During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na+(Li+)/H+ antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.

Metagenomics: Application of Genomics to Uncultured Microorganisms

Common bean (Phaseolus vulgaris) has become a cosmopolitan crop, but was originally domesticated in the Americas and has been grown in Latin America for several thousand years. Consequently an enormous diversity of bean nodulating bacteria have developed and in the centers of origin the predominant species in bean nodules is R. etli. In some areas of Latin America, inoculation, which normally promotes nodulation and nitrogen fixation is hampered by the prevalence of native strains. Many other species in addition to R. etli have been found in bean nodules in regions where bean has been introduced. Some of these species such as R. leguminosarum bv. phaseoli, R. gallicum bv. phaseoli and R. giardinii bv. phaseoli might have arisen by acquiring the phaseoli plasmid from R. etli. Others, like R. tropici, are well adapted to acid soils and high temperatures and are good inoculants for bean under these conditions. The large number of rhizobia species capable of nodulating bean supports that bean is a promiscuous host and a diversity of bean-rhizobia interactions exists. Large ranges of dinitrogen fixing capabilities have been documented among bean cultivars and commercial beans have the lowest values among legume crops. Knowledge on bean symbiosis is still incipient but could help to improve bean biological nitrogen fixation.

Diversity of Rhizobium-Phaseolus vulgaris symbiosis: overview and perspectives

The manganese and calcium ions of photosynthetic oxygen evolution.

Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the “keys” to a succession of legume “doors”. Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys.

Molecular Basis of Symbiotic Promiscuity

We compared responses to six insecticidal crystal proteins from Bacillus thuringiensis by a Cry1A-resistant strain (NO-QA) and a susceptible strain (LAB-P) of the diamondback moth, Plutella xylostella. The resistant strain showed > 100-fold cross-resistance to Cry1J and to H04, a hybrid with domains I and II of Cry1Ab and domain III or Cry1C. Cross-resistance was sixfold to Cry1Bb and threefold to Cry1D. The potency of Cry1I did not differ significantly between the resistant and susceptible strains. Cry2B did not kill resistant or susceptible larvae. By combining these new data with previously published results, we classified responses to 14 insecticidal crystal proteins by strains NO-QA and LAB-P. NO-QA showed high levels of resistance to Cry1Aa, Cry1Ab, and Cry1Ac and high levels of cross-resistance to Cry1F, Cry1J, and H04. Cross-resistance was low or nil to Cry1Ba, Cry1Bb, Cry1C, Cry1D, Cry1I, and Cry2A. Cry1E and Cry2B showed little or no toxicity to susceptible or resistant larvae. In dendrograms based on levels of amino acid sequence similarity among proteins, Cry1F and Cry1J clustered together with Cry1A proteins for domain II, but not for domain I or III. High levels of cross-resistance to Cry1Ab-Cry1C hybrid H04 show that although Cry1C is toxic to NO-QA, domain III or Cry1C is not sufficient to restore toxicity when it is combined with domains I and II of Cry1Ab. Thus, diamondback moth strain NO-QA cross-resistance extends beyond the Cry1A family of proteins to at least two other families that exhibit high levels of amino sequence similarity with Cry1A in domain II (Cry1F and Cry1J) and to a protein that is identical to Cry1Ab in domain II (H04). The results of this study imply that resistance to Cry1A alters interactions between the insect and domain II.

Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins.

Transgenic head cabbage (Brassica oleracea var. capitata), resistant to diamondback moth (Plutella xylostella) larvae, was developed through Agrobacterium tumefaciens-mediated transformation with Bacillus thuringiensis (Bt) cry genes using a modified procedure. Factors important for transformation included cabbage cultivar; preculture and coculture of explants on a callus initiation medium; use of appropriate amount; and delay in initial application of selective agents. A total of 15 independent transformed lines with over 100 plants were obtained from several transformation experiments, representing an overall transformation efficiency of ∼1%. Cabbage plants transformed with a synthetic Bt gene, cry1Ab3, were all resistant to larvae of the diamondback moth, whereas all plants transgenic for cryIIa3, a wild-type Bt gene, were susceptible. As a first step towards testing the hypothesis that reduced exposure of Bt to target insects would delay the evolution of insect resistance to Bt, cry1Ab3 expression was put under the transcriptional control of the soybean wound-inducible vspB promoter and transgenic cabbage was obtained. Insect bioassay showed that such plants were all resistant to diamondback moth even without induction for the expression of Bt.

Development of transgenic cabbage ( Brassica oleracea var. Capitata ) for insect resistance by Agrobacterium tumefaciens -mediated transformation

strains on general carbon and nitrogen sources versus mimosine, the toxin appears to be converted mostly to biomass and carbon dioxide. Most isolates able to grow on mimosine as a source of carbon and nitrogen are also able to utilize 3-hydroxy-4-pyridone, a toxic intermediate of mimosine degradation in other organisms.

https://www.ctahr.hawaii.edu/bnf/Downloads/Bibliography/Mimosine%20Catabolism%20by%20Rhizobium%20Strains,%20Soedarjo%20et%20al,%201994.pdf

Mimosine, a Toxin Present in Leguminous Trees (Leucaena spp.), Induces a Mimosine-Degrading Enzyme Activity

Thirty-seven Rhizobium isolates obtained from the nodules of leguminous trees (Leucaena spp.) were selected on the basis of their ability to catabolize mimosine, a toxin found in large quantities in the seeds, foliage, and roots of plants of the genera Leucaena and Mimosa. A new medium containing mimosine as the sole source of carbon and nitrogen was used for selection. The enzymes of the mimosine catabolic pathway were inducible and were present in the soluble fraction of the cell extract of induced cells. On the basis of a comparison of the growth rates of Rhizobium strains on general carbon and nitrogen sources versus mimosine, the toxin appears to be converted mostly to biomass and carbon dioxide. Most isolates able to grow on mimosine as a source of carbon and nitrogen are also able to utilize 3-hydroxy-4-pyridone, a toxic intermediate of mimosine degradation in other organisms.

Mimosine, a Toxin Present in Leguminous Trees (Leucaena spp.), Induces a Mimosine-Degrading Enzyme Activity in Some Rhizobium Strains.

The newly developed cultivars of soybean in Africa, known as Tropical Glycine cross (TGx), are nodulated by bradyrhizobia indigenous to African soils, here designated Bradyrhizobium spp. (TGx). Isolates of Bradyrhizobium spp. (TGx) obtained from nodules of TGx soybeans that were inoculated with soils from 65 locations in six African countries were characterized and grouped into 11 phylogenetic clusters on the basis of RFLP of the 16S rRNA gene. Five restriction enzymes (RsaI, HinfI, MspI, CfoI and HaeIII) established RFLP groups within these Bradyrhizobium spp. (TGx) isolates, which were used to construct a phylogenetic tree showing their genetic relationship with other Bradyrhizobium species. RFLP analysis indicated that Bradyrhizobium spp. (TGx) is a heterogeneous group with some isolates related to Bradyrhizobium japonicum and Bradyrhizobium elkanii strains and some to Bradyrhizobium spp. (misc.) reference strains isolated from a variety of tropical legumes. The heterogeneity within the large phylogenetic clusters was further examined through analysis of randomly amplified polymorphic DNA (RAPD) using GC-rich PCR primers. The RAPD analysis showed additional heterogeneity in the Bradyrhizobium spp. (TGx) phylogenetic clusters, which was not revealed by separations based on RFLP analysis. The Bradyrhizobium spp. (TGx) isolates were classified into effective and ineffective types based on their symbiotic performance on TGx soybean. The isolates were randomly distributed throughout the phylogenetic clusters regardless of their symbiotic effectiveness on TGx soybean.

Bradyrhizobium spp. (TGx) isolates nodulating the new soybean cultivars in Africa are diverse and distinct from bradyrhizobia that nodulate North American soybeans.

Dulal Borthakur

Papers

Cross-resistance of the diamondback moth indicates altered interactions with domain II of Bacillus thuringiensis toxins.

Development of transgenic cabbage ( Brassica oleracea var. Capitata ) for insect resistance by Agrobacterium tumefaciens -mediated transformation

Mimosine, a Toxin Present in Leguminous Trees (Leucaena spp.), Induces a Mimosine-Degrading Enzyme Activity

Mimosine, a Toxin Present in Leguminous Trees (Leucaena spp.), Induces a Mimosine-Degrading Enzyme Activity in Some Rhizobium Strains.

Bradyrhizobium spp. (TGx) isolates nodulating the new soybean cultivars in Africa are diverse and distinct from bradyrhizobia that nodulate North American soybeans.