Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide.

AIMS: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer-walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9-kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI-expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. CONCLUSIONS: Thermophilin 1277 biosynthesis genes were encoded by a 9·9-kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.

A unique lantibiotic, thermophilin 1277, containing a disulfide bridge and two thioether bridges.

AIMS: To identify the chemical structure of a bacteriocin, thermophilin 1277, produced by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277 was purified and partial N-terminal sequence analysis revealed 6 unidentified amino acids amongst 31 amino acids residues. A 2.7-kbp region containing the thermophilin 1277 structural gene (tepA) encoding 58 amino acids was cloned and sequenced. Mature thermophilin 1277 (33 amino acids) was preceded by a 25-amino acid putative leader peptide containing a double glycine cleavage motif. Peptide sequence analysis following chemical modification of thermophilin 1277 revealed that the Cys21 and Cys29 residues form a disulfide bridge and that Thr8 or Thr10 forms two 3-methyllanthionines with Cys13 or Cys32 via thioether bridges. Antimicrobial activity was disrupted by ethanethiol or reductive agent treatments, indicating that the internal amino acid modifications are crucial for the activity. CONCLUSIONS: Thermophilin 1277 from Strep. thermophilus SBT1277 belongs to the class of AII-type lantibiotics that has a disulfide and two thioether bridges. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a lantibiotic produced by a GRAS species of Strep. thermophilus; thermophilin 1277 has a unique structure containing both a disulfide bridge and two thioether bridges that are crucial for its activity.

Characterization of a bacteriocin, Thermophilin 1277, produced by Streptococcus thermophilus SBT1277.

AIMS: To assess the inhibitory activity and the influence of culture condition on the growth and bacteriocin, Thermophilin 1277, production by Streptococcus thermophilus SBT1277. METHODS AND RESULTS: Thermophilin 1277, which was produced by S. thermophilus SBT1277, showed an antimicrobial activity against several lactic acid bacteria and food spoilage bacteria including Clostridium butylicum, C. sprogenes and Bacillus cereus. Thermophilin 1277 was inactivated by proteinase K. Heating treatment did not affect the antimicrobial activity. The partially purified Thermophilin 1277 had an apparent molecular mass of 3.7 kDa. N-terminal sequence analysis revealed 15 amino acid residues that correspond with amino acid sequence of the lantibiotics bovicin HJ50 produced by Streptococcus bovis HJ50. The effects of culture condition for the bacteriocin production by S. thermophilus SBT1277 were studied. During the batch fermentation, Thermophilin 1277 was produced in M17 broth, but no bacteriocin production occurred in the sucrose-tryptone (ST) broth. Bacteriocin production was detected in pH controlled ST broth at pH values of 5.5-6.5. CONCLUSIONS: Thermophilin 1277 production from S. thermophilus strain depended on the culture conditions. Some characters and N-terminal amino acid sequence of Thermophilin 1277 differed from bacteriocins produced by S. thermophilus reported previously. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus thermophilus SBT1277 or its bacteriocin which has a wide inhibitory spectrum has a potential use as a biopreservative in dairy products.

Production, purification and characterization of reutericin 6, a bacteriocin with lytic activity produced by Lactobacillus reuteri LA6.

A bacteriocin (Reutericin 6) produced by Lactobacillus reuteri LA6, was purified by hydrophobic chromatography from the modified MRS broth (D'-MRS) with 6180-fold increase in specific activity with 14% recovery. The molecular weight of reutericin 6 was determined to be 2.7 kDa by SDS-PAGE and ESI-MS. By amino acid analysis, reutericin 6 comprised of 67% hydrophobic and polar neutral amino acids. Lanthionine was not detected. The lytic activity against Lactobacillus delbrueckii subsp. bulgaricus JCM 1002T and N1A1 B6 was detected by the decrease of both turbidity and the number of viable cells, and by leaking of beta-galactosidase.

[Inhibitory effect of Kanpo-medicines: saiboku-to, syouseryu-to, sairei-to on Dermatophagoides farinae antigen-induced IL2 responsiveness in lymphocytes from patients with bronchial asthma and their comparison].

We had previously reported that antigen-induced IL2 responsiveness by lymphocytes can be used to identify etiological allergens and to monitor the clinical activity in atopic diseases. The effect of Kanpo-Medicines; Saiboku-to, Syoseiryu-to, Sairei-to, antiallergic agents, on Dermatophagoides farinae (Df) antigen-induced interleukin 2 (IL2) responsiveness from patients with bronchial asthma was studied and compared among 3 Kanpo-medicines. Allergen-sensitized patient mononuclear cells pretreated with 30-30,000 ng/ml doses of Saiboku-to for 16 hours failed to induce responsiveness to IL2 on stimulation with Df antigen in 8 patients out of 11 (73%). The cells treated with 30 micrograms/ml of Syoseiryu-to also failed to introduce the response in 6 out of 10 (60%) with less frequency compared with Saiboku-to. Sairei-to also suppressed the response on stimulation with a 10 micrograms/ml dose alone in 1 x 10(-3)-1 x 10(-4)ng/ml doses examined in 6 patients out of 17 (35%) with much less frequency and % inhibition. Also Saiboku-to and Sairei-to not Syoseiryu-to inhibited purified protein derivatives (PPD)-induced IL2 responsiveness. However. These agents failed to suppress the Con A-induced IL2 responsiveness. Antigen-presenting adherent cells were more susceptible to any Kanpo-medicines studied rather than IL2-responding T cells except Sairei-to. These results indicated that Kanpo-medicines studied have a weak immuno-suppressive property resulting in inhibiting antigen-induced IL2-responsiveness. Suppressive effects of each Kanpo-medicine seemed to depend on combinations and doses of their elements (Syouyaku). Decreased suppressive effect of higher doses of any Kanpo-medicine was likely to be mitogenic effects of each element on T cells.

[Suppressive effect of Saibokuto on Dermatophagoides farinae (Df) antigen-induced IL2 responsiveness of lymphocytes from asthmatic children].

The effect of Saibokuto on induction of antigen specific IL2 responsiveness was studied. Saibokuto suppressed the induction of IL2 responsiveness induced by Dermatophagoides farinae (Df) antigen in peripheral blood mononuclear cells from patients with bronchial asthma. Saibokuto-pretreated adherent cells failed to present Df antigen to non-adherent responding T cells for induction of their IL2 responsiveness. The same dose of Saibokuto had no impact on non-adherent cells. Saibokuto also suppressed the induced IL2 responsiveness of lymphocytes from the same patients on stimulation wit PPD, but not with Con A. These combined data suggests that Saibokuto has a weak immunosuppressive effect on Df induced IL2 responsiveness of lymphocytes from asthmatic children. Effect of Saibokuto on induction of antigen specific IL2 responsiveness was studied. Saibokuto suppressed the induction of IL2 responsiveness induced by Dermatophagoides farinae (Df) antigen in peripheral blood mononuclear cells from patients with bronchial asthma. Saibokuto-pretreated adherent cells failed to present Df antigen to non-adherent responding T cell for induction of their IL2 responsiveness. Non-adherent cells had no impact by the same dose of Saibokuto. Saibokuto also suppressed the induced IL2 responsiveness of lymphocytes from the same patient on stimulation with PPD, but not Con A. These combined data suggest that Saibokuto has a weak immunosuppressive effect on Df induced IL2 responsiveness of lymphocytes from asthmatic children. Effect of Saibokuto on induction of antigen specific IL2 responsiveness was studied. Saibokuto suppressed the induction of IL2 responsiveness induced by Dermatophagoides farinae (Df) antigen in peripheral blood mononuclear cells from patients with bronchial asthma. Saibokuto-pretreated adherent cells failed to present Df antigen to non-adherent responding T cell for induction of their IL2 responsiveness. Non-adherent cells had no impact by the same dose of Saibokuto. Saibokuto also suppressed the induced IL2 responsiveness of lymphocytes from the same patient on stimulation with PPD, but not Con A. These combined data suggest that Saibokuto has a weak immunosuppressive effect on Df induced IL2 responsiveness of lymphocytes from asthmatic children.