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