Taxonomically Characterized and Validated Bacterial Species Based on 16S rRNA Gene Sequences from India During the Last Decade

Abstract

Microbial taxonomy dealing with identification and characterization of prokaryotes like bacteria and archaea has always been a major area of research all over the world. Exploring diversity of microbes and description of novel species with different genes and secondary compounds is of utmost importance for better future and sustenance of life. India having an enormous range of ecosystems and diverse species inhabiting these niches is considered to be one of the richest biodiversity regions of the world. During the last decade, with newer methodologies and better technology, the prokaryotic taxonomy from India has extended our inventory of microbial communities in specific niches. However, there still exist some limitations in classifying the microbes from India as compared to that is done world-over. This review enlists the taxonomic description of novel taxa of prokaryotes from India in the past decade. A total of 378 new bacterial species have been classified from different habitats in India in the last ten years and no descriptions of archaeal species is documented till date.

Electronic supplementary material

The online version of this article (10.1007/s12088-019-00845-7) contains supplementary material, which is available to authorized users.

Introduction

Among one of the richest biodiversity regions of the world, India encompasses from greater Himalayas in the north to the second largest peninsula in the south that show diverse environmental conditions across it and contain three unique biodiversity hotspots viz., Western Ghats, eastern Himalayas and the Indo-Burma region. Also, it has desert in west, world’s largest alluvial plain (Great plain of north India) and the thick forests in east. This results into richness of the diversity of both flora and fauna with respect to various environmental conditions. While the wealth of this diversity with respect to higher forms of life, viz., plants and animals is well explored and inventoried, such efforts in prokaryote diversity are recent that need lot more to be discovered and classified. The field of microbial taxonomy in India has taken a new dimension in the last decade and exponentially added the descriptions of new taxa of prokaryotes in the catalogue using polyphasic approach. Along with the description of novel species, the identification of new genes important for environment and human health has various industrial and biotechnological applications [1]. Therefore, taxonomical classification of commercially important organisms acts as a base for further studies on microbiological research. While there are quite a few research groups carrying out microbial taxonomy in India, but with the new and improved methodologies in the recent years’ microbiologists have managed to unravel the structure, function and classification of microbial communities from different ecological niches (Supplementary Table 1). In this review, prokaryotes classified in the last few years (2008–2019) have been enlisted and summarized. However, gaps still exist as evident from the review that some of the taxa are underrepresented due to several limitations. The major limitations include difficulty to culture the micro-organisms and lack of expertise. The discipline metagenomics i.e. the culture independent approach has eased the study of unculturable diversity across the country not only for bacteria [2–7] but also for archaea and viruses [8]. Also, next generation sequencing technologies have provided enormous amount of genomics and metagenomics data [9, 10] and its integration with taxonomic methods (Taxo-genomics) can now be utilized for characterization of a species [11]. Progress in classical polyphasic approach and integration of genomics and metagenomics has helped overcome the limitations and contribute significantly to taxonomical characterization of prokaryotes [12]. Further, with the genome sequences freely available in public databases, various comparative genomics studies have been done that identify the novel genes [13], characteristic differences and similarities at genus/species/strain level [14–18] and also predict their phylogenetic evolution. This has developed tremendous interest of microbiologists to identify novel species from different parts of the country as listed in this review.

Prokaryote Descriptions from India: 2008–2019

A total of 378 new microbial species/strains have been classified during the last eleven years by Indian taxonomists (Supplementary Table 1). All the characterized microbes from India broadly classify in 12 different phyla of domain bacteria namely Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Chlorobi, Candidatus, Cyanobacteria, Spirochaetes, Deinococcus-Thermus, Thermodesulfobacteria and Thermotogae. After reviewing, it was evident that there is a major dodge in classifying archaeal species when compared to bacteria as no report of archaeal characterization was found from India. However, their diversity has been explored using metagenomics [19–22]. Amongst the bacteria, the distribution of classified species suggested that nearly half of the characterized species belong to the phylum Proteobacteria (47%) (Fig. 1a). The remaining species fall into the phyla Firmicutes (20%), Actinobacteria (17%), Bacteroidetes (13%), Spirochaetes (1%) and others (2%) (Fig. 1a, b). These findings are concurrent with the global trends of taxonomy. Most of the genera in these phyla have either single species or very few species described whereas genera Streptomyces, Bacillus, Pseudomonas, Sphingobium, Sphingomonas, Rhodobacter and Rhodovulum are represented by many validly published species. Proteobacteria divided into five major classes Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Epsilonproteobacteria forms the dominant and versatile bacterial phylum with maximum number of validly classified species [23]. They comprise a vast diversity of bacterial pathogens, agriculturally important bacteria and other notable genera [24]. This dominance is due to their ubiquitous occurrence, metabolic diversity, low demand of nutrients and ease of culturing. Further, characterization of pathogenic species and genome information benefits the scientists to design new strategies to overcome their virulence [25, 26]. Also, characterizing agriculturally important bacteria is advantageous for developing efficient bio-control agents and bio-fertilizers [27]. The review of classified species belonging to proteobacteria from India indicated that these are isolated from structurally and functionally variable habitats and are categorized into wide-range of genera namely Acinetobacter, Devosia, Novosphingobium, Psychrobacter, Rhodobacter, Rhodovulum, Pseudomonas, Vibrio, Rhizobium, Shewanella, Sphingobium, Sphingomonas etc. (Supplementary Table 1).

Fig. 1.

a Pie-chart showing the distribution of characterized species from India from 2008–2019 into different bacterial phyla Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Spirochaetes and others. Others include Planctomycetes, Chlorobi, Candidatus, Cyanobacteria, Deinococcus-Thermus, Thermodesulfobacteria and Thermotogae. b Stack-plot depicting year wise distribution and classification of species from India in different phyla (2008–2019) *species characterized only till May, 2019 have been included in the list (Supplementary Table 1)

Members of phylum Actinobacteria are well-known for secondary metabolite production and are therefore significant pharmacologically as they produce an arsenal of antibiotics and maintains gut homeostasis [28]. Classification of new species belonging to phylum Actinobacteria from India majorly fall into the genera Microbacterium, Cellulosimicrobium, Mycobacterium, Leifsonia, Streptomyces, Micrococcus, Brevibacterium, Micromonospora, Corynebacterium, Tessaracoccus, Nocardia and several others (Supplementary Table 1). Likewise, species descriptions from Firmicutes are largely represented by Genus Bacillus and related genera like Paenibacillus, Alteribacillus, Pontibacillus, Pseudogracilibacillus, Fictibacillus, Solibacillus, Desertibacillus, Lysinibacillus, Geobacillus, etc. However, actinobacteria and firmicutes are not classified as frequently as proteobacteria as they are Gram-positive bacteria and require additional cell wall analysis for characterization [29]. Further, taxonomic studies from Bacteroidetes in the past decade are dominated majorly by species from genera Pontibacter and Flavobacterium and several others represented by single species only include Indibacter, Cyclobacterium, Mariniradius, Algoriphagus and Chryseobacterium. Last of all, other phyla having very few or single species classified include Planctomycetes, Chlorobi, Candidatus, Cyanobacteria, Spirochaetes, Deinococcus-Thermus, Thermodesulfobacteria and Thermotogae in the last decade (Fig. 1a, b).

Niches Explored for Prokaryote Taxonomy

As stated, India spans through ten recognizable biogeographic zones viz. trans-Himalayan region, Himalayan zone, Indian desert zone, semi-arid region, Gangetic plains, deccan plateau, western Ghats, north-east region, coastal region and Andaman and Nicobar Islands. These zones harbour a lot of unique niches and thus contribute to the biodiversity of all kind. During the last decade, prokaryotes have been isolated from many different sites with distinctive selection pressures and most outlandish were the Firmicutes, Janibacter hoylei PVAS-1 ^T, Bacillus isronensis B3W22^T, Bacillus aryabhattai B8W22^T isolated from the air samples [30]. As discussed in the previous section, Proteobacteria constitute the greatest number of prokaryotic isolates and their niches are widespread too, HCH dumpsite, solar saltern, marine water, rice fields, mangroves, lobster eggs, hotsprings, Rhizosphere, Tribolium gut, algal mat at beach, macroalgae, Ganges water, etc. [31–43]. Likewise, Firmicutes and Bacteroidetes taxonomy has been carried out at large scale and from varied niches (Supplementary Table 1). A total of six Spirochaetes have been classified specifically from the niches that are high in salt content. Such niches include Lonar soda lake, gut of a termite and a cockroach living in Rann of Kutch, solar saltern [44–48]. Actinobacteria have been commonly classified from the soil/sediment samples obtained from garden, forest, lakes, rivers, dumpsites, rhizosphere, mangroves, limestone quarry, industry effluents, ocean floor etc.[49–54]. The uncommon isolates belonging to the phylum actinobacteria include Ornithinimicrobium algicola JC311^T isolated from Ulva (green algae) [55], Micrococcus aloeverae AE-6^T isolated from leaf of Aloe barbadensis (aloe vera) [56], Microbacterium aureliae JF-6 ^T isolated from Aurelia aurita (the moon jellyfish) [57] and lastly Microbacterium telephonicum S2T63^T isolated from the screen of a cell phone [58]. Single organism belonging to each of the phylum Candidatus, Chlorobi, Cyanobacteria, Deinococcus-Thermus, Planctomycetes, Thermodesulfobacteria and Thermotogae have been variously isolated from YLD-symptomatic palms, aquaculture pond, Mandsaur soil, Parastromateus niger fish, Hydrilla plant, hydrothermal vent in the Indian Ocean and Bombay High oil production unit respectively [59–65]. Further, Indian taxonomists have also explored and classified novel species from international sites like Azores [66], Arctic [67–72] and Antarctic region [73–77] as mentioned in the Supplementary Table 1.

Major Improvements in the Methodology

The current polyphasic approach of classifying microbes using standard genotypic, phenotypic and chemotaxonomic methods has improved over the time with the integration of genomics data i.e. Taxo-genomics [11]. With the introduction of user-friendly and cost-effective sequencing platforms, vast amounts of genomic data are generated. Though, genomics approach has been in use for the last decade as an alternative to laborious experiments but recently new strategies have been described for microbial systematics to overcome the limitations of classical methods [78]. For instance, DNA-DNA hybridization (DDH) is a commonly used traditional method to determine genetic relatedness between species but then it is difficult to reproduce the results as it is labour intensive that requires special equipment. On the other end, in-silico tools like GGDC (Genome to Genome Distance Calculator) harnesses genomic information to predict genome relatedness for species delineation [79]. Earlier phylogeny was largely based on single gene approach using 16S rRNA gene sequence as molecular marker due to its ubiquitous presence. However, it has been reported that it has low resolution at species and strain as it is highly conserved [80]. In this post genomic era, development of tools like multi-locus sequence analysis (MLSA), Tetranucleotide frequency [81], Average Nucleotide Identity [82], Codon Usage Bias [83] make use of whole genome sequences that provide sufficient information for delineation of phylogenetic relationships of species being characterized. Recently, a new web server Type (Strain) Genome Server (TYGS) is developed for genome-based taxonomy and predicts phylogeny at species and sub-species level [84]. Likewise, another approach Homologous Coverage Ratio (HCR) has been introduced for inferring homology between the species using genome sequences [85]. Therefore, microbial taxonomy should be revisited and additional computational tools based on genomic information should be considered for prokaryotic classification [86].

Archaeal Diversity: The Unexplored Domain

Archaea are the most ubiquitous of all three domains of life and this calls for the justifiable classification of these organisms. The review of all the taxonomical classification of prokaryotes carried out in the last decade in India indicated a huge gap in discovering and characterizing archaea. They mark limits of life on earth and have been found variously in gut microbiomes, deep hydrothermal vents, hot-springs, mine drainages, coalfields, solar salterns, anaerobic environments, methane sediments and are abundant in common niches as well. Although, all such environments in India have been explored through metagenomic studies and they categorically state the bacterial and archaeal diversity of these niches based on single gene amplicon or metagenome data analysis [2–10, 87–94] but lack archaeal classification. The field of archaeal taxonomy is totally unexplored in India and the reason may be the lack of expertise to culture these niche specific organisms.

Applications and Research Beyond Taxonomy

The secondary metabolites produced by the bacteria have extensive commercial and clinical applications. So, India being the biodiversity hotspot promises to be hub of bacteria with capability of producing the industrially important compounds. Bacteria can evade anti-microbial compounds by various mechanisms and many of the pathogenic bacteria develop biofilm to protect themselves forming their own protective micro-environment [95]. Compounds from other bacteria, e.g. Pyocyanin from Pseudomonas, have the ability to inhibit biofilm formation [96, 97]. Along with this Pseudomonas aeruginosa CR1 has been shown to produce rhamnolipid biosurfactant [97]. P. aeruginosa CR1 has been isolated from field where Chilli was grown with high chemical inputs at Jonnalagadda, Guntur Dt. Andhra Pradesh [18].

Most of the Actinomycetes have been discovered to produce some or the other anti-bacterial compound and their metabolites are being investigated for their activity in comparison of the already discovered antibiotics. Streptomyces amritsarensis 2A^T exhibited broad-spectrum antibiotic properties and has recently been shown to inhibit microcystin synthesis by Microcystis aeruginosa [98, S269].

The Sphingomonads that have been taxonomically characterized have been further used for the genomic study of their genomes, comparative genome analysis for deducing the enzymatic pathways of HCH degradation and lastly for the purpose of bioremediation [14]. Novosphingobium lindaniclasticum LE124^T isolated from HCH dumpsite in Lucknow compiled with other 27 Novosphingobium spp. strains led to the identification of habitat-specific genes and regulatory hubs in them based on their distinct four ecological groups—rhizosphere, contaminated soil, marine, and freshwater [99]. Similarly, comparative genomics of Sphingopyxis spp. has been done to unravel their functional attributes [100].

Many such studies have taken forward the taxonomic isolates from India and their applications have been deciphered widely. Many of them have been found to be industrially, commercially and/or scientifically significant.

Conclusions

The present review affirms that taxonomy research in India is growing rapidly and has shown tremendous progress in the last decade. However there still exist gaps in prokaryotic taxonomy as reviewed that organisms from the domain Archaea have not been characterized from India. But, with the new techniques and improved culturing methods it is possible in the future to characterize archaeal species as well. The inferences from the reviewed species suggest that India harbors very diverse and distinct bacterial species with diverse applications but more efforts need to be done to explore the unknown biodiversity as compared to the other parts of the world. To conclude, application of genomics to microbial systematics has brought a revolutionary change and redefined the concept of species.

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Conflict of interest

The authors declare that they have no conflict of interest.