Bacteriocin and Quorum Sensing

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1 Bacteriocin and Quorum Sensing -Struggle for existence of lactic acid bacteria- Associate Prof. Jiro Nakayama Prof. Kenji Sonomoto Assistant Prof. Takeshi Zendo KYUSHU UNV. Lab. of MCROBAL TECHNOLOGY

2 What is Bacteriocin Peptidic compounds ribosomallysynthesized in bacteria, which show antibacterial activity against normally closely-related species or different strains of same species. Generally NO effect on eukaryotic cells Degradable by proteolytic enzyme. LAB bacteriocins are notably applicable as safe food preservatives. ndicator bacteria Bacteriocin-producing LAB

3 Class (lantibiotic) H 2 N nisin Q 1 le DHB Ala le 5 DHA Leu Ala ABU S Ala Gly 15 Val Lys ABU Leu Met Pro Gly 2 S S S 1 Gly Ala Asn Leu Lys ABU 25 Ala ABU S Ala Asn Ala Ser Val His Val DHA Lys 3 34 COOH nukacin SK-1 2 Ala-S-Ala: ランチオニン Abu-S-Ala: 3- メチルランチオニン Dha: デヒドロアラニン Dhb: デヒドロブチリン Class b (two peptide bacteriocin) Abp118α:KRGPNCVGNFLGGLFAGAAAGVPLGPAGVGGANLGMVGGALTCL Abp118β:KNGYGGSGNRWVHCGAGVGGALGAGGPWSAVAGGSGGFTSCR Class a pediocin PA-1 S S S S KYYGNGVTCGKHSCSVDWGKATTCNNGAMAWATGGHQGNHKC

4 Classification of bacteriocins produced by LAB Class Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Property Lantibiotic; small peptides containing the unusual amino acid Lan, MeLan and dehydrated amino acids Small and heat-stable Non-Lan-containing a: antilisterial activity peptides with YGNGV consensus sequence b: two-peptide bacteriocins c: other bacteriocins Large (>3kDa) heat-labile proteins Complex bacteriocins carrying lipid or carbohydrate moieties Circular peptides Bacteriocins Nisin A, Nisin Q,, Lacticin 481, Lactocin S Pediocin PA-1, Sakacin P Enterocin A, Lactococcin Q, ABP-118, Durancin EF Lacticin Q Helveticin J, Enterolysin A Millericin B Unknown Enterocin AS-48 Gassericin A

5 Bacteriocin-producing lactic acid bacteria in our laboratory Species Strain Source Bacteriocin Class Molecular Mass Antibacterial Spectrum O-1 Kitchen sink Chizuka, Yasaka Nukadoko (Fermented rice bran) QU 1 Seaweed QU 3 Corn QU 8 Carrot g56-3 Chinese cabbage KD12 Komatsuna River water Nisin Q 3327 QU 4 Corn Lactococcin Q lb 426/418 Specific QU 5 Corn Lacticin Q c 5926 Enterococcus faecium WHE81 Soft cheese Enterocin A, B la,c 4829/5463 Moderate Enterococcus mundtii QU 2 Soybean Mundticin KS lla 4287 Moderate Enterococcus durans QU 21 Flower Durancin TW-21 c 5228 Moderate Enterococcus durans QU 49 Carrot Durancin TW-49 c 5228 Moderate Lactobacillus curvatus k12-3 Flower Curvacin A a 431 Moderate Lactobacillus salivarius AC21 Chicken intestine Salivacin K21 llb 497/4283 Enterococcus faecium NKR-5-3 Pla-ra (Fermented fish) Brochocin A etc. ll 5243/etc.

6 Topics Today A variety of bacteriocins with different antimicrobial spectra and with different mode of actions, produced by isolated from corn. Diversity of bacteriocins and their immunities in enterococci Enterococcus faecium NKR-5-3 isolated from Pla-ra: Multiple bacteriocins and their production control

7 Antibacterial spectra of various L. lactis strains isolated from corn ndicator strain Producer: Bacteriocin: Micrococcus luteus Bacillus subtilis Bacillus circulans Bacillus coagulans Listeria innocua Lactobacillus sakei Lactobacillus plantarum Enterococcus faecalis Leuconostoc mesenteroides Pediococcus pentosaceus Enterococcus faecium ssp. lactis ssp. lactis Escherichia coli QU1 nisin Z Activity (AU/mL) OU4 OU5 lactococcin Q lacticin Q OU6 Unknown

8 Lactococcin Q: novel class b bacteriocin Lactococcin Qα (39 a.a.) SWGDGQGVGKAAYWVGKAMGNMSDVNQASRNRKKKH Lactococcin Qβ(35 a.a.) KKWGWLAWVEPAGEFLKGFGKGAKEGNKDKWKN - Produced by Lc. lactis QU 4 isolated from corn - Novel two-peptide (class b) bacteriocin - Synergistic action of peptides α and β - Active only against strains Zendo et al., Appl. Environ. Microbiol., 72, 3383 (26)

9 Purification of bacteriocins produced by Enterococcus faecium NKR-5-3 M17 Culture supernatant Through fraction Amberlite XAD-16 2-propanol fraction SP Sepharose < Step 1 > < Step 2 > Octyl Sepharose 1M NaCl fraction 7% EtOH fraction 7% EtOH fraction Active fraction RP HPLC Active fraction Peptide A (Class b) Peptide D (nducer) Peptide B (Class c) Peptide C (Class V)

10 Known autoinducing peptides which induce bacteriocin production nducer EntF PisN CbaX CbnS Sap-Ph AbpP Pln A PLNC8F SppP P-TX PeptideD Amino acid sequence Bacteriocin Leader peptide Mature peptide inducer Enterocin A M E E K N R L N A K QC S D Q E L K K K GG A G T - K P QGK P A S N L V E C V F S L F K K C N Piscicolin 126 MN D K K Y L K L K E C S E K K L K Q QGG N K S - V K GN P A S N L A QC V F S F F K K C Carnobacteriocin A MK K Y T R K Q L Q K GG S N - S Q GK A T S S S K C V F S F F K K C CarnobacteriocinB2 MK K T T K K Q L Q K GG S K N - S Q GK S T S S S K C V F S F F K K C Sakacin A MK L N Y E K K Q L T N K Q L K L GG - T N - R N Y GK P N K D G T C WS G F R H C ABP118 MK F E V L T E K K L QK A GG A T K - K GG F K R WQC F T F F G V C K Plantaricin EF/JK MK Q K GMK Q L S N K EMQK V GG K S S - A Y S L QMGA T A K Q V K K L F K KWGW Plantaricin NC8 MK N N K Y T E L N D QK L Q S L GG K T K - T S L M S G L Q V P H A F T K L L K A L GGH H Sakacin P MM F K K L S E K E L QK N GG MA GN S S N F H K K Q F T H R Sakacin TX M T N R K T L P K E E L K K K GG T P GG F D S GG P H V A QD V L N A K D F F K NKR-5-3 M T N R K L P K E E L K K K GG T P GG F D F S GG P H V A QD V L N A K N F F K A F F V V G G G T L G P D D H P S Q K N N K A F Peptide C (Ec. faecium)

11 Concerted control of bacteriocin production by peptide-mediated quorum sensing Peptide D P P HPK RR Peptide D A B C HPK: histidine kinase RR: response regulator

12 Concluding Remarks Even same species produces different bacteriocins with different antimicrobial spectra and with different mode of actions. (L. lactis QU3, QU4, QU5 and QU6 isolated from corn) One strain sometimes produces more than one bacteriocins. (E. durans QU21, QU49, E. faecium NKR-5-3) A immunity gene sometimes showed cross-immunity against different bacteriocin. (E. faecium JCM584) Multiple bacteriocins are sometimes regulated by one autoinducing peptide. (E. faecium NKR-5-3) Complex network organized by various bacteriocins with various antimicrobial spectrum, their immunities, and their quorum sensing control, would explain struggle for existence of LAB in complex ecosystem.