Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A

Gargis, Shaw R.; Heath, Harry E.; Heath, Lucie S.; LeBlanc, Paul A.; Simmonds, Robin S.; Abbott, Brian; Timkovich, Russell; Sloan, Gary L.

doi:10.1128/aem.01647-08

Cited by 26 publications

(29 citation statements)

References 28 publications

(25 reference statements)

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“…The zoocin A bacteriocin, which is produced by some strains of Streptococcus equi subsp. zooepidemicus, is a D-alanyl-L-alanine endopeptidase that hydrolyzes the peptidoglycan cross bridges of susceptible streptococci (9,10,47). In the S. equi strains carrying the zooA gene, the gene is always accompanied by a neighboring immunity gene termed zif.…”

Section: Discussionmentioning

confidence: 99%

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

2012

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Streptococcus pneumoniae and probably most other members of the genus Streptococcus are competent for natural genetic transformation. During the competent state, S. pneumoniae produces a murein hydrolase, CbpD, that kills and lyses noncompetent pneumococci and closely related species. Previous studies have shown that CbpD is essential for efficient transfer of genomic DNA from noncompetent to competent cells in vitro. Consequently, it has been proposed that CbpD together with the cognate immunity protein ComM constitutes a DNA acquisition mechanism that enables competent pneumococci to capture homologous DNA from closely related streptococci sharing the same habitat. Although genes encoding CbpD homologs or CbpD-related proteins are present in many different streptococcal species, the genomes of a number of streptococci do not encode CbpD-type proteins. In the present study we show that the genomes of nearly all species lacking CbpD encode an unrelated competence-regulated murein hydrolase termed LytF. Using Streptococcus gordonii as a model system, we obtained evidence indicating that LytF is a functional analogue of CbpD. In sum, our results show that a murein hydrolase gene is part of the competence regulon of most or all streptococcal species, demonstrating that these muralytic enzymes constitute an essential part of the streptococcal natural transformation system.

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Section: Discussionmentioning

confidence: 99%

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

2012

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“…Zoocin A is a bacteriolytic endopeptidase against the cell wall of sensitive bacteria produced by Streptococcus equi subsp. zooepidemicus strain 4881 (57). The cross bridge in peptidoglycan of S. equi is an L-Ala-L-Ala peptide and is susceptible to the peptidoglycan hydrolase activity of zoocin A (57).…”

Section: Discussionmentioning

confidence: 99%

Bacteriocin Protein BacL₁of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala₂-Cross-Bridged Peptidoglycan

et al. 2014

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c Bacteriocin 41 (Bac41) is produced from clinical isolates of Enterococcus faecalis and consists of two extracellular proteins, BacL 1 and BacA. We previously reported that BacL 1 protein (595 amino acids, 64.5 kDa) is a bacteriolytic peptidoglycan D-isoglutamyl-L-lysine endopeptidase that induces cell lysis of E. faecalis when an accessory factor, BacA, is copresent. However, the target of BacL 1 remains unknown. In this study, we investigated the targeting specificity of BacL 1 . Fluorescence microscopy analysis using fluorescent dye-conjugated recombinant protein demonstrated that BacL 1 specifically localized at the cell divisionassociated site, including the equatorial ring, division septum, and nascent cell wall, on the cell surface of target E. faecalis cells. E nterococcus faecalis is a commensal Gram-positive bacterium in the intestinal tract of healthy humans or animals and is also known to be an opportunistic pathogen causing various infectious diseases, including urinary infectious disease, bacteremia, infective endocarditis, and others (1-3). The infection-derived E. faecalis strains often produce various plasmid-encoded bacteriocins (4, 5).Bacteriocins are bacterial peptides or proteins with antimicrobial activities (6). Heat-and acid-stable bacteriocin peptides produced by Gram-positive bacteria are divided into class I and class II according to posttranslational modifications (7,8). Class I bacteriocins are lantibiotics that contain nonproteinogenic amino acids generated by posttranslational modification (9). Only two class I bacteriocins have been identified in enterococci: ␤-hemolysin/bacteriocin (cytolysin) and enterocin W (10-14). In contrast, most enterococcal bacteriocins belong to class II and are nonmodified antimicrobial peptides, such as AS-48, enterocin A, and others (7, 15, 16). We have found the enterococcal class II bacteriocins, including Bac21, Bac31, Bac32, Bac43, and Bac51, in clinical strains of E. faecalis or Enterococcus faecium (17-21). Unlike the low-molecular-weight peptide-type class I and II bacteriocins, heat-labile antimicrobial proteins are referred to as bacteriolysins, previously named class III bacteriocins, and show enzymatic bactericidal activity (22,23). In enterococci, the bacteriolysins enterolysin A and bacteriocin 41 (Bac41) have been identified (24-26).Bac41 was originally found expressed from the pheromoneresponsive plasmid pYI14 carried by the clinical strain E. faecalis YI14 (26,27). The Bac41-type bacteriocins were also found in the E. faecalis VanB-type vancomycin-resistant E. faecalis (VRE) outbreak strains (27). Bac41 is specifically active only against E. faecalis (26, 28). The determinant region of Bac41 contains six open reading frames (ORFs), including bacL 1 , bacL 2 , bacA, and bacI (Fig. 1A). The bactericidal activity of Bac41 is actually expressed by the two extracellular components, the bacL 1 -and bacA-encoded proteins BacL 1 and BacA (26). BacL 1 and BacA are secreted proteins that coordinately exert bactericidal activity against E. faeca...

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“…Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6,9,15,25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A.…”

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“…zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18).…”

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confidence: 99%

“…After incubation, the sample was boiled for 5 min and subjected to centrifugation to remove any undigested material. The supernatant was reduced with sodium borohydride as described previously (12). The resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography (RP-HPLC) with an HPLC system from Shimadzu (Columbia, MD) and a C 18 column (150 by 3 mm [internal diameter]; 5-m particle size [Waters, Milford, MA]) using a 0 to 25% solution B gradient applied from 5 to 90 min (0.5 ml/min; solution A, 0.1% trifluoroacetic acid [TFA] in water; solution B, 0.085% TFA in 80% acetonitrile) at room temperature.…”

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Zif, the Zoocin A Immunity Factor, Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Gargis

Heath

et al. 2009

Appl Environ Microbiol

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Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a D-alanyl-L-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three L-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.

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Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A

Cited by 26 publications

References 28 publications

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

Bacteriocin Protein BacL₁of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala₂-Cross-Bridged Peptidoglycan

Zif, the Zoocin A Immunity Factor, Is a FemABX-Like Immunity Protein with a Novel Mode of Action

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Use of 4-Sulfophenyl Isothiocyanate Labeling and Mass Spectrometry To Determine the Site of Action of the Streptococcolytic Peptidoglycan Hydrolase Zoocin A

Cited by 26 publications

References 28 publications

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

LytF, a Novel Competence-Regulated Murein Hydrolase in the Genus Streptococcus

Bacteriocin Protein BacL1of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala2-Cross-Bridged Peptidoglycan

Zif, the Zoocin A Immunity Factor, Is a FemABX-Like Immunity Protein with a Novel Mode of Action

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Bacteriocin Protein BacL₁of Enterococcus faecalis Targets Cell Division Loci and Specifically Recognizes l-Ala₂-Cross-Bridged Peptidoglycan