Assessment of cry1 gene contents of Bacillus thuringiensis strains by use of DNA microarrays

Abstract

A single Bacillus thuringiensis strain can harbor numerous different insecticidal crystal protein (cry) genes from 46 known classes or primary ranks. The cry1 primary rank is the best known and contains the highest number of cry genes which currently totals over 130. We have designed an oligonucleotide-based DNA microarray (cryArray) to test the feasibility of using microarrays to identify the cry gene content of B. thuringiensis strains. Specific 50-mer oligonucleotide probes representing the cry1 primary and tertiary ranks were designed based on multiple cry gene sequence alignments. To minimize false-positive results, a consentaneous approach was adopted in which multiple probes against a specific gene must unanimously produce positive hybridization signals to confirm the presence of a particular gene. In order to validate the cryArray, several well-characterized B. thuringiensis strains including isolates from a Mexican strain collection were tested. With few exceptions, our probes performed in agreement with known or PCR-validated results. In one case, hybridization of primary- but not tertiary-ranked cry1I probes indicated the presence of a novel cry1I gene. Amplification and partial sequencing of the cry1I gene in strains IB360 and IB429 revealed the presence of a cry1Ia gene variant. Since a single microarray hybridization can replace hundreds of individual PCRs, DNA microarrays should become an excellent tool for the fast screening of new B. thuringiensis isolates presenting interesting insecticidal activity.

FIG. 1.

Schematic representation for the cryArray showing the relative position of all oligonucleotide probes. Each keyword represents a unique probe printed in triplicate spots, and each gray block represents all probes that must be positive if a given gene is present. The incompletely separated blocks surrounding cry1Ib/c #105 and cry1Ib #081 reflect the common cry1Ib overlap between the two probes. Primary-ranked probes other than cry1 are grouped in the lower left quadrant, and secondary-ranked cry1 probes are grouped in the lower right.

FIG. 2.

Scanned images of microarrays hybridized with Cy5-labeled genomic DNAs from different known B. thuringiensis strains. Probes belonging to the same cry gene target are grouped inside the individual squares.

FIG. 3.

Scanned images of microarrays hybridized with Cy5-labeled genomic DNAs from different unknown or partially characterized B. thuringiensis strains. Probes belonging to the same cry gene target are grouped inside the individual squares.

FIG. 4.

Sequence comparison of the cry1I amplicon from B. thuringiensis to cry1I-specific immobilized cryArray probes. Differences between the cry1I gene amplified from strain IB360 and the immobilized specific secondary or tertiary cry1I -derived gene sequence are shown in large, boldface capital letters.