I went to the beach and brought back sand: a story of anoxygenic phototrophic bacteria.

Lorena Rangel

Microbial Diversity Course 2013

MBL

Introduction

Anoxygenic phototrophic bacteria

• chlorophyll-based photosynthetic energy

Overmann 2006. The Prokaryotes

www.nau.edu

• Indole-3-acetic acid (IAA)– Plant hormone

• Signaling compound – Controls root growth, tropism, and plant senescence

– Microorganism capable of interfering with IAA signals– Synthesis and Degradation of IAA by bacteria– At least five IAA biosynthetic pathways identified in bacteria– Example: Pseudomonas savastanoi produces IAA to induce tumor formation

– Examples of bacterial response to IAA exposure• Increased bacterial tolerance

– IAA exposure to nonpathogenic E.coli upregulated genes involved in adaptation to unfavorable environments (Bianco et al. 2006. Arch Microbiol 185, 373)

• Optimizing conditions for bacterial entry– IAA opens stomata, even in dark (Huang et al. 2000. Plant Sci 156, 65)

Introduction

Sippewissett Salt Marsh

DiatomsCyanobacteria

Purple Sulfur BacteriaGreen Sulfur Bacteria

Purple NonSulfur Bacteria

Overmann 2006. The Prokaryotes

PNSB

GSB

PSB

All in 10ml Marine Phototrophic Base!

Add 1mM Na succinateIlluminate at 850nm

Add 5mM Na thiosulfateAdd 1mM Na2SIlluminate at 850nm

Add 3mM Na2SIlluminate at 750nm

SHAKE TUBES

GSB

PSB PNSB

Purple Sulfur Bacteria

Green Sulfur Bacteria

• Class Gammaproteobacteria• Order Chromatiales• BChl a or b• Internal sulfur granules

• Phylum Chlorobi• BChl c, d, or e and BChl a• External sulfur granules

Purple Non-Sulfur Bacteria

• Highest metabolic flexibility of all phototrophs

• BChl a or b• Class Alphaproteobacteria

or Betaproteobacteria

GSB

PNSB

PSB

ChlorinAbsorption maxima (nm)

Whole Cells

BChl a 375, 590, 805, 830-911

BChl b400, 605, 835-850, 986-1035

BChl c 457-460, 745-755

BChl d 450, 715-745

BChl e 460-462, 710-725

BChl g 375, 419, 575, 788

590.

2258

9.75

? Rhodovulum sp. JA545Prosthecochloris

aestuarii DSM 271

Acetate

Propionate

Citrate

Hypothesis1:

Under limited light conditions PNS bacteria will not grow at rates comparable to “natural” or full light exposure.

Treatments • 24L• 16L:8D• 0L

Hypothesis2:

PNS bacteria will grow at a greater rate when provided a less complex carbon source

Treatments • 10mM Citrate• 10mM Acetate• 10mM Propionate

Propionate + Tryp

Propionate

Acetate + Tryp

Acetate

Citrate + Tryp

Citrate

Carbon Sources 24h Light

0

2

4

6

8

10

12

0 2 4 6 8 10

Days Post Inoculation

Con

cent

ratio

n (m

M)

Carbon Sources 16h Light

0

2

4

6

8

10

12

0 2 4 6 8 10

Days Post Inoculation

Con

cent

ratio

n (m

M)

Carbon Sources 0h Light

0

2

4

6

8

10

12

0 2 4 6 8 10

Days Post Inoculation

Con

cent

ratio

n (m

M)

Do PNSB make IAA?

Hypothesis3:

PNS bacteria have the ability to make indole-acetic acid.

Rhodovulum sp. JA545

Rhodovulum sp. PH10:• Indole-3-glycerol phosphate

synthase• Tryptophan synthase

Ouyang et al 2000.. The Plant Journal 24: 327-333.

Chorismate

Anthranilate

PRA

CdRP

IGP

Indole

Tryptophan

IAA

ASA GI: 402500488ASB GI: 402500495

PAT GI: 402500496

PAIGI:495666825

IGSGI: 402500497

TSAGI: 402498195

TSBGI: 495666823

Trp-independent

Trp-depen

dent

Chorismate

Anthranilate

PRA

CdRP

IGP

Indole

Tryptophan

IAA

ASA GI: 402500488ASB GI: 402500495

PAT GI: 402500496

PAIGI:495666825

IGSGI: 402500497

TSAGI: 402498195

TSBGI: 495666823

Trp-independent

Trp-depen

dent

Standard

- tryp

+ tryp

+ tryp

- tryp

C C CA A AP P P

24h 16h 0h

200µM 100µM 80µM 60µM 40µM 20µM 10µM 0µM

Standard

- tryp

+ tryp

+ tryp

- tryp

C C CA A AP P P

24h 16h 0h

200µM 100µM 80µM 60µM 40µM 20µM 10µM 0µM

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10

Day(s) Post Inoculation

Con

cent

rati

on (

uM) Citrate/NH4Cl 24h

Citrate/NH4Cl 16h

Citrate/NH4Cl 0h

Citrate/Tryp 24h

Citrate/Tryp 16h

Citrate/Tryp 0h

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10

Day(s) Post Inoculation

Con

cent

rati

on (

uM) Propionate/NH4Cl 24h

Propionate/NH4Cl 16h

Propionate/NH4Cl 0h

Propionate/Tryp 24h

Propionate/Tryp 16h

Propionate/Tryp 0h

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10

Day(s) Post Inoculation

Con

cent

rati

on (

uM) Acetate/NH4Cl 24h

Acetate/NH4Cl 16h

Acetate/NH4Cl 0h

Acetate/Tryp 24h

Acetate/Tryp 16h

Acetate/Tryp 0h

Salkowski Assay

Measures specifically for IAA, indolepyruvic acid, and indoleacetamide

Tryptophan IAA

Tryptophan IAA

5-Hydroxyindoleacetic acidIndole-3-acetaldehyde

Main Findings

Conclusions

• Enriched for GSB (Prosthecochloris), PSB and PNSB (Rhodovulum)

• Propionate encourages faster growth rates of PNSB

• PNSB may produce low concentrations of indole-like compounds

Conclusions

• Enriched for GSB (Prosthecochloris), PSB and PNSB (Rhodovulum)

• Propionate encourages faster growth rates of PNSB

• PNSB may produce low concentrations of indole-like compounds

Main Findings

Conclusions

• Enriched for GSB (Prosthecochloris), PSB and PNSB (Rhodovulum)

• Propionate encourages faster growth rates of PNSB

• PNSB may produce low concentrations of indole-like compounds

Main Findings

Conclusions

• Optimization of carbon sources for future PNSB enrichments

• First evidence of anoxygenic phototrophic bacteria producing indole

• Further work on the actual compound produced