A sandwich enzyme-linked immunosorbent assay for the Bacillus sphaericus binary toxin.

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5alpha harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362.

Isolation of Bacillus megaterium mutants that produce high levels of heterologous protein, and their use to construct a highly mosquitocidal strain.

A xylose-regulated plasmid expression system for producing high levels of recombinant proteins in Bacillus megaterium has recently been described [Appl Microbiol Biotechnol 35:594, 1991]. Using an antibiotic resistance protein as the expressed protein, we have been able to select mutant plasmids that produce increased levels of heterologous protein. The mutant plasmids show increased segregational stability and have lost the ability to be transformed into Escherichia coli. The same selection protocol has been used to isolate a mutant strain producing high levels of the Bacillus sphaericus mosquitocidal binary toxin. This strain shows toxicity to Culex quinquefasciatus larvae that is comparable toB. sphaericus 2362 and higher than a B. megaterium strain with the original expression plasmid. This approach may be generally useful for high-level regulated protein expression in B. megaterium.

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301.

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10(-5)-10(-7) exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50% of larvae (LC50) being 2.1 x 10(5) and 1.3 x 10(5) cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth.

Sequencing, chromosomal inactivation, and functional expression in Escherichia coli of ppsR, a gene which represses carotenoid and bacteriochlorophyll synthesis in Rhodobacter sphaeroides.

Sequencing of a DNA fragment that causes trans suppression of bacteriochlorophyll and carotenoid levels in Rhodobacter sphaeroides revealed two genes: orf-192 and ppsR. The ppsR gene alone is sufficient for photopigment suppression. Inactivation of the R. sphaeroides chromosomal copy of ppsR results in overproduction of both bacteriochlorophyll and carotenoid pigments. The deduced 464-amino-acid protein product of ppsR is homologous to the CrtJ protein of Rhodobacter capsulatus and contains a helix-turn-helix domain that is found in various DNA-binding proteins. Removal of the helix-turn-helix domain renders PpsR nonfunctional. The promoter of ppsR is located within the coding region of the upstream orf-192 gene. When this promoter is replaced by a lacZ promoter, ppsR is expressed in Escherichia coli. An R. sphaeroides DNA fragment carrying crtD', -E, and -F and bchC, -X, -Y, and -Z' exhibited putative promoter activity in E. coli. This putative promoter activity could be suppressed by PpsR in both E. coli and R. sphaeroides. These results suggest that PpsR is a transcriptional repressor. It could potentially act by binding to a putative regulatory palindrome found in the 5' flanking regions of a number of R. sphaeroides and R. capsulatus photosynthesis genes.

An improved suicide vector for construction of chromosomal insertion mutations in bacteria.

We have constructed an R6K-based suicide vector (pJP5603) that requires a trans supply of the pir-encoded pi protein of plasmid R6K for replication. Therefore, efficient plasmid suicide results upon transfer to bacteria not harbouring pir. The 3.1-kb vector encodes kanamycin resistance and is mobilizable. When used in conjunction with a JM109 strain carrying pir, it has nine unique restriction sites available for alpha-complementation cloning. Vector functionality was demonstrated in Rhodobacter sphaeroides.

High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors in Pseudomonas stutzeri.

A number of Escherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained in Pseudomonas stutzeri ATCC 17588. A restrictionless mutant of P. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With the E. coli cloning vector pHSG298, transformation frequencies of up to 2 x 10(7) transformants/micrograms DNA were achieved. This frequency is comparable to that obtained for CaCl2-mediated transformation of E. coli; thus, direct cloning of DNA into P. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G + C genera in cases in which E. coli is not a suitable heterologous cloning host.