Department of Microbiology Bacillus species isolated from Tungrymbai and Bekang, naturally fermented soybean foods of North East India Rajen Chettri Research Associate Department of Microbiology Sikkim University

Tungrymbai and Bekang are the indigenous fermented soybean product from Meghalaya and Mizoram states in North East India. Tungrymbai (also pronounced as Turangbai) is prepared and consumed mostly by the ethnic tribes; Khasi, Jaintias and Garos of Meghalaya whereas Bekang is prepared and consumed by Mizos of Mizoram. Bacilli are the predominant microorganism in both the products and was found at the level of 108 cfu/g. Strains of bacilli isolated from turangbai were phenotypically identified as Bacillus subtilis, B. pumilus and B. licheniformis and bacillii isolated from bekang samples were identified as B. subtilis, B. pumilus, B. licheniformis, B. sphaericus, B. brevis, B. coagulans and B. Circulans.

Fig 1. Flowsheets of preparation of Tungrymbai and Bekang

Methods Applied For Molecular Characterization of bacilli DNA Isolation: Bacterial genomic DNA was isolated according to the slightly modified method of Zhang et al. (2002). Polymerase Chain Reaction (PCR) : Amplified 16S rDNA was obtained from each strain by PCR with the following universal primers; forward 5’–AGAGTTTGATCCTGGCTCAG-3’ and reverse 5’-AAGGAGGTGATCCAGCCGCA-3’ (Weisburg et al., 1991). Amplified 16S ribosomal DNA restriction analysis (ARDRA): The restriction digestion analysis of each PCR amplified product of 16S rDNA was separately carried out with HinfI (R6205, Promega), CfoI (R6241, Promega) and RsaI (Promega). 16S – 23S rDNA intergenic transcribed spacer (ITS) – PCR analysis: The amplification of 16S–23S rDNA intergenic transcribed spacer (ITS) region was carried out. RAPD – PCR Analysis To differentiate at strain level, RAPD-PCR was carried out. Gel Electrophoresis The amplified DNA fragments was separated through gel electrophoresis. 16S rDNA sequence analysis The sequencing reactions were performed using ABI 3100 DNA sequencer (Applied Biosystems) in both direction with universal primers used for amplification.

Amplification of 16S rDNA Fig 2. Agarose gel of PCR products of various strains of Bacillus isolated from turangbai and bekang. Lane M: DNA size marker (Genei, RMBD 135, India); PC: positive control; NC: negative control Fig 3 Agarose gel of PCR products of various strains of Bacillus isolated from turangbai and bekang. Lane M: DNA size marker (Genei,RMBD 135, India); PC: positive control; NC: negative control

Gel data of ARDRA analysis of 16S rRNA gene PCR amplimers 500 200 bp 1000 Fig 4a. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Hinf I, Lane M: 100 bp DNA ladder (Genei, RMBD135). Fig 4b. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Hinf I. Lane M: 100 bp DNA ladder (Genei, RMBD135). Fig 5a. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Rsa I. M1: 100 bp DNA ladder (Genei,RMBD135), M2: 1 Kb DNA ladder (Promega, G5711). M1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 M2 bp 1000 500 200 M1 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 M2 Fig 5b. ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Rsa I. Lane M1: 100 bp DNA ladder (Genei,RMBD135), M2: 1 Kb DNA ladder (Promega, G5711).

Gel data of ARDRA analysis of 16S rRNA gene PCR amplimers bp 1000 500 200 Fig 6 (a). ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Cfo I. Lane M: 1 Kb DNA ladder (Promega, G5711). Fig 6 (b). ARDRA analysis of 16S rRNA gene PCR amplimers of various isolates of Bacillus from turangbai and bekang with restriction endonuclease Cfo I. Lane M: 1 Kb DNA ladder (Promega, G5711).

Restriction digestion profile (bp) Table 1. Grouping of Bacilli based on the band sizes of amplified ribosomal DNA restriction analysis (ARDRA) ARDRA GROUP Restriction digestion profile (bp) Strains No. of strains Hinf I Rsa I Cfo I Group I ( II ) (II ) TM1:B1, TPI:B1, TB2:B8a, TB2:B8b, TB1:B10, BD1:B1, BT:B9, BK2:B6, BK1:B15, BT:B3, TM1:B5, TS2:B13, TP1:B4a, TP1:B4b, TB2:B13, TB1:B17, BT:B17, BME:B10, BAV:B1, TB1:B16, TB2:B11, TS1:B6,TM1:B12, BT:B20, BME:B23, BK1:B24, BME:B20, TSA:B15, BD1:B22, TP2:B12 30 600 370 340 200 500 480 420 150 890 430 230 Group II (III) TP1:B2, BT:B11, BK1:B13, TP2:B10, TP1:B5, TP1:B15 6 330 180 Group III ( I ) (I ) TSB:B17, BK2:B12, TS1:B25, TSA:B4, BK2:B8, BAV:B12, TM2:B6, TS2:B24, BME:B6 9 990 400 360 580 Group IV BK1:B18 1 Group V (III ) BAV2:B6, BAV:B3 2 Group VI BT2:B18 Group VII BAV:B15

ARDRA GROUP sub-group (bp) No. of bands Strains No. of strains Table 2. Internal transcribed spacer (ITS) sub-grouping of ARDRA groups of bacilli isolated from turangbai and bekang ARDRA GROUP ITS sub-group ITS profile (bp) No. of bands Strains No. of strains Group I I A 650, 620, 500 3 TM1:B1,TB2:B8a, TB2:B8b, BD1:B1, BT:B9, BK2:B6, BK1:B15, BT:B3, TM1:B5, TP1:B4a, TP1:B4b, BT:B17, BAV:B1, TS1:B6, TM1:B12 15 I B 650, 620, 580, 500 4 TPI:B1, TB1:B10, TS2:B13,TB2:B13, TB1:B17,BME:B10,TB1:B16, TB2:B11, BT:B20, BME:B23 10 I C 680, 580, 520 TSA:B15 1 I D 620, 500 2 BK1:B24, BME:B20 Group II II A 700,670, 500 TP2:B10 II B 710, 680, 600, 550 BK1:B13, TP1:B2, TP1:B15 II C 650, 580, 530 BT:B11 II D 600,500 TP1:B5 Group III III A 710, 670, 550, 490 TSA:B4 III B 710, 670, 490 TSB:B17, TS1:B25, TM2:B6, TS2:B24, BME:B6 5 III C 490 BK2:B12, BAV:B12, BK2:B8 Group IV IV A 710, 670, 650, 490 BK1:B18 Group V V A 700, 650, 500 BAV2:B6 V B 650 BAV:B3 Group VI VI BT2:B18 Group VII VII BAV:B15

RAPD profiles bp bp bp bp M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 2324 25 26 M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 bp 1000_ 500 1000 500 Fig 7. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using OPB-18 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates. Lane M: 100 bp DNA ladder (Genei, RMBD135). Fig 8. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using OPN-13 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates Lane M: 100 bp DNA ladder (Genei, RMBD135). M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 bp M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 bp 1000 500 1000 500 Fig 9. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using E-11 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates Lane M: 100 bp DNA ladder (Genei, RMBD135). Fig 10. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using OPD-5 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates. Lane M: 100 bp DNA ladder (Genei, RMBD135). M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 1718 19 20 21 22 2324 25 26 M bp 600 500 Fig 11. RAPD profiles of major Bacillus subtilis group of isolates from turangbai and bekang using M-13 random primer. Lanes 1-26 belongs to Bacillus subtilis group of isolates Lane M: 100 bp DNA ladder (Genei, RMBD135).

GROUP I GROUP II GROUP III GROUP V GROUP VI GROUP VII GROUP IV Fig 12. Dendogram based on the UPGMA of Jaccard Similarity Coefficient (Si) of combined ARDRA and ITS profiles of various isolates of Bacillus from turangbai and bekang. Groups I and VII belongs to B. subtilis group, group II represents B. licheniformis group, groups III and IV belongs to B. cereus group, groups V and VI belongs to Lysinibacillus fusiformis group.

GROUP B GROUP A Fig 13. Dendogram based on the UPGMA of Jaccard Similarity Coefficient (Si) of combined RAPD and ITS profiles of various isolates of Bacillus subtilis (Group A and B) from turangbai and bekang.

Fig 14. Dendogram based on the UPGMA of Jaccard Similarity Coefficient (Si) of combined RAPD and ITS profiles of various isolates of Bacillus subtilis (Group A and B) from turangbai and bekang. GROUP A GROUP B

Bacillus subtilis sub sp. subtilis Table 2 . 16S rDNA sequencing results of the representative bacilli of different ARDRA groups Strain code RDP database NCBI database Closest relative S_ab score % Similarity TB2:B8 (I) Bacillus subtilis sub sp. subtilis 0.921 98% TB1:B10 0.953 99% BK1:B13 (II) Bacillus licheniformis 0.950 BK1:B18 (IV) Bacillus cereus 0.859 97% BAV2:B6 (V) Lysinibacillus fusiformis 0.947 BAV:B3 0.968 BT2:B18 (VI) 0.926 BAV:B15 (VII) 0.972 Numerals in parenthesis denotes ARDRA groups

Conclusion The Phenotypic identification of representative strains of Bacillus isolated from turangbai and bekang were reconfirmed by molecular identification tools based on ARDRA, ITS-PCR and RAPD-PCR techniques. Eight isolates (representing 7 ARDRA groups) were sequenced and their identity was confirmed as follows: Bacillus subtilis subsp. subtilis, B. licheniformis, B. cereus and Lysinibacillus fusiformis. Thank-you