Introduction

Nucleases are involved in replication, repair, restriction and recombination. These are also involved in transposition, transcription and topoisomerization, as well as RNA processing, RNA splicing, editing and interference (Mishra, 2002). The accumulation of 8-OHGua induces G:C to T:A transversion during DNA replication that is related to mutagenic events. The 8-OHGua is formed by Reactive Oxygen Species which is removed from DNA by endonucleases in DNA salvage pathways (Hui et al. 2003). Single strand specific endonuclease has been exploited in mutation studies (Tierney et al. 2005). In DNA foot printing also DNases are used which is a method of investigating the sequence specificity of DNA-binding proteins. This technique is used to study protein-DNA interactions both outside and within cells (www.wikipedia.org). DNases cleaves chromosomal DNA into  nucleosomal fragments in 180-200 bp  in the process of apoptosis and later on these fragments are broken into 20-30 nucleotides and then into single nucleotides (Sluyser Mels, 2005). Qin et al. (2005) has explored the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against dengue infection and are also found useful against moloney murine leukemia virus. Derivatives of nucleotide products are prepared which mimics the viral activities (Howell et. al., 1988). Nucleases are also candidates for the enzymes producing a tasty substance, 5′-GMP. (Kobayashi et al. 2000). Nucleases are studied from plants, insects and fungi.

In present study Serratia marcescens strains were screened through a large number of soil samples. These strains were then studied for DNase production on DNase test agar and on the basis of zone of DNA hydrolysis around the colony; efficient two strains, a pigmented and non-pigmented strains were chosen further for comparative study.

Materials and Methods

Soil Sample Collection (Gary M. Banowetz  et al., 2006)

Soil samples were collected using a 2.54-cm-diameter soil corer. The corer was cleaned after each sampling with water followed by methanol and samples were placed in zip-lock plastic bags and stored for no longer than 7 days at 6°C. (Gary M. Banowetz   et al., 2006).

Screening of DNase Producing Bacteria (Schreier, J. B.1969)

Screening was done on DNase test agar with toludine blue. Toludine blue on reaction with DNA gives sky blue color. When DNA is hydrolyzed by bacterial action; toludine becomes free and being in a free form a faint pink or colorless halos is observed around the colony.

Morphological and biochemical characterization of efficient DNase producers.

Routine microbiological methods were adopted for this purpose.

Phylogenetic study of some efficient cultures (Saitou, N. and Nei, M. (1987), Felsenstein, J., (1985), Kimura, M. (1980), Tamura, et al., (2007).

Identification by 16S rRNA sequencing is the most preferred these days. The service was provided by Chromous Biotech Pvt. Ltd. Bangalore.

Estimation of DNase (Lachica R.V.F. et.al., 1972)

The characterized Serratia marcescens strains were grown in nutrient broth containing 0.2% DNA. The broth was centrifuged at 13,500 g to separate out the cells, so that supernatant is used as source of DNases as DNases are extracellular enzymes. Wells were then cut on methyl green DNA agar by cork borer of 2 mm diameter. A measured quantity (3ml) of DNase was added by micropipette aseptically. These were then added in the wells prepared. Smaller DNase conc. formed smaller zones and higher DNase conc. formed larger zones of DNA hydrolysis as seen in photo plate NO. 3 and 4. A standard graph already prepared was referred for quantitative estimation of DNase in the broth.

Result and Discussion

Screening of DNase producing bacteria

Plate:1 Serratia marcescens on DNase testDNase Test agar with toludiene blue.   Click here to View plate

Plate:2 Serratia marcescens on  DNase Test agar with toludiene blue.   Click here to View plate

Table 1: Morphological & Biochemical Characteristics of isolates from East region.

Figure 1: The pigmented strain in above phylogenetic tree is 165 D.   Click here to View figure

Figure 2: The non-pigmented strain in above phylogenetic tree is 165 E.   Click here to View figure

Phylogenetic study of the selected cultures

The pigmented strain in above phylogenetic tree is 165 D

The non-pigmented strain in above phylogenetic tree is 165 E

Estimation of DNase   

Plate 3: and 4 Metachromatic Agar Diffusion Assay of DNase.   Click here to View plate

Serratia marcescens a pigmented strain produced maximum DNase i.e. 6.1mg/ml at pH 9 and 6.25 mg/ml at temperature of 20°C. Other non-pigmented strain produced maximum DNase at pH 9 i.e. 6.3mg/ml and at temperature 30°C maximum DNase i.e. 6.35mg/ml was produced. This has shown that a non-pigmented strain of Serratia marcescens produces more DNase than pigmented strain.

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