The ecology of Bacillus thuringiensis on the Phylloplane: colonization from soil, plasmid transfer, and interaction with larvae of Pieris brassicae

Seedlings of clover (Triflorium hybridum) were colonized by Bacillus thuringiensis when spores and seeds were co-inoculated into soil. Both a strain isolated in the vegetative form from the phylloplane of clover, 2810-S-4, and a laboratory strain, HD-1, were able to colonize clover to a density of about 1000 CFU/g leaf when seeds were sown in sterile soil and to a density of about 300 CFU/g leaf in nonsterile soil. A strain lacking the characteristic insecticidal crystal proteins produced a similar level of colonization over a 5-week period as the wild type strain, indicating that crystal production was not a mitigating factor during colonization. A small plasmid, pBC16, was transferred between strains of B. thuringiensis when donor and recipient strains were sprayed in vegetative form onto leaves of clover and pak choi (Brassica campestris var. chinensis). The rate of transfer was about 0.1 transconjugants/recipient and was dependent on the plant species. The levels of B. thuringiensis that naturally colonized leaves of pak choi produced negligible levels of mortality in third instar larvae of Pieris brassicae feeding on the plants. Considerable multiplication occurred in the excreted frass but not in the guts of living insects. Spores in the frass could be a source of recolonization from the soil and be transferred to other plants. These findings illustrate a possible cycle, not dependent on insect pathology, by which B. thuringiensis diversifies and maintains itself in nature.

Multiple-locus sequence typing analysis of Bacillus thuringiensis recovered from the phylloplane of clover (Trifolium hybridum) in vegetative form

The chromosomal genotype, as judged by multi locus sequence typing, and the episomal genotype, as judged by plasmid profile and cry gene content, were analyzed for a collection of strains of Bacillus thuringiensis. These had been recovered in vegetative form over a period of several months from the leaves of a small plot of clover (Trifolium hybridum). A clonal population structure was indicated, although greater variation in sequence types (STs) was discovered than in previous collections of B. cereus/B. thuringiensis. Isolates taken at the same time had quite different genotypes, whereas those of identical genotypes were recovered at different times. The profiles of plasmid content and cry genes generally bore no relation to each other nor to the STs. Evidently, although relatively little recombination was occurring in the seven chromosomal genes analyzed, a great deal of conjugal transfer, and perhaps recombination, was occurring involving plasmids. A clinical diarrheal isolate of B. cereus and the commercial biopesticide strain HD-1 of B. thuringiensis, both included as out-groups, were found to have very similar STs. This further emphasizes the role of episomal elements in the characteristics and differentiation of these two species.

Changes in the properties of Bacillus thuringiensis after prolonged culture in a rich medium

AIM: To investigate the effect of repeated culture in a rich medium on certain genetic, metabolic, pathogenic and structural characteristics of fresh isolates of Bacillus thuringiensis. METHODS AND RESULTS: Four strains of B. thuringiensis, which had been isolated in vegetative form from leaf surfaces, were grown for 500 generations in batch culture in a rich medium. One of the strains, S4g, differed from the parent in the following respects: greater cell width; changed plasmid profile; complete loss of ability to produce delta-endotoxins; loss of ability to produce beta-exotoxin and disruption of vip3 gene; radically different fatty acid composition; and altered metabolic activity. Two of the other evolved strains (S1g and S6g) showed differences in fatty acid profiles compared with the parents. Genetic finger-printing showed that there were also mutations in the cry genes of two of the evolved strains (S1g and S2g). The delta-endotoxins of strain S6g were significantly less toxic to the larvae of Pieris brassica compared with those of the parent and it also differed in the plasmid content. CONCLUSION: Radical and unpredictable changes can occur in fresh isolates of B. thuringiensis when subjected to growth in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first analysis of a Gram positive and biotechnologically significant bacterium after repeated laboratory culture. It is of great relevance to the biotechnological exploitation of B. thuringiensis that prolonged growth of environmental isolates on laboratory culture media can have profound effects on their structure, genome and virulence determinants.

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).

Recovery of Bacillus thuringiensis in vegetative form from the phylloplane of clover (Trifolium hybridum) during a growing season

Two media were developed which specifically allow the cultivation of Bacillus thuringiensis while it is in the vegetative as opposed to the spore form. Using these media B. thuringiensis was shown conclusively for the first time to exist in an active form on the phylloplane. The profile of its appearance in vegetative and spore form was followed over a growing season on clover (Trifolium hybridum) in the field. Three simultaneous and sudden rises and declines of both spore and vegetative cell densities were observed. The most common other spore-former on these leaves was Bacillus cereus but the fluctuations in appearance of these two very closely related species were not co-incident. Using specific PCR primers a considerable diversity of cry toxin gene types was found in isolates that had been recovered in vegetative form ([`]vegetative isolates') with the majority possessing multiple [delta]-endotoxin genes while some had only one of those tested. Bioassays against a lepidopteran insect of purified [delta]-endotoxins showed that they were no more potent than those from a laboratory-adapted strain. PCR primers for an internal region of the vip3A gene produced amplification in 70% of the vegetative isolates compared to 25% of the laboratory-adapted strains tested.

Multiple dextranases from the yeast Lipomyces starkeyi

The soil yeast Lipomyces starkeyi (NCYC 1436) secretes dextranase activity into the growth medium. Resolution of a dextranase-active protein fraction by SDS-PAGE produced three protein bands, of 66 kDa, 68 kDa and 78 kDa, and isoelectric focusing of the same fraction resulted in seven protein bands, of pIs 3.50, 3.85, 4.20, 4.80, 4.85, 5.00 and 5.30. Dextranase activity was demonstrated for all the isoelectric forms, and for the 78 kDa species in the presence of SDS. Amino acid compositions of the 66 kDa, 68 kDa and 78 kDa protein bands were determined, and the N-termini of the 66 kDa and 78 kDa protein bands were sequenced: the first two amino acids at the N-terminus of each protein were alanine and valine, respectively; an alanine-valine pair is seen early in the N-terminal coding sequences of the dextranases and the isopullulanase produced by the phylogenetically disparate organisms contributing to glycosyl hydrolase family 49.

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E(mM) for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of alpha-helix (56%) with lesser amounts of random coil (21%), beta-turn (13%) and beta-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% alpha-helix, 33.7% random coil and 10.5% beta-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60 degrees C for 5 min; thereafter by cooling at 1 degrees C min(-1) to 45 degrees C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60 degrees C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330-350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.