Table S1. Effect of strain PA14 mutations in two-component regulatory system kinases and response regulators on cytotoxicity towards human bronchial epithelial cells. In general 3 or more experiments were performed assessing cytotoxicity (measured as LDH release after 8 hours of infection); results are expressed as the mean ± standard error expressed as a percentage of cytotoxicity caused by WT.
| PA14 locus ID | PAO1 homolog | Gene name | Description | Mean % of WT ± standard error |
| Mutants with increased cytotoxicity | ||||
| PA14_07840 | PA0601 | agtR | Two-component response regulator; contains a CheY-like receiver domain; amine uptake | 119.9 ± 4.2 |
| PA14_52260 | PA0928 | gacS | Sensor/response regulator hybrid; multi-host virulence through regulation of small regulatory RNAs RsmZ and RsmY | 134.3 ± 4.7 |
| PA14_49170 | PA1180 | phoQ | Two-component sensor kinase PhoQ | 121.1 ± 5.7‡ |
| PA14_45590 | PA1458 | Probable two-component sensor kinase; putative homolog of E. coli chemotaxis regulator CheA | 144.3 ± 6.3 | |
| PA14_33780 | PA2388 | fpvR | Probable Fe2+-dicitrate sensor kinase | 121.6 ± 7.9 |
| PA14_30650 | PA2586 | gacA | Response regulator; multi-host virulence through regulation of small regulatory RNAs RsmZ and RsmY | 144.7 ± 15.8 |
| PA14_26810 | PA2882 | Probable two-component sensor kinase; has binding domain homologous to that found in sensors of C4-dicarboxylates | 139.3 ± 15.7 | |
| PA14_16470 | PA3704 | wspE | CheA-type sensor kinase; c-di-GMP regulation | 134.5 ± 10.4 |
| PA14_62530 | PA4725 | cbrA | Two-component sensor kinase; required for carbon-nitrogen balance and control of catabolite repression | 158.8 ± 9.1 |
| PA14_62540 | PA4726 | cbrB | Two-component response regulator; required for carbon-nitrogen balance and control of catabolite repression | 140.8 ± 9.7 |
| PA14_67680 | PA5125 | ntrC | Two-component response regulator; regulates use of carbon and nitrogen | 122.6 ± 4.1 |
| PA14_67670 | PA5124 | ntrB | Two-component sensor kinase; regulates use of carbon and nitrogen | 117.1 ± 5.8 |
| PA14_70790 | PA5364 | Probable two-component response regulator | 130.4 ± 9.1 | |
| PA14_72740 | PA5512 | mifS | Two-component sensor kinase; regulates biofilm development | 119.6 ± 13.2 |
| Mutants with decreased cytotoxicity | ||||
| PA14_05320 | PA0408 | pilG | Type IV pilus response regulator; required for pilus extension and twitching motility | 28.8 ± 4.4 |
| PA14_05330 | PA0409 | pilH | Type IV pilus response regulator; required for pilus retraction | 25.8 ± 1.5 |
| PA14_50220 | PA1097 | fleQ | Flagella major transcriptional regulator; cyclic-di-GMP responsive; potential FleSR modulator | 77.6 ± 12.7 |
| PA14_50200 | PA1098 | fleS | Two-component sensor kinase; required for hook and basal body protein biosynthesis for flagellum assembly | 20.6 ± 6.7‡ |
| PA14_50180 | PA1099 | fleR | Two-component response regulator; required for hook and basal body protein biosynthesis for flagellum assembly | 12.3 ± 0.8‡ |
| PA14_16500 | PA3702 | wspR | Two-component response regulator with GGDEF domain; c-di-GMP regulation | 16.8 ± 3.7 |
| PA14_12780 | PA3948 | rocA1 | Two-component response regulator; cyclic-di-GMP regulation | 53.2 ± 0.9 |
| PA14_60260 | PA4547 | pilR | Two-component response regulator; required for pilus expression and therefore for type IV pilus biosynthesis | 14.6 ± 4.5 |
| PA14_68230 | PA5165 | dctB | Two-component sensor kinase; regulates a C4-dicarboxylate transport system with DctD | 14.9 ± 3.5 |
| PA14_69470 | PA5261 | algR | Alginate biosynthesis regulatory protein | 33.2 ± 11.7 |
| No major change in cytotoxicity | ||||
| PA14_00430 | PA0034 | Probable two-component response regulator | 108.9 ± 5.1 | |
| PA14_01860 | PA0150 | Probable transmembrane sensor kinase | 101.4 ± 3.0 | |
| PA14_02250 | PA0178 | cheA | Probable two-component sensor kinase | 107.5 ± 4.1 |
| PA14_02260 | PA0179 | Probable two-component response regulator | 103.1 ± 4.1 | |
| PA14_06060 | PA0463 | creB | Two-component response regulator; catabolism and motility | 94.2 ± 3.3 |
| PA14_06070 | PA0464 | creC | Two-component sensor kinase; catabolism and motility | 109.9 ± 5.4 |
| PA14_07820 | PA0600 | agtS | Two component ensor kinase; amine uptake | 95.9 ± 7.0 |
| PA14_54510 | PA0756 | Probable two-component response regulator | 103.3 ± 1.3 | |
| PA14_54500 | PA0757 | Probable two-component sensor kinase | 109.4 ± 4.9 | |
| PA14_52240 | PA0930 | pirS | Two-component sensor kinase; iron acquisition | 109.9 ± 3.5 |
| PA14_49440 | PA1157 | Probable two-component response regulator | 107.5 ± 3.8 | |
| PA14_49420 | PA1158 | Probable two-component sensor kinase | 113.8 ± 5.7 | |
| PA14_49180 | PA1179 | phoP | Two-component response regulator; regulates cytotoxicity and resistance to cationic peptides | 104.4 ± 1.8 |
| PA14_49160 | PA1243 | Probable sensor kinase /regulator hybrid | 94.0 ± 8.5 | |
| PA14_47390 | PA1301 | Probable transmembrane sensor kinase | 111.2 ± 8.2 | |
| PA14_46980 | PA1336 | aauS | Two-component sensor kinase; amino acid uptake | 110.1 ± 0.3 |
| PA14_46850 | PA1347 | Probable transcriptional regulator | 106.6 ± 1.3 | |
| PA14_46370 | PA1396 | Probably two-component sensor kinase | 98.5 ± 3.5 | |
| PA14_46060 | PA1422 | gbuR | Transcriptional regulator | 95.5 ± 4.5 |
| PA14_45880 | PA1437 | Probable two-component response regulator | 106.7 ± 3.1 | |
| PA14_45870 | PA1438 | Probable two-component sensor kinase | 111.6 ± 4.5 | |
| PA14_43350 | PA1636 | kpdD | Two-component sensor kinase; potassium transport regulation | 109.8 ± 4.5 |
| PA14_42220 | PA1727 | mucR | Two component sensor kinase; alginate regulation | 113.0 ± 4.5 |
| PA14_41490 | PA1785 | nasT | Response regulator; nitrate/nitrite assimilation | 103.2 ± 2.6 |
| PA14_41270 | PA1798 | parS | Two-component sensor kinase; resistance to cationic peptides | 93.9 ± 8.5 |
| PA14_41260 | PA1799 | parR | Probable two-component response regulator; resistance to cationic peptides | 107.7 ± 4.3 |
| PA14_40570 | PA1851 | Probable two-component response regulator | 104.4 ± 1.9 | |
| PA14_38970 | PA1976 | ercS' | Probable two-component sensor kinase; ethanol oxidation | 112.7 ± 5.1 |
| PA14_38900 | PA1980 | eraR | Probable two-component response regulator; ethanol oxidation | 98.7 ± 5.0 |
| PA14_37980 | PA2051 | Probable transmembrane sensor kinase | 103.9 ± 5.7 | |
| PA14_36420 | PA2177 | Probable histidine sensor kinase | 100.6 ± 4.6 | |
| PA14_32580 | PA2479 | Probable two-component response regulator | 107.3 ± 4.7 | |
| PA14_31960 | PA2523 | czcR | Two-component response regulator; metal and imipenem resistance | 107.4 ± 4.1 |
| PA14_31950 | PA2524 | czcS | Two-component sensor kinase; metal and imipenem resistance | 107.5 ± 1.0 |
| PA14_30830 | PA2572 | Probable two-component response regulator | 111.9 ± 5.2 | |
| PA14_30700 | PA2583 | Probable sensor kinase/response regulator hybrid | 100.3 ± 6.5 | |
| PA14_29740 | PA2656 | Probable two-component sensor kinase | 101.6 ± 6.5 | |
| PA14_29360 | PA2687 | pfeS | Two-component sensor kinase; iron acquisition | 88.0 ± 4.8 |
| PA14_27940 | PA2798 | Probable two-component response regulator | 108.6 ± 3.4 | |
| PA14_27810 | PA2809 | copR | Two-component response regulator; copper resistance | 106.4 ± 5.3 |
| PA14_27800 | PA2810 | copS | Two-component sensor kinase; copper resistance | 93.7 ± 2.3 |
| PA14_27550 | PA2824 | sagS | Two-component sensor/regulator hybrid; motile/sessile switch, activates Gac/Hpt/Rsm system | 88.9 ± 8.6 |
| PA14_24720 | PA3044 | rocS2 | Two-component sensor kinase; c-di-GMP regulation | 96.9 ± 4.5 |
| PA14_24710 | PA3045 | rocA2 | Two-component response regulator; c-di-GMP regulation | 103.2 ± 4.0 |
| PA14_24350 | PA3077 | cprR | Probable two-component system regulatory protein; cationic peptide resistance | 112.8 ± 9.5 |
| PA14_24340 | PA3078 | cprS | Probable two-component sensor kinase; cationic peptide resistance | 98.4 ± 6.2 |
| PA14_22960 | PA3191 | gtrS | Probable two-component sensor kinase; virulence | 107.3 ± 4.4 |
| PA14_22940 | PA3192 | gltR | Two-component response regulator; virulence | 98.6 ± 2.6 |
| PA14_22730 | PA3206 | Probable two-component sensor kinase | 102.5 ± 6.3 | |
| PA14_21700 | PA3271 | Probable two-component sensor kinase | 105.9 ± 11.3 | |
| PA14_20820 | PA3343 | Probable two-component response regulator | 108.8 ± 7.1 | |
| PA14_20780 | PA3346 | Probable two-component response regulator | 105.7 ± 3.9 | |
| PA14_20000 | PA3409 | hasS | Probable Fe2+-dicitrate sensor kinase; diguanylate cyclase | 102.5 ± 5.7 |
| PA14_17670 | PA3604 | erdR | Two-component response regulator; ethanol oxidation | 99.4 ± 10.1 |
| PA14_16350 | PA3714 | Probable two-component response regulator | 110.9 ± 5.3 | |
| PA14_13740 | PA3878 | narX | Two-component sensor kinase; nitrate respiration, biofilm, motility | 100.5 ± 10.9 |
| PA14_12820 | PA3900 | fecR | Probable transmembrane sensor protein | 110.9 ± 5.6 |
| PA14_12820 | PA3946 | rocS1 | Two-component sensor kinase; c-di-GMP regulation | 103.4 ± 7.4 |
| PA14_12810 | PA3947 | rocR | Antagonist of rocA1 response regulator; c-di-GMP regulation with EAL domain | 93.5 ± 1.9 |
| PA14_11680 | PA4032 | Probable two-component response regulator | 106.0 ± 2.6 | |
| PA14_11630 | PA4036 | Probable two-component sensor kinase | 110.9 ± 3.8 | |
| PA14_10770 | PA4112 | Probable sensor/regulator hybrid | 97.4 ± 9.4 | |
| PA14_09690 | PA4196 | bfiR | Probable two-component response regulator; biofilm formation | 90.8 ± 9.9 |
| PA14_09680 | PA4197 | bfiS | Two-component sensor kinase; biofilm formation | 97.5 ± 2.9 |
| PA14_55780 | PA4293 | pprA | Two-component sensor kinase; outer membrane permeability and resistance | 108.8 ± 4.1 |
| PA14_55810 | PA4296 | pprB | Probable two-component response regulator; outer membrane permeability and resistance | 100.5 ± 6.8 |
| PA14_56950 | PA4381 | colR | Probable DNA-binding response regulator; polymyxin resistance | 103.8 ± 2.5 |
| PA14_57140 | PA4396 | Probable two-component response regulator | 103.0 ± 10.0 | |
| PA14_57170 | PA4398 | Probable two-component response regulator | 105.5 ± 12.7 | |
| PA14_58300 | PA4493 | roxR | Two-component response regulator; regulates production of cyanide insensitive oxidase | 107.0 ± 3.8 |
| PA14_58320 | PA4494 | roxS | Two-component sensor kinase; regulates production of cyanide insensitive oxidase | 108.1 ± 6.4 |
| PA14_60250 | PA4546 | pilS | Two-component sensor kinase; type IV pili production | 113.5 ± 5.3 |
| PA14_63150 | PA4776 | pmrA | Two-component response regulator; cationic peptide resistance | 104.3 ± 2.3 |
| PA14_63160 | PA4777 | pmrB | Two-component sensor kinase; cationic peptide resistance | 92.0 ± 10.8 |
| PA14_64050 | PA4843 | gcbA | Probable two-component response regulator; diguanylate cyclase | 108.8 ± 3.0 |
| PA14_64880 | PA4983 | Probable two-component response regulator | 108.5 ± 5.4 | |
| PA14_68250 | PA5166 | dctD | Two-component response regulator; C4-dicarboxylate metabolism | 97.1 ± 3.6 |
| PA14_68680 | PA5199 | amgS | Two-component sensor kinase; membrane stress, aminoglycoside resistance | 91.5 ± 3.3 |
| PA14_70750 | PA5360 | phoB | Two component response regulator; phosphate regulation and quorum sensing | 105.9 ± 3.5 |
| PA14_70760 | PA5361 | phoR | Two-component sensor kinase; phosphate regulation and quorum sensing | 108.8 ± 3.3 |
| PA14_72380 | PA5483 | algB | Two-component response regulator; alginate production | 96.1 ± 3.2 |
| PA14_72390 | PA5484 | kinB | Two-component sensor kinase; alginate production | 93.6 ± 5.6 |
| PA14_59770 | rcsB | Two component response regulator; cup fimbria and biofilm formation | 104.0 ± 5.2 | |
| PA14_59790 | pvrR | Two component response regulator; EAL domain, c-di-GMP regulation, cup fimbria and biofilm | 103.6 ± 4.7 | |
| PA14_59800 | pvrS | Two component sensor kinase; c-di-GMP regulation, cup fimbria and biofilm | 110.0 ± 4.2 | |
†Mutants are listed in order of the PAO1 homolog. Mutants not in the screen included PA1637 kpdE—response regulator with KpdD, potassium transport; PA1786 nasS—sensor with nasT, nitrate/nitrite assimilation; PA2686 pfeR—response regulator with pfeS, iron acquisition; PA0929 pirR—response regulator with pirS, iron acquisition; PA3345 htpB—response regulator with sagS, activates Gac/Htp/Rsm system; PA3879 narL—response regulator with narX, nitrate respiration, biofilm, motility; PA3974 ladS—sensor, virulence, biofilm formation, cytotoxicity; PA4380 colS—sensor with colR, polymyxin resistance; PA4101 bfmR—response regulator with bfmS, biofilm development; PA4102 bfmS—sensor with bfmR, biofilm development; PA4856 retS—sensor, virulence, biofilm formation, cytotoxicity; PA4959 fimX—sensor (orphan?); contains GGDEF and EAL domains; cytotoxicity; PA5200 amgR—response regulator with amgS, membrane stress and aminoglycoside resistance; PA5262 fimS—sensor with algR, virulence, alginate biosynthesis, motility, biofilm; PA5511 mifR—response regulator with mifS, biofilm development; PA14_59780 rcsC—sensor with rcsB, biofilm formation. ‡The phenotype for these mutants was different between PA14 and PAO1 as described in the text.