1. *Percentage of database sequence that matches exactly to query sequence
Table 2. Evidence of Colonizing Mechanism
Searching for the Mechanism for the Colonizing Ability of Pseudomonas fluorescens L :
Resulting in Take-All Decline
Jennifer Butt Bellevue College
Introduction
There are three main fungal pathogens that can infect wheat plants, Rhizoctonia solani AG-8, Rhizoctonia oryzae, and Gaeumannomyces graminis var. tritici (Ggt), the cause of the disease Take-All and the focus of this research.
Ggt is a fungus that infects the roots of plants and prevents water from being absorbed by the plant. Traditional farming methods such as tilling, crop rotation, burning and the use of pesticides to reduce the presence of the fungus can be harmful to the environment and/or may not be economically viable for the farmer to sustain. After a period of 5-10 years of planting wheat in an afflicted plot, a natural phenomenon called Take-All Decline will begin improve crop yield.
Pseudomonas fluorescens, a bacterium that is present naturally in the soil, competes with the fungus and produces 2, 4-diacetylphlorogucinol (DAPG), an anti-fungal. The research in this lab will sequence the genome of Pseudomonas fluorescens L because it can colonize well in the soil and survive in extreme conditions, where other species of Pseudomonas could not.
Figure 3 represents the core genome of Pseudomonas fluorescens species. By determining if the genes in the DNA insert are unique to L5.1-96, we can discover the mechanism for colonization. Using a plasmid containing an insert of Pseudomonas fluorescens L5.1-96’s genome in a clone of an E. coli bacterium, we will sequence the DNA of L and search for the mechanism responsible for creating successful bacteria colonies in the soil using BLAST. These methods will answer our research questions of what makes L a good colonizer, whether the comparison shows other good colonizers as a match for our sequence (Figure 2) and if the gene in question is within the core genome (Figure 1).
Results
Disscussion
The research goals of this project are to find a mechanism that could explain Pseudomonas fluorescens L ’s ability to colonize well in the soil, whether the gene we sequence is in the core genome of Pseudomonas spp. and if the mechanism is found in other good colonizers.
Of the 10 sequences analyzed, two clones do not match to other fluorescens strains and can be considered mechanisms of colonization (Table 2). N08 matched Pseudomonas sp. UW4 with the tBLASTx protocol. Top hits in the BLAST did not match other Pseudomonas fluorescens spp. and this may indicate that this gene is involved in colonization ability, but will require further study to confirm.
Clone N12 compares to a hypothetical protein in the database and the tBLASTx results match Pseudomonas brassicacearum strain DF41 (Table 2). Further analysis is needed to determine the function of the hypothetical protein and whether it is related to colonization ability. Because it does not match with other Pseudomonas fluorescens spp. in the database, it may be important in L5.1-96’s ability to colonize and survive in the soil.
All sequences also had a match for Pseudomonas brassicacearum subsp. NFM421 (Table 1), and may require that Pseudomonas fluorescens L will need to be reclassified as Pseudomonas brassicacearum L
Figure 2. Pseudomonas fluorescens Colonizing Ability
From the chromatogram results represented in Finch TV, the nucleotide sequence was analyzed and compared to NCBI database organisms and proteins using BLAST (BLASTn, BLASTx, tBLASTx). The top two matches for each BLAST protocol and clone are represented below. Identity refers to percentage of matching bases between the query and the database.
Table 2. Evidence of Colonizing Mechanism
Clone #
Primer
BLASTn
BLASTx
tBLASTx
N 08
SR2
Pseudomonas fluorescens F113
histidine utilization repressor [Pseudomonas fluorescens]
Pseudomonas sp. UW4
N 12
Pseudomonas brassicacearum strain DF41
hypothetical protein [Pseudomonas fluorescens]
Table 1. BLAST Results
Clone #
Primer
BLASTn Species
BLASTn Identity*
BLASTx Proteins
BLASTx Identity*
tBLASTx Species
N 06
SL1
Pseudomonas brassicacearum subsp. NFM421
96%
MULTISPECIES: XRE family transcriptional regulator [Pseudomonas]
99%
Pseudomonas fluorescens F113
90%
XRE family transcriptional regulator [Pseudomonas sp. GM49]
SR2
98%
MULTISPECIES: 5, 10-methylene-tetrahydrofolate dehydrogenase [Pseudomonas]
95%
methenyltetrahydrofolate cyclohydrolase [Pseudomonas kilonensis]
N 08
urocanate hydratase
83%
92%
MULTISPECIES: urocanate hydratase [Pseudomonas]
HTH-type transcriptional repressor DasR [Pseudomonas fluorescens]
100%
93%
histidine utilization repressor [Pseudomonas fluorescens]
Pseudomonas sp. UW4
N 10
acriflavine resistance protein B [Pseudomonas fluorescens]
sensor histidine kinase [Pseudomonas fluorescens]
88%
histidine kinase [Pseudomonas brassicacearum]
N 12
hemerythrin [Pseudomonas fluorescens]
MULTISPECIES: hemerythrin [Pseudomonas]
MULTISPECIES: hypothetical protein [Pseudomonas]
76%
Pseudomonas brassicacearum strain DF41
89%
hypothetical protein [Pseudomonas fluorescens]
N 18
diguanylate phosphodiesterase [Pseudomonas fluorescens]
77%
91%
MULTISPECIES: diguanylate phosphodiesterase [Pseudomonas fluorescens]
Materials and Methods
Genomic Library
Created by Amplicon Express with a total of 18,432 clones each containing the plasmid: pSMART LC Kan, and insert: kilo base pairs (kpb)
Cell Culture
Each clone incubated at 37°C overnight in an LB broth with Kanamyacin to promote E. coli growth
Plasmid Isolation
Each plasmid was isolated from the E. coli cell using alkaline lysis with QIAprep Spin Miniprep Kit
Sequencing Reaction
Each insert was sequenced using BigDye® Terminator Cycle Sequencing Kit (with two primers: SL1 & SR2)
Sequencing Reaction Clean-up
BigDye XTerminator® Purification Kit emoved unincorporated ddNTPs and salts from the sequenced DNA
Sequencing Analysis
Nucleotide sequence d by 3130 Genetic Analyzer
Finch TV & BLAST Analysis
Matching nucleotide sequences to database nucleotide and protein sequences (NCBI)
Figure 1
*Percentage of database sequence that matches exactly to query sequence