Detection of New cry Genes of Bacillus thuringiensis by Use of a Novel PCR Primer System

Pedro A. Noguera; Jorge E. Ibarra

doi:10.1128/AEM.00797-10

DOI: 10.1128/AEM.00797-10

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FIG. 1.
SEM images of spore-crystal complexes from the three strains where the new cry gene types were found. (A) LBIT-113, harboring the cry58Aa1 gene; (B) LBIT-979, harboring the cry57Aa1 gene; (C) LBIT-980, harboring the cry59Aa1 gene. spr, spore; cry, parasporal crystal. Bars, 1 μm.
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FIG. 2.
SDS-polyacrylamide gel showing the new Cry protein types (arrows), identified by their molecular masses and by MALDI-TOF MS and Micromass Q-TOF micro-MS analyses. Lane 1, Cry58Aa1 from the LBIT-113 strain; lane 2, Cry57Aa1 from the LBIT-979 strain; lane 3, Cry59Aa1 from the LBIT-980 strain; lane M, PageRuler Plus prestained molecular size markers (Fermentas).

Tables

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TABLE 1.

Universal primers designed from the conserved blocks of the three-domain cry genes and results of in silico analysis of their efficiency^a

Set	Block	Sequence	SPE (%)	AE (%)
1	1	5′-TATGCWCAAGCWGCCAATYTWCATYT-3′	90	90
	5	5′-GGRATAAATTCAATTYKRTCWA-3′
2	2	5′-TTTAGATATTGTTGCAWTATKKYC-3′	68	98
	5	5′-GGRATAAATTCAATTYKRTCWA-3′
3	1	5′-TATGCWCAAGCWGCCAATYTWCATYT-3′	46	100
	4	5′-CATAACGTAGWYTTAYCTKAWT-3′

↵ a SPE, single-pair efficiency; AE, accumulated efficiency.

TABLE 2.

Some features from the 27 selected atypical strains of B. thuringiensis subjected to amplification by the three-set primer system and the highest hits of their amplicons with the known cry genes

LBIT strain no.	Serotype	Origin	Morphology	Gene with highest hit (% identity^a)
LBIT strain no.	Serotype	Origin	Morphology	Set 1	Set 2
LBIT-104	entomocidus	Mexico	Flat squares	cry1Aa (99)	cry21Aa (96)
				cry1Ia (99)
LBIT-113	entomocidus	Mexico	Flat squares	cry1Ia (99)	cry21Ba (25)
LBIT-129	thuringiensis	Mexico	Amorphous	cry1Aa (99)	cry1Ia (99)
				cry1Ba (99)
				cry1Ia (99)
LBIT-133	Unknown	Mexico	Amorphous	No amplification	cry21Aa (96)
LBIT-154	thuringiensis	Mexico	Bipyramidal	cry1Aa (99)	cry1Ab (99)
LBIT-154					cry21Ba (95)
LBIT-275	thuringiensis	Mexico	Bipyramidal	cry1Ab (99)	cry1Ab (99)
				cry1Ia (99)	cry1Ia (99)
LBIT-279	thuringiensis	Mexico	Bipyramidal	cry1Ab (99)	cry1Ab (99)
					cryIa (99)
LBIT-442	Unknown	Mexico	Collapsed balloons	cry1Ia (99)	cry5Ba (95)
					cry1Ia (99)
LBIT-596	darmstadiensis	Argentina	Elongated bypiramidal	No amplification	cry5Ba (98)
LBIT-597	kenyae	Argentina	Small spheres	cry1Ka (43)	cry1Ia (99)
				cry1Ia (99)
LBIT-599	tohokuensis	Argentina	Collapsed balloons	cry1Ab (99)	cry1Ab (99)
				cry1Ba (99)	cry1Ia (99)
				cry1Ia (99)
LBIT-772	fukuokaensis	China	Collapsed balloons	cry1Ia (99)	cry5Ba (80)
					cry1Ia (99)
LBIT-824	canadensis	Kenya	Small spheres	cry7Ba (38)	cry5Ba (96)
LBIT-935	japonensis	Denmark	Collapsed balloons	cry1Ca (63)	cry5Ba (93)
LBIT-937	japonensis	Guyana	Collapsed balloons	cry24Aa (34)	cry5Ba (96)
LBIT-953	mexicanensis	Japan	Small spheres	cry1Ca (63)	cry7Ba (38)
					cry5Ba (95)
LBIT-955	mexicanensis	Thailand	Small spheres	cry1Ca (63)	cry21Aa (25)
LBIT-956	monterrey	Spain	Collapsed balloons	No amplification	cry7Ba (38)
LBIT-960	amagiensis	South Korea	Small spheres	No amplification	cry5Ba (95)
LBIT-976	higo	Canada	Small spheres	cry27Aa (99)	cry7Ba (38)
				cry20Aa (69)
LBIT-979	kim	Denmark	Small spheres	cry48Aa (28)	No amplification
LBIT-980	kim	Spain	Collapsed balloons	cry24 (34)	cry1Ia (99)
				cry1Ia (99)
LBIT-985	iberica	Guyana	Collapsed balloons	No amplification	cry7Ba (38)
					cry4Ba (61)
LBIT-1003	Unknown	Mexico	Collapsed balloons	No amplification	cry1Da (63)
					cry5Ba (95)
LBIT-1004	Unknown	Mexico	Collapsed balloons	No amplification	cry4Ba (61)
LBIT-1045	Unknown	Mexico	Polyhedral	cry1Ia (99)	cry1Ia (99)
LBIT-1189	Unknown	Mexico	Bipyramidal	cry1Ca (63)	cry1Ab (97)
				cry1Ka (69)
				cry1Ab (97)

↵ a Percent identity with the known cry genes.

TABLE 3.

Some features of the seven complete new cry gene sequences selected from the previously sequenced amplicons by their low levels of identity to the known three-domain Cry proteins^a

Strain	GenBank accession no.	Size		Highest hit		BTTNC no.
Strain	GenBank accession no.	nt	aa	Gene	% identity	BTTNC no.
LBIT-113	FJ770568	2,514	837	cry21Ba	28	Cry58Aa1
LBIT-597	FJ770570	3,522	1,173	cry8Ea	61	Cry8Ka2
LBIT-824	FJ770571	3,393	E	cry7Ab	56	Cry8-like
LBIT-976	FJ884065	2,298	765	cry20Aa	63	Cry20Ba1
LBIT-979	FJ770569	1,830	609	cry48Aa	27	Cry57Aa1
LBIT-980	FJ770572	1,998	665	cry24Aa	35	Cry59Aa1
LBIT-1189	FJ884067	2,097	699	cry1Bc	70	Cry1Ma1

↵ a nt, number of nucleotides in the gene; aa, number of amino acids in the protein; BTTNC no., code assigned by the B. thuringiensis Toxin Nomenclature Committee; E, the nucleotide sequence was not translated due to multiple stop codons in the sequence.