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Title: Phylogenetic distribution of phenotypic traits in bacillus thuringiensis analyzed by multilocus sequence typing

item Blackburn, Michael - Mike
item Martin, Phyllis
item Kuhar, Daniel
item Farrar, Robert
item Gundersen-Rindal, Dawn

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2013
Publication Date: 6/10/2013
Citation: Blackburn, M.B., Martin, P.A., Kuhar, D.J., Farrar, R.R., Gundersen, D.E. 2013. Phylogenetic distribution of phenotypic traits in bacillus thuringiensis analyzed by multilocus sequence typing . PLoS One. 8(6):e66061.

Interpretive Summary: Bacteria such as Bacillus thuringiensis (Bt) can be used to control insect pests, but it is often difficult to predict which ones will be effective. A number of methods have been used for classifying Bt, such as determining what chemical compounds the bacteria can utilize for growth (substrate utilization), or by determining the relatedness of different strains based on variations in certain genes common to all strains (sequence typing). We have combined these two approaches by determining the genetic relationships of Bts with 15 common patterns of substrate utilization. Our results show that there is significant variation in substrate utilization among strains that are considered identical by the current sequence typing method, suggesting that sequence typing may not allow Bts with clear differences to be distinguished from each other. Our results also allow us to estimate the abundance of certain types of Bt in the environment; Bt kurstaki and Bt israelensis, used to control caterpillar pests and mosquitoes respectively, appear to be to most abundant Bt varieties. The work also reveals previously unknown types of Bt that appear to be relatively abundant in nature. This work will benefit researchers studying the distribution of Bts in the environment, and those seeking to identify new traits that improve the effectiveness of Bt in insect control.

Technical Abstract: Strains from a collection of 3,639 diverse Bacillus thuringiensis isolates were classified based on phenotypic profiles resulting from six biochemical tests, including production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). Strain representatives from the 15 most common phenotypic profiles were subsequently selected and subjected to phylogenetic analyses based on the multilocus sequence typing (MLST) scheme of Priest et al. (2004). Fifty eight isolates representing 15 common phenotypes were found to be distributed among 16 sequence types (STs), including 5 new STs. Approximately 70% of the isolates belonged to either ST 8 (17 isolates), ST 171 (10 isolates), ST 23 (8 isolates), or ST 16 (7 isolates), which appeared to correspond to the classical B. thuringiensis (Bt) varieties kurstaki, finitimus, morrisoni and israelensis, respectively. In addition, ST 240 (3 isolates) and ST 549 (3 isolates) were common in our sample. The ST 8 isolates displayed the greatest apparent phenotypic variation; 7 of the 15 phenotypes analyzed were found to include a representative of ST 8. While there was substantial phenotypic variation within a given ST, certain phenotypes appeared highly correlated with particular STs. For example, isolates with the phenotypes TLUAE, TS, and LSAE were consistently found to be in ST 8, ST 23 and ST 171, respectively. Combining the MLST results with the frequencies of phenotypic combinations in our collection suggested that that the B. thuringiensis varieties mentioned above, particularly israelensis and kurstaki, represent the most abundant varieties of Bt in soil.