Culturable and Culture-Independent Bacterial ...

Microb Ecol DOI 10.1007/s00248-014-0476-4

ENVIRONMENTAL MICROBIOLOGY

Culturable and Culture-Independent Bacterial Diversity and the Prevalence of Cold-Adapted Enzymes from the Himalayan Mountain Ranges of India and Nepal Siddarthan Venkatachalam & Vasudevan Gowdaman & Solai Ramatchandirane Prabagaran

Received: 7 January 2014 / Accepted: 28 July 2014 # Springer Science+Business Media New York 2014

Abstract Bacterial diversity of soil samples collected from different geographical regions of Himalayan mountains was studied through culturable (13 samples) and cultureindependent approaches (5 samples based on abundance of diversity indices in each ecological niche). Shannon–Wiener diversity index and total bacterial count ranged from 1.50±0.1 to 2.57±0.15 and 7.8±1.6×105 to 30.9±1.7×105 cfu ml−1 of soil, respectively. Based on morphology and pigmentation, 406 isolates were selected by culturing in different cultivable media at various strengths and concentrations. All the strains were subjected to amplified ribosomal DNA restriction analysis and the representative isolates from each cluster were chosen for 16S rRNA gene sequence-based identification. Soil habitat in Himalayan foot hills was dominated by the genera Arthrobacter, Exiguobacterium, Bacillus, Cedecea, Erwinia, and Pseudomonas. Five 16S rRNA gene libraries from the selected five samples yielded 268 clones and were grouped into 53 phylotypes covering 25 genera including the genus of Ferribacterium, Rothia, and Wautersiella, which were reported for the first time in Himalayan tracks. Principal coordinates analysis indicates that all the clone libraries were clearly separated and found to be significantly different from each other. Further, extracellular investigation of cold-active enzymes showed activity of cellulase (23.71 %), pectinase (20.24 %), amylase (17.32 %), phytase (13.87 %), protease (12.72 %), and lipase (23.71 %) among the isolates. Four isolates namely Exiguobacterium mexicanum (BSa14), Exiguobacterium sibiricum (BZa11), Micrococcus antarcticus (BSb10), and Electronic supplementary material The online version of this article (doi:10.1007/s00248-014-0476-4) contains supplementary material, which is available to authorized users. S. Venkatachalam : V. Gowdaman : S. R. Prabagaran (*) Molecular Microbiology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India e-mail: [email protected]

Bacillus simplex (BZb3) showed multiple enzyme activity for five different types of enzymes. In addition, various genera like Exiguobacterium, Erwinia, Mycetecola, Cedecea, Pantoea, and Trichococcus have also shown novel hydrolytic enzyme activity in the Himalayan foothills.

Introduction The Himalayas is one of the important major biodiversity hotspot regions in the world which covers many hills from northern to north eastern part of India and Nepal. The diversity of flora and fauna of the Himalayas varies drastically with respect to climate conditions, rainfall, altitude, and soils. Particularly at higher altitudes, where snow exists throughout the year which extends life for distinct plants, animals, and microbial communities. The Himalayas also has several unique habitats such as glacial ice, permafrost, tundra wetlands, tundra soil, sub-glacial soil, peri-glacial soil, and periglacial lakes [1]. Most of the microbiota in this region are cold-adapted [2–12]. Northern and north eastern parts of high altitude hills contain numerous glaciers. Most of these glaciers were considered for microbial diversity through 16S rRNA gene-based sequencing approach of both culturable and culture-independent methods [1, 13–17, 2, 18, 19]. Several novel bacterial species such as Planococcus stackebrandtii [3], Kocuria himachalensis [16], Dietzia kunjamensis [20], Exiguobacterium himgiriensis [21], Actinoalloteichus spitiensis [22], Paenibacillus glacialis [23], Dyadobacter hamtensis [24], Bacillus cecembensis [25], Exiguobacterium indicum [26], Leifsonia kafniensis [27], Cryobacterium roopkundense [28], and Leifsonia pindariensis [29] were identified from these regions. However, while considering the geography of the Himalayas, previous studies have covered only a small portion of the vast Himalayan range. Thus,

S. Venkatachalam et al.

many hills and sub-hills of Himalayan mountain ranges have yet to be explored by the microbiological studies. Psychrophilic and psychotropic organisms from these regions have a wide scope for industrial applications. In particular, the enzymes produced from these microorganisms are of economic significance in biotechnology, agriculture, and medicine [30, 31]. These cold-active enzymes have immense biotechnological potential. With this background, the present study focuses on bacterial diversity towards bioprospecting for a battery of cold-active enzymes from different geographical locations of the Himalayas.

mesophilic (25–40 °C) bacteria [2, 6] based on the growth at different temperature (data not shown). Diversity Analysis With a view to compare the cultivable bacterial diversity among the samples, a wide range of diversity indices were used to study the bacterial communities. Shannon–Wiener Diversity Index was used to calculate Shannon index (H0) and its evenness. Similarly, the Simpson’s dominance index (D) and its equitability index [32–34] were studied using online tool (http://www.changbioscience.com/ genetics/shannon.html) [35, 36].

Materials and Methods Genotypic Characterization of Isolates Site Description and Sampling The Himalayan mountains ranges are spread along five countries namely, India, Pakistan, China, Nepal, and Bhutan. Most of these regions are in the deep forest with different climatic conditions. A total of 13 samples were collected from the Himalayan foot hills in India and Nepal (Fig. 1). Plant debris, if any, were removed from the top surface of the soil and the samples were collected in sterile 50 mL tubes. Subsequently, they were placed in coolers and transferred to Bharathiar University, Coimbatore, Tamil Nadu, for further studies. Isolation and Cultivation of Bacteria For the cultivation of bacteria, different media such as nutrient agar (peptone 5 g L−1, beef extract 3 g L−1, NaCl 5 g L−1, agar 20 g L−1; pH 7.0), Antarctic bacterial medium (peptone 5 g L−1, yeast extract 2 g L−1, 20 g L−1 agar; pH 7.0), tryptic soy agar (tryptone 17 g L−1, soya meal 3 g L−1, dextrose 2.5 g L−1, NaCl 5 g L−1, K2HPO4 2.5 g L−1, agar 20 g L−1; pH 7.0), starch yeast peptone agar (starch 2 g L−1, yeast extract 0.8 g L−1, peptone 0.1 g L−1, agar 15 g L−1; pH 7.0), and soil extract agar (glucose 1 g L−1, dipotassium phosphate 0.5 g L−1, soil extract 17.75 g L−1, agar 15 g L−1; pH 7.0) were used at two different strengths (1× and 0.5×) to isolate diverse bacterial morphotypes (Table 1). One gram of the soil sample was suspended in 9 mL of 0.9 % (w/v) NaCl solution and left on shaker for 2 h at 15 °C. Upon serial dilution, 100 μL of samples was plated on different cultivation media as described and incubated at five different temperatures (4, 10, 15, 20, and 37 °C) observed for up to 15 days [25]. Based on the colony morphology and pigmentation, distinct bacterial isolates were selected and subsequently pure cultured. The isolated strains were categorized into psychrophilic (4–20 °C), psychrotolerant (4–37 °C), and

Genomic DNA was isolated from bacterial cultures grown in Nutrient broth (Hi Media, India) using standard protocol [37]. The 16S rRNA gene was amplified using universal primers 27F (5′ GAGTTTGATCCTGGCTCAG 3′) and 1492R (5′ ACGGCTACCTTGTTACGACTT-3′) [38]. The reaction mixture (20 μL) consisted of 0.5 U of Taq polymerase (Chromous Biotech, India), 2 μL of 10× buffer, 1.5 mM of MgCl2, 0.4 mM of dNTP mix, 10 pmol of each primer, and 2 μL of template DNA (∼50 ng). The polymerase chain reaction (PCR) program was set as follows: initial denaturation at 94 °C for 5 min followed by 30 cycles of 1 min at 94 °C, 1 min at 48 °C, and 2 min at 72 °C, and a final extension cycle at 72 °C for 10 min in Master thermal cycler (Eppendorf, Germany). Amplified Ribosomal DNA Restriction Analysis Amplified ribosomal DNA restriction analysis (ARDRA) was performed to select the unique representative isolates based on the restriction profiling of MspI enzyme (New England Biolabs, USA). The 16S rRNA gene PCR product was digested with 5 U of the enzyme MspI in a total reaction volume of 15 μL for 4 h at 37 °C. The enzyme was inactivated by heating at 65 °C for 15 min, and the reaction products were analyzed by agarose (2.5 %, w/v) gel electrophoresis in TAE buffer containing 1 μg mL−1 of ethidium bromide and viewed on Gel Imaging System (Alpha Digidoc, USA). The ARDRA profiles of different isolates were compared manually using DNA Ladder, (100 bp) from New England BioLabs, USA (cat. no. N3231S). The observed DNA polymorphisms were scored as dominant markers and converted to a binary matrix. The data was used to derive similarity measures in terms of Jaccard’s coefficient in all possible pair wise combinations. The similarity matrix was used for cluster analysis using UPGMA method in NTSys-PC 2.1 software package (Exeter Publishing, Setauket, NY USA).

Bacterial Diversity of Himalayan Mountains Fig. 1 Map showing sampling area in Himalayan mountain ranges

Extraction, Purification of Metagenomic DNA, and 16S rRNA Gene Amplification For metagenomic analysis, about 0.5 g soil samples of the five different Himalayan regions was used for DNA extraction. Total genomic DNA was extracted with FastDNA®SPIN Soil Kit (MP Biomedicals, USA) according to the manufacturer’s instructions. The DNA quality was assessed by agarose gel electrophoresis and quantified using Nano-Drop spectrophotometer (Thermo Scientific, USA). For nested 16S rRNA gene PCR, about 20 ng of DNA was used as template and amplified using universal primers. The first and second round PCR generated ∼1,500 and ∼1,060 bp products, respectively. The amplified PCR product was purified using Puregene kit (Fermentas, USA) and ligated into pTZ57R/T vector (Fermentas, USA) according to the manufacturer’s instructions and then transformed to Escherichia coli DH5α-

competent cells. Recombinant clones were selected based on blue white screening, and the plasmids were isolated from all recombinants and PCR was performed using pUC18/M13 forward and reverse primers (initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 48 °C for 1 min, extension at 72 °C for 1 min, and a final extension of 72 °C for 10 min) in Master thermal cycler (Eppendorf, Germany) in order to confirm the presence of insert and subsequently representative clones were sequenced in each library. Phylogenetic Analysis of 16S rRNA Gene Sequences The representative isolates of each operational taxonomic unit (OTU) from all the samples were selected and the PCR amplicons were purified by agarose gel extraction kit (Qiagen, USA). The amplicons were then sequenced by using

BPLa1-24; BPLb1-14; BPLc1-5; BPLd1-3 BPLe1-5 BCKa1-12; BCKb1-6; BCKc1-5; BCKd1-7; BCKe1-4 BMa1-9; BMb1-11; BMc1-3 BSAa1-12; BSAb1-4; BSAc1-5; BSAd1-2; BSAe1-3

BDa1-16; BDb1-12; BDc1-8; BDd1-4 BSa1-14; BSb1-9; BSc1-6; BSd1-7; BSe1-2 BZa1-12;BZb1-9; BZc1-9; BZd1-4 BBa1-9; BBb1-5; BBc1-2; BBd1-3;BBe1-4 BGa1-8; BGb1-5; BGc1-5; BGd1-1 BCa1-4; BCb1-3; BCc1-3; BCd1-4 BKKa1-12; BKKb1-2; BKKc1-6; BKKd1-3; BKKe1-5 BKa1-14; BKb1-11; BKc1-5; BKd1-7; BKe1-10 BPa1-9; BPb1-13; BPc1-4; BPd1-2; BPe1-1

51 34 23 26 28°45′ N, 83°56′ E 27°38′ N, 85°59′ E 26°11′ N, 91°14′ E 26°14′ N, 91°18′ E Soil sediments, small pebbles Sandy, small pebbles, vegetation Small sediments soil Moisture, brownish, fine granules BPL BCK BM BSA Phewa Lake, Nepal Chitlang, Katmandu, Nepal Monapur, Barpeta, Assam Senga, Assam

17 °C 15 °C 14 °C 14 °C

40 38 34 23 19 14 28 47 29 34°07′ N, 74°52′ E 34°03′ N, 74°53′ E 33°33′ N, 76°59′ E 32°30′ N, 75°13′ E 34°02′ N, 74°21′ E 34°33′ N, 76°07′ E 32°05′ N, 76°16′ E 31°56′ N, 77°06′ E 28°10′ N, 84°14′ E Peats, brown soil sediment Yellow podzolic soils Sediments of rocky Meadow soils, granular in nature Clay loam, texture, fine granular River soil sediment River soil sediment Brownish, soft, sparse vegetation Sandy, silty alluvial soil BD BS BZ BB BG BC BKK BK BP Dal Lake, Srinagar Suru valley, Ladakh, Kashmir Zanskar, Jammu and Kashmir Banihal pass, Kashmir Gulmarg, Baramula Chamba, Himachal Pradesh Kangra, Himachal Pradesh Kullu valley, Himachal Pradesh Pokhara, Nepal

7 °C 6 °C 6 °C 7 °C 10 °C 10 °C 9 °C 10 °C 17 °C

No. of morphotypes Sampling location Sample description Sample ID

Temperature

universal primers [38]. DNA sequences were checked and edited carefully by SeqMan and EditSeq software (DNAStar, Madison, WI, USA). All the sequences were analyzed and chimeric sequences were removed using DECIPHER software [39]. Aligned sequences were subjected to BLAST analysis in Eztaxon server [40]. The phylogenetic tree was constructed using MEGA 5.0 software [41] by retrieving all type strains of nearest neighbors of the isolate. All the 16S rRNA gene sequences were aligned using the CLUSTAL_X program [42], and neighbor-joining, maximum parsimony, and maximum likelihood algorithms were used for the construction of phylogenetic tree. Bootstrap analysis was performed employing 1,000 replicate data sets in order to evaluate the confidence limits of the branching.

Cold-Active Extra Cellular Enzyme Activity

Sampling site

Table 1 Sampling locations, description of the isolate morphotype, and designation of strains from the Himalayan foot hills

Strain numbers


Cellulase, amylase, lipase, phytase, protease, and pectinase enzyme activities were tested qualitatively on diffusion agar plates containing the specific substrate by incubating at 4 and 20 °C for 4–7 days. Cellulase activity was done on carboxymethyl cellulose (CMC) agar, which contains 0.2 % NaNO3, 0.1 % K2HPO4, 0.05 % MgSO4, 0.05 % KCl, 0.2 % CMC sodium salt, 0.02 % peptone, and 1.5 % agar. Upon incubation, the plates were flooded with Gram’s iodine, and the zone of clearance around the bacterial colonies represented the positive cellulase producers [43]. The starch agar plates were prepared with 0.2 % yeast extract, 0.5 % peptone, 0.1 % MgSO4, 0.1 % NaCl, 0.02 % CaCl2, 0.1 % soluble starch, and 1.5 % agar (pH 7.0). Amylolytic isolates were selected by flooding the starch agar plates with Gram’s iodine solution (2.0 g KI and 1.0 g iodine in 300 mL distilled water). Isolates with distinct clear zone around the colonies were identified as amylase producers [44]. Lipase activity was detected on the enrichment medium containing 0.5 % peptone, 0.3 % yeast extract, 4 mL of olive oil, tributryin 1 mL, and 1.5 % agar (pH 7.2). The colonies with clear hydrolysis zones were marked as lipase producers [45]. Screening of phytase producers was done on a medium containing 1.5 % glucose, 0.3 % (NH4)2SO4, 0.05 % KCl, 0.05 % MgSO 4 ·7H 2 O, 0.01 % NaCl, 0.01 % CaCl 2 ·2H 2 O, 0.001 % FeSO 4 , 0.001 % MnSO 4 (pH 6.5) with 0.5 % sodium phytate (Sigma). The sodium phytate hydrolyzing capacity of the isolates was recognized by zone of clearance around the colonies [46]. For protease activity, 0.3 % casein and 0.5 % gelatin were used as substrate along with 0.2 % yeast extract and 1.5 % agar powder, (pH 7.0) [47]. For pectinase activity, 0.2 % pectin was used as substrate along with 0.3 %

Bacterial Diversity of Himalayan Mountains

Results and Discussion

environmental stress condition [51] thereby facilitating survival of those bacteria in cold conditions [52, 31]. A wide range of diverse cold-tolerant bacteria was isolated in the present study where the sampling locations experienced not only cold temperatures [53, 11, 18] but also drastic annual temperature fluctuations (4 to 30 °C).

Soil Samples Description

Diversity Analysis

Thirteen soil samples were collected from the Himalayan foot hills and belonged to the regions of Jammu and Kashmir, Himachal Pradesh, Assam (India), and Nepal (Table 1). The color of soil samples varied from black to brownish. Soil textures were found to be clay, sandy, or with small pebbles. Besides, river soil sediments from a few regions were light brown sticky and blended with pebble-like structures (Table 1).

Among the isolated strains, 76 % were Gram-positive, which were predominant as observed in tropical forest diversity. Diversity indices are used to compare the differences between the communities and to analyze the richness and evenness of a particular species in a defined community [54]. Shannon– Wiener diversity (H′), its evenness (EH), and Simpsons diversity index (D) along with its equitability (ED) were studied. The Shannon–Wiener diversity index reflects both phylotypes richness and evenness which is a good overall measure of diversity. The Shannon richness (H′ index) ranged from 1.50± 0.1 to 2.57±0.15 and its evenness (EH) ranged from 0.885± 0.04 to 0.974±0.01. The D index and ED ranged from 0.080± 0.03 to 0.211±0.05 and 0.746±0.03 to 0.914±0.01, respectively (Table 2). While the highest bacterial diversity (Shannon–Wiener diversity H=2.57±0.15) was found at Phewa Lake (BPL), the lowest (H=1.50±0.1) was Chamba (BC) region. The diversity indices found in the present study was in accordance with previous reports [1, 2, 6] from Himalayas.

KH2PO4, 0.6 % Na2HPO4, 0.2 % NH4Cl, 0.5 % NaCl, 0.01 % MgSO4·7H2O, and 1.5 % agar [48].

Isolation and Cultivation of Bacteria From the 13 soil samples, 406 morphologically divergent bacterial strains were isolated with different nutritional media of two strengths (Table 1). Bacterial count in the soil samples varied from 7.8±1.6×105 to 30.9±1.7×105 cfu ml−1 of soil. Most of the isolates in all the collected soil samples were found to be Gram-positive. Among the isolates, 253 were pigmented, in which pale yellow was predominant, followed by fluorescent green and pale orange. A majority of the isolates were highly pigmented, which is in accordance with earlier studies on microbiota of Antarctic glacial ice, soil [49, 50], and Puruogangri ice core [12]. Lowering of the cultivation temperature of bacteria resulted in a concomitant increase in carotenoid production, which is attributed to membrane stabilization, which plays a significant role in adapting to

ARDRA Profiling and Molecular Identification of Bacterial Isolates To select unique representatives for sequencing, all the 406 isolates were subjected to 16S rRNA gene amplification

Table 2 Total bacterial abundance and diversity index values from the samples Sample ID

Total bacterial count (×105) (cfu ml−1)

Species richness (S)

Shannon–Wiener diversity index

EH index

Simpson’s (D) index

ED index

BD BS BZ BB BG

11.7±0.97 25.2±0.80 18.7±3.2 7.8±1.6 24.4±2.4

7±0.8 10±0.5 12±0.8 8±0.3 11±1.2

1.78±0.18 2.26±0.09 2.44±0.23 1.84±0.08 2.32±0.13

0.886±0.04 0.953±0.08 0.974±0.04 0.938±0.09 0.895±0.05

0.148±0.01 0.108±0.05 0.086±0.07 0.148±0.08 0.082±0.06

0.814±0.07 0.787±0.04 0.860±0.03 0.892±0.02 0.908±0.01

BC BKK BK BP BPL BCK BM BSA

7.9±2.0 17.9±1.9 26.8±2.1 17.8±1.0 30.9±1.7 11.0±2.0 11.3±1.2 13.9±1.7

6±1.0 11±0.9 12±1.4 7±1.0 14±0.5 7±0.3 5±0.8 10±1.2

1.50±0.1 2.16±0.15 2.33±0.05 2.21±0.18 2.57±0.15 1.89±0.08 1.62±0.25 2.1±0.11

0.933±0.01 0.934±0.02 0.885±0.04 0.974±0.01 0.964±0.01 0.963±0.03 0.893±0.01 0.944±0.01

0.211±0.05 0.098±0.05 0.118±0.04 0.098±0.11 0.080±0.03 0.150±0.08 0.194±0.07 0.126±0.08

0.746±0.03 0.817±0.04 0.914±0.01 0.845±0.02 0.906±0.14 0.840±0.02 0.808±0.02 0.840±0.05

BKK

BC

BG

BB

BZ

BS

BD

Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod to cocci Cocci Rod Rod Rod Rod Cocci Rod Rod

Rod Rod Rod Rod Rod Rod Rod Rod to cocci Spherical Rod Rod Rod Rod Rod

99.5 97.8 98.8

Arthrobacter oryzae KV-651T (AB279889) Phycicola gilvus SSWW-21T (AM286414) Mycetocola manganoxydans MB1-14T (GU217690) + + +

99.7 100 98.9 99.7 99 99.9 99.3 99.9 99.8 98.1 99.1 99.9 99.7 100 99.9 99.9 99.6 99.5

Erwinia billingiae LMG 2613T Y13249 Arthrobacter koreensis CA15-8T (AY116496) Bacillus amyloliquefaciens 1034 DSM 7T (FN597644) Arthrobacter aurescens DSM 20116T (AJ512504) Bacillus safensis FO-036bT (AF234854) Erwinia rhapontici ATCC 29283T (U80206) Bacillus sonorensis NRRL B-23154T (AF302118) Erwinia billingiae LMG 2613T (Y13249) Brevibacterium frigoritolerans DSM 8801T (AM747813) Arthrobacter ureafaciens DSM 20126T (X80744) Planomicrobium glaciei 423T (EU036220) Exiguobacterium artemiae 9ANT (AM072763) Brevundomonas olei (GQ250440) Bacillus kochii WCC 4582T (FN995265) Arthrobacter ilicis DSM 20138T (X83407) Trichococcus pasteurii KoTa2T (X87150) Bacillus aryabhattai B8W22T (EF114313) Bacillus amyloliquefaciens 1034 DSM 7T (FN597644)

– + + + + − + – + + + + − + + + + +

100 99.8 99.7 99.8 100 99.7 99.8 99.9 99.8 100 100 99.8 99.3 100

Bacillus muralis LMG 20238T (AJ628748) Pseudomonas graminis DSM 11363T (Y11150) Bacillus asahii MA001T (AB109209) Exiguobacterium aurantiacum DSM 6208T (DQ019166 ) Pseudomonas vancouverensis ATCC 700688T (AJ011507) Exiguobact erium mexicanum 8NT (AM072764) Arthrobacter psychrophenolicus AG31T (AJ616763) Rhodococcus baikonurensis GTC 1041T (AB071951) Micrococcus antarcticus T2T (AJ005932) Arthrobacter oxydans DSM 20119T (X83408) Arthrobacter oxydans DSM 20119T (X83408) Brevibacterium frigoritolerans DSM 8801T (AM747813) Exiguobacterium sibiricum 255-15T (CP001022) Bacillus simplex NBRC 15720T (AB363738)

Actinobacteria Actinobacteria Actinobacteria

Firmicutes Actinobacteria Firmicutes Firmicutes Firmicutes

Proteobacteria Actinobacteria Firmicutes Actinobacteria Firmicutes Proteobacteria Firmicutes Proteobacteria Firmicutes Actinobacteria Firmicutes Firmicutes

Firmicutes Proteobacteria Firmicutes Firmicutes Proteobacteria Firmicutes Actinobacteria Actinobacteria Actinobacteria Actinobacteria Actinobacteria Firmicutes Firmicutes Firmicutes

16S rRNA gene sequence Division similarity in %

+ − + + − + + + + + + + + +

Cell morphology Gram stain Nearest Phylogenetic neighbor

Pale yellow Rod Yellow Rod to cocci Fluorescent green Rod

White Yellow White Pale yellow White White Yellowish-cream Pale white White Creamy yellow Yellow to orange Pale orange White Pale white Pale yellow White White White

BZb9 BZc7 BZc8 BZd1 BBa9 BBa3 BBe3 BBd1 BGa5 BGb1 BGb4 BGb5 BGc3 BGc4 BGc5 BCa1 BCc3 BCd4

BKKa5 BKKa6 BKKb2

Pale white Yellow White Orange Pale yellow Pale orange Glossy yellow White Yellow Pale red Pale red White Orange White

BDa14 BDb2 BDb11 BDc2 BDd4 BSa14 BSa12 BSb3 BSb10 BSc6 BSe1 BZa2 BZa11 BZb3

Sample ID Representative isolate namea Colony color

Table 3 Identification of the 67 bacterial strains isolated from the Himalayan range of India and Nepal, based on BLAST analysis of the 16S rRNA gene sequences


a

Yellowish orange beige Pale yellow Light beige Yellow White Yellow Pale yellow White White Creamy-yellow Cream yellow Creamy white Fluorescent green Pale yellow

BPLd1 BCKb5 BCKc2 BCKd1 BCKd2 BCKe2 BMa8 BMb3 BMb4 BMc1 BSAa5 BSAa10 BSAc1 BSAd1 BSAc4

Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod Spherical Rod Cocci Rod Rod to cocci

Rod Rod Rod Rod to Cocci Spherical Rod Rod Rod Rod Rod Rod Rod Rod Cocci to Rod Rod Rod Rod Rod + – + – + – + + – + + + + − +

+ + – + + + − + – + − + + + + + − + 99.8 98.9 99.7 99.7 99 99.9 99.9 97.7 99.9 99.9 99.9 99.6 99.7 99.3 99.7 99.8 99.1 100 100 99.3 99.9 99.5 99.7 99.9 99.8 99.4 99.9 99.6 99.3 99.9 99.4 99.9 99.9

Exiguobacterium soli DVS3YT (AY864633) Pantoea rwandensis LMG 26275T (JF295055) Arthrobacter globiformis NBRC 12137T (BAEG01000072) Pantoea rodassi LMG 26273T (JF295053) Arthrobacter antarcticus SPC26T (AM931709) Enterobacter cancerogenus LMG 2693T (Z96078) Arthrobacter ramosus CCM 1646T (AM039435) Arthrobacter nitroguajacolicus G2-1T (AJ512504) Cedecea neteri GTC1717T (AB086230) Bacillus pumilus ATCC 7061T (ABRX01000007) Arthrobacter sulfonivorans ALLT (AF235091) Arthrobacter equi IMMIB L-1606T (FN673551) Arthrobacter defluvii 4C1-aT (AM409361) Pseudomonas moraviensis CCM 7280T (AY970952) Arthrobacter chlorophenolicus A6T (CP001341)

Firmicutes Proteobacteria Actinobacteria Proteobacteria Actinobacteria Proteobacteria Actinobacteria Actinobacteria Proteobacteria Firmicutes Actinobacteria Actinobacteria Actinobacteria Proteobacteria Actinobacteria

Actinobacteria Actinobacteria Proteobacteria Actinobacteria Actinobacteria Actinobacteria Proteobacteria Firmicutes Proteobacteria Firmicutes Proteobacteria Firmicutes Actinobacteria Firmicutes Firmicutes Firmicutes Proteobacteria Firmicutes

16S rRNA gene sequence Division similarity in %

Arthrobacter ramosus CCM 1646T (AM039435) Agromyces cerinus subsp. nitratus ATCC 51763T (AY277619) Kluyvera cryocrescens ATCC 33435T (AF310218) Arthrobacter psychrolactophilus B7T (AF134179) Arthrobacter sulfonivorans ALLT (AF235091) Micrococcus lactis DW152T (FN673681) Enterobacter soli LF7aT (CP003026) Exiguobacterium indicum HHS31T (AJ846291) Cedecea davisae DSM 4568T (AF493976) Exiguobacterium indicum HHS31T (AJ846291) Cedecea neteri GTC1717T (AB086230) Bacillus acidicola 105-2T (AF547209) Arthrobacter polychromogenes DSM 20136T (X80741) Planomicrobium koreense JG07T (AF144750) Bacillus cereus ATCC 14579T (AE016877) Bacillus weihenstephanensis WSBC 10204T (Z84578) Pseudomonas baetica a390T (FM201274) Bacillus pseudomycoides DSM 12442T (ACMX01000133)

Cell morphology Gram stain Nearest Phylogenetic neighbor

In each isolate, type of media was used represented by a—nutrient agar, b—Antarctic bacterial medium, c—tryptic soy agar medium, d—starch yeast peptone agar, e—soil extract agar

BSA

BM

BCK

BPL

BP

BK

Yellow Yellow White Pale yellow Creamy-yellow Yellow White Orange Pale white Orange Pale white Pale white Pale yellow Yellow to orange White Cream white Florescent White

BKKd3 BKKe5 BKa8 BKb7 BKc1 BKd2 BKe4 BPa9 BPb8 BPc2 BPe1 BPLa1 BPLa7 BPLa14 BPLa18 BPLa23 BPLb3 BPLb4

Sample ID Representative isolate namea Colony color

Table 3 (continued)



Fig. 2 Distribution of different culturable phylotypes from the studied sampling regions

followed by restriction digestion with MspI enzyme. ARDRA profiling was carried out to eliminate sibling strains among the isolates in each sample. Based on previous studies [55, 56], analysis revealed a high level of genetic diversity among the isolates which narrowed the 406 isolates into 67 ARDRA haplotypes assuring at least 67 different species (Supplementary Figs. 1–13). Phewa Lake (BPL) sample yielded a maximum of eight haplotypes which correlated to the high diversity index (H′) values of these regions. Among the samples BZ and BG, five haplotypes were found, followed by BS which yielded six haplotypes. Samples BC and BM were found to be less diverse with four common haplotypes. These results correlate the relationship between diversity indices obtained from these sample regions on distribution of different genera. On the basis of ARDRA profiling, a representative isolate from each cluster of each samples were sequenced and the nucleotide sequences were deposited in the NCBI GenBank database (Accession numbers: KF387654 to KF387720). Culturable Bacterial Diversity The nearest phylogenetic neighbor of all the 67 representative isolates were identified through BLAST analysis of the 16S rRNA gene sequences in “Eztaxon-e” database and phylogenetic tree construction. The study revealed 17 different genera (Table 3) which belonged to four divisions (Fig. 2) namely Firmicutes, Actinobacteria, α-Proteobacteria, and γProteobacteria. Bacterial diversity was found to be maximum (38.8 %) in the division of Firmicutes, in which 15 clusters were formed among the genera Bacillus, Exiguobacterium, Brevibacterium, Trichococcus, and Planomicrobium (Fig. 3b). This was followed by 11 clusters of Actinobacteria (38.8 %) spreading among the genera Arthrobacter, Rhodococcus, Micrococcus, Phycicola, Mycetecola, and Agromyces in the phylum (Fig. 3a). The

remaining 22.5 % were shared by α-Proteobacteria and γProteobacteria which include the genera Brevundomonas, Pseudomonas, Erwinia, Kluyvera, Enterobacter, Cedeceae, and Pantoea (Fig. 3c). Our results were in concurrence with the previous study in Pindari glacier, where Firmicutes, Actinobacteria, and Proteobacteria were the most common phylum present in the Himalayan tracks [2]. Bacteria belonging to the phylum Firmicutes were predominantly found in the soil samples collected from the regions of Dal Lake (BD), Zanskar (BZ), Gulmarg (BG), Chamba (BC), and Phewa Lake (BPL). Majority of Actinobacteria were present in Suru Valley (BS), Kangra (BKK), Kullu Valley (BK), and Senga (BSA) regions. Proteobacteria dominated in the regions of Chitlang (BCK), Pokhara (BP), and Banihal (BB). However, Chamba (BC) region had low diversity of cultivable bacteria spreading over only two genera (Fig. 2). The bacterial diversity from these regions was comparable with the soil microbial diversity of Puruogangri ice regions [13, 10, 12] with respect to the presence of phyla Actinobacteria and Proteobacteria. These phylotypes were also distributed in Antarctic habitats which include aquatic microbial mats [57–60], soils from Antarctic continentals [61–65], and the sub-Antarctic islands sediment samples [66–72]. While comparing the bacterial diversity from Kafni, Roopkund, and Pindari glaciers of the Himalayas, the present study commonly shared Bacillus simplex, Phycicola gilvus, Arthrobacter psychrolactophilus, Arthrobacter sulfonivorans, and Micrococcus lactis [14, 1, 2]. Further genera Pseudomonas, Exiguobacterium, Plannococcus, and Pantoea have been found previously in various geographical regions of Himalayas [73, 74, 3]. Studies on Western Himalayas especially the Siachen glacier revealed the coverage of 39 % of γ-Proteobacteria followed by Actinobacteria (16 %) and Bacteroidetes (6 %) [6]. On the contrary, in our study, we have observed equal distribution of phyla Actinobacteria (38.8 %) and Firmicutes (38.8 %) from our sampling regions. Yet another biodiversity hotspot of India, namely the Western Ghats of Southern India, has revealed the presence of genera Micrococcus, Enterobacter, Arthrobacter, Kluyvera, Pseudomonas, Bacillus, and Pantoea [75, 76]. This significantly endorses the distribution a few common genera over the Indian subcontinent in the biodiversity hot spots. In the present study, through culturable approach, many predominant phyla were described from the sampling site. This predominance may be due to the richness of the particular genera in a habitat or because of the capability of growing well in the provided media and cultivation conditions. In addition, the diversity would be limited to those bacteria, which can grow in


given conditions. Cultivation also imposes some selection so that the relative abundances may not be entirely comparable to the actual community in the habitat. This could be one of the demerits of cultivable approach, whereas in culture independent analysis, it is possible to elucidate the complete picture of microbial community from any habitat.

clones of BZ library yielded 13 haplotypes according to the similarity of the banding patterns obtained through ARDRA. Similarly, 16S rRNA libraries of the other samples namely BG, BK, BPL, and BSA yielded nine, eight, eight, and ten haplotypes, respectively (Supplementary Fig. 13-18). One representative clone from each cluster of each library was sequenced using pUC18/M13 forward and reverse primers.

Culture-Independent Bacterial Diversity Phylogenetic Analysis of 16S rRNA Gene Libraries Bacterial diversity of the five selected soil samples namely BZ, BG, BK, BPL, and BSA with high values of diversity indices were chosen for culture-independent studies. A total of 268 recombinant clones from the five 16S rRNA gene libraries were subjected to ARDRA analysis. Among them, 58

Fig. 3 a Neighbor-joining tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of the soil bacterial isolates among Gram-positive bacteria. Aquifexpyrophilus Kol5aT was taken as an outgroup. b Phylogenetic analysis of Firmicutes c Neighbor-joining tree based on the 16S rRNA gene sequences showing the phylogenetic relationship of the soil bacterial isolates among Gramnegative bacteria. Methanococcus thermolithotrophicus DSM 2095Twas taken as an outgroup.Bootstrap values (expressed as percentage of 1,000 replications) greater than 50 % are given at nodes. Dark circle indicates the clades that were conserved when neighbor-joining, maximum-parsimony, and maximum-likelihood methods were used to construct phylogenetic trees. The bar represents 0.05 substitutions per alignment position in the tree

a

0.05

A total of 53 representative clones were sequenced and the nucleotide sequences were deposited in the NCBI GenBank database (Accession numbers: KF800830-KF800879 and KF914181-KF914184). The nearest phylogenetic neighbor


b

Fig. 3 (continued)


c

0.05

Fig. 3 (continued)

of all the 16S rRNA gene sequences were analyzed through BLAST analysis in “EzTaxon-e” database (http://eztaxon-e. ezbiocloud.net/ezt_identifyQ) and phylogenetic tree was constructed (Fig. 4a, b). About 81.5 % of the clone sequences among the five libraries could be grouped with the nearest phylogenetic neighbor. The clones of 16S rRNA gene library of BZ comprised the phyla Firmicutes, Actinobacteria, α-Proteobacteria, βProteobacteria, γ-Proteobacteria, and Bacteroidetes

(Fig. 5). Among them, Firmicutes (31 %) was found to be predominant. Besides, two clones in this library were found to be uncultured bacteria (Fig. 5). Sample BG library contained the phyla Firmicutes, Actinobacteria, α-Proteobacteria, β-Proteobacteria, and Acidobacteria. Phyla Actinobacteria and α-Proteobacteria were found to be predominant in the BK sample library, followed by Bacteroidetes. The BPL library was conquered by Proteobacteria viz., α, β, and γ, followed by Firmicutes


and Bacteroidetes. Phyla β-Proteobacteria was found to be predominant in sample BSA followed by Actinobacteria, αProteobacteria, and γ-Proteobacteria. In conclusion, among all the five libraries (Fig. 5), phyla Actinobacteria was predominant followed by αProteobacteria, β-Proteobacteria, Firmicutes, γProteobacteria, Bacteroidetes, and Acidobacteria. Clone libraries revealed the presence of 25 genera, namely Nocardioides, Wautersiella, Mucilaginibacter, Brevundimonas, Sphingobium, Streptomyces, Sphingomonas, Terrimonas, Phycicola, Massilia, Gynumella, Rothia, Schlegelella, Arthrobacter, Acinetobacter, Ferribacterium, Anoxybacillus, Pelosinus, Methylobacterium, Propionivibrio, Steroidobacter, Micrococcus, Pelomonas, Acidobacteria, and Planococcus. However, about 18.5 % of the clones (MBK6, MBK27, MBK37, MBSA14, MBZ12, MBZ51, MBPL39, MBG21, MBG29, and MBG35) exhibited 1, which explained 86.97 % of the total variability among the Fig. 4 a Neighbor-joining tree based on the metagenomic 16S rRNA gene sequences showing the phylogenetic relationship among five clone library (BZ, BG, BK, BPL, and BSA). Bootstrap values (expressed as percentage of 1,000 replications) greater than 50 % are given at nodes. Dark circle indicates the clades that were conserved when neighbor-joining, maximumparsimony, and maximum-likelihood methods were used to construct phylogenetic trees. Methanococcus thermolithotrophicus DSM 2095T was taken as an out-group. The bar represents 0.05 substitutions per alignment position in the tree b Neighbor-joining tree of Actinobacteria


a


c

0.01 Fig. 4 (continued)

five clone libraries (BZ, BG, BK, BPL, and BSA), where PC1 and PC2 contributed 50.64 and 36.33 % of total variance, respectively. The PCoA analysis revealed the important axes along which samples varied which showed the distinctness of bacterial communities among the libraries (Fig. 6). Throughout this study, the calculation of diversity indices (Table 4) and UniFrac analysis were performed with the unique sequences of their respective libraries to determine the bacterial distribution and phylogenetic relationship within the samples. Clone libraries BZ, BSA, and BG showed high abundance and evenness of bacterial species, which were correlated by the distribution of different phylotypes among the cloned sequences. However BK and BPL libraries contained less diversity and more evenness. The BZ sample showed more

evenness in diversity index analysis with its diverse results obtained from cultivable and culture-independent analysis. This illustrates that this sample can be explored for identification of novel organisms using specific media and growth conditions. Further analysis with more number of clones would help in revealing the community structure and the ecological role of bacteria. Often, the diversity data obtained by the cultureindependent approach was different from those obtained by culturable approach. However, occasionally genera like Arthrobacter and Bacillus were commonly found in Zanskar (BZ) region. Cultivation method involves the use of different nutritional conditions at various temperatures, which would support growth of diverse organisms, but still many organisms remain unrepresented. The isolated genera which belonged to


Fig. 5 Pie chart showing the distribution of phylotypes among the 16S rRNA gene libraries

the phylum Firmicutes and Actinobacteria were fast cultivars, whereas few members of Proteobacteria are uncultivable even with different cultivation media and conditions [73]. The significant discrepancy between bacterial community compositions using culturable and culture-independent methods was revealed through many previous studies in various environments [87-89, 77]. The differences might be due

to the use of synthetic media that were different from the in situ habitats [90]. Furthermore, the culture-independent studies revealed different phylotypes in the sampling regions than the culturable method. Therefore, a combination of both cultivable and culture-independent approaches is the perfect way to describe the bacterial diversity of any natural habitat.


Fig. 6 a–c PCoA analysis of bacterial communities obtained with weighed and normalized UniFrac using the 16S rRNA gene sequences among five libraries (BZ, BG, BK, BPL, and BSA)

Extracellular Hydrolytic Enzyme Activity In recent years, scientific communities have been focusing on the cold-active enzymes from microbial sources, which have Table 4 Statistical indices calculated for five clone libraries

potential applications in biotechnology, agriculture, and medicine [91, 31]. In the present study, the representative isolates were screened for their extracellular hydrolytic enzyme activity, viz., cellulase, amylase, lipase, protease, and pectinase.

Library name

No. of OTUs

Shannon index

Simpsons index

Evenness

Chao 1

BZ BG BK BPL BSA

13 11 9 9 11

3.94 3.70 2.69 3.02 3.23

0.99 0.97 0.88 0.92 0.94

0.92 0.89 0.62 0.71 0.78

689.5 219.75 26.62 34.66 50


Fig. 7 a Distribution of extracellular enzyme activity among the isolates. b Extracellular hydrolytic enzyme activity by the representative isolates in percentage

About 23.71 % of the representative isolates showed cellulase activity, followed by 20.24, 17.32, 13.87, 12.72, and 12.13 % for pectinase, amylase, phytase, protease, and lipase activity, respectively (Fig. 7a). Phylum Firmicutes and Actinobacteria showed predominant activity for the enzyme cellulase and pectinase (Fig. 7b). Four representative isolates, namely Exiguobacterium mexicanum (BSa14), Exiguobacterium sibiricum (BZa11), Micrococcus antarcticus (BSb10), and Bacillus simplex (BZb3) showed multiple enzyme activity for five different types of enzymes. Ten representative isolates (BDa14, BDb2, BSc6, BSe1, BGa5, BGb1, BPc2, BPe1, BPLa14, and BPLd1) exhibited hydrolytic activity for four enzymes. A large number (19) of isolates (BDc2, BZc7, BZc8, BZd1, BGc3, BGc5, BKKa5, BKa8, BKb7, BKc1, BKe4, BPa9, BPLa23, BPLb4, BCKc2, BMb3, BSAa5, BSAc1, and BCa1) showed enzyme activity for three different enzymes. Thirty-three isolates exhibited activity for ≤2 different enzymes. These results clearly indicated the potential of the isolated bacteria, which can be further scaled up for mass level enzyme production in industries. Previous studies [30, 92] on Himalayan tracks for the bioprospecting of cold-active enzymes of cellulase, amylase, and protease showed predominant activity by genus Pseudomonas, Bacillus, Arthrobacter, and Brevundomonas. Our results were almost concurrent with the same. In addition, Exiguobacterium, Erwinia, Mycetecola, Cedecea, Pantoea, and Triochococcus isolated in our study also showed some novel hydrolytic enzyme activity. In summary, we have studied bacterial diversity through cultivable approach in 13 soils from different geographical regions of the Himalayas, in which psychrotolerant and psychrophilic bacteria were found to be predominant. In addition, we first documented various taxons which include Erwinia, Mycetecola, Agromyces, Kluyvera, and Enterobacter from the foot hills of Himalayas through cultivable method. The 16S rRNA gene library was constructed for the five samples, which showed significant amount of diversity indices. To the best of our knowledge, the genus Ferribacterium, Rothia, and Wautersiella were identified from the Himalayas for the first time through our study. Further, all the isolates were screened

for cold-adapted enzymes, which resulted in novel hydrolytic enzyme activity in various genera like Exiguobacterium, Erwinia, Mycetecola, Cedecea, Pantoea, and Trichococcus. By and large, this study has provided a better understanding of the microbial ecology and phylogeny of bacteria and predominance of cold-adapted enzyme producing bacteria present in the Himalayan tracks. Acknowledgments The authors express their gratitude to the Department of Science and Technology, Government of India (GOI) for the grant (vide reference No. SR/FT/LS-032/2008) and support by University Grants Commission (GOI) through the Special Assistance Program (SAP) (vide reference NO. F. 3-9/2007-SAP-II). The authors also acknowledge Dr. Muthu Krishnan, Assistant Professor, Department of Linguistics, Bharathiar University, for proof reading the manuscript. Conflict of Interest The authors declare that they have no conflict of interest

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