Peptide Information 3908

1-Adamantanecarbonitrile
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These strains were obtained from the bacterial culture collection of Dr. Overnight cultures of S.

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The inhibition zones in mm were measured from the edge of the S. The bacteriocin-producing strains were tested by PCR for the presence of the sslA structural gene previously identified in the bacteriocin-producing strain S. PCR reactions consisted of This medium, which increases bacteriocin production by S.

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The acetonitrile and TFA were removed by a rotary evaporator prior to analyzing the fractions for bacteriocin activity using the spot test plate assay. The purified bacteriocin preparation 0. Bacteriocin activity was detected using an overlay of soft agar medium inoculated with the indicator strain S. Nisin A Sigma-Aldrich was used as a positive control. The susceptibility of the purified bacteriocin to heat, pH, and enzymatic treatments was determined using the spot test plate assay and S.

To evaluate its susceptibility to extreme pHs, the bacteriocin solution was adjusted to pH 2 or 11 with 0. Several swine pathogens were tested using the spot test plate assay to evaluate the inhibitory spectrum of the purified bacteriocin.

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The effect of the purified bacteriocin in combination with penicillin G or amoxicillin was evaluated using the checkerboard technique [ 16 ]. A cell suspension of S. Wells with no bacteria or compounds were used as controls. Bacterial growth was assessed visually. The lowest concentration at which no growth occurred was considered the MIC.

Two independent experiments were performed to ensure reproducibility. The ability of the purified bacteriocin to permeabilize the cytoplasmic membrane of S. The fluorescence resulting from the binding of the dye to the bacterial DNA was recorded using a Synergy 2 microplate reader BioTek Instruments, USA every 3 min for 20 min with the excitation and emission wavelengths set at nm and nm, respectively. PBS was used as a negative control. The bacteriocin band, which was located based on the migration of molecular weight markers and the detection of bacteriocin activity, was excised and transferred into a microtube.

Ethanethiol derivatization of post-translationally modified amino acids of the PVDF-blotted bacteriocin was carried out as previously described by Meyer et al.

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Published S. The putative bacteriocin gene cluster that was identified was used to design primers for PCR amplification using genomic DNA extracted from S. Bacterial strains that produce either identical or similar bacteriocins exhibit cross-immunity. Therefore, the presence of cross-immunity between bacteriocin producers is generally indicative of relatedness between their bacteriocins. To evaluate cross-immunity, S. The susceptibility of S.

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Briefly, a h bacterial culture in THB was diluted in fresh broth medium to obtain an optical density at nm OD of 0. Wells with no bacteria or no antibiotics were used as controls. The MIC values were determined in three independent experiments. Two of the ten strains of S. Given that we previously purified and characterized a lantibiotic suicin 90— produced by a strain of S. As shown in Fig. The gdh gene used as control was amplified in both strains. This confirmed that the bacteriocin produced by S. We thus selected this strain for further analysis.

Lane 1, DNA molecular weight markers; lanes 2 and 4, S. We used a plate diffusion assay to test S. These strains belonged to either ST1, ST25, or ST28, which are known to be highly, moderately, and slightly virulent in animal models, respectively [ 19 ] unpublished data. No relationship was observed between specific STs and higher or lower susceptibility. Three independent experiments were performed. Bacteriocin activity recovered from the reversed-phase HPLC column eluted in a single peak.

An overlay of the polyacrylamide gel with the indicator strain MGGUS3 correlated bacteriocin activity with the protein band Fig. Panel A. Gel stained with Coomassie blue.

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Panel B. Gel used to detect antibacterial activity by an overlay with the indicator strain S. Lane 1: molecular weight markers; Lane 2: nisin A; Lane 3: suicin The purified suicin was subjected to various treatments, and its activity was evaluated using the spot test plate assay to determine its stability Table 2. In addition, storage at room temperature for one week did not reduce the antibacterial activity.

Extreme pHs 2 and 11 had no effect on the stability of suicin Lastly, proteolytic enzymes trypsin, chymotrypsin, proteinase K had no detectable effect on antibacterial activity. To determine its inhibitory spectrum, suicin was tested against various Gram-positive and Gram-negative bacterial species known to cause infections in swine. As shown in Table 2 , suicin was only active against S.

The MIC of suicin against S. Suicin had a MIC of 0. We used the checkerboard technique to determine whether suicin in combination with antibiotics had a synergistic or additive effect against S. Suicin in association with penicillin G or amoxicillin gave a FICI of 1, which indicates an additive effect. The ability of suicin to permeabilize the membrane of the indicator strain S.

No increase in fluorescence occurred with the negative control.

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Three assays were performed. The purified bacteriocin was derivatized with ethanethiol and was then subjected to Edman degradation. An analysis of the first ten amino acids of the purified bacteriocin yielded the following sequence: Ala 1 -Gly 2 -Ser 3 -Gly 4 -Phe 5 -Val 6 -Lys 7 -X 8 -Leu 9 -X 10 -, where the X residues are modified cysteines, serines, or threonines. G7SEC9 produced by S.

An analysis of the S. Based on the gene locus in S.

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The cluster contained nine ORFs involved in bacteriocin production. More specifically, the cluster contained ORFs for the suicin precursor suiA , a synthetase involved in lantibiotic modification suiM , an ABC transporter suiT , a response regulator suiR , a sensor histidine kinase suiK , and four immunity proteins suiF , suiE , suiG , and suiI. Based on the structural gene suiA of suicin , the inferred amino acid sequence of the leader peptide contained 24 amino acids, while that of mature unmodified peptide consisted of 33 amino acids Fig.

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Immunity peptides are known to confer specific protection against the cognate bacteriocin [ 8 ]. As expected, since the bacteriocin produced by S. Given that the bacteriocin-producing S.

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The strain was less sensitive to ampicillin with a MIC of 0. Antibiotics are commonly used in the swine industry to prevent disease and promote growth [ 3 , 4 ]. Consequently, S. Since S. One strategy to overcome this problem is to search for new antimicrobial agents. LeBel et al. Suicin 90— is a lantibiotic that shares a high degree of homology with nisin U, which is produced by Streptococcus uberis. In the present study, we identified a novel bacteriocin produced by a S. In a complex environment colonized by a wide range of bacterial species such as the respiratory tract, bacteriocins give the producing bacteria a unique advantage that helps to ensure their persistence.

On the one hand, the bacteriocin was active against all S.