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Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10.

Zschüttig A, Zimmermann K, Blom J, Goesmann A, Pöhlmann C, Gunzer F - PLoS ONE (2012)

Bottom Line: Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S.Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay.Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Dresden, Germany.

ABSTRACT
Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.

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Growth curve of E. coli MDS42 with pAZ15 or pGS1 showing A600(A) and colony count of viable cells (B).pAZ15 is a vector containing mcsS under control of the L-arabinose-induced araC PBAD activator-promoter. Induction with L-arabinose of the respective cultures, as indicated in the legend to panel A and B, was carried out after 90 minutes. pGS1 is the empty vector with the araC PBAD activator-promoter. E. coli MDS42 with pAZ15 shows a significant reduction of its A600 as well as of its colony counts after induction with L-arabinose (red squares) compared to growth without induction (green triangles) and also with the vector devoid of mcsS (pGS1, blue rhombi). Data are the mean±SD of three independent experiments.
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pone-0033351-g005: Growth curve of E. coli MDS42 with pAZ15 or pGS1 showing A600(A) and colony count of viable cells (B).pAZ15 is a vector containing mcsS under control of the L-arabinose-induced araC PBAD activator-promoter. Induction with L-arabinose of the respective cultures, as indicated in the legend to panel A and B, was carried out after 90 minutes. pGS1 is the empty vector with the araC PBAD activator-promoter. E. coli MDS42 with pAZ15 shows a significant reduction of its A600 as well as of its colony counts after induction with L-arabinose (red squares) compared to growth without induction (green triangles) and also with the vector devoid of mcsS (pGS1, blue rhombi). Data are the mean±SD of three independent experiments.

Mentions: In order to directly demonstrate microcin S activity against a susceptible E. coli strain, mcsS was cloned into pGS1 resulting in the plasmid pAZ15 (Table 1). In this construct, mcsS expression is controlled by an araC PBAD activator-promoter, rendering microcin S expression inducible by L-arabinose. When E coli MDS42 [23] growing in liquid culture after being transformed with pAZ15 was treated with 0.2% v/v L-arabinose at the beginning of the logarithmic phase after 1.5 h, A600 remained almost stable around 0.3 while absorbance of control cultures increased constantly (Fig. 5A). Counts of colony forming units (CFU) taken at various time points during this experiment revealed a sharp drop in the number of viable bacteria in the L-arabinose induced culture of E. coli MDS42+pAZ15 while CFU of the other cultures rose to a maximum of 1.4 × 109/ ml (Fig. 5B).


Identification and characterization of microcin S, a new antibacterial peptide produced by probiotic Escherichia coli G3/10.

Zschüttig A, Zimmermann K, Blom J, Goesmann A, Pöhlmann C, Gunzer F - PLoS ONE (2012)

Growth curve of E. coli MDS42 with pAZ15 or pGS1 showing A600(A) and colony count of viable cells (B).pAZ15 is a vector containing mcsS under control of the L-arabinose-induced araC PBAD activator-promoter. Induction with L-arabinose of the respective cultures, as indicated in the legend to panel A and B, was carried out after 90 minutes. pGS1 is the empty vector with the araC PBAD activator-promoter. E. coli MDS42 with pAZ15 shows a significant reduction of its A600 as well as of its colony counts after induction with L-arabinose (red squares) compared to growth without induction (green triangles) and also with the vector devoid of mcsS (pGS1, blue rhombi). Data are the mean±SD of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316575&req=5

pone-0033351-g005: Growth curve of E. coli MDS42 with pAZ15 or pGS1 showing A600(A) and colony count of viable cells (B).pAZ15 is a vector containing mcsS under control of the L-arabinose-induced araC PBAD activator-promoter. Induction with L-arabinose of the respective cultures, as indicated in the legend to panel A and B, was carried out after 90 minutes. pGS1 is the empty vector with the araC PBAD activator-promoter. E. coli MDS42 with pAZ15 shows a significant reduction of its A600 as well as of its colony counts after induction with L-arabinose (red squares) compared to growth without induction (green triangles) and also with the vector devoid of mcsS (pGS1, blue rhombi). Data are the mean±SD of three independent experiments.
Mentions: In order to directly demonstrate microcin S activity against a susceptible E. coli strain, mcsS was cloned into pGS1 resulting in the plasmid pAZ15 (Table 1). In this construct, mcsS expression is controlled by an araC PBAD activator-promoter, rendering microcin S expression inducible by L-arabinose. When E coli MDS42 [23] growing in liquid culture after being transformed with pAZ15 was treated with 0.2% v/v L-arabinose at the beginning of the logarithmic phase after 1.5 h, A600 remained almost stable around 0.3 while absorbance of control cultures increased constantly (Fig. 5A). Counts of colony forming units (CFU) taken at various time points during this experiment revealed a sharp drop in the number of viable bacteria in the L-arabinose induced culture of E. coli MDS42+pAZ15 while CFU of the other cultures rose to a maximum of 1.4 × 109/ ml (Fig. 5B).

Bottom Line: Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S.Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay.Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology and Hygiene, TU Dresden, Dresden, Germany.

ABSTRACT
Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10.

Show MeSH
Related in: MedlinePlus