The Alchemy of Becoming: Characterizing the Re-
evoltuion BamBCE- Mutants
BAM Complex
β – barrel Outer Membrane Proteins in Escherichia coli
P4
Nascent MatureOMP
AssembledPorin
Sec
Cyt
BamB
Bam
C
Bam
D
BamA
BamE
PrecursorOMP
P5
P3
P2
P1
Chaperones(SurA, Skp)
Per
Gram-negative Bacteria
OM
IM
Misra 2012 ISRN Mol Biol
What is the minimal amount of BAM A Complex you can create in E coli?
How can E coli compensate for loss of the other BAM proteins?
QUESTIONS
Retro-evolution of BAM Complex
BamABCDE BamACD (Conditional Lethal)
BamA*CD
ΔBamBE
BamA*CD
ΔBamC
BamA*D (Conditonal Lethal)
BamA*D
X*
VRGY(F)
R541 (E780)
Q440 (N631)
T285 (F474) G444 (G635)
Q458 (G649)(A652)
L6 loop
Tellez Jr. & Misra 2012 J BacteriolLeonard-Rivera & Misra 2012 J Bacteriol
E. coli (ancestral proteobacteria)
Significance of Retro and Re-evolution of BAM Complex
Retro-evolution
Re-evolution
OmpACF
1 .83 .81 .60 .47
OmpA 1 .78 .83 .51 .60OmpC 1 1 .97 .89 .79OmpF 1 .82 .84 .34 .17
OmpACF
1 .92 1.28
.95 .1.47
OmpA 1 .77 1 .83 1.37
OmpC 1 1 1.39
1.1 2
OmpF 1 .50 1.14
.87 .11
OmpCOmpFOmpA
OmpCOmpFOmpA
A*BCDE+
A*CDE+
A*BCD+
A*CD+
A*D+
A*D+
ABCDE+
1-1 1-2 3-8 4-1
A*BCDE+
A*CDE+
A*BCD+
A*CD+
A*D+
OMPs A*D 1-1 1-2 3-8 4-1OMPs
My story begins with 6 Mutants…..
1D 2C3B
4A4C4D
MacConkey
Resistant
λ Resistant
λ Sensitive
LBA λ Streak
Minimal Plate 30̊ / 48hr
OmpCOmpFOmpA
OmpCOmpFOmpA
OmpCOmpFOmpA
Gel 1
126 1D 2C 3 B
4A 4C 4D1827
126 1D 2C 3 B
4A 4C 4D1827
126 1D 2C 3 B
4A 4C 4D1827
Gel 3Gel 2
SDS Urea PAGE
SDS-PAGE Gel Averages per Outer Membrane Protein
1827 126 1D 2C 3B 4A 4C 4D 0
0.5
1
1.5
2
2.5
Omp C
Omp C
1827 126 1D 2C 3B 4A 4C 4D 0
0.2
0.4
0.6
0.8
1
1.2
Omp F
Omp F
1827 126 1D 2C 3B 4A 4C 4D 0
0.2
0.4
0.6
0.8
1
1.2
1.4
Omp A
Omp A
1827 126 1D 2C 3B 4A 4C 4D0
5
10
15
20
25
30Disk Diffusion Assay
Average Diameter
NOVOBIOCIN
0 50 100 150 200 250 300 3500
20
40
60
80
100
120
140
160
1827 Av126 Av1D Av4A Av
NPN Efflux Assay
Slopes:1827: -3.0063(a.u.)/(s) ± 0.393252 126: -2.07445 (a.u.)/(s) ±
0.3864541D: -4.69265 (a.u.)/(s) ±
0.7953734A: -3.77925 (a.u.)/(s) ±
0.451426
WESTERN BLOT
STAINED MEMBRANE
BAM A
‘CONTROL’
126
1D 2C 3B 4A 4C 4D1827
126
1D 2C 3B 4A 4C 4D1827
1827 126 1D 2C 3B 4A 4C 4D0
2
4
6
8
10
12
BAM A Intensity Analysis
Strains
Inte
nsity
INT*
mm
2
WESTERN BLOT
STRAINS BAM A Control1827 8.663435 5.967747126 9.712198 5.67631D 8.338653 5.3149192C 7.181835 5.4975073B 6.618693 5.0068624A 7.571512 4.8238554C 7.863744 5.5024014D 8.292057 4.677809
126
1D 2C 3B 4A 4C 4D1827
STAINED MEMBRANE
WESTERN BLOT
TolC
126
1D 2C 3B 4A 4C 4D1827
126 1D 2C 3B 4A 4C 4D18
27
Future Plans
• PCR •Western Blot