Isolation and Molecular Level Identification of DNase Producing Halophilic Bacillus cereus Family Isolates from Marine Sediment Sample

Halophilic bacteria are excellent source of enzymes not only salt stable but also it can withstand and carry out reactions very efficiently under extreme conditions. Screening of bacteria from different hyper saline environment in Kanyakumari Coast, South India led to the isolation of total 111 culturable heterotrophic bacteria. Screening of Halophiles for the DNase production led to 23 isolates and from which maximum DNase producing 4 strains were selected through DNase well diffusion method. The potential isolate KVCMST-8A 12 showed maximum zone of inhibition at 35mm after 72 hours, which were able to grow optimal in media with 2-40% of salt. According to its phenotypic characteristics and comparatively partial 16S rRNA sequence, the halophilic KVCMST-8A 12 was identified as member of Bacillus cereus family.


INTRODUCTION
Marine environment is the prime reservoir of biological diversity and the marine microorganisms are recognized to be rich source of novel compounds. In India about 1000 natural products were derived from marine microbes (Suthindhiran et al., 2010). Marine microorganisms are mostly unexplored and marine environment is rich source of microorganisms producing novel and efficient compounds (Sujatha et al., 2005). Microbes form the topmost source of industrially important enzyme producers due to their rapid doubling time when compared to plants or animals (Kumar and Takagi, 1999). The ability of marine bacteria to synthesis variety of enzymes with good stability at higher temperature, alkaline conditions and high salinity (Berdy 2005) are attractive phenomenon to accomplish extremophilic organisms in biotechnological processes.
DNase catalyses the hydrolysis of deoxyribonucleic acid by breaking down phosphodiester bonds (Nishino and Marikawa, 2002), consequently DNase is considered to have a potential role in DNA utilization and nutrient cycling in the environment (Mulcahy et al., 2010). The balance of extracellular DNA in the marine environment is largely regulated by DNase, which is important for the functioning of deep sea ecosystems (Dell anno and Danovaro 2005). The role of DNases in cellular metabolism of nitrogen and phosphorous suggests that DNase secretion may also play an important role in the fate of extracellular DNA in the natural environment, particularly with regarded to the dynamics and stability of marine aggregates (Aisha et al., 2019).
Halophiles are the most likely source of enzymes, because not only is their enzyme salt tolerant but also may be active at high temperature, and pH values (Gomez and Steiner 2004). The isolation of moderate halophiles with capacity to produce extracellular enzymes will provide the possibility to have optimal activities at different salt concentrations (Ventosa et al., 1998). Halophiles are now gaining more access to industrial microbiology and biotechnology because halophiles grow at high salt concentration and this minimize the risk of contamination during cultivation (Oren, 2006), also it has great potential uses in industrial processes (Ventosa and Nieto, 1995).
Marine bacteria, especially the benthic microbes could utilize the DNA from dead and degraded organism as sole source of energy. Hence, the attempt to isolate the marine bacteria with potential and unique DNase activity from sediments off Kanyakumari coast, Southern tip of India was initiated. This is a preliminary report on the isolation and characterization of marine bacteria with DNase activity in the largely untapped Kanyakumari coastal areas.

METHODOLOGY Isolation of marine bacteria
Sediment samples were collected from 3 sampling points, off coast Kanyakumari and transferred aseptically to lab. The samples were serially diluted and plated on Zobell marine agar and incubated at room temperature for 48 h. The bacterial isolates were selected at random on the basis of their prominence and distinctness in colony morphology and pigmentation. The isolates were screened for their capacity to produce DNA hydrolyzing activity on DNase test agar plate.

DNase well diffusion method
The well diffusion method was followed for DNase test employing the standard procedure (Sanchez and Colom, 2010). Petri plates were prepared by pouring 20 ml of DNase test agar and allowed to solidify. The 48 h culture broths inoculated with the bacterial isolates were added to the wells on the agar. After incubation at 37°C for various time points (24 h, 48 h, & 72 h) the zone of inhibition was measured.

In vitro DNase assay
The activity of DNase was studied by the reaction of cleavage of calf thymus DNA (20µg/ml) in 10mM Tris -HCl buffer ( pH 7.5) containing 2.5mM MgCl 2 , 0.1mM CaCl 2 , and DNase (0.1-2U) at 33°C for 1-15 min. The reaction mixture was supplemented with EDTA (final concentration 2mM) and incubated at 65°C for 10 min. The degradation products were analyzed by electrophoresis in 1% agarose gel stainined with ethidium bromide.

DNase Activity Gel System
For substrate gel, the methodology of Shikawa et al., 1994 was followed. In brief, after SDS -PAGE, the gel was washed with 10mM Tris -HCl (pH 7.8) / 5mM mercaptoethanol at 50°C for 1 h to remove SDS, and then with 10 mM Tris -HCl (pH 7.8), at 4°C overnight. Then the gels were incubated in 10 mM Tris -HCl (pH 7.8), containing 1mM mercaptoethanol and indicated concentration of MgCl 2 , CaCl 2 at 37°C for 3 h. When the gel was stained with 0.5µg/ml ethidium bromide, apparent endonuclease activity was detected as dark areas on fluorescent background by UV transilumination of the gel.

Effect of NaCl concentration on DNase production of potential isolate
The activity of crudes in potential strains KVCMST-6A 20, KVCMST-6A 23, KVCMST-7A 6 and KVCMST-8A 12 in presence of NaCl (2-40%) was measured by standard assay method and DNase activity through well diffusion method.

Characterization of isolate
Characterization of the isolate was carried out by traditional biochemical methods and molecular characterization of the 16S rRNA gene of the potential isolate was done through commercial sequencing and the organism after analysis with CLUSTAL X, the phylogenetic relationship was established using phylip and MEGA 4 and submitted to Genbank.

RESULT AND DISCUSSION
Marine environment is bestowed with high microbial consortium and biologically productive ecosystem inspite of the extreme environmental conditions. To thrive in these extreme conditions of physical and chemical changeovers, the marine bacteria have developed strategies to adapt to this environment. To maintain their structure and physiology, they are compelled to produce specialized metabolites and potent extracellular hydrolytic enzymes to assimilate the nutrients available around them. Although utilization of dissolved proteins has been investigated to some extent (Hollibaugh and Azam, 1983), the mechanism of utilization of dissolved macromolecules in the marine environment has not been extensively studied. Few references are available on the rapid hydrolysis of extracellular DNA in the marine environment by cell associated and extracellular nuclease. In the present work, the bacterial isolates obtained from the sediment samples collected from various depths off coast Kanyakumari was analyzed for their ability to produce extracellular enzyme, DNase.

Isolation of marine microbe (s)
In the deep sea environment, sediments form a reservoir of microbes with enzymatic action with potential biotechnological applications. Kaboyashi et al., 2008 reported a number of cultivable aerobic microbes with variety of enzyme activities from the deep sea floor sediment off shore Shimokita Penishla, Japan. In a view to isolate microbes with novel enzymes activities KVCMST-6A 3 28(b) 3.

Screening for extracellular DNase
The culture filtrate of bacterial isolates positive on DNase test agar were analyzed for extracellular DNase production employing agar well diffusion method. Although, all the 23 isolates demonstrated extracellular DNase activity, 2 strains from sampling point 6A designated as KVCMST-6A 20, KVCMST-6A 23, one strain from sampling point 7A denoted by KVCMST-7A 6, and yet another strain from sampling point 8A named KVCMST-8A 12 were shown to produce maximum DNase activity and these strains were used for further studies (Table. 2). DNase test  agar is generally used for screening of microbes for DNase production. A thermophilic fungus was isolated and found to produce extracellular DNase employing this method (Landry et al., 2014).

Time -Course study for DNase production
Based on the preliminary screening, four strains that produce maximum zone of > 30mm on 72 hours (Fig. 1) were further analyzed for production of extracellular deoxyribonuclease activity at different time points on DNase test agar employing agar well diffusion method. Test agar plates were incubated 72 h to analyze the production of DNase and the zone measured at 24, 48, and 72 hours. The zone of DNase activity by the strains KVCMST-6A 20, KVCMST-6A 23, KVCMST-7A 6 and KVCMST-8A 12 were shown in Table. 3. Among the four strains, KVCMST-8A 12 demonstrated a prominent zone right from 24 hours up to 72 hours compared to other strains. However, the other three strains KVCMST-6A 20, KVCMST-6A 23 and KVCMST-7A 6 were not to be excluded as they produce a zone on par with KVCMST-8A 12.

In vitro DNase activity
DNA cleavage is one of the important mechanism to arrest the growth of bacteria and viruses, in control of diseases particularly cancer, there is a considerable interest in development of enzymes suitable to cleave DNA. The selected strains KVCMST-6A 20, KVCMST-6A 23, KVCMST-7A 6, and KVCMST-8A 12 were subjected to DNA cleavage assay on prokaryotic bacterial DNA to demonstrate the in vitro DNase activity employing electrophoretic technique. When the crude extracts of the four isolates were incubated with DNA in tris buffer for 30 minutes and electrophoresed on 0.8% agarose, there was clear cut degradation of DNA (Fig. 2, lane 3 to 6) compared to the control DNA (lane 2) and extract without sample DNA (lane 1). The DNA cleavage potential of the isolates was evidenced

Substrate gel analysis of DNase
For demonstration of in vitro enzyme activities, substrate gel substrate system plays a vital role. It helps not only to locate the presence of enzyme, but also indicates whether the enzyme is in active state, thus to isolate and purify this enzyme, substrate gel analysis was carried out. In activity gel analysis, the crude extracts of the bacterial isolates KVCMST-6A 20, KVCMST-6A 23, KVCMST-7A 6, and KVCMST-8A 12 were separated by SDS polyacrylamide gel in which Calf thymus DNA was incorporated. After electrophoresis, the gel was washed and incubated in appropriate buffer and DNase activities were revealed by    staining with ethidium bromide. The enzyme activity which appeared as dark band (Fig. 3) was shown in arrow marks.

Effect of NaCl on growth and DNase production of potential strains
There are only few reports on halophilic nuclease production by halophiles, including Micrococcus variants, Var halophilus (Kamekura and Onishi 1978) and Bacillus halophilus (Onishi et al., 1983, Ventosa 2015. In order to know the halophilic nature and capacity of DNase production of all strains it is necessary to check the growth and DNase production at various NaCl concentrations (Table. 4 to Table. 7). The isolates were grown at various NaCl concentrations and the overnight grown culture was centrifuged and supernatant was assayed for DNase production on toludine blue DNA agar, DNA hydrolysis zone were noted. The strains exhibited maximum DNase production and growth at 7.5% except KVCMST-8A 12 it showed maximum DNase production at 8% (1.36M) concentration but the strain was capable of producing DNase upto 40% NaCl concentration tested (6.81M) (Fig.4). Onishi et al., 1983 identified a moderate halophile, Bacillus sp N23-2 produced an extracellular nuclease during the growth in 1 to 2M NaCl. KVCMST-8A 12 is a true halophilic microbe as it possess extracellular deoxyribonuclease even at 6.81M.

Identification of strain
Having known that the crude extracts from the bacterial isolates were (KVCMST-6A 20, KVCMST-6A 23, KVCMST-7A 6 and KVCMST-8A 12) capable of producing extra cellular DNase and its in vitro DNase activity have been demonstrated both in in vitro DNase activity on agarose gel and activity gel analysis on SDS-PAGE, it is necessary that the taxonomical identity of the bacterial isolates have to be established. Therefore, employing various biochemical tests (Table.3 and Table.4), these isolates were tentatively identified as Bacillus Sp belonging to Bacillus cereus group. They were gram positive (Fig.8) anaerobic, spore forming, rod shaped organisms with smooth, convex, round, glossy and white colony morphology. From which KVCMST-8A 12 tolerated up to 40% NaCl and growth occurs on Mac Conkey agar. All strains showed positive for hydrolysis of starch, casein and DNA, but negative for hydrolysis of gelatin, urease, indole and negative for oxidase.
On the basis of phenotypic characteristics and the comparison of partial 16S rRNA gene sequences, all the isolates KVCMST-6A 20, KVCMST-