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75 50 25 37 15 10 6 7 8 5 1 2 3 4 2.7 0 2.9 2 3.3 3.61 2. 06 0.0 4 0.2 0.9 2 1.8 3 OD 600 : Time (hr) : Media : LB + 0.4% NO 3 - 21% O 2 0 O 2 : ΔlasB mutant Elastase PAO1 Fig. S1. Aerobic growth of a ΔlasB mutant and SDS-PAGE analysis of its culture supernatants. The mutant bacteria grown overnight in LB at 37 °C were inoculated at 1:100 in LB + 0.4 % NO 3 - , and growth was monitored by measuring OD 600 . Aliquots of the culture were harvested every hour and protein contents present in each culture supernatant (CS) were analyzed by SDS- PAGE. For a comparison, SDS- PAGE of the PAO1 CS harvested after 8 hr aerobic growth was shown to the right. The protein band that corresponds to the mature elastase is shown with an arrow.
description
21% O 2. O 2 :. Media :. LB + 0.4% NO 3 -. OD 600 :. 0.04. 0.2. 0.92. 1.83. 2.06. 2.70. 2.92. 3.3. 3.61. Time (hr) :. 0. 1. 2. 3. 4. 5. 6. 7. 8. 75. 50. 37. Elastase. 25. 15. 10. Δ lasB mutant. PAO1. - PowerPoint PPT Presentation
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2.70 2.92 3.3 3.612.060.04 0.2 0.92 1.83OD600 :
Time (hr) :
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ΔlasB mutant
Elastase
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Fig. S1. Aerobic growth of a ΔlasB mutant and SDS-PAGE analysis of its culture supernatants. The mutant bacteria grown overnight in LB at 37 °C were inoculated at 1:100 in LB + 0.4 % NO3
-, and growth was monitored by measuring OD600. Aliquots of the culture were harvested every hour and protein contents present in each culture supernatant (CS) were analyzed by SDS-PAGE. For a comparison, SDS-PAGE of the PAO1 CS harvested after 8 hr aerobic growth was shown to the right. The protein band that corresponds to the mature elastase is shown with an arrow.
Fig. S2. The effect of anaerobiosis on the elastase secretion of various P. aeruginosa strains including clinical isolates. (A) Strains indicated at the top were grown in LB plus 0.4 % (w/v) NO3
- either aerobically (21% O2) or anaerobically (0% O2) for 18 hrs. The level of elastase was analyzed by SDS-PAGE. PA14 and PAK are non-mucoid pathogenic strains, while FRD1 is a mucoid laboratory strain derived from a CF patient. Pneu1, 2 and 3 are non-mucoid isolates from Korean pneumonia patients. (B) MTT cell viability assay of A549 human epithelial cells treated with cell-free culture supernatants (CS) of indicated strains. A549 cells were treated with the same set of CSs as in panel A. Experimental conditions are identical to those described for Fig. 1A. *p<0.01 vs. treatment with anaerobic CS.
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Gene name Primer sequences (5'-3')
mvfRF : ATCAAGCAGGACAACGCGGAR : GCAGGGAGGCATTGCACAAC
rsaLF : ACAGCCCCAAAACATGGCCTR : GGGCAGGTTCTCGCCATTCT
vqsRF : AGCAGATCGCGCTGTTCGAGR : CTCCTCCCTGACCGCATCCT
rhlRF : GCGCTTTCACATCGACCAGGR : GCGGTGGTGTATTCGTCCCA
rhlAF : GCGCTTTCACATCGACCAGGR : GCGGTGGTGTATTCGTCCCA
PA3904F : GGTAGGTAGCGTCGGCACCCR : GGCATTGCAGGCACAGCTTC
PA4677F : CGAAGCGGTGTTCACCAAGCR : GCCGTAACGCTGCAACAACC
xcpPF : GCGCGGACGACATTACAAGCR : CGAGTCTTCTTCGGCGGGTT
pqsAF : CCACTCCGCTGGACGACAACR : GCAGCATGTGCGAGGGAATC
pqsCF : ATCTGTTCGGCTTCCTCGCCR : CGCACTCCATCTGCGAATCC
lasBF : CCGCAAGACCGAGAATGACAR : CTTCCCACTGATCGAGCACT
lasIF : TTCAAGGAGCGCAAAGGCTGR : GTTCTTCAGCATGTAGGGGC
rhlIF : CTTCATCGAGAAGCTGGGCTR : AGGTAGGCGAAGACGTCCTT
rpoDF : AAGGCCCTGAAGAAGCACGGR : GATCGGCATGAACAGCTCGG
Table S1. Primers used for qRT-PCR
