Novel cost-effective method of screening soils for the presence of mosquito-pathogenic bacilli.

AIMS: The aim was to simplify the cumbersome conventional process of isolating virulent bacilli, which involves isolating all bacilli strains from a source followed by screening for strains that are effective for bio-control of mosquito vectors. METHODS: A new simplified technique involving eight steps was devised for screening soil samples for the presence of mosquito-pathogenic bacilli before isolating individual strains. RESULTS: Using the new technique, we obtained eight bacilli strains (KSD1-8) showing pathogenic activity against mosquito larvae from three out of 10 soil samples screened. These strains were characterized, identified and the main bioassay tests were performed with three most promising strains (KSD-4, KSD-7 and KSD-8), and their pathogenic activity against Anopheles stephensi Liston, Culex quinquefasciatus, Say and Aedes aegypti Linnaeus compared well with commercial reference strains of B. thuringiensis israelensis and B. sphaericus. SIGNIFICANCE AND IMPACT OF THE STUDY: The new technique of screening soil samples for the presence of virulent pathogenic strains of bacilli against mosquito larvae proved quick, efficient and cost effective.

Identification of a meta-cleavage pathway for metabolism of phenoxyacetic acid and phenol in Pseudomonas cepacia AC1100.

Pseudomonas cepacia strain AC1100 grows luxuriantly on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) but does not utilize phenoxyacetic acid. After long-term selective pressure on phenoxyacetic acid, mutants designated as strain PAA, capable of utilizing phenoxyacetic acid as well as phenol, emerged spontaneously at a frequency of 1 x 10(-8). A deletion mutant strain PT88, which is devoid of a part of 2,4,5-T metabolic pathway, generated neither phenoxyacetic acid utilizing nor phenol-utilizing mutants. The wild type (Wt) strain AC1100 and all its mutants utilized benzoate via ortho-cleavage pathway. However, only mutant strain PAA harbored the whole set of enzymes required for utilization of phenol via meta-cleavage pathway. The results suggest that Wt strain AC1100 carries silent genes for meta-cleavage pathway which are expressed in strain PAA enabling it to utilize phenoxyacetic acid and phenol. Gene activation is presumed to be due to the translocation of insertion elements.

Cloning and characterization of a chromosomal DNA region required for growth on 2,4,5-T by Pseudomonas cepacia AC1100.

A series of spontaneous 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) nonmetabolizing mutants of Pseudomonas cepacia AC1100 were characterized to be defective in either 2,4,5-T uptake or conversion of this compound to 2,4,5-trichlorophenol (2,4,5-TCP). Two of these mutants, RHC22 and RHC23, were complemented for growth on 2,4,5-T using an AC1100 genomic library constructed in the cosmid vector pCP13. Recombinant cosmids isolated from the complemented mutants contained a 27.5-kb insert which frequently underwent various-sized deletions in Escherichia coli. Hybridization studies showed this DNA to be of chromosomal origin and totally deleted in RHC22, RHC23 and other similar mutants. Complementation analyses of RHC22 with a series of subcloned fragments and spontaneously deleted derivatives of the recombinant cosmid pRHC21 showed the 2,4,5-T (tft) genes to occur within an 8.9-kb region. Pseudomonas aeruginosa cells transformed with this DNA acquired the ability to convert 2,4,5-T to 2,4,5-TCP. The genetic determinant for this function was further localized within a 3.7-kb region. This DNA, in the absence of other sequences from the 8.9-kb tft gene region allowed RHC22 cells to metabolize 2,4,5-T, but at low rates which were insufficient to support growth. Copies of the insertions sequence element IS931 were identified either adjacent to or within this tft gene region in the genomes of two independent wild-type AC1100 isolates. Preliminary evidence suggests that these sequences either facilitate or are required for growth on 2,4,5-T and hence may be implicated in the genetic evolution of the 2,4,5-T metabolic pathway.

Upstream regulatory sequence for transcriptional activator XylR in the first operon of xylene metabolism on the TOL plasmid.

Transcription of the first operon coding for m-xylene-degrading enzymes on the TOL plasmid of Pseudomonas putida is activated by the xylR gene product in the presence of m-xylene. The operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an RNA polymerase containing a sigma factor NtrA (RpoN or sigma 54). Deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xylE gene on a plasmid. Their promoter activities were analyzed in Escherichia coli by monitoring catechol 2,3-dioxygenase activity, the xylE gene product. A cis-acting DNA element was identified, which is required for activation of the operon promoter by XylR protein in the presence of the inducer. This regulatory sequence of about 40 base-pairs in length was located 150 base-pairs upstream from the transcription start site. Analysis of the mutants containing insertions between the upstream regulatory sequence and the promoter sequence demonstrated strong dependence of the activation upon helical periodicity of DNA. The regulatory sequence functioned in the inverse orientation or at a distance of more than 1 x 10(3) base-pairs upstream from the promoter though less efficient. These results indicated that this upstream regulatory sequence might be the binding site for XylR protein. DNA-loop formation through protein-protein interaction between XylR protein attached to the upstream sequence and the NtrA-containing RNA polymerase bound by the promoter sequence was suggested for activation of the operon transcription. A sequence similar to the regulatory sequence of the first operon of xylene metabolism was found in the upstream region of the xylS gene, which is also activated by XylR protein in the presence of m-xylene.

Repeated sequences including RS1100 from Pseudomonas cepacia AC1100 function as IS elements.

Several lines of evidence were obtained that the previously identified, repeated sequence RS1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences for a newly identified 3400 bp repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.

Characterization and nucleotide sequence determination of a repeat element isolated from a 2,4,5-T degrading strain of Pseudomonas cepacia.

Pseudomonas cepacia strain AC1100, capable of growth on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), was mutated to the 2,4,5-T- strain PT88 by a ColE1::Tn5 chromosomal insertion. Using cloned DNA from the region flanking the insertion, a 1477-bp sequence (designated RS1100) was identified which was repeated several times on the wild-type chromosome and was also present on AC1100 plasmid DNA. Various chromosomal fragments containing this sequence were cloned and their nucleotide sequence was determined. Examination of RS1100 revealed the presence of 38-39-bp terminal inverted repeats immediately flanked by 8-bp direct repeats. The translated sequence of the single large open reading frame of RS1100 showed structural similarity to the phage Mu transposase and other DNA-binding proteins. Thus the AC1100 repeated sequence has several structural features in common with insertion sequence elements. Three copies of RS1100 were mapped near 2,4,5-t genes encoding degradation of 5-chloro-1,2,4-trihydroxybenzene, an intermediate in 2,4,5-T degradation. Neither RS1100 nor the 2,4,5-t genes hybridized to DNA isolated from Pseudomonas strains, including P. cepacia, suggesting that both gene fragments may be of foreign origin recruited in strain AC1100. The origin of these two DNA segments as well as the role played by RS1100 in the recruitment of 2,4,5-t genes in AC1100 are presently under investigation.

Cloning, physical mapping and expression of chromosomal genes specifying degradation of the herbicide 2,4,5-T by Pseudomonas cepacia AC1100.

A genomic library of total DNA of Pseudomonas cepacia AC1100 was constructed on a broad-host-range cosmid vector pCP13 in Escherichia coli AC80. A 25-kb segment was isolated from the library which complemented a Tn5-generated, 2,4,5-trichlorophenoxyacetic acid-negative (2,4,5-T-) mutant, P. cepacia PT88. This mutation was partially characterized and appeared to be lacking functional enzyme required for metabolism of an intermediate of the 2,4,5-T pathway, recently identified as 5-chloro-1,2,4-trihydroxybenzene [Chapman et al., Abstr. Soc. Environ. Toxicol. Chem. USA 8 (1987) 127]. A simple colorimetric assay was developed to detect the presence of this active enzyme in intact cells and was used to determine the expression of complementing genes. Subcloning experiments showed that a 4-kb BamHI-PstI fragment and a 290-bp PstI-EcoRI fragment, separated by 1.3-kb, were required for complementation. Both fragments are identified to be chromosomal in origin. Hybridization studies using the subcloned fragments revealed that in addition to a Tn5 insertion, mutant PT88 contained an extensive chromosomal deletion accounting for its 2,4,5-T- phenotype. The cloned fragments did not show homology to plasmid DNAs carrying degradative genes for toluene, naphthalene and 3-chlorobenzoate.