Team:MichiganState/Results

Results


Isolation and Characterization of Endophytic Bacteria

Our team isolated and characterized 39 strains of endophytic bacteria harbored in the roots of the wild grass Arrhenatherum elatius and the cultivated bioenergy crop switchgrass (Panicum virgatum). These strains were evaluated for resistance to chloramphenicol and streptomycin using the Kirby-Bauer test (Susceptible, Intermediate, or Resistant). Additionally, strains were tested for ACC utilization by plating on DF-ACC agar plates. We measured strain growth rates, with OD600 10X dilution after 18 hrs incubation at 30°C. Risk group was determined based on classification by 16S amplification and sequencing.


Table 1: Characteristics of evaluated strains.

KB Antibiotic Key: R = resistant; S = susceptible; I = intermediately susceptible/resistant
ACC Utilizer Key: Y = yes (does utilize ACC); N = no (does not utilize ACC)
Risk Groups: classified according to this resource.
ID KB Chlor KB Strep ACC utilizer Synonym OD600 after 18 hrs at 30°C, 10X dilution Risk group
1A1T-03 Pseudomonas corrugata/brassciacearum N 0.227 1
1A1T-04 Pseudomonas fluorescens/mandelii N 0.051 1
1C20-02 Pseudomonas fluorescens/mandelii N 0.03 1
UAP1-01 Pseudomonas baetica/moraviensis N 0.226 1
UBP2-01 Pseudomonas baetica/moraviensis N 0.181 1
UBP2-02 Pseudomonas putida/koreensis N 0.152 1
FP0-02 Pseudomonas sp. N 0.178
FBP2-01 N FT0-01 0.16
FBP3-01 N 0.338
UCP1-01 Pseudomonas mohnii N 0.102 1
UCP1-02 Pseudomonas baetica/moraviensis N 0.161 1
UCP1-03 Enterobacter asburiae N 0.336 1
UCP2-01 Pseudomonas moraviensis/baetica N 0.191 1
UP0-02 Burkholderia stabilis Y 0.086 2
FT0-01 S S N FBP2-01 0.227
UT0-01 Pseudomonas baetica N 0.139 1
UCP2-02 Pseudomonas putida/koreensis I S N 0.301 1
1C2P-04 Pseudomonas rhodesiae I I Y 1
FCP2-01 Enterobacter ludwigii S S N
FCP1-02 Acinetobacter calcoaceticus R S N 2
FCP1-01 Pseudomonas fluorescens N 1
UAP1-02 Enterobacter/Klebsiella N
UAP2-01 Pseudomonas vancouverensis/mohnii/ fluorescens R I N 1
FP0-01 Acinetobacter calcoaceticus/rhizosphaerae N 2
UP0-01 Lysinibacillus sp. R R N
1A1T-02 Pseudomonas sp. I S Y
UP0-04 Burkholderia stabilis Y 2
UP0-03 Enterobacter/Klebsiella N
1C2P-01 Pseudomonas sp. R S N
1A1T-01 Pseudomonas sp. Y
2FAP1-02 Pseudomonas sp. S Y
2UAP1-02 Pseudomonas sp. S Y
2FP0-01 Bacillus sp. S N
2UCP1-01 S N
2UBP1-01 Bacillus mycoides S N
2FP0-02 Pseudomonas sp. S N
2UT0-01 Pseudomonas sp. S Y
2FCP1-01 Bacillus mycoides S N
2FAP1-01 Pseudomonas sp. S N
2UAP1-01 Pseudomonas sp. S N
2FT0-01 Pseudomonas sp. S N
Figure 1. ACC utilization assay. UPO-02 (upper-right quadrant on left plate) is positive for ACC utilization.
ACCD assay plates

Transformation of endophytic bacteria

After a series of transformation attempts, a transformation protocol was developed which could be consistently used. About 25-50% of colonies on later plates are transformants.

Figure 2. A) FCP2-01 transformed with GFP (left) vs wild-type (right) under blue light with orange filter. B) Transformed colonies on an agar plate.

Plant persistence tests

A key test of the effectiveness of an endophyte to impact its host if the endophyte can persist in and on the plant. FCP2-01 was isolated from the roots, and therefore we examined ability for the endophyte to inhabit the roots of Brachypodium distachyon. Seeds were inoculated by submerging in bacterial culture, then grown axenically in glass culture tubes in a growth chamber. Confocal micrographs were acquired at intervals of 1 week for 1-5 weeks post-inoculation. Bacteria were observed on roots at weeks 3 and 4 most clearly, but overwatering may have been responsible for no visible colonies in week 5.

Figure 3. 3 weeks post inoculation on plant roots. A) FCP2-01 forming large colonies about 2 mm long in B. distachyon roots B) Detail of panel A. C) FCP2-01 growing between plant cells on the root surface. D) Detail of colonies on root epidermis.
4 weeks colonization
Figure 4. 4 weeks post inoculation on plant roots. A) FCP2-01 on B. distachyon roots. B) FCP2-0 colonizing root hairs. C) FCP2-01 growing on epidermis of the root. D) Enlarged image of panel C.
4 weeks colonization

Next Steps

  • Demonstrate expression and activity of acdS in FCP2-01
  • Develop a regulation system based on abscisic acid to turn on only in drought
  • Inoculate B. distachyon and measure effects of FCP2-01 expressing acdS on plant growth and yield under osmotic stresses
  • Test transformation protocol on additional endophytic isolates of grasses
  • Evaluate ability for endophytic bacteria to enter roots, and how endophytic fungi may affect this process
  • Test species specificity of FCP2-01 and ability to horizontally transmit between a plant and a neighbor
  • Pathogenicity screens of FCP2-01 and more complete evaluation of antibiotic resistance
  • Review genomes of Enterobacter ludwigii for insights into plasmid stability and other factors affecting biotechnology potential
  • Field trials of FCP2-01 (if the strain is cleared as safe) to test persistence and physiological effects in real-world applications

Project Achievements

Successful Results
  • Isolation and screening of endophytic bacterial isolates provided a diversity of strains and for synthetic biology experimentation.
  • A transformation protocol was developed for a endophytic bacteria, which provides a chassis for affecting plant physiology without engineering the plant
  • Reinfection and persistence were observed in a distantly related grass 51.6 ± 7.9 (SD) million years since divergence (Vicentini et al. 2008). FCP2-01 is capable of stably expressing a fusion protein of acdS and GFP for at least 4 weeks in the plant.
  • Modeling
Unsuccessful results
  • Most transformations did not work. We were unable to transform and express any of our vectors in Pseudomonas spp. Attempts at transformation via conjugation were also unsuccessful.
  • Due to timeline disruptions by unviable seeds, we were unable to evaluate the effect of engineered FCP2-01 on plant growth
  • None of our engineered strain could grow with ACC as the only available nitrogen source. It is possible that the attachment of a GFP tag (used for identifying transformed colonies and visualizing bacteria in and on roots) disrupted the quaternary structure of the ACC deaminase tetramer.

References

  • Vicentini A, Barber JC, Aliscioni SS, Giussani LM, Kellogg EA. The age of the grasses and clusters of origins of C4 photosynthesis. Global Change Biology. 2008 Dec;14(12):2963-77.