2244 Chiang Mai J. Sci. 2018; 45(6)

Chiang Mai J. Sci. 2018; 45(6) : 2244-2255 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper

Characterization of a Halotolerant Archaeon larsenii HA8 Isolated from Solar Saltern for Halocin Production Monika Chaudhary [a], Bhakti Bajpai [b], Kapil Suthar [c] and Jyoti Saxena* [d] [a] Department of Bioscience and Biotechnology, Banasthali University, Distt. Tonk 304022, Rajasthan; Present address: 13836 Jefferson Park Dr, Apt 9102, Herndon, VA-20171, USA. [b] Department of Biotechnology, ARIBAS, New Vallabh Vidhya Nagar-388121, Gujarat. [c] Zydus Cadila, Ahemdabad, Gujarat, India. [d] Biochemical Engineering Department, B.T. Kumaon Institute of Technology, Dwarahat 263653, Uttarakhand, India. * Author for correspondence; e-mail: [email protected]

Received: 7 January 2017 Accepted: 12 October 2017

ABSTRACT Halocins are a new class of potentially useful antibiotics produced by halophilic bacteria and . Only a limited number of halocins have been described so far. This paper is an attempt to characterize halocin produced by haloarchaeal strain HA8 isolated from a solar saltern in Pachpadra, India. The strain was identified as Haloferax larsenii by sequencing of partial fragments of 16S rDNA amplicon. On the basis of antagonistic activity, the strain HA8 was selected as producer strain against an indicator strain HA3. The correlation between growth curve and halocin production profile indicated that maximum halocin production occurred during transition from exponential to stationary phase. The optimum culture conditions for halocin production were found to be: pH 8; temperature 30-40°C; salt concentration 2.5 M. Halocin production was stimulated by UV irradiation, maximum percent induction was 35% after 90 sec of UV exposure. Halocin HA8 was resistant to trypsin digestion but sensitive to proteinase K and was moderately stable in isopropanol. Moreover, it was inactivated at low salt concentrations but was stable at high salt concentrations and no change in activity was observed after its storage at 4°C. It can be concluded that antimicrobial peptide halocin HA8 showed many useful characteristics which can be exploited in industries.

Keywords: archaea, halocin, Haloferax larsenii., hypersaline, pachpadra saltern

1. INTRODUCTION The term bacteriocin is applied to a wide usually closely related to the producer range of biologically active proteins of organism. These antimicrobial proteins have bacterial origin that exhibit antimicrobial been described not only in bacteria but also properties against other bacterial species, among (extreme halophiles) or Chiang Mai J. Sci. 2018; 45(6) 2245

crenarchaeota (hyperthermophiles) [1-2]. from Halobacterium salinarum [21], C8 from are the dominant heterotrophic Halobacterium sp. [13] and KPS1 from Haloferax population in hypersaline environments with volcanii [22] were purified and characterized 3-5 M salt concentrations such as salt lakes, but the pace is much slower than required. salt ponds and marine salterns [3-6]. Recently, Kumar and Tiwari reported a new Discovery and identification of natural cytocidal halocin HA1 from Halofeax larsenii antimicrobial products from new sources HA1 [23] and in another study they such as extreme environments therefore characterized halocin H3 produced by play an important role in the uncovering of Haloferax larsenii HA3 [24]. Kanekar et al. novel drug leads and drug development isolated Haloferax larsenii from rocky substrate process [7]. for bacteriorhodopsin production [25]. The production of antimicrobial Looking at the need to identify and proteins, named halocins, is a widely characterize the new halocins, the present distributed feature among haloarchaea [8]. work was undertaken on six haloarchaeal Halocins are particularly interesting as they strains isolated from Pachpadra saltern, have been used to probe cell membrane an unexploited site in Jodhpur, India. structure and the production and maintenance A potential halocin producer was identified of energetic ion gradients across the on the basis of 16S rRNA gene partial membrane, hence can also be used in sequencing. treatment of reperfusion injury [9]. Besides, they have a role in human medicine as 2. MATERIALS AND METHODS controlling and chemotherapeutic agents 2.1 Organisms against human and animal pathogens [10]. Six halophilic archaeal strains viz. HA1, Also, halocins are under investigation as HA3, HA4, HA8, HA9 and HA10, isolated antimicrobials for use in controlling spoilage from water and sludge samples of Pachpadra during industrial processes, preserving agents salterns, located between latitude 25.9290° in food preparations and in some cases, North and longitude 72.2466° East, were leather production [11]. screened for halocin production. The Despite their ubiquity, only limited isolation of haloarchaea was done on 23 % halocins so far have been described in Saline Water (SW) Modified Growth Medium detail [12-14] and many more are waiting (MGM) described by Smith [26]. Plates to be discovered. Based on antagonistic were incubated at 37±2 °C in plastic bags to studies, different types of halocins have avoid drying up of the medium for 2 weeks. been identified; of these three have been The isolates were purified and stored. characterized fully and found to demonstrate HA8 strain was selected for further studies great heterogeneity in size, salt dependence, and identified on the basis of 16S rRNA gene and spectrum of activity [15-17]. A few sequence. The culture has been deposited in halocins such as halocin, H4, H6 and R1 department of Bioscience and Biotechnology, have been characterized at the protein level Banasthali University, India. [18-19] and halocin H4 has been characterized at both the gene and mRNA transcript 2.2 Halocin Assay level [15]. Later, a few more halocins such To test the activity level and spectrum of as S8 from a haloarchaeal strain S8a [17], halocin, the indicator (target) strain was H1 from Haloferax mediterranei M2a [20], R1 grown in modified Sehgal and Gibbons 2246 Chiang Mai J. Sci. 2018; 45(6)

complex medium [27] until stationary phase, 2.5 Characterization of Halocin then 0.4 ml (4×108 cells ml-1) of the culture Halocin HA 8 was subjected to the was added to 20 ml of the same medium following tests: (i) heat treatment at 60, 70, 80 containing 1.2 % (w/v) agar, which had been and 100 °C for 10, 20, 30, 40, 50 and 60 min melted and kept at 50 °C to prepare the plates. (ii) treatment with equal volume of organic In these plates, the wells of one cm in solvents such as methanol, ethanol and diameter were aseptically punched in the isopropanol at room temperature for lawn of the indicator strain. Next, 0.05 ml 60 min (iii) desalting using dialysis bag culture supernatant from halocin producer (HiMedia, India, 5000 MWCO) and (iv) strain was loaded in the wells, whereas, the incubation of desalted halocin (obtained by uninoculated medium in a separate well dialysis against distilled water) with various served as a control. The plates were then concentrations of proteinase K (100 μg incubated at 37±2 °C in sealed plastic bags. mL-1, 400 μg mL-1, 1 mg mL-1 and 2 mg The clear zone of inhibition around the mL-1) and trypsin (200 μg mL-1, 400 μg mL-1, well indicated the halocin activity. The 600 μg mL-1, 1mg mL-1 and 2 mg mL-1) for following formula was used to calculate 60 min at 37 °C. the halocin activity in terms of assay units For desalting, 1 mL supernatant (fresh AU ml-1. AU ml -1= (inhibition halo diameter halocin) was taken in a dialysis bag and - well diameter)/2 dialyzed against distilled water only (without To study antagonistic activity, the isolate NaCl), 1 and 3 M NaCl solutions. After 6 h (HA8) was treated as producer as well as of dialysis, it was checked for inhibitory target strain. activity. In case of dialysis against distilled water and 1 M salt solution, the NaCl 2.3 Correlation of Halocin Production concentration of dialyzed solution was with Growth Phase reconstituted to 2 M and again the inhibitory Two ml of active culture was inoculated activity was determined, -1 in halophilic broth (g L ) MgCl2.7H2O 20.0, In all the cases, halocin activity was

K2HPO4 5.0, CaCl2.2H2O 0.1, yeast extract checked before and after the treatment. 5.0, NaCl 2.5M, pH=7.5 and incubated at The salt concentration of the assay buffer was 37°C±2°C on an orbital shaker at 200 rpm. 1M. Samples were drawn at 6 h interval for determination of halocin activity and growth. 2.6 Induction of Halocin Production by Absorbance of the sample was read at UV Treatment 600 nm and then centrifuged at 7500 rpm The isolate was grown in 100 ml for 10 min. The supernatant was used to halophilic broth (2.5 M NaCl) up to check the halocin activity on target plate. exponential phase, and then centrifuged. The pellet was collected, resuspended in 2.4 Optimization of Culture Conditions 20 ml phosphate buffered saline and exposed for Growth and Halocin Production to UV rays (254 nm, distance 30 cm from Culture conditions viz. pH (4-9), UV lamp) for 0, 30, 60 and 90 sec. Four ml temperature (30-50 °C) and NaCl concentration sample after UV exposure from each (0-5 M) were optimized for growth and suspension was inoculated in fresh medium halocin production. (40 ml) for growth and halocin production. Chiang Mai J. Sci. 2018; 45(6) 2247

2.7 Storage Stability product was sent for sequencing to Bioroutes The culture supernatant containing halocin Life Sciences, Karnal, India. was stored at 4 °C and activity was measured The partial sequences of 16S rRNA gene up to 5 days. obtained for the isolated strains were aligned to those available in EMBL/GenBank 2.8 DNA Isolation, PCR Amplification database to search for the homology with and 16S rDNA Homology the nearest archaeal sequences. The multiple Genomic DNA from potential halocin alignment of the sequences was performed producer viz. HA8 was obtained using with the ClustalW software and phylogenetic bacterial HiPurA and yeast genomic DNA trees were constructed by the Neighbor Miniprep purification kits (Himedia joining method in the Molecular Evolutionary Laboratories Pvt. Ltd., Cat. No: MB505) Genetics Analysis (MEGA 6). following the manufacturer’s instructions. All the DNA extractions were performed 3. RESULTS AND DISCUSSION on fresh grown cultures and DNA 3.1 Antagonistic Activity of Haloarchael thus obtained was stored in deep fridge Strains for further processing. The DNA stock To select potent halocin producers and samples were quantified using Nano-drop indicator strains, a preliminary antagonistic spectrophotometer (Bioroutes Life Sciences) study was done with a total of six halophilic at 260 and 280 nm using the convention that archaeal strains. The data presented in one absorbance unit at 260 nm wavelength Table 1 show that all the isolates except for equals 50 μg DNA per ml. Purity of DNA HA3 exhibited various degree of antagonism. was judged on the basis of optical density HA10 inhibited the growth of all the other ratio at 260/280 nm. Amplification of isolates but reverse antagonism was not 16S rDNA was done in a Thermal Cycler observed. HA8, HA1 and HA9 also showed (Bio-Rad, make) taking haloarchaeal good antimicrobial spectrum, the number of specific primers which were designed isolates inhibited by each of the strains using DNA Star software. The primers used were 4, 3 and 2, respectively, whereas HA4 for the amplification of 16S rDNA were: showed antagonism against only HA9. forward primer 5’-CTGGTAGTCCACG On the basis of differential antimicrobial CCGTAAA-3’ and reverse primer 5’-GCT spectrum it can be concluded that halocins CGTTACGGGACTTAACC-3’. produced by all the six isolates were different. The steps in PCR included: initial Meseguer et al. and Torreblanca et al. denaturation at 95 °C for 3 min, 30 cycles conducted two non-overlapping antagonistic of 95 °C for 30 s, 59.6 °C for 45 s, 72 °C studies on 79 and 68 isolates and found for 30 s and a final extension step of inhibitory activity in 98% of strains, whereas 10 min at 72 °C. The amplified product in our study 83% strains showed this activity was identified by its electrophoretic mobility [31, 8]. It was inferred from the antimicrobial with reference to marker and documented spectrum of selected strains that different by gel documentation system (SynGene, Gene halocins were produced by these strains. Genius Bio Imaging System, U.K.). The PCR 2248 Chiang Mai J. Sci. 2018; 45(6)

Table 1. Antagonistic activity of all the strains against each other. Target Halocin activity (AU mL-1) HA1 HA3 HA4 HA8 HA9 HA10 Producer HA1 - 230±18 250±17 NI 230±22 NI HA3 NI - NI NI NI NI HA4 NI NI - NI 90±7 NI HA8 110±9 200±19 200±15 - 230±20 NI HA9 NI- 100±8 130±10 NI - NI HA10 150±10 230±20 200±18 130±9 250±21 - NI- no inhibition, - target and producer are same Results are mean of three experiments±SD

For further characterization of halocin, (Micrococcus sp.) and HA 8 showed marked Haloferax strain viz. HA8 was selected as difference. Micrococcus sp. showed the presence producer strain against HA3 as an indicator of FAME (Rf values 0.7), whereas, spot of

(target) strain on the basis of its better GDEM (Rf 0.18 and 0.32) typical of archaea antagonistic activity. Though halocin from was observed in HA8. Kumar et al also strain HA10 showed very good antimicrobial reported the presence of GDEM (glycerol property but the organism was extremely diether Moieties) in Haloferax larsenii HA1 slow growing, hence it was not selected for but were absent in halophilic bacteria [36]. further studies. 3.3 Biochemical Characterization 3.2 Organism As for biochemical tests (Table 2), HA 8 The organism was Gram-negative showed positive oxidase and catalase coccoi, red pigmented at higher salt reactions; hydrolyzed starch and gelatin but concentration; sensitive to bacitracin and did not show the hydrolysis of casein and novobiocin but resistant to penicillin, DNase; Indole negative; H2S was not ampicillin, chloramphenicol, ciprofloxacin, produced from thiosulfate; reduction of erythromycin, neomycin and streptomycin, nitrate and nitrite were observed. Though no growth on taurocholic acid was observed. acid production is common feature among These properties, together with the colony halobacteria, acid was produced only from pigmentation and requirement for high salt dextrose and maltose and sugars used as the concentrations, suggest that organism sole source of carbon were mannitol, sucrose, belonged to archaeal family, glycerol, fructose, mannose, glucose, sorbitol, [28-29]. Enache et al. also used the same raffinose and starch. Ribose, cellobiose, strategy to distinguish between halophilic fructose, xylose and lactose were not utilized bacteria and archaea [30]. by the organism. Similarly, Hfx. larsenii strain Haloarcharea are characterized by the ZJ206T showed acid production from presence of ether linked lipids in the cell maltose only and little acid production wall as against ester linkages present in from glucose, fructose and sucrose. Xylose, halobacteria. In this study, a comparison of arabinose, lactose, mannitol and galactose lipid profile of cell walls of halophilic bacteria were not utilized during growth [3]. Chiang Mai J. Sci. 2018; 45(6) 2249

Table 2. Biochemical characterization of The correlation between growth curve Haloferax H8. and halocin production profiles indicated that the maximum halocin production was Test HA8 during transition from exponential to Oxidase + stationary phase. Thus, the isolate seemed to Catalase +++ be growth dependent and probably needed DNase - large amount of nutrients for halocin Starch hydrolysis 1.5 production. There are mixed reports about Halo zone (cm) the growth phase during which maximum Gelatin hydrolysis 1.2 halocin production occurs. Pasic and his team Halo zone (cm) mates found the maximum activity in the Casein hydrolysis - exponential phase and it was found to be Indole - growth dependent [14]. Whereas, Platas et al. H S production - 2 and O’Connor and Shand observed that Nitrate reduction + most of the halocin produced in the culture N gas + 2 supernatant was either at the beginning or at Tween 80 + the transition into stationary phase except Halocin + for H1 whose activity was first detectable during mid-exponential phase [20-21]. 3.4 Correlation of Halocin Production The same pattern was demonstrated by withGrowth Price and Shand in halocin S8 [17]. In contrast, The growth and halocin activity Shand and Leyva found first detectable of producer was followed up to 142 h halocin activity when cultures of the (Figure 1). HA8 showed exponential growth producing cells entered the stationary phase from 6 to72 h approximately, after that the of growth [32]. These findings corroborate stationary phase ensued. The induction of with our results observed for HA8 strain. halocin production was approximately at 6 h, which exponentially increased up to 3.5 Optimization of Culture Conditions 72 h, and then it remained constant in the for Halocin Production stationary phase. pH The growth of HA8 was observed in the pH range from 5.0 to 9.0. As evident from Figure 2, the producer strain showed maximum biomass accumulation at pH 7.5 i.e. 1.1 g 100 mL-1. In general the acidic pH supported very little biomass production. Our isolate showed two different pH optima for growth and halocin production, halocin production was more at pH 8.0 as compared to pH 7.5. In the present study neutral to slightly alkaline pH was found to be better for growth, suggesting that HA 8 belonged Figure 1. Correlation of halocin activity and to neutrophilic group however, more halocin growth of HA8 strain. was produced in alkaline pH. Pasic et al. 2250 Chiang Mai J. Sci. 2018; 45(6)

reported the halocin activity from Haloarchaeon Sech7a to be optimum at pH 7.5 [14]. Our findings almost corroborated with the above study; for HA8 the optimum pH was slightly alkaline i.e. 8.0. The pH of the samples collected from study site was around 8.0, which could be the reason for better halocin production at alkaline pH. Natrinema sp. BTSH10 also showed maximum halocin production at pH 8.0 and showed sharp Figure 3. Effect of temperature on growth, decline in production on either side, i.e. at pH biomass and halocin activity of HA8 strain. values 7.0 and 9.0 [37].

3.7 Salt Concentration Salt concentration plays crucial role during growth and halocin production by halophiles. The isolate did not grow in the absence of NaCl and the maximum NaCl concentration tolerated was 5 M, indicating that it is an obligate extreme halophile [38]. The maximum growth as well as halocin production, however was observed at 2.5 M. The biomass produced was 0.9 g 100 mL-1 and halocin activity was 250 AU mL-1 Figure 2. Effect of pH on growth, biomass (Figure 4). Other workers also observed formation and halocin activity of HA8 strain. the salt dependence of halocin production. The H-4 and H-5 halocins showed best 3.6 Temperature production at 20 % (3.5 M) NaCl, while 8 % The isolate showed maximum growth at (1.5 M) NaCl was optimum in case of H-1, 30 °C and the biomass produced was 1.0 g H-3, H-6 and H-7, and for H-2 it was 15 % 100 mL-1. As depicted in Figure 3, it showed (2.5 M) [33-34]. Haloarchaeon Sech7a was slightly more halocin production at 40 °C reported to have optimal halocin activity in as compared to 30 °C. The study site is a medium containing 3.4 M NaCl with pH situated in arid zone of Rajasthan, India value 7.5 [14]. where environmental temperature reaches up to 50 °C in summers. The selected isolate 3.8 Induction of Halocin Activity by UV showed in vitro survival in the temperature Treatment range of 20-55 °C which explains the best The production of many bacteriocin halocin activity at a little higher temperature (halocin) is stimulated by treatment of the i.e. 40 °C. Bajpai et al. observed that for producer strain with UV light. In our study, Haloferax sp. HA10, also isolated from there was a general increase in halocin solar saltern, the optimum temperature for production with increase in UV exposure growth and amylase production was 37 °C time; maximum percent induction was 35% [6]. after 90 sec of UV exposure (Table 3). Chiang Mai J. Sci. 2018; 45(6) 2251

The organism is overproducing halocin in 3.9 Characterization of Halocin response to UV exposure. This is in contrast Temperature to the findings of Hescox and Carlberg who To see the effect of high temperature on deduced that halobacteria in general show halocin stability, culture supernatant containing unusual tolerance to radiation [35]. Meseguer halocin was exposed to temperatures from and Rodriguez-Valera did not find induction 60-100 °C for different time periods. As of H4 halocin production upon UV exposure shown in Table 4, halocin from HA8 strain [18]. During the study UV exposure stimulated showed complete loss of activity after 50 min the halocin production which is in contrast treatment at 60 °C. There was less than 50% to the findings of Hescox and Carlberg who reduction in inhibitory activity when exposed deduced that halobacteria in general show to 70 °C for 20 min, however it could unusual tolerance to radiation [35]. not tolerate exposure to 80 °C or above. There are reports of other halocins being more thermostable than the ones under investigation. Torreblanca et al. found that halocin H-6 was stable after heating to 90 °C for 10 min [16]. After heat treatment at 100 °C the activity was reduced by 50 % but only autoclaving at 121 °C for 30 min destroyed it completely. Li and his group also showed that halocin C8 did not lose any activity after boiling (at about 100 °C) for 1 h [13]. Figure 4. Effect of different NaCl concentrations on growth, biomass formation 3.10 Proteinase K and Trypsin Treatment and halocin production by HA8 strain. To study the stability of halocin against proteolytic cleavage two proteases, one specific (trypsin) and another non-specific Table 3. Effect of UV rays on halocin HA8 (proteinase K), were used (Table 5). Halocin production. showed resistance to trypsin digestion. Maximum reduction in halocin activity was Control UV Halocin Activity observed at 2 mg mL-1 concentration i.e. (AU mL-1) Treatment AU Percent 47 %, however the activity was completely Time (in sec) mL-1 Induction lost when halocin was treated with proteinase 170 30 190±12 11 K. Similar to our results Li et al. found that 60 215±17 26 halocin C8 was sensitive to proteinase K 90 230±20 35 but not to trypsin treatment [13]. Earlier, Torreblanca et al. found that halocin H-6 was ± Results are mean of three experiments SD. stable in trypsin at 5 mg mL-1 concentration; however pronase at 400 mg mL-1 completely abolished its inhibitory activity [16]. 2252 Chiang Mai J. Sci. 2018; 45(6)

Table 4. Effect of temperature on stability of halocin HA8 Control* Treatment 60 °C 70 °C AU/ml Time AU/ml Percent AU/ml Percent (min) Reduction Reduction 150 10 130±8 13 110±6 26 20 120±9 20 80±4 46 30 90±6 40 - - 40 70±4 53 - - 50 - - - - * control was maintained at 30 °C, - no activity; results are mean of three experiments ±SD

Table 5. Effect of trypsin on halocin HA8.

Control Concentration Trypsin AU mL-1 μg mL-1 AU mL-1 Percent Reduction 150 200 180±9 5 400 180±7 5 600 150±10 21 1000 110±7 42 2000 100±5 47

Results are mean of three experiments ±SD

3.11 Tolerance to Organic Solvents and that halocin C8 retained its activity in ethanol Storage Stability and methanol, whereas, reduction in halocin The stability of halocin in three organic activity was observed even with 50% solvents was studied. It showed moderate isopropanol [13]. stability only in isopropanol up to 30 min i.e. 30 AU mL-1, in comparison to control 3.12 Salt Dependence of Halocin showing 75% reduction in activity; ethanol The inhibitory activity of halocin was or methanol demonstrated adverse effect completely lost after dialysis against distilled where the activity was abolished completely water and 1M salt solution but at 3 M salt after just 15 min exposure. However, Kumar solution there was slight loss of activity after et al. demonstrated no effect on halocin dialysis; it still had 170 AU mL-1 activity as activity after the treatment with organic compared to 180 AU mL-1 before dialysis. solvents [36]. The observations regarding stability of As regarding the storage stability, no halocin at high salt concentrations suggested change in halocin activity was observed after that halocin in general is inactivated at low its storage at 4 °C for up to 5 days. Therefore, ionic concentrations, as observed by other it can be deduced from the results that workers in similar studies. The environment halocin from the isolate HA8 was quite stable. in which haloarchaea occur are aquatic In contrast to our results, Li et al. observed hypersaline, therefore their biomolecules are Chiang Mai J. Sci. 2018; 45(6) 2253

adapted to elevated salt concentrations. 3.13 Molecular Characterization using Halocins already characterized by other 16 S rDNA sequencing workers differed in their ionic stability, R1, The genus level identification of HA8 was H6, Ha1 and S8 halocins were salt dependent, done by amplification and sequencing of 16S whereas H4 lost its antimicrobial activity rDNA followed by sequence analysis using when it was dialyzed below 50% sea water MEGA 4.0 software. The sequence similarity [17]. Platas and co-workers showed that analysis of partial sequence of 16S rRNA dialysis of halocin H-1 against lower salt gene (480 bp) by the FASTA network service concentration produced an immediate revealed that the isolate shared 98% sequence decrease in its activity [20]. Halocin HA8 similarity with Haloferax species. As shown was also stable up to 5 days of storage which in the phylogenetic tree, the alignment placed shows that it can be used commercially. the isolate HA8 close to Haloferax larsenii Recently, Karthikeyan et al. found that the (Figure 5). The gene sequence was submitted optimal conditions for maximal halocin to GenBank and the Accession no. is production by Natrinema sp. were 42 °C, pH HM368367. 8.0 in Zobell’s medium containing 3 M NaCl [37].

Figure 5. Phylogenetic tree derived from the partial 16S rRNA gene sequences showing the position of Haloferax sp. HA8.

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