Biochar quieting of microbial chatter varies with production conditions

Transcription

1 Biochar quieting of microbial chatter varies with production conditions Caroline Masiello, Ye Chen, Xiaodong Gao, hirley Liu, helly Cheng, Matt Bennett, Jenn Rudgers, Dan Wagner, Kyriacos Zygourakis, and Joff ilberg Departments of Biochemistry & Cell Biology, Bioengineering, Chemical & Biomolecular Engineering, Earth cience, Ecology & Evolutionary Biology

2 Microbes use cell-cell communication to control behaviors Low density Quorum density Higher density = signal A diffusible signal () is synthesized continuously by each bacterium When QUORUM is achieved, accumulates and flips molecular switch that changes biosynthesis Newly synthesized molecules accumulate and affect cell behaviors

3 Diverse behaviors are coupled to population density (growth & health) Biofilm formation ymbiosis Virulence N 2 NH 3 Quorum sensing Legumes (Rhizobium leguminosarum) (Bradyrhizobium japonicum) (inorhizobium meliloti) pore formation Carrot/Potato (Erwinia carotavora) Maize (Pantoea stewartii) Fruit/nut (Agrobacterium tumefaciens) Onion (Burkholderia cepacia) Lettuce (Pseudomonas aeruginosa) Reviews = Annu Rev Phytopath. 2003, 41:455; Annu.Rev.Cell Dev.Biol. 2005, 21:319; oil Biol. 2008, 15: 265

4 Chemical languages vary among species Bacteria (MW = 300 to 2300 amu) Fungi Acylhomoserine lactones Oligopeptides Farnesol Tyrosol dimethoxycinnamate A factor Other: furanosyl borate diesters, quinolones, indole Annu. Rev. Genet : Eukar. Cell :

5 A single microbial species can have parallel conversations Low population density Quorum density Higher density 1 2 = signal = signal Multiple signals (1 & 2) synthesized by each bacterium 2 When QUORUM is achieved, flips different molecular switch that controls biosynthesis reactions (can be different densities) Newly synthesized molecules accumulate and affect different cell behaviors ome N 2 fixing symbionts for plants (e.g., Rhizobium leguminosarum) uses five distinct signals to make decisiosn!

6 Other organisms can silence conversations by making signal-degrading enzymes Potatoes Virulence by Erwinia carotovora requires quorum signal (). No virulence when organisms present that degrade signal used by Erwinia. Nature (2001) 411,

7 Above ground plant biomass Percent root colonization Like quorum quenching organisms, biochar can alter cell growth orhgum root (+Fungi) Glomus intraradices ymbiosis Parasitism ( ) (+BC) ( ) (+BC) Biochar, 400 C (+N nutrient) LeCroy et al. oil Biology Biochemistry :

8 Hypothesis 1. Biochars sorb some of the signals used for microbial cell-cell communication (focus on 12C acyl homoserine lactones, AHL). 2. AHL sorption occurs on the time course of microbial signaling and gene expression. 3. Biochar production conditions influence the extent to which biochar quiets microbial signaling. 4. Biosensors can rapidly assess biochar effects on signaling and provide insight into biological observations.

9 Programming Escherichia coli to report on one acyl homoserine lactone (AHL) 1. Insert DNA that encodes an AHL-dependent transcriptional regulator (LasR) from Pseudomonas aeruginosa DNA lasr gene LasR protein DNA gfp 2. Insert DNA encoding green fluorescent protein (GFP) under LasR regulation +AHL 3. Use AHL addition to switch LasR ON so that it binds DNA, drives GFP synthesis, and makes cells green (which are easy to image) LasR (+AHL) gfp GFP

10 Relative fluorescence Escherichia coli display AHL-dependent fluorescence [AHL] E. coli XL1 (+receiver plasmid) Add AHL to E. coli cultures E. coli XL1 AHL, nm

11 trategy for comparing effects of different biochars on AHL availability [AHL] that makes cells green 1 µm in 1 ml Biologically relevant concentration of AHL within the environment biochar Incubate AHL w. varying [biochar] 1hr incubation Add soluble fraction to E. coli Time is near the doubling rate of microbes within rich nutrient conditions

12 urface area (m 2 /g) low pyrolyzed mesquite was used to study effects of production conditions [AHL] that makes cells green Hardwood slow-pyrolyzed at a range of temperatures 1 µm in 1 ml biochar Incubate AHL w. varying [biochar] 1hr incubation Add soluble fraction to E. coli Temperature, C

13 Relative fluorescence 700 C biochar inhibits AHL-induced fluorescence N Data 6 [AHL] that makes cells green 1 µm in 1 ml biochar Incubate AHL w. varying [biochar] 1hr incubation % by mass is comparable to level that elicits biological effects within soils Add soluble fraction to E. coli mg charcoal/ml

14 Relative fluorescence Production conditions alter biochar inhibition of cellular fluorescence B Data 6 D Data 6 F Data 6 H Data 6 J Data 6 Pyrolysis L temperature N Data 6 Data C 350 C 400 C 450 C 550 C 600 C 700 C mg/ml charcoal Weakest inhibition half-maximal inhibition at 50 mg/ml char trongest inhibition half-maximal inhibition at 1 mg/ml char

15 Relationship between surface area, production temperature, and GFP expression 300 C biochar (mg/ml) needed for GFP inhibition (50%) 350 C Exponential fit (R 2 = 0.99) 400 C 450 C 550 C 600 C 700 C biochar surface area, m 2 /g

16 Absorbance Microbial growth is not altered by charcoal treated water 300 C 350 C 400 C 450 C 600 C 700 C 550 C mg/ml charcoal incubated with AHL for 60 minutes uggests that these charcoal aren t releasing compounds that mimic AHL. (would be interesting to screen biochars for such compounds)

17 Relative fluorescence Inhibition occurs on the time scale of microbial signaling and gene expression F D B [AHL] that makes cells green Data 6 Data 6 Data 6 1 µm in 1 ml C 600 C 700 C 1 1 biochar Incubate AHL w. varying [biochar] min incubation Add soluble fraction to E. coli mg/ml charcoal Note: Under optimal growth conditions (37 C, rich medium), Escherichia coli doubles every minutes

18 An addressable cell-cell signaling system for evaluating hard-to-image biochars ender () cell Receiver (R) cell AHL LasI Cherry AHL LasR +AHL P lasr gfp GFP R R R R 10 mm 20 mm 30 mm

19 37 mm Agar plate assay for quantifying biochar inhibition of microbial chatter Empty agar Agar & biochar Receiver colony #1 ender colony Receiver colony #2 14 mm 14 mm 8 mm 10 mm 8 mm

20 Cells are added to plates, grown for 18 hours, and then imaged LasR +AHL P lasr gfp GFP Receiver cell colony #1 AHL Empty agar AHL LasI ender cell colony Cherry AH L Agar +biochar LasR P lasr gfp Receiver cell colony #2 Chatter inhibition = = ratio green on right:left

21 Biochars inhibit chatter to varying extents! Agar plate #1 Agar plate #2 Agar plate #3 300 C 0.24 Agar plate #1 Agar plate #2 Agar plate #3 700 C 0.02

22 Conclusions Wood biochars disrupt cell-cell communication mediated by AHL signals other signals from bacteria/fungi? Inhibition of coordinated cell behaviors (GFP expression) varied 10 fold other biochars? aged biochr? Biochar effects on gene expression needs to be better characterized tradeoffs? symbionts vs pathogens? ynthetic biology is a simple way to build biosensors to compare biochars better reporters? Unclear how biochars impact cell-cell communication when mixed with soils additive effects? non-additive? relationship to short term vs long term priming?

23 Questions?

24