Use of Genomic Approaches in Understanding the Role 16 of Actinomycetes as PGP in Grain Legumes Mamta Sharma, Avijit Tarafdar, and Raju Ghosh Abstract The advancement in molecular technologies has given a breakthrough to explore the untapped and novel microbial isolates for characterization in every aspect as we can consider microbes as an important primary natural store house for key secondary metabolites and enzymes. Actinomycetes are the most fruitful source of microorganisms for all types of bioactive second- ary metabolites, including agroactive-antibiotic molecules that are best recognized and most valuable for their role in agriculture and industries. In agriculture, actinomycetes are used as biocontrol agents against some pests and pathogenic organisms as well as plant growth-promoting (PGP) agents for crops. Use of different molecular methods, e.g., metagenomics, metatranscriptomics, genetic fingerprinting, proteogenomics, and metaproteomics, are more significant for classifying and discovering the immense diversity in microbial population and for understanding their interactions with other abiotic and biotic environmental elements. The opportunity of accessing inexpensive sequencing techniques has led to the assemblies of copious genomic data for actinomycetes, such as Streptomy- ces and related species, with the goal of discovering novel bioactive meta- bolic and their utility as PGP; however, the use of actinomycetes in agriculture using genomic approaches is in its initial stages. Keywords Actinomycetes • Plant growth-promotion • Grain legumes • Whole genome sequence • Molecular technologies 16.1 Introduction M. Sharma (*) • A. Tarafdar • R. Ghosh International Crops Research Institute for the Semi-Arid The analysis of microbial communities with Tropics (ICRISAT), Hyderabad, Telengana 502 324, the recent advances in culture-independent India molecular techniques, including sequencing e-mail: [email protected] # Springer Science+Business Media Singapore 2016 249 G. Subramaniam et al. (eds.), Plant Growth Promoting Actinobacteria, DOI 10.1007/978-981-10-0707-1_16 250 M. Sharma et al. technologies and genomics information, has 16.2 Role of Molecular Approaches begun a new era of microbial ecology. Multiple for Identification techniques in molecular approaches based on of Actinomycetes direct analysis of lipids, proteins, and nucleic acids from environmental samples have uncov- For identification and characterization of any ered structural and functional information about biological organism, nucleic acid-based molecu- microbial communities. Molecular techniques, lar approach is considered the most powerful such as genetic fingerprinting and whole genome approach and provides significant information sequencing (WGS), are important tools for dis- about the organisms and the relationship with covering, characterizing the diversified microbial others (Kumar et al. 2014a). In past decades, population, and understanding their chemistry classification and identification of organisms by with other abiotic and biotic factors in environs. approach of molecular systematics was based on Molecular and advanced technologies have a nucleic acid hybridization studies. Gradual intro- massive role in investigating the knowledge by duction of nucleic acid-based sequencing exploring the actinomycetes across the microbial techniques in molecular systematics has been world. Advancement of WGS has made a scien- proved to be authentic (O’Donnell et al. 1993). tific breakthrough, which unchains the under- Following the polymerase chain reaction standing of latent biochemical and molecular (PCR) technology, for better separation of the topographies of uncultured microbe present in PCR products in polyacrylamide matrix, urea- composite environs. The tactic of WGS of formamide denaturing gradient gel electrophore- microbes bare the chemistry of the cryptic sis (DGGE) (Myers et al. 1985) and temperature clusters of biosynthetic-related genes that are gradient gel electrophoresis (TGGE) (Riesner sometimes present but hidden, because those et al. 1989) were well adapted in the laboratory are not well recognized for producing any bioac- for studying microbial ecology. Later, for getting tive secondary metabolites (Fraser et al. 2002). In a sequence-based DNA fingerprint of microbial recent days, a total number of six genera of populations, temperature gradient gels were actinomycetes viz. Corynebacterium, Mycobac- found to be quite promising (Muyzer 1999). terium, Arthrobacter, Frankia, Rhodococcus, Heuer et al. (1997) used DGGE and TGGE to and Streptomyces have enough information study the genetic diversity of actinomycetes in about complete genome sequences to extempo- different soils and to monitor shifts in their rize the core analysis of potential secondary abundances in the potato rhizosphere. They metabolite and gene diversity (James and used actinomycetes group-specific primers for William 2013). Utilizing inexpensive sequencing the direct amplification of 16S rDNA and an techniques has led to the gathering of enormous indirect nested PCR approach using a forward genome sequencing data for Streptomyces and actinomycetes group-specific primer and a related species (Liu et al. 2013) with the goal of reverse bacterial primer, followed by PCR with discovering novel bioactive metabolites. This two bacterial primers. Use of DGGE or TGGE of chapter summarizes recent progress in the poten- the products obtained with the nested PCR made tial applications of actinomycetes using genomic it possible to estimate the abundance of the approaches in agriculture. How they can be com- actinomycetes populations relative to the abun- bined for a comprehensive evaluation of dance of the other bacteria present in the soil. actinomycetes has been illustrated with example Sequence of 16S rDNA in systematic as well studies. as phylogenetic studies of actinomycetes and 16 Use of Genomic Approaches in Understanding the Role of Actinomycetes as... 251 other bacteria is another most commonly used 16.3 Utility of Advanced Genomic approach. Primarily, the study of identification Approaches in Actinomycetes and evolution of actinomycetes based on 16S rDNA has been initiated by amplifying the 16S The genomic technologies are promising tools to rRNA gene implying PCR strategy and followed explore the untapped and novel microbial by direct sequencing of amplified DNA fragments isolates for characterization in all aspects as we (Shiva 2001). Generally, thereafter, the obtained can consider its significance as natural store sequences are further explored in global database house of excellent enzymes and bioactive sec- using bioinformatics tools to find out the identity ondary metabolites. Fluorescence-activated cell and genetic information of query sequence if sorting (FACS) technology is a quick method for available, followed by analysis of phylogenetic separating the cells in a suspension on the basis correlation which reveals the identification of the of fluorescence and cell size. This technology actinomycetes up to the genus level and an over- can be performed for isolation of actinomycetes view on evolutionary aspect. cell from a complex microbial population. Gel In a study by Intra et al. (2011), 16S rRNA gene MicroDrop technology is an efficient enzyme- sequences were used to identify the diversified fluorescence technology, basically used for actinomycetes groups exists in a collection of detection of positive clones by capturing of emit- environmental samples. Detailed comparison of ted fluorescence from catalytic broke down of 16S rRNA gene of 30 actinomycetes isolates biotinylated substrate by specific top articular determined that majority of the isolates (87 %) enzymes present in the positive clone (Short within the environmental samples belong to the et al. 2003). genus Streptomyces spp., whereas one each Recently, high throughput screening (HTS) belongs to Saccharopolyspora and Nocardiopsis technique is considered to be rapid and economi- and two to Nocardia. However, 16S rRNA cal for classifying of any microbial population by sequencing does not always give clear resolution enzymatic characterization, but it is extended to distinguish between closely related genera very little for actinomycetes. HTS consists of (Girard et al. 2013). To order the actinomycetes, drop-based microfluid platform and gives an they presented a novel method based on the array data of insoluble substrates specific for conserved sequence of genes SsgAandSsgB the desired enzymes (Chang et al. 2013). Com- proteins. The wide conserve feature between pared with all HTS-based methods, currently the members of the same genus in amino acid proteomics approach is well accepted in the way (aa) sequence of the SsgB, e.g., only one aa varia- of discovering new microbial flora and fauna. tion was found in between all the SsgB Wang et al. (2012a) first reported on Streptomy- orthologues of identified in Streptomyces,whereas ces sp. products α-glucosidase inhibitor miglitol it has low sequence identity as 40–50 % even using HTS method. They considered 12 actino- between genera of closely related morphologically mycete strains as to be producers of complex actinomycetes, provides concrete data α-glucosidase inhibitors in which strain PW409 for better resolution in classification systems. showed effective inhibitory and was used for Recently, real-time PCR (RT-PCR) technology is fermentation and separation of bioactive com- being used for the specific detection and quantifi- pound using HPLC. In mass spectrometry, two