Engineering D-glucose utilization in Azospirillum brasilense Sp7 promotes rice root colonization.

Bacteria of the genus Azospirillum include several plant associated bacteria which often promote the growth of their host plants. Although the host range of Azospirillum brasilense Sp7 is much wider than its close relative Azospirillum lipoferum 4B, it lacks the ability to efficiently utilize D-glucose for its growth. By comparing the genomes of both the species, the genes of A. lipoferum 4B responsible for conferring D-glucose utilization ability in A. brasilese Sp7 were identified by cloning individual or a combination of genes in a broad host range expression vector, mobilizing them in A. brasilense Sp7 and examining the ability of exconjugants to use D-glucose as sole carbon source for growth. These genes also included the homologs of genes involved in N-acetyl glucosamine utilization in Pseudomonas aeruginosa PAO1. A transcriptional fusion of the 5 genes encoding glucose-6-phosphate dehydrogenase and 4 components of glucose phosphotransferase system were able to improve D-glucose utilization ability in A. brasilense Sp7. The A. brasilense Sp7 strain engineered with D-glucose utilization ability showed significantly improved root colonization of rice seedling. The improvement in the ability of A. brasilense Sp7 to colonize rice roots is expected to bring benefits to rice by promoting its growth. KEY POINTS: • Genes required for glucose utilization in Azospirillum lipoferum were identified. • A gene cassette encoding glucose utilization was constructed. • Transfer of gene cassette in A. brasilense improves glucose utilization and rice root colonization..

Cometabolism of Ethanol in Azospirillum brasilense Sp7 Is Mediated by Fructose and Glycerol and Regulated Negatively by an Alternative Sigma Factor RpoH2.

Azospirillum brasilense is a plant growth-promoting rhizobacterium that is not known to utilize ethanol as a sole source of carbon for growth. This study shows that A. brasilense can cometabolize ethanol in medium containing fructose or glycerol as a carbon source and contribute to its growth. In minimal medium containing fructose or glycerol as a carbon source, supplementation of ethanol caused enhanced production of an alcohol dehydrogenase (ExaA) and an aldehyde dehydrogenase (AldA) in A. brasilense. However, this was not the case when malate was used as a carbon source. Inactivation of aldA in A. brasilense resulted in the loss of the AldA protein and its ethanol utilizing ability in fructose- or glycerol-supplemented medium. Furthermore, ethanol inhibited the growth of the aldA::Km mutant. The exaA::Km mutant also lost its ability to utilize ethanol in fructose-supplemented medium. However, in glycerol-supplemented medium, A. brasilense utilized ethanol due to the synthesis of a new paralog of alcohol dehydrogenase (ExaA1). The expression of exaA1 was induced by glycerol but not by fructose. Unlike exaA, expression of aldA and exaA1 were not dependent on σ54. Instead, they were negatively regulated by the RpoH2 sigma factor. Inactivation of rpoH2 in A. brasilense conferred the ability to use ethanol as a carbon source without or with malate, overcoming catabolite repression caused by malate. This is the first study showing the role of glycerol and fructose in facilitating cometabolism of ethanol by inducing the expression of ethanol-oxidizing enzymes and the role of RpoH2 in repressing them. IMPORTANCE This study unraveled a hidden ability of Azospirillum brasilense to utilize ethanol as a secondary source of carbon when fructose or glycerol were used as a primary growth substrate. It opens the possibility of studying the regulation of expression of the ethanol oxidation pathway for generating high yielding strains that can efficiently utilize ethanol. Such strains would be useful for economical production of secondary metabolites by A. brasilense in fermenters. The ability of A. brasilense to utilize ethanol might be beneficial to the host plant under the submerged growth conditions.

Dicarboxylate Transporters of Azospirillum brasilense Sp7 Play an Important Role in the Colonization of Finger Millet (Eleusine coracana) Roots.

Azospirillum brasilense is a plant growth-promoting bacterium that colonizes the roots of a large number of plants, including C3 and C4 grasses. Malate has been used as a preferred source of carbon for the enrichment and isolation Azospirillum spp., but the genes involved in their transport and utilization are not yet characterized. In this study, we investigated the role of the two types of dicarboxylate transporters (DctP and DctA) of A. brasilense in their ability to colonize and promote growth of the roots of a C4 grass. We found that DctP protein was distinctly upregulated in A. brasilense grown with malate as sole carbon source. Inactivation of dctP in A. brasilense led to a drastic reduction in its ability to grow on dicarboxylates and form cell aggregates. Inactivation of dctA, however, showed a marginal reduction in growth and flocculation. The growth and nitrogen fixation of a dctP and dctA double mutant of A. brasilense were severely compromised. We have shown here that DctPQM and DctA transporters play a major and a minor role in the transport of C4-dicarboxylates in A. brasilense, respectively. Studies on inoculation of the seedlings of a C4 grass, Eleusine corcana, with A. brasilense and its dicarboxylate transport mutants revealed that dicarboxylate transporters are required by A. brasilense for an efficient colonization of plant roots and their growth.

Comprehensive Metabolomic Fingerprinting Combined with Chemometrics Identifies Species- and Variety-Specific Variation of Medicinal Herbs: An Ocimum Study.

Identification of plant species is a crucial process in natural products. Ocimum, often referred to as the queen of herbs, is one of the most versatile and globally used medicinal herbs for various health benefits due to it having a wide variety of pharmacological activities. Despite there being significant global demand for this medicinal herb, rapid and comprehensive metabolomic fingerprinting approaches for species- and variety-specific classification are limited. In this study, metabolomic fingerprinting of five Ocimum species (Ocimum basilicum L., Ocimum sanctum L., Ocimum africanum Lour., Ocimum kilimandscharicum Gurke., and Hybrid Tulsi) and their varieties was performed using LC-MS, GC-MS, and the rapid fingerprinting approach FT-NIR combined with chemometrics. The aim was to distinguish the species- and variety-specific variation with a view toward developing a quality assessment of Ocimum species. Discrimination of species and varieties was achieved using principal component analysis (PCA), partial least squares discriminate analysis (PLS-DA), data-driven soft independent modelling of class analogy (DD-SIMCA), random forest, and K-nearest neighbours with specificity of 98% and sensitivity of 99%. Phenolics and flavonoids were found to be major contributing markers for species-specific variation. The present study established comprehensive metabolomic fingerprinting consisting of rapid screening and confirmatory approaches as a highly efficient means to identify the species and variety of Ocimum, being able to be applied for the quality assessment of other natural medicinal herbs.

Rapid metabolic fingerprinting with the aid of chemometric models to identify authenticity of natural medicines: Turmeric, Ocimum, and Withania somnifera study.

Herbal medicines are popular natural medicines that have been used for decades. The use of alternative medicines continues to expand rapidly across the world. The World Health Organization suggests that quality assessment of natural medicines is essential for any therapeutic or health care applications, as their therapeutic potential varies between different geographic origins, plant species, and varieties. Classification of herbal medicines based on a limited number of secondary metabolites is not an ideal approach. Their quality should be considered based on a complete metabolic profile, as their pharmacological activity is not due to a few specific secondary metabolites but rather a larger group of bioactive compounds. A holistic and integrative approach using rapid and nondestructive analytical strategies for the screening of herbal medicines is required for robust characterization. In this study, a rapid and effective quality assessment system for geographical traceability, species, and variety-specific authenticity of the widely used natural medicines turmeric, Ocimum, and Withania somnifera was investigated using Fourier transform near-infrared (FT-NIR) spectroscopy-based metabolic fingerprinting. Four different geographical origins of turmeric, five different Ocimum species, and three different varieties of roots and leaves of Withania somnifera were studied with the aid of machine learning approaches. Extremely good discrimination (R2 > 0.98, Q2 > 0.97, and accuracy = 1.0) with sensitivity and specificity of 100% was achieved using this metabolic fingerprinting strategy. Our study demonstrated that FT-NIR-based rapid metabolic fingerprinting can be used as a robust analytical method to authenticate several important medicinal herbs.

Maximizing genetic gain through unlocking genetic variation in different ecotypes of kalmegh (Andrographis paniculata (Burm. f.) Nee).

Andrographis paniculata, commonly known as kalmegh is among the most popular medicinal herbs in Southeast Asia. It is widely cultivated for medicinal purposes. The bioactive molecule, Andrographolide accumulated in herb leaves has immense therapeutic and economic potential. However, comprehensive information regarding genetic diversity is very limited in this species. The present study assessed genetic diversity between and within the six populations (ecotypes) of twenty-four kalmegh accessions using multiple datasets (agro-morphological traits, phytochemical traits, and genic markers). This is the established report where EST-SSR (Expressed sequence tags-Simple Sequence Repeat) markers have been used to unlock genetic variation in kalmegh. Here, we identified and developed ninety-one metabolic pathway-specific EST-SSR markers. Finally, 32 random EST-SSR primer pairs were selected for genetic diversity assessment. Multivariate analysis to unveil the agro-morphological, phytochemical and genotypic variability was helpful in discriminating various germplasms studied in the present study. Among all the morphological discriptors used in present study, days to fifty percent flowering and dry herb yield were found as potential selection index for AP genetic improvement. Hierarchical cluster analysis built with agro-morphological data identified three major groups. However, corresponding analysis with phytochemical and molecular data generated two clear-cut groups among the studied individuals. Moreover, the grouping of individuals into different clusters using multiple datasets was geographically independent, and also showed inconsistency in grouping among agromorphological, phytochemical and molecular dataset based clusters. However, joint analysis using agro-morphological, phytochemical and genotypic information generated two genetic groups, which could be a valuable resource for identifying complementary crossing panels in the kalmegh breeding program. The accessions AP7, AP13, AP5, AP3 belong to cluster I and accessions AP17, AP18 belong to cluster II could be utilized as potential donors for high dry herb yield and andrographolide content, respectively in different selective breeding programs of AP. Thus, our results provided useful information about the overall genetic diversity and variation in economic traits useful for initiating selective breeding programs for contrasting traits of interest and maximizing genetic gain in kalmegh.