Evaluation of an rRNA-targeted oligonucleotide probe for the detection of mosquitocidal strains of Bacillus sphaericus in soils: characterization of novel strains lacking toxin genes

Abstract

Colonies of Bacillus sphaericus on primary isolation have been identified using an oligonucleotide probe targeted to a specific region of the 16S rRNA. Of 3440 colonies from soil samples from Brazil, 57 hybridized to the probe in colony blots but when purified DNA was used in slot‐blots the probe was more specific and only 27 isolates hybridized. Of these, 20 strains were confirmed as members of DNA homology group IIA (potential mosquitocidal strains) by ribotyping and isoenzyme analysis. However, none of these strains was toxic to Anopheles or Culex larvae, nor did they contain recognized toxin genes. This is the first demonstration of such non‐pathogenic strains of B. sphaericus DNA homology group IIA and their common occurrence suggests that pathogenicity is not an important contribution to the success of these bacteria in the environment. Similar screening of strains from Scottish soils indicated that B. sphaericus DNA homology group IIA strains were less common in this habitat and none were recovered on this occasion.

Distribution and characterization of mosquitocidal toxin genes in some strains of Bacillus sphaericus

“…DNA manipulations and bioassays. Chromosomal DNA was prepared, digested with HindIII (for btx hybridizations) or ClaI (for mtx hybridizations), transferred to nylon membranes, and hybridized to probes for the binary and mosquitocidal toxin genes as described previously (3,11). For sequencing, btx DNA was amplified from chromosomal DNA of strains LP1-G, 9002, IAB 881, IAB 872, and PR-1 with primers 1711 forward (5Ј-CCTATAACTAATCCACT TACGC) and 2710 reverse (5Ј-CAGTTTTTGTCTCTTTAGAGCC), which are complementary to regions 1690 to 1711 and 2731 to 2710, respectively, of the btx gene sequence (4).…”

“…Many high-toxicity strains synthesize both Mtx and the binary toxin, while others synthesize only the binary toxin (12). Low-toxicity strains synthesize only Mtx or neither toxin (8,11).…”

Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis

“…Chromosomal DNA was digested with HindIII, electrophoresed and transferred to nylon membranes as described previously [19]. Digoxigenin-labelled probes were prepared by PCR using the nhe and hbl primers with the reaction supplemented with DIG-11-dUTP according to the manufacturer's instructions (Boehringer).…”

Common occurrence of enterotoxin genes and enterotoxicity inBacillus thuringiensis

“…Chromosomal DNA was digested with Hin dIII, electrophoresed and transferred to nylon membranes as described previously [19]. Digoxigenin‐labelled probes were prepared by PCR using the nhe and hbl primers with the reaction supplemented with DIG‐11‐dUTP according to the manufacturer's instructions (Boehringer).…”