Article Figures & Data
Figures
- Fig. 1.
Immunodetection of RcsA in lon mutants. Equal amounts of protein from whole-cell extracts were boiled in sample buffer, fractionated on a 14% tricine-SDS-polyacrylamide gel, and analyzed by immunoblotting with preabsorbed polyclonal antiserum specific to RcsA protein. Lanes: 1, SG20781 (lon+); 2, SG20780 (Δlon); 3, JT1900 (class III lon); 4, JT1916 (class II lon); 5, JT1920 (class I lon).
- Fig. 2.
Immunodetection of SulA in lon mutants. Cultures were treated with UV light as described in Materials and Methods. Samples were treated as described for Fig. 1 except that antiserum specific to SulA was used. Lanes: 1, SG20780 (Δlon), no UV; 2, SG20781 (lon+), no UV; 3, SG20781 (lon+), with UV; 4, SG20780 (Δlon), with UV; 5, JT1900 (class III lon), with UV; 6, JT1916 (class II lon), with UV; 7, JT1920 (class I lon), with UV.
- Fig. 3.
Comparison of the predicted coiled-coil structure (generated with COILS version 2.2 [39, 40, 48]) to the predicted domain structure of E. coli Lon protease.
Tables
- Table 1.
Bacterial strains and plasmid used in this study
Straina or plasmid Relevant genotype Reference(s), source, or construction E. coli strains CAG12017 lon+zba-3054::Tn10 52 DDS90 lon+ rcsA+rcsA90::lacZ D. Sledjeski; 47, 54 JM12 lon E240K (class III)zba-3054::Tn10 This study JM64 lon G384D (class II)zba-3054::Tn10 This study JM98 lon G374R D483N (class I)zba-3054::Tn10 This study JT1900 lon E240Kzba-3054::Tn10 cpsB10::lacZ SG20781 + P1(JM12) JT1916 lon G384Dzba-3054::Tn10 cpsB10::lacZ SG20781 + P1(JM64) JT1920 lon G374R D483Nzba-3054::Tn10 cpsB10::lacZ SG20781 + P1(JM98) JT2029 proCYA221 zaj-403::ΔTn10 rcsA+ rcsA90::lacZ DDS90 + P1(SG1030) JT2036 lon E240Kzba-3054::Tn10 Δgal-165 SG21020 + P1(JM12) JT2037 lon G384Dzba-3054::Tn10 Δgal-165 SG21020 + P1(JM64) JT2038 lon G374R D483Nzba-3054::Tn10 Δgal-165 SG21020 + P1(JM98) JT2046 Δlon-510 rcsA+rcsA90::lacZ JT2029 + P1(SG4144) JT4000 Δlon-510 SG1030 + P1(SG4144) MS100 lon E240Kzba-3054::Tn10 rcsA+rcsA90::lacZ DDS90 + P1(JM12) MS101 lon G384Dzba-3054::Tn10 rcsA+rcsA90::lacZ DDS90 + P1(JM64) MS102 lon G374D D483Nzba-3054::Tn10 rcsA+rcsA90::lacZ DDS90 + P1(JM98) SG1030 F− Δlac araD proCYA221 zaj-403::ΔTn10 60 SG4144 Δlon-510 44 SG20250 lon+Δlac 29 SG20780 Δlon-510 cpsB10::lacZ 4 SG20781 lon+cpsB10::lacZ 4 SG21020 lon+ Δgal-165 S. Gottesman SG21155 Δlon Δgal-165 S. Gottesman Plasmid pATC400 pBR322-rcsA+ 58 ↵a All strains except CAG12017 and SG4144 are derived from MC4100 (ΔlacU169 araD flbB rel).
- Table 3.
Phenotypic descriptions of the effects of three classes of lon mutation on the stability of RcsA and SulA protein
Relevant genotype (strainsa) RcsA SulA Mucoidyb cpsB10::lacZ (β-galactosidase activity)c MMS phenotyped Filament formation 8 h after UV exposuree lon+(SG20250 or SG20781) − 5 R No filaments (single cells) Δlon (JT4000 or SG20780) + 418 S Extremely long filaments Class Ilon (JM98 or JT1920) + 575 S Extremely long filaments Class II lon (JM64 or JT1916) +/− 117 S/R Medium-length filaments Class IIIlon (JM12 or JT1900) + 230 R No filaments (single cells) ↵a All strains are MC4100 derivatives. Strains assayed for β-galactosidase activity containcpsB10::lacZ.
↵b Assessed visually. −, nonmucoid; +, mucoid.
↵c β-Galactosidase assays were carried out as described by Miller (45). Values represent the average of three independent assays.
↵d Determined on LB agar containing 0.05% MMS. R, resistant; S, sensitive; S/R, intermediate phenotype.
↵e Assayed as described in Materials and Methods.
- Table 4.
Effects of class I, II, and III lon mutations on rcsA expression as measured byrcsA90::lacZ activity
Relevant genotype (straina) β-Galactosidase activityb Fold change from: lon+ Δlon lon+ (DDS90) 12 0.0045 Δlon (JT2046) 2,641 220 Class Ilon (MS102) 5,137 428 2.0 Class IIlon (MS101) 1,220 100 0.46 Class IIIlon (MS100) 4,658 388 1.8
