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The Alchemy of Becoming: Characterizing the Re-evoltuion BamBCE- Mutants

BAM Complex barrel Outer Membrane Proteins in Escherichia coli

P4

Nascent MatureOMP

AssembledPorin

SecCyt

BamBBamCBamDBamABamEPrecursorOMP

P5

P3

P2

P1Chaperones(SurA, Skp)PerGram-negative Bacteria

OM

IMMisra 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 ComplexBamABCDE BamACD (Conditional Lethal)

BamA*CD

BamBEBamA*CD

BamCBamA*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 BacteriolE. coli (ancestral proteobacteria)

Significance of Retro and Re-evolution of BAM ComplexRetro-evolutionRe-evolutionOmpACF1.83.81.60.47OmpA1.78.83.51.60OmpC11.97.89.79OmpF1.82.84.34.17

OmpACF1.921.28.95.1.47OmpA1.771.831.37OmpC111.391.12OmpF1.501.14.87.11

OmpCOmpFOmpAOmpCOmpFOmpA

A*BCDE+A*CDE+A*BCD+A*CD+

A*D+

A*D+ABCDE+1-11-23-84-1

A*BCDE+A*CDE+A*BCD+A*CD+

A*D+OMPsA*D1-11-23-8

4-1OMPs

My story begins with 6 Mutants..1D 2C3B

4A4C4D

MacConkey Resistant

Resistant Sensitive

LBA Streak

Minimal Plate 30 / 48hr

OmpCOmpFOmpA

OmpCOmpFOmpA

OmpCOmpFOmpA

Gel 11261D2C3B4A4C4D18271261D2C3B4A4C4D18271261D2C3B4A4C4D1827Gel 3Gel 2SDS Urea PAGE

SDS-PAGE Gel Averages per Outer Membrane Protein

NOVOBIOCIN

NPN Efflux AssaySlopes: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 BLOTSTAINED MEMBRANE

BAM A

CONTROL1261D2C3B4A4C4D18271261D2C3B4A4C4D1827

WESTERN BLOTSTRAINSBAM AControl18278.6634355.9677471269.7121985.67631D8.3386535.3149192C7.1818355.4975073B6.6186935.0068624A7.5715124.8238554C7.8637445.5024014D8.2920574.677809

1261D2C3B4A4C4D1827

STAINED MEMBRANE

WESTERN BLOT

TolC1261D2C3B4A4C4D18271261D2C3B4A4C4D1827

Future Plans PCR Western Blot