Bc17 And Cyt B150 A Fools Game

WHY CAN STRAIN BC -17 GROW AEROBICALLY AND NOT

PHOTOSYNTHETICALLY?

WHY DO ONLY SOME OF THE H 217Q I MUTATIONS TURN GREEN

WHEN GROWN ANAEROBICALLY?

The bc1 complex and cyt b150: The role of the bc1 complex in the light and oxygen regulation

of the expression of the photosynthetic apparatus of R. sphaeroides

or

R. sphaeroides strain BC-17

The bc1 complex is formed by four sub-units: cytF, cytB, and cytC as part of the fbc operon on

chromosome I.

The fourth sub-unit cytQ, is encoded elsewhere on chromosome I.

R .sphaeroides strain BC17 is a kanamycin insertion mutant for the fbc operon

Genome map of R. sphaeroides

cytQ mapped to ~

2 o’clock on I

cyt FBC

From: http://mmg.uth.tmc.edu/sphaeroides/genome_map/phys_gen_map.pdf

Role of the bc1 complex: 1994

The bc1 complex is thought to provide reduced cytochrome c to aa3

under aerobic conditions, and cbb3 under micro-aerophilic conditions

García-Horsman JA, et al., J Bact., 1994, 176 (18) 5587–5600.

cbb3 oxidase: overview

Expressed maximally under micro-aerophilic or anaerobic conditions

Four non-identical sub-units CcoN, CcoO, CcoP (core), CcoQ (subunit IV)

Oxioreductase cbb3 is upstream of the prrA/prrB two –component regulatory system

cbb3 inversely regulates the PrrBA system via an unknown signal from the CcoQ subunit (RdxBH?)

The prrA/prrB regulates the expression of the bacteriochlorophyll synthesis loci in a positive manner

ccoNOQP

From: http://mmg.uth.tmc.edu/sphaeroides/genome_map/phys_gen_map.pdf

Genome map of R. sphaeroides

Much Mutational Work on ccb3 :1998

Double mutations of ccb3 with both thePrrAB system or the rdxBH gene show affecton photosynthetic expression as follows:

ccb3 deletion : spectral complexesexpressed (CCOP1)

ccb3 deletion with the PrrAB deletion:lack of any photosynthetic complexesunder any growth condition (PRRA2,PRRB1)

Filled bar: LHC II (850-800)

Open bar LHC I (875)

Aerobic

Anaerobic

O’Gara J, Eraso JM , S Kaplan J Bact., 1998, 180 (16) 4044–4050


The bc1 complex reducescytochrome c2 providing reductantto cbb3

Aerobic conditions: O2 is the finalelectron acceptor

Anaerobic conditions the electronflow controls the ratio of thecartenoids SE (LHII) to SO (LHI)

Low light: SE SO

Dark + O2 : SE

+ DMSO: SE SO

SE – spheroidene (yellow)

SO – spheroidenone (red)

Yeliseev & Kaplan, J. Bact., 1996 ,178: 5877 – 5883; Oh & Kaplan, Biochemistry, 1999, 38: 2688-2696


Oh J & S Kaplan, The EMBO J, 2000, 19 (16) 4237 - 4247

The bc1 complex is shown to provide reductant to either aa3 or cbb3 via cytochrome c2 or cy via mutational analysis (R. sphaeroides): 2001

Daldal, et.al., J Bact. 2001, 2013–2024.

Anaerobic Growth Scheme for R. sphaeroides: 2005

Happ, et.al., Mol. Micro., 2005, 903 - 914

H217 Mutants of the QI site of the bc1 complex

Gray & Daldal (1994) H217D, R, and L in R. capsulatus

D and R grow photohetrotrophically with 60% of wild type bc1 levels, L is non-photosynthetically competent with 10% of wt bc1 levels

R. capsulatus was grown on MYPE media (LB for Rhodobacter genus) and authors noted some cases of reversion.

Hacker & Gennis (1993) H217A, complex assembled, cyt bH oxidation blocked in R. sphaeroides strain BC-17

Grown on sistrom media (defined media), no mention of reversion

published but personal communication indicates there was a difficulty in growing this mutation.

DMSO „Rescues‟ QI Site Mutations

Recent mutations constructed in R. sphaeroides consist of H217D, F, G, K, R, W, and Y The D, G, K, R, and Y mutants are photosynthetically competent, yet will revert to H in

the presence of oxygen or when grown photosynthetically on defined media (sistrom) with succinate as a carbon source.

The F and W mutations are photosynthetically incompetent when grown on defined media, and are lethal to the organism when oxygen is present.

The H217N and H217Q mutations both have been claimed to be highly detrimental to the organism (Hacker (Q), Padden (N).

Personal note: The H217N mutation would never stabilize in R. sphaeroides, initial selection after mating always yielded revertant or no colonies on the mating selection plate.

DMSO „rescues‟ the 1st class of mutations, but the amount needed increases with the „severity‟ of the mutation (R,K < D,Y <G <W <F <N,Q)

DMSO will also rescue W, but the organism remains photosynthetically incompetent and sickly.

DMSO has yet to be shown to be able to „rescue‟ the H217F mutant

DMSO decreases the amount of spectral complexes formed, 2000

2.4.1: WT

CCOP1: ccoP mutant

CCOP1/FNRL:

ccoP and fnrL mutant

PPS1: ppsR mutant

Oh J & S Kaplan, The EMBO J, 2000, 19 (16) 4237 - 4247

DorR represses cycA in the presence of DMSO

Tavano C., Comolli J., Donohue T., Microbio., 2004, 150 (6), 1893 - 1899 .

Model of the DMSO Reductase System: 1998

Mouncey & Kaplan, J. Bact., 1998, 1951-1961

Photosynthetic spectral complexes: 2007A: Aerobic; B. Anaerobic + DMSO

deletion

wildtype

mutant

Kim et. al., J. Bact., 2007, 5617-5625.

mutant

Roh, J. H. et al. J. Biol. Chem. 2004, 9146-9155

Expression patterns of genes encoding components of the electron transport chain and other redox active proteins: 2004

R. sphaeroides strain BC-17

Can not grow photosynthetically due to loss of reductant being passed to cbb3

Can grow aerobically using quinol oxidase (Qxt) in place of the bc1 complex

Null mutants for the cbb3 gene turn green in the presence of oxygen

AppA/PpsR Regulatory System

PpsR is a repressor of many of the genes responsible for bacteriochlorophyll synthesis

AppA is an anti-repressor that block PpsR binding

AppA responds to the redox state of the quinone pool and blue light

Zeilstra-Ryalls, J., et, al.,1998, J. Bact.,180:2801-2809.

bc1 complex and the b150 form

The bc1 complex has three potentiometric midpoints observed during a redox titration: -90, 50 and 150 mV

Two of these three are ascribed to the two hemes present: -90 (bL) and 50 (bH) (see Fig I.)

The third midpoint b150 is proposed to be the „high-potential‟ form of the bH cytochrome arising from a redox state between the quinone and bH at the Qi site.

The question is what is the nature of this redox state? What is the contribution of each species?

How do the species interact with each other to form the 150 mv signal?

Does the Em of either species „tune‟ to the conditions present?

Is the b150 form a product only of the QI:bH couple or are additional conditions necessary? Redox poise, pH, mutations in the QI pocket

Gray & Dadal, Biochemistry, 1994, 723 - 733

R. capsulatus on MYPE

R. capsulatus H217 Mutant Data

Authors mention “Plots of the absorption change at various ambient redox potentials shows the spectrum of cytochrome b150 appears identical to that of ferro-cytochrome bH with a single maximum in the α band at 560 nm”

A: 180 mV

B: 180 mV + 10 μM AA

C: 100 mV

D: 100 mV + 10 μM AA

cytochrome b red.

cytochrome c ox.

The values from the Table in slide 24 were used in a program provided

by Crofts which attempted to model the contribution of each species

of the bc1 complex to the spectra observed under experimental

conditions seen in slide 27.

Conclusion: The program successfully recreated the curves for the WT,

H217D and H217R mutants. However, the program failed to reproduce

the curves measured for the H217L mutation. (slide 23)

pMTS H217R

H217D H217L

H217 mutations affect the formation of the chromatophore

The lack of „depth‟ in the 542nm signal (seen below) can indicate either a lack of c2, ora lack of BRCs. When grown in the presence of DMSO the level of cytochrome c2 isdecreased, and a irregular electron flow through the cbb3 oxidase will lead to adecrease in spectral complex formation by decreasing the amount of PrrA, a enhancerfor photosynthetic gene expression. Both pathways lead to a decrease in spectralcomplex formation. From the image above it is clear the H217 mutation affects theexpression of the genes responsible for the „greening‟ of the chromatophore.

Heavy fraction of WT

chromatophore prep

Heavy fraction of H217x

chromatophore prep

Conclusion

The cyt b150 form has never been „uncoupled‟ from the central physiology of the Rhodobacter species. The intact

chromatophore is needed to study the cyt b150 form and the H217 mutations are difficult to study because the amino

acid is essential. The cells will revert or die unless DMSO is provided. This has the effect of vastly reducing the spectral

complexes of the chromatophore and the amount of cytochrome c2. For these reasons it is not clear if cyt b150 is

exclusively due to the interaction of QI and bH, or does the surrounding physiology of the chromatophore play a role?

Any and all mutations in the bc1 complex will have the potential to affect the assembly of the chromatophore, by altering the

activity of the PrrAB system. The chromatophore is the unit used to study the affect of the mutation, how can any

conclusions be readily drawn if the unit of measurement fluxes along with the change?

To solve this problem the BC17 strain needs to be re-done in a DorR- / PpsR- background (Donohue 2004, Kaplan 2009).

This combination of mutants will remove the repressor for photosynthetic gene expression and cytochrome c2

expression. This will eliminate the stress on chromatophore formation for originating from the cbb3 complex under

anaerobic conditions. This strain (BC 18?) will restore the full complement of photosynthetic complexes to the

chromatophore and allow the H217 mutations, and all other bc1 complex mutations, to be studied uncoupled from the

central Rhodobacter physiology.

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.0040

-0.0035

-0.0030

-0.0025

-0.0020

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

BC17 pRK415 H217K 6 flash 10 M AntiA 10 M Myxo

pH 7.0 ascorbate reduction aerobic

Y A

xis

Title

Time

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.0040

-0.0035

-0.0030

-0.0025

-0.0020

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

BC17 pRK415 H217D 6 flash AntiA 10 M

pH 7.0 ascorbate reduction, aerobic

Y A

xis

Title

Time

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.0040

-0.0035

-0.0030

-0.0025

-0.0020

-0.0015

-0.0010

-0.0005

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

BC17 pRK415 H217D 6 flash No Inh

pH 7.0 ascorbate reduced aerobic

Y A

xis

Title

Time

cytc

cytbH

RC

bLold

H217D

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

H217G 10 M AntiA 10 M Myxo

1 mM KCN, 1 mM ascorbate, 5 M val., 5 M nig.

Y A

xis

Title

X Axis Title

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

H217G no inh.

1 mM KCN, 1 mM ascorbate, 5 M val., 5 M nig.Y

Axis

Title

X Axis Title

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

Y A

xis

Title

X Axis Title

H217G no inh.


H217G

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

H217R no inh.


Y A

xis

Title

X Axis Title

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

H217R 10 M AntiA


Y A

xis

Title

X Axis Title

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

H217R 10 M AntiA 10 M Myxo


Y A

xis

Title

X Axis Title

cytc

cytbH

RC

bLold

H217R

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.007

-0.006

-0.005

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

BC17 pRK415 H217Y No Inh

6 flashes pH 7.0 ascorbate reduction aerobic

Y A

xis

Title

Time

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.007

-0.006

-0.005

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

BC17 pRK415 H217Y 10 M AntiA


Y A

xis

Title

Time

cytc

cytbH

RC

bLold

-0.2 0.0 0.2 0.4 0.6 0.8 1.0

-0.007

-0.006

-0.005

-0.004

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

BC17 pRK415 H217Y 10 M AntiA 10 M Myx


Y A

xis

Title

Time

cytc

cytbH

RC

bLoldH217Y

Technology

Bc17 And Cyt B150 A Fools Game