Ann Microbiol (2015) 65:431–441 DOI 10.1007/s13213-014-0876-1

ORIGINAL ARTICLE

Predominance of Bacillus sp. in soil samples of the southern regions of Western Ghats, India

Gowdaman Vasudevan & Venkatachalam Siddarthan & Prabagaran Solai Ramatchandirane

Received: 5 August 2013 /Accepted: 12 March 2014 /Published online: 17 April 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract The aim of this study was to determine the bacterial samples assessed by both cultivation-dependent and diversity in soils of the southern region of the Western Ghats, a cultivation-independent methods. ‘biodiversity hotspot’, and thereby futher our understanding of the microbial communities in this ecological niche. The Keywords Western Ghats . Diversity index . 16S rRNA . diversity and phylogeny of bacterial populations in soil sam- ARDRA . Metagenomic DNA . Bacillus species ples collected from various locations of the Tamil Nadu and Kerala regions of Western Ghats were compared using both cultivation-dependent and cultivation-independent methods. Introduction A total of 171 bacterial strains were isolated based on their morphological characteristics and their diversity indices cal- India is considered by UNESCO (2012) to have two of the culated. The distinctive amplified ribosomal DNA restriction ‘mega diversity hotspots’ in the world, namely, the Himalayas analysis (ARDRA) pattern of each isolate was determined, and the Western Ghats. The Western Ghats is a forested tract and representative isolates were then subjected to 16S rRNA of relatively smooth but very old mountain range that extends gene sequencing. On the basis of their sequence similarity, the from Central Maharashtra to the southern tip of Kerala. The isolates were distributed among three different genera belong- region supports a wide range of endemic taxa with nearly ing to Firmicutes (83.3 %), Proteobacteria (8.3 %) and high 1,500 species of flowering plants (38 %), 126 species of G+C Gram-positive bacteria (8.3 %). The highest and the amphibians (78 %), 157 species of reptiles (62 %), 508 species lowest values for the diversity indices were obtained for of birds (4 %) and 137 species of mammals (12 %) identified metagenomic DNA extracted from isolates BWGA and to date (Bawa et al. 2007). The diversity of flora and fauna of BVP, respectively; these were used for 16S rRNA gene library the Western Ghats has been reasonably well documented, but construction and analysis. Based on their phylogenetic analy- rather fewer studies have examined fungal diversity in the area sis, the predominant members of the habitat were found to (Raviraja 2005) and fewer yet have looked at the diversity of belong to the phylum Firmicutes (84.62 %). Firmicutes was bacteria. Soil bacteria are an essential component of the biotic the dominant bacterial phylum detected by both approaches, community in natural forests and are largely responsible for but the culture-independent approach detected a considerably ecosystem functioning due to their participation in most nu- higher number of uncultivable bacteria. In conclusion, in our trient transformations (Hackl et al. 2004). Although the main study of the bacterial diversity of this Western Ghats region, diversity of life has been proven to be microbial, the vast we fund that the genus Bacillus was predominant among the majority of soil bacteria still remain unidentified because only a minor percentage of naturally occurring microorganisms can be cultured (Pace 1997). Electronic supplementary material The online version of this article The diversity of microorganisms and the role they play ‘in (doi:10.1007/s13213-014-0876-1) contains supplementary material, ’ which is available to authorized users. situ remains largely unknown. For almost a century, culture- dependent methods used to study bacterial diversity have been : : * G. Vasudevan V. Siddarthan P. Solai Ramatchandirane ( ) continuously optimized to detect diverse microorganisms. Molecular Microbiology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India However, only a small proportion of the bacteria present in e-mail: [email protected] extreme environments are readily cultivable, limiting further, 432 Ann Microbiol (2015) 65:431–441 more detailed study. In addition, culture-dependent methods been not many reports on bacterial diversity. Therefore, the are selective and biased based on the type of media used, the aim of our study was to estimate bacterial diversity and nutrients provided and the culture conditions. This has led in identify the predominant soil bacteria in the southern regions more recent decades to the development of culture- of the Western Ghats using both culture-dependent and ad- independent methods to study the enormous diversity of un- vanced culture-independent methods in order to obtain an cultivable organisms and emphasized the need for comple- overview of the bacterial diversity. Although the culture- mentary approaches for the analysis of soil bacterial diversity independent approach is considered to be the superior tech- (Vaz-Moreira et al. 2011). nology for studying bacterial diversity, culture-dependent The relatively recent application of molecular methods to methods are considered to potentially be of more practical investigate unculturable microbes from diverse environments importance (Burmølle et al. 2009). has altered commonly accepted views of microbial diversity. Research based on the extraction of total community DNA from environmental samples followed by PCR, cloning and sequencing of 16S rRNA genes has become conventional, Materials and methods often comprising one of the first steps in the study of the bacterial diversity of an environment of importance (Oline Site description and sampling et al. 2006). The recovery and analysis of 16S rRNA genes directly from environmental DNA provides a means for in- The present-day Western Ghats constitute a continuous vestigating microbial populations in any habitat, eliminating chain of small to medium-sized mountain ranges running the dependence on the isolation of pure cultures (Amann et al. between 8 and 21°N latitude and 73 and 77 °E longitude. 1995b; Ahmad et al. 2009). The southern part of the Western Ghats region encom- Only limited investigations have been carried out on bac- passes the states of Tamil Nadu and Kerala, where many terial diversity in various forest ecosystems of the Western reserve forests can be found. Soil samples from 12 differ- Ghats. The southern regions of the Western Ghats are mostly ent sampling sites throughout the southern regions of located in the states of Tamil Nadu and Kerala. These two Western Ghats (Fig. 1) were obtained by first removing states are characterized by their abundance of forested areas, the top surface soil and then collecting samples at a soil which are largely unexplored to the fullest. To date, there have depth of about 10 cm. A small amount of each sample was

Fig. 1 Experimental area depicting Western Ghats and the locations of the sampling sites Ann Microbiol (2015) 65:431–441 433 used for serial dilution plating, and the remaining was Sigma-Aldrich, St. Louis, MO). The ARDRA profiles of stored at 4 °C until further use. different isolates were compared manually and the observed DNA polymorphisms scored as dominant markers and con- Isolation and morphological characterization of bacteria verted to a binary matrix. The data were used to derive similarity measures in terms of Jaccord’s coefficient in all Enumeration of soil bacteria was achieved by serial dilu- possible pairwise combinations. The similarity matrix was tion and plating on different types of solid media at various used for cluster analysis using the UPGMA method in the strengths, such as nutrient agar (NA), yeast starch agar and NTSys-PC 2.1 software package (Exeter Publishing, soil extract agar (SEA) [see Electronic Supplementary Setauket, NY). Material (ESM) Table 1] (Bianchi and Armand 1982). Following incubation at 37 °C for 24–72 h, the isolates Extraction and purification of metagenomic DNA were selected based on their cell morphology, colony mor- phology, Gram reaction, among others, and the results Soil DNA was extracted from 5 g of each soil sample with documented. 13 ml of extraction buffer (100 mM Tris–HCl pH 8; 100 mM EDTA pH 8, 100 mM sodium phosphate, 1.5 M NaCl, 1 % Diversity analysis CTAB) and incubated for 30 min at 37 °C before being centrifuged at 6,000 rpm for 5 min. The supernatant was A range of diversity indices (ESM Table 2) were used to collected and used for DNA isolation as described by Zhou characterize the bacterial community studies, including the et al. (1996). Metagenomic DNA consists of humic acids and ubiquitous Shannon index, the evenness index derived from other phenolic compounds that inhibit its downstream appli- the former and Simpson’s dominance index and its associated cations. Hence, purification is important for further process- equitability index (McCaig et al. 1999;ChoandKim2000; ing. Purification was preceded by treatment with a modified Dunbar et al. 2000). method of Ausubel et al. (1987).

Genotypic characterization of isolates Construction of library and sequence analysis

Genomic DNA was isolated from bacterial cultures grown in Purified PCR products were ligated with pTZ57R/T vector Luria Bertani (LB) broth (Hi Media, Mumbai, India) using (Fermentas) following the manufacturer’s instructions. The standard protocols (Sambrook et al. 1989). The 16S rRNA ligation reaction was carried out overnight at 4 °C, following genes were amplified using the primers 27F (5′-GAG TTT which the ligated mixture was transformed into competent GAT CCT GGC TCA G-3′) and 1492R (5′-ACG GCT ACC Escherichia coli DH5α cells using the calcium chloride- TTG TTA CGA CTT-3′) (Lane et al. 1985) in a reaction mediated heat shock method. Transformants were obtained volume containing 10× buffer, 25 mM MgCl2,4mM on LB agar plates containing ampicillin, X-Gal (5-bromo-4 dNTPs, 0.5 U Taq polymerase (Fermentas, Thermo chloro 3-indolyl-β-D-galactopyranoside) and isopropyl-β-D- Scientific, Vilnius, Lithuania), 10 μmol of each primer and thio-galactopyranoside. Positive clones were selected using 50–100 ng template DNA. Thermocycling was performed blue/white screening, and successful insertion of the vector using a Mastercycler® Pro (Eppendorf, Hamburg, was confirmed by PCR in the Mastercycler® Pro thermal Germany); the cycling program consisted of an initial dena- cycler using a cycling program of one cycle of initial dena- turation at 96 °C for 5 min, followed by 30 cycles of denatur- turation at 96 °C for 5 min, followed by 30 cycles of denatur- ation at 94 °C for 1 min, annealing at 48 °C for 1 min and ation at 94 °C for 1 min, annealing at 48 °C for 1 min and extension at 72 °C for 1 min, with a final extension of 72 °C extension at 72 °C for 1 min, with a final extension of 72 °C for 10 min) in Master thermal cycler. for 10 min. Sequence analysis was performed using M13/pUC forward and reverse primers at Macrogen, Seoul, South Amplified ribosomal DNA restriction analysis Korea.

Amplified ribosomal DNA restriction analysis (ARDRA) was Phylogenetic analysis of 16S rRNA gene sequences performed to select the unique representative isolates based on restriction pattern prior to sequencing. The 16S rRNA gene The taxonomical identity of each isolate was assigned through product was digested with 3 U each of the restriction enzymes BLAST searches against the 16S ribosomal database of TaqIandHhaI (New England Biolabs, Ipswich, MA) in a total GenBank (www.ncbi.nlm.nih.gov). Based on our 16S rRNA volume of 10 ul for 4–16 h at 37 °C and 65 °C respectively. gene sequence-based comparative analysis of isolates and The digested products were electrophoresed in a 2 % agarose type strains, we constructed a phylogenetic tree following gel and viewed on a gel imaging system (Alpha Digidoc, pairwise and multiple sequence alignment by neighbor- 434 Ann Microbiol (2015) 65:431–441

joining method. The phylogeny inference method, maximum Wiener index (H′) and its evenness (EH) and Simpsons diver- parsimony method and maximum likelihood method were sity index (D) and its equitability (ED). The values of the H′ applied to assess dendrogram reliability and stability using index and of EH ranged from 1.93 to 2.93 and from 0.84 to the software MEGA 5.0 (Tamura et al. 2011). Nucleotide 0.96, respectively. The values of the D index and of ED ranged sequences of the isolates and clones have been submitted to from 0.05 to 0.21 and from 0.84 to 0.96, respectively (ESM GenBank under accession numbers KC595863–880; Table 3). The highest and lowest values of the diversity AB813001–016; JX860327–341, AB872125–143; indices were obtained for isolates BWGA and BVP, AB890370–371; AB893352–354 and KF910780–785. respectively. Employing universal eubacteria primers, we amplified the 16S rRNA gene to further explore the diversity of the cultiva- Results and discussion ble soil bacterial community. The amplified product upon restriction digestion with Taq1andHha1 generated several Cultivable bacterial diversity digestion patterns which could be used for diversity studies. However, ARDRA patterns for individual restriction enzymes A total of 171 morphologically different bacterial isolates generated fewer fragments; consequently, the banding pat- were identified from 12 sampling sites using different types terns of both enzymes were combined for analysis. of nutritional media (Bianchi and Armand 1982) and pre- Using the restriction digestion patterns of the isolates (171 served as glycerol stock. Of these strains, 84 and 16 % were isolates), we constructed a dendrogram with NTSys-PC soft- Gram-positive and Gram-negative, respectively (data not ware (ESM Fig. 1). A minimum of three representatives from shown). The diversity of the isolates was determined based each cluster was selected for identification (48 isolates; on the values of various diversity indices, i.e. the Shannon– Fig. 2a, b) in order to obtain data that was not biased due to

Fig. 2 a Amplified ribosomal DNA restriction analysis (ARDRA) 25 BVP4, 26 BVP7. b ARDRA profile of sequenced isolates. Lanes: M profile of the sequenced isolates. Lanes: M 100-bp marker, 1 BWGA1, 100-bp marker, 1 BVP11, 2 BVP12, 3 BVE11, 4 BGL11, 5 BGL12, 6 2 BWGA3, 3 BWGA4, 4 BWGA8, 5 BWGA13, 6 BWGA16, 7 BGL13, 7 BGL17, 8 BGL18, 9 BSS9, 10 BSS1, 11 BMU4, 12 BMU5, 13 BWGB18, 8 BWGB19, 9 BWGB21, 10 BWGB24, 11 BWGB25, 12 BMU6, 14 BMU8, 15 BMU9, 16 BMA4, 17 BMA6, 18 BMA7, 19 BWGB27, 13 BKA1, 14 BKA5, 15 BKA7, 16 BKA8, 17 BKA10, 18 BWA6, 20 BWA9, 21 BWA13, 22 BWA14 BKS4, 19 BKS6, 20 BKS7, 21 BKS10, 22 BKB4, 23 BKB14, 24 BVP2, Ann Microbiol (2015) 65:431–441 435 similar banding patterns amongst the isolates. Oravecz et al. dominant species in soil have been reported previously, such (2004) suggested that the ARDRA patterns for universal as B. benzoevorans, which constituted a majority of bacteria in eubacterial primers are complex; in our study, we found with Dutch soil, and B. macroides and B. insolitus, which were only minor differences and similar banding patterns among found to be dominant in Scottish soil (McCaig et al. 2001). the samples studied. The diversity of cultivable bacteria as determined by the Phylogenetic analysis of the isolates revealed that the iso- 16S rRNA gene sequence analysis revealed the occurrence of lates belonged to bacterial taxa Firmicutes, Proteobacteria three divisions, the predominant being Firmicutes (83.3 %), and high G+C bacteria. To study the diversity of species followed by Proteobacteria (8.3 %) and high G+C Gram- associated with the soil samples, we constructed a phyloge- positive bacteria (8.3 %). Of the 171 isolates studied netic tree from the 16S rRNA gene sequences (Fig. 3). All 83.64 % were Gram-positive and the rest were Gram- isolates in phylum Firmicutes, with one exception, were found negative organisms (data not shown). Amongst the phylum to belong to genus Bacillus (Table 1), with the exception being Firmicutes, the genus Bacillus was the dominant group and BGL11, which was identified as Staphylococcus sciuri. was represented by nine species (sequence similarity 99– Agrobacterium tumefaciens and Stenotrophomonas 100 %; Table 1). Staphylococcus was yet another significant maltophilia in the phylum Proteobacteria were distributed in representative of this phylum (99 %). the subgroups Alphaproteobacteria and We used three types of complex media at various strengths Gammaproteobacteria, respectively. In addition, a few iso- for the isolation of the soil bacteria, namely, NA, LB and lates (BVP12, BG17, BG18 and BSS1) represented the less SEA. Although these media can support the growth of diverse abundant high G+C bacteria; these were identified as microorganisms, there is always the possibility that some Brachybacterium rhamnosum, Micrococcus luteus and isolates were not cultivable under our isolation conditions. Microbacterium hominis (Table 1). Hugenholtz et al. (1998) The diversity and function of microorganisms remain mostly reported that Firmicutes and Proteobacteria are well repre- unclear, as in general only a small proportion of the bacteria sented by cultivable organisms, with members of Firmicutes present in these environments are readily cultivable (Amann accounting for 90 % of all cultivated bacteria, which is similar et al. 1995a; Schleifer 2004). Based on the results of our study, to ourresults. it would appear that the majority of bacteria which grew on The Bacillus group, which belongs to the phylum these common nutritional media are of a single community, as Firmicutes, were found to be predominant in all of the samples reflected in the 16S rRNA gene sequence identity. The diver- analyzed. Members of Bacillus are low G+C Gram-positive sity in the cultivable isolates confirmed that only a small bacteria which are ubiquitous in nature. Members of Bacillus fraction of the bacterial population was recovered on the have been isolated from various environments, such as fresh- culture media used. Bacteria which were uncultivable could water, saline water, soil, plants, animals and air (Pignatelli be members of taxa for which the growth conditions were not et al. 2009). They are well documented in terms of phenotypic adequate (Ruckmani and Chakrabarti 2011). It has been characterization and have been found to be metabolically shown that culture-independent approaches allow the detec- diverse. This group contains the endospore-forming bacteria, tion of such taxa (Vaz-Moreira et al. 2011) and provide a better the lactic acid bacteria and Gram-positive cocci. insight into the diversity of microbial communities. Overrepresentation of Gram-positive bacteria can also be observed in culture collection centers (Ahmad et al. 2009). Our phylogenetic analysis of the samples revealed the Culture-independent analysis dominance of Bacillus species. Amongst the sequenced iso- lates, ten clustered with the Bacillus megaterium strain NBRC Analysis of 16S rRNA genes from soil metagenomic DNA 15308 with 99 % similarity. Among the remaining isolates, 12 provides vital analytical data on microbial populations in any belonged to the B. cereus group (B. cereus, B. thuringiensis habitat and eliminates any dependence on the isolation of and B. anthracis) of which six clustered with B. thuringiensis cultures (Ward et al. 1992;Amann1995; Ahmad et al. ATCC 23842 with 100 % similarity and six clustered with 2009). In the our study, bacterial diversity analysis using a B. cereus JCM 2152 with 99 % similarity (Fig. 3). Another ten culture-independent method was carried out on the BVP and isolates showed 100 % similarity with the B. subtilis group, BWGA samples because of their extreme diversity indices. which formed clusters with B. subtilis and B. pumilus BVP showed dominance towards a single species, while (Table 1). In addition, isolate BGL11, identified as BWGA showed more evenness of species distribution as Staphylococcus sciuri, showed 100 % similarity with DSM revealed by a cultivable method. Metagenomic DNA was 20345 strain, which again belonged to phylum Firmicutes. isolated and 16S rRNA gene amplification was carried out The diversity results based on cultivable isolates revealed to generate two 16S rRNA gene libraries using the TA cloning that Bacillus was the dominant genera distributed in the south- method. The recombinant clones were randomly selected ern regions of Western Ghats. Similar occurrences of such from both libraries for identification. 436 Ann Microbiol (2015) 65:431–441

Fig. 3 Neighbor-joining tree based on the 16S rRNA gene sequences replications) of >50 % are given at the nodes. Filled circles indicates the showing the phylogenetic relationship of the 38 soil bacterial isolates clades that were conserved when the neighbor-joining, maximum parsimo- among 22 members of the genus Bacillus, Agrobacterium and ny and maximum likelihood methods were used to construct the phyloge- Brachybacterium. Bootstrap values (expressed as percentage of 1,000 netic trees. Mycoplasma alkalescens was used as the out-group organism

All of the clones identified from the BVP library clones clustered with B. thuringiensis and one each clus- belonged to the phylum Firmicutes and showed high sim- tered with B. cereus and B. acidicola (Table 2). The phy- ilarity with Gram-positive Bacillus species. Eleven clones logenetic relationships of the sequenced clones were deter- were phylogenetically related to Bacillus anthracis, where- mined using the neigbhour-joining method and are given as three clones clustered with B. weihenstephanensis,two as a dendrogram in Fig. 4). Ann Microbiol (2015) 65:431–441 437

Table 1 Characteristics of cultivable strains isolated from soil samples collected throughout the Western Ghats together with their taxonomic affiliation based on 16S rRNA gene

Isolate name Colony color Cell Gram Division Nearest phylogenetic % Accession morphology stain neighbor Similarity number

BWGA 1 Pale white Rod + Firmicutes Bacillus flexus 99 AB813007 BWGA 3 Pale white Rod + Firmicutes Bacillus thuringiensis 100 AB813003 BWGA 4 White Rod + Firmicutes Bacillus pumilus 99 AB813001 BWGA 8 Pale yellow Rod + Firmicutes Bacillus megaterium 99 AB813012 BWGA 13 Pale yellow Rod + Firmicutes Bacillus cereus 99 AB872141 BWGA 16 Pale white Rod + Firmicutes Bacillus simplex 99 KC595864 BWGB 18 White Rod + Firmicutes Bacillus pumilus 99 AB813005 BWGB 19 Pale white Rod + Firmicutes Bacillus pumilus 99 AB872138 BWGB 21 Pale white Rod + Firmicutes Bacillus sphaericus 99 AB872140 BWGB 24 Pale white Rod + Firmicutes Bacillus thuringiensis 100 AB813006 BWGB 25 White Rod + Firmicutes Bacillus megaterium 99 AB872142 BWGB 27 Pale white Rod + Firmicutes Bacillus subtilis 99 KC595863 BKA 1 White Rod + Firmicutes Bacillus anthracis 99 KC595876 BKA 5 Pale white Rod + Firmicutes Bacillus simplex 99 AB813011 BKA 7 White Rod + Firmicutes Bacillus megaterium 99 AB872128 BKA 8 White Rod + Firmicutes Bacillus simplex 99 AB872129 BKA 10 Pale white Rod + Firmicutes Bacillus flexus 99 AB813004 BKS 4 White Rod + Firmicutes Bacillus anthracis 99 KC595875 BKS 6 White Rod + Firmicutes Bacillus megaterium 99 AB872132 BKS 7 Pale white Rod + Firmicutes Bacillus megaterium 99 KC595867 BKS 10 Pale white Rod + Firmicutes Bacillus megaterium 99 AB872133 BKB 4 Pale white Rod + Firmicutes Bacillus pumilus 99 AB872130 BKB 14 Pale white Rod + γ-Proteobacteria Stenotrophomonas maltophilia 99 AB872131 BVP 2 Pale yellow Rod + Firmicutes Bacillus megaterium 99 KC595869 BVP 4 Pale yellow Rod + Firmicutes Bacillus pumilus 99 AB813008 BVP 7 Yellow Rod + Firmicutes Bacillus subtilis 99 KC595866 BVP 11 Pale white Rod + Firmicutes Bacillus megaterium 99 AB813002 BVP 12 Pale white Cocci + High G+C Gram+ve Micrococcus luteus 100 AB872139 BVE 11 Pale yellow Rod – γ -Proteobacteria Stenotrophomonas maltophilia 99 AB813016 BGL 11 Pale white Rod + Firmicutes Staphylococcus sciuri 100 AB813014 BGL 12 Pale white Rod + Firmicutes Bacillus cereus 99 AB872125 BGL 13 Pale white Rod + Firmicutes Bacillus sphaericus 99 AB872126 BGL 17 White Rod + High G+C Gram+ve Brachybacterium rhamnosum 99 AB813015 BGL 18 Pale white Cocci + High G+C Gram-negative Micrococcus luteus 100 AB872127 BSS 9 White Rod + Firmicutes Bacillus subtilis 99 KC595868 BSS 1 Pale white Rod + High G+C Gram-positive Microbacterium hominis 99 AB872137 BMU 4 Pale yellow Rod + Firmicutes Bacillus thuringiensis 99 KC595874 BMU 5 White Rod + Firmicutes Bacillus flexus 99 KC595873 BMU 6 White Rod + Firmicutes Bacillus flexus 99 KC595872 BMU 8 Pale yellow Rod + Firmicutes Bacillus thuringiensis 100 KC595871 BMU 9 Pale white Rod + Firmicutes Bacillus thuringiensis 99 KC595870 BMA4 White Rod – α-Proteobacteria Agrobacterium radiobacter 100 AB872134 BMA6 Pale white Rod + Firmicutes Bacillus thuringiensis 99 AB872135 BMA7 Pale white Rod + Firmicutes Bacillus pumilus 99 AB872136 BWA 6 Pale white Rod + Firmicutes Bacillus flexus 99 KC595865 BWA 9 Pale white Rod – α-Proteobacteria Agrobacterium radiobacter 100 KC595880 BWA13 Yellow Rod + Firmicutes Bacillus cereus 99 KC595879 BWA14 Pale white Rod + Firmicutes Bacillus simplex 99 KC595878 438 Ann Microbiol (2015) 65:431–441

Table 2 Clones of the BVP (Valparai) and BWGA (Maruthamalai) library to its nearest phylogenetic neighbor

Clone Taxonomic group Nearest phylogenetic neighbour Number of nucleotides % Similarity Accession number

CVP1 Firmicutes Bacillus anthracis 1,496 99 % JX860327 CVP3 Firmicutes Bacillus cereus 1,456 99 % JX860328 CVP7 Firmicutes Bacillus thuringiensis 1,543 99 % JX860329 CVP15 Firmicutes Bacillus anthracis 1,461 99 % AB893354 CVP27 Firmicutes Bacillus weihenstephanensis 1,502 99 % JX860330 CVP33 Firmicutes Bacillus anthracis 1,499 99 % JX860331 CVP42 Firmicutes Bacillus thuringiensis 1,454 99 % JX860332 CVP49 Firmicutes Bacillus anthracis 1,536 99 % JX860341 CVP54 Firmicutes Bacillus acidicola 1,542 99 % JX860340 CVP60 Firmicutes Bacillus weihenstephanensis 1,518 99 % JX860333 CVP65 Firmicutes Bacillus anthracis 1,459 99 % JX860334 CVP78 Firmicutes Bacillus weihenstephanensis 1,438 99 % JX860335 CVP85 Firmicutes Bacillus anthracis 1,513 99 % JX860337 CVP93 Firmicutes Bacillus anthracis 1,528 99 % KF910781 CVP95 Firmicutes Bacillus anthracis 1,519 99 % KF910782 CVP96 Firmicutes Bacillus anthracis 1,526 99 % KF910783 CVP116 Firmicutes Bacillus anthracis 1,516 99 % KF910784 CVP123 Firmicutes Bacillus anthracis 1,527 99 % KF910785 CWG4 Firmicutes Bacillus anthracis 1,495 99 % JX860338 CWG5 β-Proteobacteria Ralstonia mannitolilytica 1,432 99 % JX860339 CWG9 Unidentified Uncultured bacterium 1,014 89 % AB893353 CWG47 Unidentified Uncultured bacterium 1,505 95 % AB890371 CWG65 Firmicutes Bacillus weihenstephanensis 1,518 99 % KF910780 CWG71 Firmicutes Bacillus anthracis 1,517 99 % AB893352 CWG102 Unidentified Uncultured bacterium 1,270 96 % AB890370

Similarly, there was a predominance of members of the varying between 89 and 96 %. Such a low sequence similarity phylum Firmicutes among the BWGA library clones, with indicates that these three clones may represent new taxa, four clones showing a high similarity to Bacillus anthracis. possibly at a higher taxonomic hierarchy (Ruckmani and The phylum Gammaproteobacteria was the other group iden- Chakrabarti 2011). The remaining clone, CWG5, showed tified in the BWGA library, represented by clone CWG5 99 % sequence similarity with Ralstonia mannitolilytica. which was identified as Ralstonia mannitolilytica. The three Bacillus thuringiensis, B. cereus and B. antharacis were well clones CWG9, CWG47 and CWG102 could not be classified represented (16 clones) in the clone libraries and showed 99 % into any taxonomic phyla. However, CWG9 clustered within sequence similarity with three strains (BWGA3, BWGA13 Firmicutes, while CWG47 and CWG102 formed a separate and BKA1) which had been isolated using the cultivable cluster (Fig. 5). The phylogenetic relationship of the se- approach. The two 16S rRNA gene libraries BVP and quenced clones and their similarities based on BLAST analy- BWGA showed substantial differences in the distribution of sis are shown in Table 2. The ubiquitous nature of Bacillus phyla between them. species in the environment was apparent from the results The BVP sample exhibited a high dominance of Bacillus obtained through the phylogenetic analysis of clones from sp. from both the culture-dependent and culture-independent the BVP library, as explained previously by Pignatelli et al. analyses. However, using the culture-dependent approach we (2009). identified only Bacillus sp. in the BWGA sample (Table 1), The 16S rRNA clones examined in these two libraries while the presence of uncultured bacteria was revealed in the showed major associations with the Firmicutes group. culture-independent analysis (Table 2). The relative abun- Among these 25 clones, 21 belonged to Bacillus sp. with a dance of Bacillus clones was different among the libraries sequence similarity of 99 %; of the remaining four clones, analyzed: some clones, identified as B. cereus, B. threewereplacedinthegroupof‘uncultured bacteria’ thuringiensis and B. acidicola, were typical of the BVP sam- (CWG9, CWG47 and CWG102) with a sequence similarity ple but were not found in the BWGA sample. One possible Ann Microbiol (2015) 65:431–441 439

Fig. 4 Neighbor-joining tree based on the 16S rRNA gene sequences Cladesthat were conserved when neighbor-joining, maximum parsimony showing the phylogeneticrelationship of 18 BVP clones among the ten and maximum likelihood methodswere used to construct the phylogenet- members of the genus Bacillus. Bootstrap values (expressed aspercentage ic trees. Methanococcus thermolithotrophicus wasusedasthe of 1,000 replications) of >50% are given at the nodes. Filled circles outgrouporganism. explanation for this difference is that strains isolated from the culture-dependent methods. Previous studies by Smit et al. BVP soil tended to belong to a single group based on the (1997) on soil bacteria found large differences between the diversity index analysis of cultivable strains when compared results obtained by culture-dependent and culture- to BWGA sample, which is reflected in their species distribu- independent methods. In our study, apart from the difference tion in the culture-independent analysis. Thus, the richness of in revealing uncultured bacteria, both methods were able to Bacillus species in the BVP soil sample can be studied as a detect Bacillus sp. belonging to phylum Firmicutes.Using means of exploring and identifying novel enzymes and other culture-dependent and culture-independent methods, Felske metagenomic genes for bioprospecting for use in industrial et al. (2003) reported that Bacillus species constitute a processes (Schallmey et al. 2004). On the other hand, the majority of the bacteria in Dutch soil. It would therefore BWGA sample could be a source for the exploration and appear that members of the Firmicutes dominate the soil identification of uncultured organisms, as indicated by the ecosystem, which is consistent with our finding of Bacillus culture-independent analysis, with the aid of specific media being the most frequently isolated species from our soil and growth conditions. samples. Studying bacterial diversity by our culture-independent Our results imply that members of phlyum Firmicutes method revealed a different population from that found using could to be the major contributors to the bacterial population 440 Ann Microbiol (2015) 65:431–441

Fig. 5 Neighbor-joining tree based on the 16S rRNA gene sequences the nodes. Filled circles Clades that were conserved when the neighbor- showing the phylogenetic relationship of seven BWG clones among 12 joining, maximum parsimony and maximum likelihood methods were members of the genus Bacillus and Ralstonia. Bootstrap values used to construct the phylogenetic trees. Methanococcus (expressed as percentage of 1,000 replications) of >50 % are given at thermolithotrophicus was used as the out-group organism

in the soil samples evaluated, although more quantitative The results of our study indicate that it is possible to evaluations are required. Furthermore, culture-independent explore the diversity of cultivable and culture-independent analysis for diversity can be used to ascertain unidentified soil bacteria using the 16S rRNA gene-based approach. The phylum, which cannot be done using cultivable methods. implementation of the 16S rRNA gene-based approach has Thus, culture-independent analyses could explain the diversi- expanded the field of microbial ecology. The phylogenetic ty of these communities and illustrate community structure as position of environmental bacterial populations in the evolu- well as describe the ecological role of bacteria in the forest tionary tree can now be determined and traced very precisely soil. using this approach, even in these complex ecosystems. This It is possible to assess bacterial communities using approach will be suitable to increase our knowledge of the both culture-dependent and culture-independent methods; physiological, biochemical, genetic and molecular properties however, no one single method provides a feasible mea- of soil bacteria. Eventually the diversity studies revealing the sure of the bacterial community under study, as each predominance of Bacillus species in southern regions of method can only give a partial picture of microbial Western Ghats will provide a reservoir for exploring potential diversity. Zhang et al. (2008) suggested that different novel enzymes and other biotechnologically important prod- methods that complement each other should be used to ucts from Bacillus species. obtain a more complete picture of bacterial diversity. Therefore, a combination of both approaches, i.e., culture-dependent and and culture-independent, would Acknowledgments The study was supported by a grant from Depart- ment of Science and Technology (DST), India vide the grant SR/FT/LS- be the ideal approach to study the bacterial diversity of 032/2008. V. Gowdaman gratefully acknowledges the University Re- any natural habitat. search fellowship awarded to him by Bharathiar University. Ann Microbiol (2015) 65:431–441 441

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