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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