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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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Circular diagram of megaplasmid pSYM1.The outer circle indicates (to scale) the genetic organization of ORFs within the plasmid. The direction of transcription of each ORF is indicated. The middle circle indicates the GC content, and the inner circle indicates the GC skew. The 10 kb region of pSYM1 that is different from pMAS2027 is marked. The image was constructed using cgview [41]. CDS = coding sequences.
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pone-0033351-g002: Circular diagram of megaplasmid pSYM1.The outer circle indicates (to scale) the genetic organization of ORFs within the plasmid. The direction of transcription of each ORF is indicated. The middle circle indicates the GC content, and the inner circle indicates the GC skew. The 10 kb region of pSYM1 that is different from pMAS2027 is marked. The image was constructed using cgview [41]. CDS = coding sequences.

Mentions: The EPEC in vitro adherence assay was then repeated with all genomotypes of the probiotic drug Symbioflor 2, which are E. coli G1/2, G3/10, G4/9, G5, G6/7 and G8. Surprisingly, we could show that E. coli G3/10 also significantly inhibited EPEC adherence efficiency (Fig. 1B) indicating a bactericidal activity of that strain. The genome of E. coli G3/10 was sequenced in our laboratory (unpublished data). However, neither during manual editing of the automatically annotated sequence nor with a BLAST analysis, could any coding sequences of a known microcin be identified within the genome of E. coli G3/10. Microcins are often encoded by plasmids [5]. E. coli G3/10 contains a large conjugative plasmid pSYM1 (Fig. 2), having a size of 50.6 kb. The plasmid is 99% identical to plasmid pMAS2027 of uropathogenic E. coli MS2027 [22]. However, it additionally contains a 10 kb insertion fragment, which carries only uncharacterized and unnamed genes. To identify the origin of E. coli G3/10s bactericidal action we first tried to cure the strain from its megaplasmid pSYM1. However, while using several common curing procedures such as treatment with mitomycin C or heat, we failed to remove pSYM1 from E. coli G3/10. Therefore, the plasmid was transferred to E. coli G4/9 by conjugation. To allow screening of conjugants, we first integrated an ampicillin resistance cassette into pSYM1 resulting in pSYM1-ST76An (Table 1). E. coli G4/9 transformed with pSYM1-ST76An was able to inhibit EPEC adherence significantly (Fig. 1C). It logically followed that plasmid pSYM1 carries genes responsible for the observed effect. Then, we cloned a 4.7 kb subfragment of plasmid pSYM1 into pBR322, resulting in plasmid pAZ6 (Table 1), which was subsequently transformed into E. coli G4/9. We demonstrated that pAZ6 enables E. coli G4/9 to inhibit EPEC adherence efficiency significantly (Fig. 1C), indicating that the 4.7 kb fragment of pSYM1 (Fig. 3) is responsible for the EPEC adherence inhibition effect of E. coli G3/10. BLAST analysis of automatically annotated open reading frames (ORFs) in this segment of pSYM1 revealed small homologies to characterized proteins or protein families belonging to microcin-encoding operons. Nevertheless, the microcin itself remained undetected. For this reason, we cloned three genes, named mcsI, mcsA and mcsB in the following, into pBR322 leading to pAZ8 (Table 1). Small ORFs upstream of this operon (Fig. 3), which were candidate microcin-encoding genes, were cloned into pACYC184 and subcloned into E. coli G4/9 pAZ8. With this strategy we successfully managed a sequential plasmid-based identification and characterization of potential microcin-coding regions together with the respective microcin-helper proteins. Only E. coli G4/9, containing plasmids pAZ8 and pAZ9 (Table 1) together, significantly inhibited EPEC adherence, whereas E. coli G4/9 pAZ8 affected EPEC adherence similar to that of E. coli G4/9 wild-type (Fig. 1C). We therefore concluded that the small gene cloned into pAZ9, which we termed mcsS, encodes a novel E. coli microcin, named microcin S in the following. Truncated mcsS (pAZ12; Table 1) resulting from an alternative ORF did not show microcin activity in the in vitro adherence assay (Fig. 1C).


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)

Circular diagram of megaplasmid pSYM1.The outer circle indicates (to scale) the genetic organization of ORFs within the plasmid. The direction of transcription of each ORF is indicated. The middle circle indicates the GC content, and the inner circle indicates the GC skew. The 10 kb region of pSYM1 that is different from pMAS2027 is marked. The image was constructed using cgview [41]. CDS = coding sequences.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316575&req=5

pone-0033351-g002: Circular diagram of megaplasmid pSYM1.The outer circle indicates (to scale) the genetic organization of ORFs within the plasmid. The direction of transcription of each ORF is indicated. The middle circle indicates the GC content, and the inner circle indicates the GC skew. The 10 kb region of pSYM1 that is different from pMAS2027 is marked. The image was constructed using cgview [41]. CDS = coding sequences.
Mentions: The EPEC in vitro adherence assay was then repeated with all genomotypes of the probiotic drug Symbioflor 2, which are E. coli G1/2, G3/10, G4/9, G5, G6/7 and G8. Surprisingly, we could show that E. coli G3/10 also significantly inhibited EPEC adherence efficiency (Fig. 1B) indicating a bactericidal activity of that strain. The genome of E. coli G3/10 was sequenced in our laboratory (unpublished data). However, neither during manual editing of the automatically annotated sequence nor with a BLAST analysis, could any coding sequences of a known microcin be identified within the genome of E. coli G3/10. Microcins are often encoded by plasmids [5]. E. coli G3/10 contains a large conjugative plasmid pSYM1 (Fig. 2), having a size of 50.6 kb. The plasmid is 99% identical to plasmid pMAS2027 of uropathogenic E. coli MS2027 [22]. However, it additionally contains a 10 kb insertion fragment, which carries only uncharacterized and unnamed genes. To identify the origin of E. coli G3/10s bactericidal action we first tried to cure the strain from its megaplasmid pSYM1. However, while using several common curing procedures such as treatment with mitomycin C or heat, we failed to remove pSYM1 from E. coli G3/10. Therefore, the plasmid was transferred to E. coli G4/9 by conjugation. To allow screening of conjugants, we first integrated an ampicillin resistance cassette into pSYM1 resulting in pSYM1-ST76An (Table 1). E. coli G4/9 transformed with pSYM1-ST76An was able to inhibit EPEC adherence significantly (Fig. 1C). It logically followed that plasmid pSYM1 carries genes responsible for the observed effect. Then, we cloned a 4.7 kb subfragment of plasmid pSYM1 into pBR322, resulting in plasmid pAZ6 (Table 1), which was subsequently transformed into E. coli G4/9. We demonstrated that pAZ6 enables E. coli G4/9 to inhibit EPEC adherence efficiency significantly (Fig. 1C), indicating that the 4.7 kb fragment of pSYM1 (Fig. 3) is responsible for the EPEC adherence inhibition effect of E. coli G3/10. BLAST analysis of automatically annotated open reading frames (ORFs) in this segment of pSYM1 revealed small homologies to characterized proteins or protein families belonging to microcin-encoding operons. Nevertheless, the microcin itself remained undetected. For this reason, we cloned three genes, named mcsI, mcsA and mcsB in the following, into pBR322 leading to pAZ8 (Table 1). Small ORFs upstream of this operon (Fig. 3), which were candidate microcin-encoding genes, were cloned into pACYC184 and subcloned into E. coli G4/9 pAZ8. With this strategy we successfully managed a sequential plasmid-based identification and characterization of potential microcin-coding regions together with the respective microcin-helper proteins. Only E. coli G4/9, containing plasmids pAZ8 and pAZ9 (Table 1) together, significantly inhibited EPEC adherence, whereas E. coli G4/9 pAZ8 affected EPEC adherence similar to that of E. coli G4/9 wild-type (Fig. 1C). We therefore concluded that the small gene cloned into pAZ9, which we termed mcsS, encodes a novel E. coli microcin, named microcin S in the following. Truncated mcsS (pAZ12; Table 1) resulting from an alternative ORF did not show microcin activity in the in vitro adherence assay (Fig. 1C).

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