Bacteriocin Activities Of Bacillus Thuringiensis BRC-ZLL5 And BRC-XQ6

Bacteriocins,a kind of peptides or proteins producing through the ribosomes synthesis mechanism in the metabolic processes of some bacteria,were not likely to be resistant by intestinal pathogenic bacteria,and did not have teratogenesis and accumulation toxicity.Hence,they are considered to be good biological preservatives with huge commercial potential.Bacteriocins are active against many kinds of intestinal pathogenic bacteria including Listeria monocytogenes and Staphylococcus aureus.Discovering new bacteriocins can therefore be helpful in developing new drugs,promoting the research,development and applications.Bacillus thuringiensis(Bt)is a kind of extensive microbial pesticides.Besides insecticidal crystal proteins(ICPs),it can produce many kinds of bacteriocins(thuricin)in different sizes with antibacterial or bactericidal activities.In this study,15 out of 94 Bt strains were resistant to Listeria monocytogenes(Lm),determined by improved method "agar-well diffusion method".Of them,Bt BRC-ZLL5 and BRC-XQ6 were chosed for further studies and the corresponding indicator bacteria were Lm 100625 and Lm 100626 respectively.Bt BRC-ZLL5 and BRC-XQ6 have no antibacterial activities against 4 probiotic bacteria.By testing the remaining 40 strains of pathogenic bacteria and 7 Bt standard strains used in this study,BRC-ZLL5 was resistant to Listeria,Pseudomonas aeruginosa and Streptococcus.faecalis,and BRC-XQ6 was active against Listeria and Staphylococcus epidermidis.It is well-known that most of the Bt bacteriocins inhibited Lm and Bacillus cereus,and some of them were resistant to Streptococcus,Staphylococcus aureus,Streptococcus pyogenes,P.aeruginosa,Vibrio cholera and Clostridium botulinum.Each strain was not resistant to itself and the 7 Bt standard strains.Herein,the Bt strains should contained immune factors against bacteriocins produced with themselves.The neutralized cell-free fermentation supernatant of BRC-ZLL5 showed the antibacterial effects against Lm 100625 during the 6 h-18 h incubation,peaking at 12h and BRC-XQ6 showed different antibacterial effects against Lm 1006256 during the 4 h-72 h incubation,peaking at 56 h.However,most of the bacteriocins reached their peaked expressions at the 30 h incubation.The bacteriocins produced by BRC-ZLL5 and BRC-XQ6,detected by the tricine-SDS-PAGE,were separately partially purified with 70%and 50%saturated ammonium sulphate precipitation.The molecular weights of vast bacteriocins were 1-14.5 kDa.The bacteriocins with molecular weights less than 10 kDa were considered as peptides and those were equal or greater than 10 kDa,regarding as proteins.Numerous Bt bacteriocins were resistant to heat(80-120?),stable within pH 1-11,and lost activities after protease K and chymotrypsin treatments.Bactericidal and bacteriolytic activities were the most common mode of actions of Bt bacteriocins while few of them appeared only antibacterial.Via tricine-SDS-PAGE and antibacterial activity detection of in situ on gels,the bacteriocins of BRC-ZLL5 and BRC-XQ6 were about 10 kDa and 20 kDa.The BRC-ZLL5 bacteriocins was antibacterial,pH tolerant(pH3-9)and heat-resistant(120°C for 30 min).Its antimicrobial activity was lost after treatment with proteinase K and chymotrypsin.Therefore,according to the classification of bacteriocins,the BRC-ZLL5 bacteriocins was a peptide and refered to a class I or class II bacteriocins.Since 10 to 30 kDa bacteriocins are defined as bacteriocin-like inhibitory substances(BLIS),the 20 kDa bacteriocins of BRC-XQ6,which was bactericidal and bacteriolytic,was likely to be a BLIS sensitive to pH and temperature.In Bacillus,the pleiotropic regulator plcR regulated most of known virulence factors.This research showed that plcR mutant strain Bt 407?plcR had a broader spetra of antibacterial activities compared with Bt 407,and the antibacterial activities of the former were 1.3 to 2.7 times of those of the latter.The internal and external secretion tests showed that the bacteriocins produced by Bt 407 and Bt 407?plcR were secreted into the fermentation,and were not accumulated in the cytoplasm.According to known plcR gene sequences reported in NCBI,two plcR gene sequences of BRC-ZLL5 and BRC-XQ6 including upstream and downstream regions of the gene,were cloned and sequenced.They showed 97%and 95%homology with known plcR sequences.The deletion mutation BRC-ZLL5-?plcR and BRC-XQ6-?plcR will be constructed by homologous recombination,promoting the researchs on regulating mechanisms of plcR.