-HA antibody, and the lower with anti-DnaK and anti-maltose binding protein (MBP) antibodies to identify cytoplasmic (C) and periplasmic (PP) fractions.
- FIG. 7.
ygiW and ompF mutants are sensitive to polymyxin B. Strains with the indicated gene deleted were incubated with 8 μg of polymyxin B/ml and plated for CFU. The data are reported as percent survival relative to the wild type (WT). Single and double asterisks represent P values on triplicate samples, as determined by a Student t test (P < 0.01 and P < 0.001, respectively).
Tables
- TABLE 1.
S. enterica serovar Typhimurium strains
Strain Genotype and/or description Source or reference SL1344 hisG xyl rpsL (wild-type serovar Typhimurium) 58 CSD221 14028s pKD46 Charlie Kim CSD257 galE::Tn10 Bruce Stocker FL416 SL1344 phoP::Cm 20 KDE445 SL1344 ydeI::Cm 20 KDE517 SL1344 rcsB::Cm This study KDE565 SL1344 invA::Cm This study KDE602 SL1344 ydeIΔ This study KDE612 SL1344 ydeI::HA::Kan This study KDE642 SL1344 ydeI::HA This study KDE662 SL1344 phoP::Cm, rcsBΔ This study KDE666 SL1344 pmrA::Cm This study KDE686 SL1344 with pYdeI-HA This study KDE735 SL1344 ompD::Cm This study KDE737 SL1344 yddG::Cm This study NMM748 SL1344 ompD::3xFLAG::Kan This study KDE755 SL1344 ydeIΔ ompD::Cm This study KDE759 SL1344 ydeI::HA ompD::3xFLAG::Kan This study NMM764 SL1344 ompD::3xFLAG This study KDE769 SL1344 ydeI::HA ompD::3xFLAG This study NMM800 SL1344 ompE/phoE::Cm This study NMM801 SL1344 ompF::Cm This study NMM802 SL1344 ompN::Cm This study NMM803 SL1344 STM1530::Cm This study NMM805 SL1344 ompC::Cm This study NMM806 SL1344 ygiW::Cm This study MCP847 SL1344 ompD::3xFLAG, with pGEX-3x This study MCP848 SL1344 ompD::3xFLAG, with pYdeI-HA This study - TABLE 2.
PCR primers used in this study
Primera Sequence (5′→3′)b invA-P1 TGAAAAGCTGTCTTAATTTAATATTAACAGGATACCTATAGTGTAGGCTGGAGCTGCTTC invA-P4 ATATCCAAATGTTGCATAGATCTTTTCCTTAATTAAGCCCATGGGAATTAGCCATGGTCC invA-5′PCR GCAGAACAGCGTCGTACTAT invA-3′PCR CGGAACGAACTAATTCAGCG ompC-P1 ATAAAAAAGCAATAAAGGCATATAACAGAGGGTTAATAACGTGTAGGCTGGAGCTGCTTC ompC-P4 AAAAAGGGCCCGCAGGCCCTTTAGCAACATCTTTTGCTGAATGGGAATTAGCCATGGTCC ompC-5′PCR GTTAACCAGTAAGCAGTGGC ompC-3′PCR TACGCCGGAATAAGGCATGA ompD-P1 GTTGAGGAAACACGCTAAGAAAATTATAAGGATTATTAAAGTGTAGGCTGGAGCTGCTTC ompD-P2 GCCCTGAAAGGACTGGCTTTGTATTCAGACTACAACAAAAATGGGAATTAGCCATGGTCC ompD-5′PCR AAACGCCTCGTTTAACAATG ompD-3′PCR TACATCAAGAGAAAAAGCCA ompD-3xFLAG-5′ ACCGACAACATCGTTGCTGTTGGTCTGAACTACCAGTTCGACTACAAAGACCATGACGGT ompD-3xFLAG-3′ CCAGTGAACGTCTGCACGGCATACTCCTTATGACCGAGTCCATATGAATATCCTCCTTAG ompF-P1 GCAGGTGTCATATAAAAAAACCAATGAGGGTAATAAATAGTGTAGGCTGGAGCTGCTTC ompF-P4 AAGTCCTGTTTTTGAGGCATAAAACAAAGGGGTCTGCTGAATGGGAATTAGCCATGGTCC ompF-5′PCR CGGAATTTATTGACGGCAGT ompF-3′PCR GAGATAAAAAAACAGGACCG ompN-P1 CAATCTTTTGCAAATAAGTTAAGTTTTTAAGGATAAAAAAGTGTAGGCTGGAGCTGCTTC ompN-P4 GCCCGCCGAAACGGCGGGCTTGAGAAGAATTAATGAATAAATGGGAATTAGCCATGGTCC ompN-5′PCR TCAACGAATCTGTAGAAGTT ompN-3′PCR GTGGTGATGAAAAAAGAAAA phoE-P1 TCCCGACAAATCATAGCGCGTAATTAAAACAGGAATGGAAGTGTAGGCTGGAGCTGCTTC phoE-P4 ATGCCTGATGGCGCAGCGCCATCAGGCACAATGCGACTTAATGGGAATTAGCCATGGTCC phoE-5′PCR TTCCTGTTTTTTACCGGGTT phoE-3′PCR GTCGGCATAACCCTGCTGCC pmrA-P1 CCGCAGATGATATTCTGCAACCGTGCAGGAGACTAAGCGAGTGTAGGCTGGAGCTGCTTC pmrA-P4 GAAGGGTCATCGCTCTTCGCTGAAAACGCATCAGGCTCACATGGGAATTAGCCATGGTCC pmrA-5′PCR ACAAACGACGTATTACCAGG pmrA-3′PCR TGTCAGCATTAAACGCTGGC STM1530-P1 TTGCGGAAAGTCCAAAAATAAGACAAATAAGGCATATAAAGTGTAGGCTGGAGCTGCTTC STM1530-P4 AAGGAGGGTATCCCTAAACTGTCTTAATCAGCAAGTTTTAATGGGAATTAGCCATGGTCC STM1530-5′PCR GACAGTTCCGCGAGCAGGCT STM1530-3′PCR CAGTGACACAGTTTTGGGAA yddG-P1 AACGCTAATAGGGCTTGTTGCCATCGTTCTGTGGAGCACGGTGTAGGCTGGAGCTGCTTC yddG-P4 GCGGGTCGATTTAATATCAATATCGGCCCGCCCGTCGCTAATGGGAATTAGCCATGGTCC yddG-5′PCR AGCATGACATCACAAAAAGC yddG-3′PCR ATGAAGAGTGTCAAGGTACA ydeI-HA-5′ GCGAAGGAACCGCTTGTTCGCGTGAACCGACTGCAAAAATATCCGTATGATGTTCCT ydeI-HA-3′ CGCTTTTACAAGACCCATGGTATCAGGCCAGCCGCGTGCCCATATGAATATCCTCCTTAG ygiW-P1 ATTAAATGGATCTGAAACGACATGAAAGGGAAAAGTAATCGTGTAGGCTGGAGCTGCTTC ygiW-P4 GGGCGGCTGTGAATCCTGACCGATCTTGCGCAATGTGGGAATGGGAATTAGCCATGGTCC ygiW-5′PCR AAGTTGTTAAGGATTACCTT ygiW-3′PCR TGTGGCCTATTCTGCCCACC ↵a The primer name indicates the gene or operon deleted or the epitope tag, followed by the primer binding site or epitope tag. P1, P2, and P4 indicate priming sites in pKD3 or pKD4 plasmids from Datsenko and Wanner (15). 5′PCR and 3′PCR refer to PCR primers used to screen deletion mutants and correspond to serovar Typhimurium genomic sequences. Primer families are divided by lines of space.
↵b Underlined sequences denote priming sequences for P1, P2, or P4 sites (15) or epitope tag sites in pSU315 (HA) or pSUB11 (3xFLAG) for 5′ epitope tag primers (65).
