S. P. S. Khanuja

Symbiotic and galactose utilization properties of phage RMP64-resistant mutants affecting three complementation groups inRhizobium meliloti

Random Tn5 insertional mutants were induced inRhizobium meliloti Rmd201, a streptomycin-resistant mutant of AK631 (which is itself a compact colony morphology mutant of the wild-type strain Rm41), and screened for sensitivity to a set of 16 phages. Out of 3000 mutants 240 were found to be phage-resistant. The phage-resistant mutants were separable into six groups on the basis of their sensitivity pattern against test phages. Nodulation tests on alfalfa showed that although all the phage-resistant mutants induced root nodules, 7 mutants out of 12 of a class resistant to phage RMP64 (Sxf-) induced atypical nodules that were ineffective in nitrogen fixation (Fix-). The aberrant nodules were small, white, contained only a few bacteria and no bacteroids, and phenotypically resembled nodules elicited by already knownexoB, exoH, ndvA andndvB mutants ofR. meliloti. Spontaneous mutants selected for resistance to RMP64 also fell into two groups: Fix+ and Fix-. Genetic complementation tests between the Sxf- mutants defined three genessxfA, sxfB andsxfC, of whichsxfA andsxfB comprise an operon. These also demonstrated thatsxfA, sxfB andsxfC must be located on the same replicon. All the Sxf- mutants were Calcofluor-positive, like their parent strains Rmd201 and AK631. Characterization of carbohydrate metabolism of the mutants revealed that while thesxfA (Fix-) andsxfB (Fix+) mutants utilized galactose as sole carbon source,sxfC (Fix-) mutants did not. It has been concluded thatsxf A, sxfB andsxfC are new genetic loci and thatsxfA andsxfC have roles in nodule invasion and development.

First report of a 16SrVI group phytoplasma associated with witches'-broom disease on Withania somnifera.

Withania somnifera (L.) Dunal is cultivated in India as an important medicinal cash crop. The whole plant is of great importance in the Indian system of medicine and pharmaceutical industries, but the roots are the main source of active alkaloids. Some of the important alkaloids are tro-pine, pseudotropine, somniferine, colin, withaferin A, withanoides, and a few flavanoides. Typical disease symptoms include phyllody, little leaf, dense clusters of highly proliferating branches with shortened internodes, and resulting witches-broom. The disease was first observed in and around Lucknow, Uttar Pradesh Province, India during January and February 1992. On the basis of symptoms, transmission electron microscopy (TEM), and antibiotic treatment, the causal organism was identified as a phytoplasma (4). The disease is now spreading to other parts of the country (Gujrat, Haryana, Madhya Pradesh, Punjab, and Rajasthan provinces) with a high disease incidence (70%). In this report, molecular characterization and taxonomic position of the associated phytoplasma is reported. Total genomic DNA was extracted from healthy and infected plants with a modified cetyltrimethylammoniumbromide (CTAB) buffer method. The samples were assayed for the presence of phytoplasma using polymerase chain reaction (PCR) with universal phytoplasma primers P1/P6 (2) for amplification of ribosomal 16S rDNA. PCR product was diluted by 1:200 and used directly as DNA template for nested PCR with primers R16F2n and R16R2 (1). Results showed the presence of an expected 1.5-kb rDNA fragment amplified with the direct PCR and a 1.2-kb product of the nested PCR from infected W. somnifera samples. No PCR product was observed in the healthy counterparts. The PCR assay confirmed the presence of phytoplasma as causal agent. The PCR product was cloned with TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and isolated plasmids were again assessed by restriction enzyme (EcoRI) digestion before sequencing. Purified plasmids were sequenced. Partially sequenced nucleotide sequence analysis of 16SrRNA gene cloned from W. somnifera phytoplasma showed high similarity with several isolates of the 16SrVI group of phytoplasmas. The highest nucleotide matching (99 and 98%) was observed with Centaurea solstitialis virescence phytoplasma (Genbank Accession No. AY270156) and Periwinkle little leaf phytoplasma (PPL-Bd; Genbank Accession No. AF 228053) reported in Italy and Bangladesh, respectively. In restriction fragment length polymorphism (RFLP) analysis, AluI, EcoRI, HhaI, HincII, KpnI, and Sau3AI (Promega, Madison, WI; 5 U per reaction) were used for comparison of restriction pattern of present/reference phytoplasma and with that previously reported (3). The present phytoplasma produced identical restriction profile to those of periwinkle infected by PPL-Bd (periwinkle little leaf phytoplasma, Bangladesh, group 16SrVI). On the basis of PCR studies, absence of virus particles under TEM in infected samples, RFLP analysis and nucleotide sequence matching with previously characterized phytoplasma, this phyto-plasma is classified as a member of Clover proliferation group (16SrVI). To our knowledge, this is the first report of a phytoplasma belonging to 16Sr VI group from W. somnifera. References: (1) S. Deng and C. Hiruki. J. Microbiol. Methods 14:53, 1991. (2) D. E. Gundersen and I.-M. Lee. Phytopathol. Mediterr.35:144, 1996. (3) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (4) M. Zaim and A. Samad. Plant Sci. 109:225,1995.

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd301. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.

Construction of a Gene Bank of Azorhizobium IRBG-46: Isolation of hup Genes

Genomic DNA from an efficient Hup+Sesbania-Azorhizobium strain IRBG-46 was isolated, partially digested with EcoRI and fractionated on a 10–40% sucrose density gradient to obtain DNA fragments in the size range of 15–23 kb. In order to isolate hup genes from this strain, a gene bank was constructed in Escherichia coil HB101 using a mobilizable plasmid vector pRK290 having a EcoRI cloning site. Approximately 2x104 Tc-resistant transformants were pooled to constitute the gene bank. Using 12.9 kb EcoRI fragment of cosmid pHU52 as a heterologous hup probe, a total of 2,000 clones were screened by colony hybridization. Five positive clones confirmed by secondary screening and ex planta uptake hydrogenase activity were identified. An insert size in the range of 15–22 kb was revealed by restriction analysis with EcoRI. These five recombinant plasmids containing Hup-determlnants of Azorhizobium IRBG-46 have been designated as pSRH1, pSRH2, pSRH3, pSRH4 and pSRH5. These plasm ids were transferred into Hup-Cicer-Rhizobium strain Rcd 301 to check the expression of hup genes in the new genetic background. In the transconjugants so obtained, the hup genes were found to express under ex planta conditions, and uptake hydrogenase activity ranged from 134 to 392 nmol H2 taken up per h per mg protein.

Cloning of Rhizobium meliloti Locus sxf C Involved in Symbiosis and Phage Sensitivity

A symbiotic mutant of Rhizobium meliloti Rmd438 (sxf C:: Tn5) which was phage resistant against RMP64, failed to utilize galactose as carbon source as reported earlier (21). The Bg/ll gene bank of wild type R. meliloti was mobilized into Rmd438 and a clone pSP676 which complemented for phage sensitivity was isolated. In order to characterize this clone, a Bg/ll and EcoRI map was constructed. The insert of 13.2 kb had three Bg/ll fragments of 4.0, 3.6 and 5.6 kb in this order. All three fragments were subcloned on the vector pRK290 and mobilized into Sxf-mutants. The complementation for phage sensitivity, symbiosis and galactose utilization properties are discussed.

Thermo-Inducible Expression of δ Endotoxin Gene of Bacillus thuringiensis HD1 Derived under Lambda PL Promoter in Escherichia coli

The insecticidal crystal protein gene cryIA(a) from Bacillus thuringiensis HD1 has been cloned as a single 3.765 kb Ndel fragment on the expression vector pRE1. The pBR322 based clone pES1 was digested with restriction endonuclease Ndel and the 3.765 kb fragment carrying the intact gene was eluted and cloned on pUC18 to confirm its functional integrity. This Ndel fragment was then cloned on the vector pRE1 carrying strong promoter PL of lambda upstream to the cloning site. The recombinant construct pUSR14.1 carried crystal protein (CP) gene under PL and was temperature inducible at 42°C in MZ1 host strain of Escherichia coli because of temperature sensitive CI857 gene carried by it as lysogen. Dilution based insect bioassays showed hyper-expression of toxin in these constructs. SDS-PAGE analysis indicated that polypeptides corresponding to 132 kD and 66 kD bands of HD1 endotoxin constituted 20.1% of the total soluble protein in this recombinant strain to be delta-endotoxin.

Detection of loci in theleu region ofRhizobium meliloti chromosome

A multi-marked strain ofRhizobium meliloti was developed by the co-mutation method and employed to contribute to the genetic map ofR. meliloti chromosome. Seven loci were placed at 5 sites in theleu region in the orderman-aba, fix, leu-cro-azt, ost-thi.

Research and Development on Artemisia annua in India

Artemisia annua (family Asteraceae) is an important medicinal plant. It produces an array of secondary metabolites, but it is mainly known for the antimalarial phytomolecule, artemisinin, which is an endoperoxide sesquiterpene lactone. Artemisinin and artemisinin-based combination therapies (ACTs) are globally used as antimalarials. Presently, the plant remains the only commercial source of artemisinin. After its introduction in India in the early 1980s, A. annua has been subjected to intensive research by Indian scientists. This chapter presents a snapshot of the research carried out on A. annua in Indian laboratories over the years.

Studies on Symbiotic Properties of Purine Auxotrophs of Strain Rmd 201 of Rhizobium Meliloti

The knowledge of the process of infection thread formation and establishment of functional nodules still remains obscure at molecular level. Yet studies on various rhizobial mutants clearly imply that certain deficiencies of bacteria can severely limit normal symbiotic process.

Heat Shock Tolerant Mutants of Rhizobium meliloti Forming Ineffective Nodules

Six heat shock tolerant mutants of Rhizobium meliloti Rmd201 were isolated through transposon Tn5 mutagenesis. The symbiotic assays of these mutants with alfalfa plants, showed four of these mutants to be affected in nitrogenase effectivity also. These four mutants could be classified into two separate complementation groups hssA and hssC through R-prime mediated merodiploid constructions. The hssC mutant Rmd1040 also showed poor interaction with phages indicating surface alterations. The results indicated possible involvement of these loci in symbiosis as well as heat shock response.