Nesterenkonia Sp. an Alkali-Tolerant, Moderate Halophilic Actinobacterium Isolated from Sambhar Salt Lake

Nesterenkonia Sp. an alkali-tolerant, moderate halophilic Actinobacterium isolated from Sambhar Salt lake

*Archana Gaur and Arti Prasad

Dept. of Zoology, M.L.S.U, Udaipur

Abstract: Sambhar lake (27o 58 N, 75o 55 E), the largest inland halo-alkaline lake of India was explored for Halophilic microbial diversity. Salt-loving extremophiles, called halophiles, are highly specialized for living in such environments. The salinity and pH of the lake ranged from 0.53–162.21 ppt, 6.54 to 8.87 respectively during the course of work. The study led to the isolation of an Aerobic Gram-positive, non-motile, moderately halophilic, alkali-tolerant, actinobacterium. Phenotypic and biochemical analysis followed by phylogenetic approach based on 16s r-RNA gene sequence showed that the isolate fell within the radiation of species of the genus Nesterenkonia as the strain showed highest similarity with Nesterenkonia halophila strain YIM 70179 (99%) and Nesterenkonia halobia (99%). ARDRA using restriction enzymes MSP 1 and Sau 3A1, was done to ascertain its genetic diversity among other isolates. The study pioneered in isolation and molecular characterization of Nesterenkonia sp. from brine of Sambhar salt lake. The gene bank accession no. for the isolate is KJ932668. Key words: Nesterenkonia, Halophiles, Actinobacterium, Extremophile, Restriction enzymes.

Introduction Halophiles have a worldwide distribution and have been isolated from a wide variety of habitats, including areas of both low and high salt concentrations (Ramos-Cormenzana 1993). Typical sites of halophile isolation have included salted hides or foods, unpurified salt crystals, saline soils, saltern ponds, saline lakes and oceans (Ventosa et al. 1998). Moderate Halophilic bacteria are microbe able to grow optimally in media containing 3 to 15 % salts (Kushner and Kamekura, 1988). This group is very heterogeneous with respect to their taxonomic distribution. Within the moderate Halophilic heterotrophic bacteria gram-negative group dominates hypersaline

*Author for Correspondence. E-mail: [email protected]

www. ijamb2002.com 12 ARCHANA GAUR AND ARTI PRASAD environments and the gram-positive strains have been studied in less detail (Mota et al., 1997). Nesterenkonia is the genus of moderately halophilic, gram positive non-motile, coccus, isolated from solar salterns. The genus Nesterenkonia was first proposed by Stackebrandt et al. (1995) with the reclassification of Micrococcus halobius (Onish & Kamekura, 1972) as Nesterenkonia halobia. The description of the genus was later emended by Collins et al. (2002) and Li et al. (2005). The remarkable adaptability to saline environment makes the Nesterenkonia an important group in the microbial community of saline habitat (Zhi et al., 2008). Saline lakes are most common in the arid and semiarid regions of the Rajasthan, because two preconditions for the formation of salt lakes occur there most frequently: evaporation exceeding precipitation and the presence of endorheic drainage basins. The Sambhar, a recognized saline lake and Ramsar site, boasts peculiar physical and chemical characteristic. It is considered athalassohaline as its salt composition derives from the dissolution of minerals of geological origin (Rodriguez-Valera, 1993). Similarly to other hypersaline ecosystems, the lake is subjected to drastic physico-chemical conditions including high salinity, high radiation (UV) and strong changes in temperatures and dryness which make it a relevant study target for microbiologists. The lake has always been exploited for salt production and microbial community remained understudied. The present study was an effort to unveil the Halophilic microbial diversity of the lake. The study resulted in the isolation of moderate Halophilic strain of Genus Nesterenkonia.

Material and Methods

Study Site:-Sambhar Salt Lake Sambhar lake (27o 58 N, 75o 55 E) is the largest inland halo-alkaline lake of India. The lake is located along National Highway 8 in Rajasthan, 96 km south west of the city of Jaipur (Northwest India) and 64 km north east of Ajmer . It encircles historical Sambhar Lake Town. The lake receives water from an endorheic basin with 5700 square km catchment area. The lake is actually an extensive saline wetland, with water depths fluctuating from as few as 60 cm (24 in) during the dry season to about 3 meters (10 ft) after the monsoon season. It occupies an area of 190 to 230 square kilometers, based on the season. It is an elliptically shaped lake 35.5 km long with a breadth varying between 3 km and 11 km. It is located in Nagaur and Jaipur districts and it also borders the Ajmer district. The circumference of the lake is 96 km, surrounded on all sides by the Aravali hills. The Sambhar lake basin is divided by a 5.1 km long dam made of sand stone. The eastern area of the dam is 80 km² and comprises salt reservoirs, canals and salt pans. After salt water reaches a certain concentration, it is released from the west side to the eastern side by lifting dam gates. Salt evaporation ponds produce 196,000 tons of salt every year, which equals 8.7% of India's salt production (Jain, 2005).

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 19 No. 2 July- Dec. 2016 Nesterenkonia Sp. An alkali-tolerant, moderate halophilic Actinobacterium…….. 13

Fig:1- Map of Sambhar salt lake (Adopted from Yadav and Sarin ,2009)

Sample collection and Microbial study Water samples were collected in sterile containers on monthly basis from Jhapok Sampling station. Sample were subjected to enrichment and further isolation procedures to recover aerobic moderately halophilic microorganisms in medium containing (per liter): NaCl, 200/250g;

MgCl2.6H2O 13g; MgSO4.7H2O, 20g; KCl, 4g; CaCl2.2H2O,1g; NaBr, 0.5g; , NaHCO3, 0.2g; yeast extract, 5g; tryptone, 8g; and glucose, 1g. pH was adjusted to 7.2 to 8 with sterile NaOH following Hedi et al. (2009). These plates were incubated for 5-7 days at 37OC and colonies appearing on the plate post incubation were re-streaked several times to get pure culture.

Morphological and Biochemical Identification Morphological and Biochemical characteristics of the selected isolate were observed with special emphasis on colonial elevation, pigment, opacity, consistency and motility. Gram staining was carried out by using acetic acid fixed samples, as described by Dussault (1955) and 3% KOH test was also performed to ascertain Gram properties. All the biochemical tests were done at 10% salt concentration in media of following composition (per liter) 4g of KCl, 13g of MgCl2.6H2O, 1g of CaCl2. 2H2O, 20g of MgSO4.7H2O, 0.5g of NaBr, 0.2g of NaHCO3) was used in all the biochemical test media. Oxidase reaction was performed according to Kovacs (1956). Catalase was determined by adding H2O2 to each strain culture (after 12 to 18 hour incubation) on solid medium. Sugar fermentation, Citrate utilization, urease, Indol production, and Voges-Proskauer

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 19 No. 2 July- Dec. 2016 14 ARCHANA GAUR AND ARTI PRASAD tests were performed using standard procedures. Exo-enzyme production for starch, Casein and lipid hydrolysis were tested by plate assay technique.

Molecular Characterization

DNA Extraction and 16S rRNA gene amplification The genomic DNA was extracted from the isolated strain using standard phenol: chloroform method (Sambrook el al., 1989). The isolated DNA templates were subjected to 16s r-DNA amplification, using the primers 18F and 1492R and optimized PCR reaction and thermal cycling conditions as described by Weisburg et al. (1991).

DNA sequencing and Phylogenetic analysis DNA sequencing of the isolated and amplified sample was carried out at Xceleris Ahmedabad. The phylogenetic analysis was performed by employing MEGA (version 5.1) software packages (Kimura, 1980) after multiple alignments of nearly complete 16S rRNA gene sequence (1383 nt.) of isolate SL-19 with those of other recognized Nesterenkonia species database sequences by CLUSTAL _X (Thompson et al., 1997) . Clustering was performed by using the neighbor-joining method (Saitou and Nei, 1987). Bootstrap analysis was used to evaluate the tree topology of the neighbor-joining data by performing 1000 resamplings (Felsenstein, 1985).

Nucleotide sequence accession numbers The amplified and Aligned 16s gene sequence was deposited in GenBank with accession numbers KJ932668.

Result and Discussion Sambhar Salt lake is a unique ecosystem and heaven for halophiles. Studies on the lake resulted in retrieval of microbes belonging to all the three domains, Archaea, Bacteria, and Eucarya (Upasani and Desai, 1990). During the study the most remarkable and obvious feature of sambhar lakes was its red/orange/pink-purple coloration, that appeared at various stages of salt concentration. This color is imparted by the extreme halophilic archaea bacterial pigments called carotenoids and bacterioruberins that protect their delicate cells from the intense sunlight (Shun Ichi, 1996). Halophiles are also widely spread within the domain Bacteria. The present work was focused on Halophilic bacteria of the lake and led to the isolation of a moderate Halophilic, alkaliphilic, aerobic bacterial strain. This new isolate was designated as SL-19 on the name of source lake. The bacterial cells of isolate Sl-19 were Gram positive and formed white, opaque, circular, convex, glossy colonies with entire margin and low elevation. The strain showed luxuriant growth between 7-15% salt concentrations, though it tolerated 1-30% NaCl in the culture media. The colony color at low salinity was light purplish pink that turns to creamish white as the salinity was raised above 10%. Along with high salt concentration it can withstand high pH as the growth was

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 19 No. 2 July- Dec. 2016 Nesterenkonia Sp. An alkali-tolerant, moderate halophilic Actinobacterium…….. 15 observed in 7-9 pH range. The strain was tested positive for Catalase and while negative for Lipid, starch and casein hydrolysis and H2S and Urease production. It failed to utilize Citrate, and ferment glucose, arabinose, mannose, mannitol, maltose. Optimum growth was recorded at temperature range of 28-32oC. Rodrigues-Valera et al., (1980a) mentioned that temperature is a major factor determining the competition between red Archaea and colorless bacteria in the intermediate salinity range, The Archaea being favored at high temperatures. Halophilic bacteria and archaea overlap in the salinity range of 250-320 g/l (Rodrigues-Valera et al., 1981).

Plate-2: Colony morphology Plate-3: +ve Catalase Plate-4:-ve H2S production of Isolate SL-19 Test Test on TSI-Agar In order to get better understanding of isolate SL-19 molecular approach was employed. PCR amplification followed by cloning of the 16S rDNA genes derived from the DNA extracted from microbes/environmental samples and subsequent phylogenetic analysis of the cloned sequences have enhanced the ability to assess naturally occurring biodiversity in many environments (Amann et al. 1995). The results of sequencing were subjected to a BLAST search on NCBI site to obtain some of the best matching sequences. The isolate SL-19 showed 99% similarity with Nesterenkonia halophila strain YIM 70179 and Nesterenkonia halobia, establishing that it is a member of Genus Nesterenkonia. All Nesterenkonia species are Gram positive, Non- encapsulated, non-endospore forming, Chemo-organotrophic, aerobic, Mesophilic, moderately halophilic or halotolerant. Few species are reported to be alkaliphilic or alkalitolerant. They contain peptidoglycan of the A4α type, mycolic acids are absent and have DNA G+C contents in the range 64−72 mol%. The phospholipids are diphosphatidylglycerol, Phosphatidylglycerol and phosphatidylinositol. The cellular fatty acids are iso and anteiso-branched fatty acids with anteiso- C15:0 and C17:0 predominating (Stackebrandt et al., 1995; Li et al., 2005). The group is also known for its high adaptability to saline environment (Zhi et al., 2008). The genus Nesterenkonia was first proposed by Stackebrandt et al. (1995). This genus belongs to the family Micrococcaceae within the phylum Actinobacteria, and is most closely related to the genera Micrococcus, Arthrobacter and Kocuria (Stackebrandt et al., (1995). Currently, the genus comprises 13 recognized species (Govender et al. ,2013) : Nesterenkonia halobia, Nesterenkonia

INDIAN JOURNAL OF APPLIED MICROBIOLOGY Vol. 19 No. 2 July- Dec. 2016 16 ARCHANA GAUR AND ARTI PRASAD lacusekhoensis , Nesterenkonia halotolerans , Nesterenkonia xinjiangensis, Nesterenkonia lutea, Nesterenkonia sandarakina , Nesterenkonia aethiopica, Nesterenkonia jeotgali, Nesterenkonia halophila, Nesterenkonia flava, Nesterenkonia alba sp. nov., Nesterenkonia sp. SA-3 and Nesterenkonia massiliensis. Most of these species were isolated from hypersaline or alkaline habitats such as saline soil, solar salt, seafood, soda lake or alkaline wastewater, however Nesterenkonia massiliensis sp. nov., strain NP1T was isolated from human feces (Edouard et al., 2014). Bacterial halophiles isolated from Sambhar lake vary widely in physiological properties, including aerobic and anaerobic chemo-heterotrophs, photo-autotrophs, photo-heterotrophs, and chemo-lithotrophs. Halophilic anoxygenic sulfur oxidizing bacteria have also been isolated from Sambhar lake brine by Upasani (2008). According to Valera et. al, 1981, in saline lakes very small salt loving microorganisms form the base of lake’s ecological food pyramid. These ecosystems would not exist if there were not halophilic bacteria that have established an ideal habitat in the saline lakes and their extremely salty environment.

Plate: 5 Gel Image showing ARDRA using restriction enzymes MSP-1 and Sau 3A1

Conclusion The Halophilic microorganisms inhibiting Sambhar salt lakes are considered to be extremophiles, which are well equipped to balance the osmotic pressure of the environment and resist the denaturing effects of salts. The lake is a treasure of unique and unexplored microbes. The present work is probably first report of isolation and phylogenetic characterization of Genus Nesterenkonia from Sambhar salt lake. The exploration of more halophiles with new and useful applications will in turn deepen our understanding of the functioning of hypersaline ecosystems also.

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Nesterenkonia lacusekhoensis EL-30 (NR 028928.1) Nesterenkonia flava (EF680886.1) Nesterenkonia alba (EU566871.1) Nesterenkonia aethiopica DSM 17733 (NR 042989.1) Nesterenkonia xinjiangensis strain (YIM70097) SL-19 (KJ932668) Nesterenkonia halobia DSM 20541 (NR 026197.1) Nesterenkonia halophila YIM 70179 (NR 043205.1) Nesterenkonia halophila(AY820953.1) Nesterenkonia luteus YIM 70081 (AY588278.1) Nesterenkonia sp. SA-3 (JF937438) Nesterenkonia halotolerans YIM70084 (NR 029073.1) Nesterenkonia sandarakina YIM 70009 (NR 029119.1) Nesterenkonia jeotgali(AY928901.1) Micrococcus luteus(AJ536198.1) Bacillus halophilus DSM 4771T (AJ243920)

0.02 Plate:5 - Phylogenetic tree showing the relationships between strain Sl-19 and related bacteria based on 16S rRNA gene sequences alignment analysis. Bootstrap values from 1 000 replicates are included, the scale bar corresponds to 0.005-estimated nucleotide substitution per sequence.

Acknowledgement The Authors are thankful to Council of Scientific and Industrial Research (CSIR), New Delhi for financially supporting the study.

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