Preparation of intestinal sample
For isolation of bacteria, intestinal sample was prepared from fish purchased from a local market. Entire digestive tract of the fish was removed by aseptic surgery and its external surface was thoroughly washed with autoclaved distilled water and then sterilized using 70% v/v ethanol. Internal contents of the digestive tract were then squeezed out and collected in a beaker. Inside of the digestive tract was then rinsed well with sterile water which was also added to the internal contents.
Isolation of bacteria
Bacteria present in the intestinal sample were isolated as previously described with minor modifications [15]. Briefly, 100 μl of the intestinal sample and its 10-fold serial dilutions (100 through 10−6) were spread on nutrient agar (NA; 5 g/L peptone, 3 g/L yeast extract, 5 g/L NaCl, 18 g/L agar; pH 7) and Luria-Bertani (LB) agar (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, 18 g/L agar; pH 7) [24] plates and incubated at 30°C for 24 to 48 h. All morphologically distinct colonies were selected and streaked on fresh NA and LB agar plates to obtain pure cultures [25].
Preparation of culture stock
Cells from the colony of pure culture was inoculated to nutrient and LB broth and incubated at 37°C. After 24 h of growth, 500 μL of the culture was transferred to a cryo-vial, sterile glycerol was added to the final concentration of 20% v/v and preserved at -80°C for further analysis.
Culture conditions
The isolates were routinely maintained in LB media at 30°C, unless otherwise noted. Each isolate was revived from its glycerol stock by transferring cells to 2 to 5 ml LB broth by a sterile loop and grown overnight in an orbital shaker at 180 rpm at 30°C. 1% v/v of this activated overnight culture was transferred to 10 ml fresh broth, incubated at 30°C for 24 h and used for subsequent analysis.
Screening for proteolytic activity
To detect presence of extracellular proteolytic activity, 10 ml of a 0.8 OD600 culture of each isolate was spot-inoculated on the surface of skim milk agar media (5 g/L peptone, 2.5 g/L yeast extract, 1 g/L dextrose, 28 g/L skim milk powder, 18 g/L agar; pH 7) as well as NA and LB media each supplemented with 1% (w/v) skim milk powder and incubated at 30°C for 24 to 48 h. Isolates that showed zones of clear halo surrounding the colonies were considered positive for protease production.
Morphological, cultural and biochemical characterization
Determination of morphological, cultural and biochemical properties of the isolates and their fermentation of various carbohydrates were carried out by methods described previously [25, 26].
16S rRNA gene amplification and sequencing
16S rRNA gene of each isolate was amplified from its genomic DNA using GoTaq G2 Hot Start Master Mix (Promega) and the purified PCR products were sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit according to a previous report [15].
Sequence deposition
The 16S rRNA genes sequenced in the present study have been deposited in the GenBank database under the accession numbers OK287066 to OK287072.
Taxonomic analysis
Taxonomic annotation of the proteolytic isolates was carried out by analysis of their 16S rRNA gene sequences with nucleotide BLAST of NCBI [27], RDP classifier and seqmatch [28] and Silva ACT: Alignment, Classification and Tree Service [29]. All parameters were set to default values with the only exception in BLAST search where the “Max target sequences” was set to 1000. Phylotypes in the BLAST searches were determined by considering the query coverage, percent identity, maximum and total scores, and the total number of hits obtained for the query sequence against a particular genus or species. Organisms with an ambiguous taxonomic description such as enrichment culture clones, uncultured bacteria or unclassified bacteria were not taken into consideration [31]. NCBI taxonomy browser was followed to obtained taxonomic hierarchy of the isolates [32].
Phylogenetic analysis
Phylogenetic analysis of the isolates was performed essentially as previously described [33]. 16S rRNA gene sequence of the isolates, and 700 bp of their nearest type strains, and the top hit strains in BLAST results were aligned by Muscle or ClustalW algorithms in Molecular Evolutionary Genetics Analysis (MEGA) software, version X [34]. The closest type strain for each isolate was found by using EzBioCloud’s 16S-based ID [35], and their sequences were collected from the EzBioCloud database having the accession numbers CP001628, LASD01000006, FLYB01000015, JJMH01000057, HQ888847, BAMA01000316, LDJN01000038. Two additional strains used in the alignment for each isolate were selected from the top hits in BLAST search results and their sequences were obtained from GenBank database with the accession numbers MW198159.1, MT509874.1, MT509997.1, MK033338.1, MN420979.1, MH341969.1, MT533939.1, MT033093.1, MK571729.1, MK640708.1, KY913809.1, EU307934.1, MT555731.1, MT649753.1. A phylogenetic tree of the aligned sequences was built by maximum likelihood (ML) method using Tamura-Nei algorithm with 100 bootstrap replications and by neighbor joining (NJ) method using Maximum Composite Likelihood algorithm in MEGA as described in [31].
Determination of substrate specificity
Ability of the proteolytic isolates to hydrolyze casein, gelatin and bovine serum albumin (BSA) was examined based on the formation of clear halos around colonies streaked on NA and LB media supplemented with 1% (w/v) of each substrate as described above.
Estimation of relative activity
To determine relative proteolytic activity, the isolates were grown on media containing 1% (w/v) of casein, gelatin or BSA at 30°C for 48 h. The diameter of the zone of clearance and that of the colonies were measured. Relative activity (RA) was then calculated using the formula, RA = (colony diameter + halo zone diameter)/colony diameter [15].
Statistical analysis
All experiments were performed at least three times separately, averaged and the standard deviation was generated. The data were presented as the mean ± standard deviation displayed as error bars.