This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sass, P.
Right arrow Articles by Bierbaum, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sass, P.
Right arrow Articles by Bierbaum, G.
Agricola
Right arrow Articles by Sass, P.
Right arrow Articles by Bierbaum, G.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, January 2007, p. 347-352, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01616-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Lytic Activity of Recombinant Bacteriophage {phi}11 and {phi}12 Endolysins on Whole Cells and Biofilms of Staphylococcus aureus{triangledown}

Peter Sass and Gabriele Bierbaum*

Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany

Received 12 July 2006/ Accepted 27 October 2006


arrow
ABSTRACT
 
The recombinant {phi}11 endolysin hydrolyzed heat-killed staphylococci as well as staphylococcal biofilms. Cell wall targeting appeared to be a prerequisite for lysis of whole cells, and the combined action of the endopeptidase and amidase domains was necessary for maximum activity. In contrast, the {phi}12 endolysin was inactive and caused aggregation of the cells.


arrow
INTRODUCTION
 
Infections caused by Staphylococcus aureus and Staphylococcus epidermidis still play a major role in human and animal disease. In particular staphylococcal biofilms on indwelling devices are difficult to treat due to their inherent antibiotic resistance (5, 7, 26). In this context, it is important to develop alternative treatment strategies to combat staphylococcal infections, particularly in view of the ability of staphylococci to acquire resistance to commonly used antibiotics (10, 23, 29). Phage lysins, or endolysins, have received considerable attention as possible antimicrobial agents against gram-positive bacteria and have been applied to a variety of pathogens, such as Bacillus anthracis (21), Streptococcus pneumoniae (9), and S. aureus (25). Endolysins are active against dead cells and even living, planktonic cells (9, 25), but their ability to lyse the complex structure of staphylococcal biofilms has not yet been investigated. In this approach, we cloned and heterologously overexpressed the lysis genes of the bacteriophages {phi}11 and {phi}12 of S. aureus NCTC8325 in Escherichia coli for subsequent analysis of the lytic activity of the enzymes and their single subdomains on cell walls, whole cells, and biofilms. Knowledge of the lytic activity of both endolysins is limited. Their nucleotide sequences have been published (16), and the {phi}11 endolysin has been shown to possess a D-alanyl-glycyl endopeptidase and an N-acetylmuramyl-L-alanine amidase activity on crude cell walls of S. aureus OS2 (24).


arrow
Sequence comparison, cloning, and overexpression of {phi}11 and {phi}12 endolysins.
 
The {phi}11 and {phi}12 endolysin sequences were BLAST searched against the NCBI protein database. Both endolysins are modular enzymes which consist of three distinct domains coding for an N-terminal CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain with hydrolytic function, a central amidase domain (N-acetylmuramyl-L-alanine amidase), and a C-terminal SH3b domain, which is involved in cell wall recognition (1). In spite of their similar domain architecture, these endolysins show low sequence identity (26.9%).

The endolysin genes of the bacteriophages {phi}11 (open reading frame [ORF] 53; 1,473 bp; accession number NC_004615) and {phi}12 (ORF 49; 1,455 bp; accession number NC_004616) were amplified by PCR from genomic DNA of S. aureus NCTC8325, using the primers listed in Table 1. In order to test the activity of the {phi}11 endolysin subunits, the endopeptidase unit ({phi}11endo, amino acids [aa] 1 to 180) and the amidase unit ({phi}11ami, aa 180 to 371) as well as each unit plus the cell wall binding domain ({phi}11endo/CBD, aa 1 to 180/371 to 490, and {phi}11ami/CBD, aa 180 to 490) were constructed separately (Fig. 1). In addition, the cell wall binding module was deleted from the {phi}11 endolysin ({phi}11endo/ami, aa 1 to 371). The amplification products were cloned into the multiple cloning site of the expression vector pET22b (Novagen) without the pelB leader tag to inhibit protein transport to the periplasm of the expression host. The resulting plasmids, pETer{Delta}11, pETer{Delta}12, pETendo11, pETendoCBD11, pETami11, pETamiCBD11, and pETendo/ami11, were used to overexpress each endolysin as a C-terminal six-His-tagged fusion protein. After subcloning of the plasmids in E. coli JM109, E. coli BL21(DE3) was used as a host for expression of each six-His-tagged endolysin. Expression cultures were grown in Luria-Bertani (LB) broth containing ampicillin (40 µg/ml) to an optical density at 600 nm (OD600) of 0.6. Then protein expression was induced by addition of isopropyl-ß-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. Expression cultures were harvested after 4 h followed by protein purification steps under native conditions via nickel-nitrilotriacetic acid affinity chromatography (Fig. 2). Protein purification was also performed with cells harboring the empty vector, and the eluate served as a control in the activity tests.


View this table:
[in this window]
[in a new window]

  		TABLE 1. Bacterial strains, plasmids, and primers used in this study


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		FIG. 1. Recombinant murein hydrolases of the staphylococcal bacteriophage {phi}11. A. Schematic overview of the {phi}11 endolysin modules illustrating the constructs tested in this study. The {phi}11 endolysin features a modular design which consists of an N-terminal endopeptidase domain (endo), a central amidase domain (ami), and a C-terminal cell wall binding domain (CBD). The full-length and deletion constructs of the {phi}11 endolysin were overexpressed as six-His fusion proteins. B. Lytic activity of the staphylococcal bacteriophage {phi}11 endolysin domains.


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		FIG. 2. SDS-polyacrylamide gel electrophoresis analysis of {phi}12 and {phi}11 endolysins and derived proteins purified by nickel-nitrilotriacetic acid affinity chromatography. The purified proteins were analyzed by 15% SDS-polyacrylamide gel electrophoresis and stained with PageBlue protein staining solution (Fermentas) according to the manufacturer's instructions. Lane M1, Fermentas Page Ruler unstained protein ladder; lanes M2, Fermentas prestained protein molecular weight marker; lane 1, {phi}12 endolysin (full length); lane 2, {phi}11 endolysin (full length); lane 3, {phi}11endo/ami; lane 4, {phi}11endo/CBD; lane 5, {phi}11ami/CBD; lane 6, {phi}11endo; lane 7, {phi}11ami.


arrow
Lytic activity of {phi}11 endolysin modules.
 
The lytic activities of the full-length {phi}11 endolysin and its deletion variants (each applied in a concentration of 20 µg/ml) were examined photometrically at 600 nm employing Staphylococcus simulans 22 cell walls (purified with sodium dodecyl sulfate [SDS]) (4) resuspended in incubation buffer (50 mM Tris-HCl, 100 mM NaCl; pH 7.5) to an OD600 of ~0.3. All lysis experiments were performed in triplicate. The full-length enzyme showed efficient lysis of the peptidoglycan compared to the control (empty-vector eluate), and both the endopeptidase and the amidase module plus cell wall binding domain ({phi}11endo/CBD and {phi}11ami/CBD) were active and able to lyse cell walls. However, the full-length enzyme was more active than the isolated subdomains, and only the combination of {phi}11endo/CBD and {phi}11ami/CBD (20 µg/ml each) restored full lytic activity (Fig. 3). The effect of the truncation was even more pronounced with whole cells. To this end, cells of S. aureus NCTC8325 were grown overnight, diluted in incubation buffer to an OD600 of approximately 0.3, and pasteurized for 10 min at 80°C. After addition of equivalent amounts of endolysin (20 µg/ml), the lytic activity was determined as before. In contrast to the full-length protein (Fig. 4), the isolated amidase or endopeptidase domains were hardly active against heat-killed cells (data not shown). In conclusion, the two catalytic domains of the enzyme have to be combined with each other to cleave the cell wall of intact cells. Thus, the {phi}11 endolysin seems to belong to a group of endolysins which act as multifunctional hydrolases (2). Recently, the lytic enzyme of phage {phi}WMY of Staphylococcus warneri M (LysWMY) was reported to show strong similarities in its domain architecture to the {phi}11 endolysin (35). In contrast to the {phi}11 endolysin, LysWMY retained its full activity even when both the amidase and cell wall binding domains had been deleted. This result indicates that combined action of different domains does not seem to be an obligatory characteristic even among closely related murein hydrolases.


Figure 3
View larger version (21K):
[in this window]
[in a new window]

  		FIG. 3. Activity of the domains of the multifunctional {phi}11 endolysin. The lytic activities of purified enzyme samples of the full-length and truncated {phi}11 endolysin (20 µg/ml each) were examined using purified cell walls of S. simulans 22. The full-length enzyme ({blacktriangleup}) was able to lyse the substrate efficiently compared to the protein eluate of the empty-vector control (•). The truncated enzymes {phi}11endo ({square}) and {phi}11ami ({diamond}) did not show mentionable lytic activity. The truncated enzymes containing the cell wall binding domain, {phi}11endo/CBD ({blacksquare}) and {phi}11ami/CBD ({blacklozenge}), showed enhanced lytic activity but still worked less effectively than the full-length enzyme. The lytic activity could be restored by the parallel use of {phi}11endo/CBD and {phi}11ami/CBD ({triangleup}).


Figure 4
View larger version (24K):
[in this window]
[in a new window]

  		FIG. 4. The {phi}11 endolysin lyses whole cells of several staphylococci. Light scattering data showed that the {phi}11 endolysin ({blacktriangleup}; 20 µg/ml) rapidly decreased the optical density of heat-inactivated S. aureus NCTC8325, S. aureus Wood 46, S. aureus Cowan 1, S. aureus Newman, S. epidermidis O-47, and S. simulans 22 cultures compared to the protein eluate of the empty-vector control (•). Its lytic activity was comparable to the effect observed after addition of lysostaphin ({blacksquare}; 5 µg/ml), which is a highly efficient glycyl-glycine endopeptidase. After deletion of the cell wall targeting domain, the {phi}11 enzyme ({phi}11endo/ami, {triangleup}; 20 µg/ml) showed a significantly reduced activity, which indicates a fundamental role of the cell wall binding domain in efficient cell lysis.

Purified cell walls were a much more sensitive substrate for the enzymes than heat-killed cells were, since they showed the residual activities of the single subdomains much more clearly. In spite of this, the {phi}12 endolysin did not show any activity with S. simulans 22 cell walls (data not shown).


arrow
Substrate recognition is essential for full lytic activity.
 
Substrate recognition mediated by the cell wall binding domain, which is homologous to the C-terminal domain of lysostaphin (20), appeared to be necessary for high catalytic efficiency. Lytic activity of the {phi}11 endolysin on heat-killed staphylococcal cells was abolished after deletion of the cell wall binding domain ({phi}11endo/ami) (Fig. 4), while it retained nearly all of its lytic activity on SDS cell walls (data not shown). The single domains, {phi}11endo and {phi}11ami, showed significantly reduced lytic activity on SDS cell walls in the absence of the cell wall binding domain (Fig. 3). These findings are similar to the results obtained with lysostaphin and ALE-1, a glycyl-glycine endopeptidase homologous to lysostaphin, which showed a significant reduction in lytic activity on autoclaved staphylococci or viable cells, respectively, after deletion of their C-terminal cell wall targeting domains (1, 22), which bind to cross-linked peptidoglycan and recognize the Gly5 interpeptide cross bridge (12, 22). Our results suggest that a similar targeting mechanism exists for the {phi}11 endolysin and that, in this respect, the phage enzyme closely resembles its staphylococcal counterparts.


arrow
Activities of the endolysins with different staphylococcal strains.
 
We next analyzed the lytic activities of the {phi}11 and {phi}12 endolysins on whole cells of several staphylococcal strains. In addition to S. aureus NCTC8325, the full-length {phi}11 endolysin rapidly lysed heat-killed cells of S. aureus Wood 46, S. aureus Cowan 1, S. aureus Newman, S. epidermidis O-47, and S. simulans 22 (Fig. 4). Its lytic activity was equivalent to that of highly purified lysostaphin (5 µg/ml) (Dr. Petry Genmedics GmbH, Reutlingen, Germany).

Unlike {phi}11, the {phi}12 endolysin was not able to hydrolyze heat-killed cells of S. aureus Wood 46, S. aureus Cowan 1, or S. aureus Newman. However, after addition of the {phi}12 endolysin an increase in optical density of the cultures was observed (Fig. 5). This was accompanied and most probably caused by an aggregation of the cells that was macroscopically visible. This phenomenon was earlier described by Takano et al. (31), who investigated the influence of synthetic peptides derived from the S. aureus major autolysin Atl on autolysis. Sequence comparison with the homologous amidase-3 domains of the S. aureus bacteriophages {phi}SA 2MW, L54a, {phi}SLT, PVL, 96, 3a, 53, 77, ROSA, and {phi}ETA and amidases of Staphylococcus haemolyticus JCSC1435 and Staphylococcus epidermidis RP62a identified an amino acid exchange at position 260 in the amidase-3 domain of the {phi}12 endolysin. This exchange introduces a histidine and therefore an additional positive charge in a position that is occupied by glutamine, glutamate, or asparagine in the other staphylococcal enzymes. Whether this amino acid exchange or an inadequate folding of the His-tagged protein is involved in the loss of hydrolytic activity is part of current investigations. Most probably, the {phi}12 endolysin in our test system is unable to exert efficient hydrolytic activity on staphylococcal murein, while retaining its ability to bind the cell wall. The substrate binding ability of the enzyme may then lead to the enhanced adhesion between the cells.


Figure 5
View larger version (11K):
[in this window]
[in a new window]

  		FIG. 5. Increase of optical density after addition of {phi}12 endolysin. Unlike the {phi}11 enzyme, the {phi}12 endolysin was not able to lyse heat-killed cells of any tested staphylococcal strain. Upon addition of {phi}12 endolysin (15 µg/ml) we detected an obvious increase of optical density of S. aureus Wood 46 (•), S. aureus Cowan 1 (x), and S. aureus Newman ({blacktriangleup}). The protein eluate of empty-vector control ({blacklozenge}) showed no effect, whereas the control with lysostaphin ({blacksquare}; 5 µg/ml) lysed all staphylococcal strains.


arrow
{phi}11 endolysin eliminates S. aureus NCTC8325 biofilm on artificial surfaces.
 
Finally, we determined the influence of the {phi}11 and {phi}12 endolysins on staphylococcal biofilms by a modified biofilm plate assay (33). To this end, an overnight culture was diluted 1:200 in tryptic soy broth medium supplemented with 0.25% D-(+)-glucose to a final volume of 200 µl in each well of a 96-well polystyrene microtiter plate (Nunc, Wiesbaden, Germany). The plates were incubated for 24 h or 48 h at 37°C under aerobic and O2-limited conditions, respectively. Afterwards, the wells were washed twice with incubation buffer. The biofilm-containing wells were then filled with incubation buffer plus endolysin. Fresh medium and incubation buffer alone or incubation buffer plus lysostaphin or lysozyme was employed as a control. Lysostaphin was used as a positive control, since it is able to lyse S. aureus and S. epidermidis biofilms (33). Following incubation for 2 hours at 37°C, the wells were washed again and stained with 0.1% safranin.

We found that the {phi}12 endolysin showed no hydrolytic activity on biofilms of S. aureus NCTC8325 and S. epidermidis O-47. In contrast, after addition of the {phi}12 endolysin we noticed an enhanced staining of the biofilms (Fig. 6). This effect is consistent with the results of the experiments performed with heat-killed cells and may be explained by a murein hydrolase-mediated adhesion of staphylococci (14, 15). In contrast, the purified {phi}11 endolysin was able to eliminate biofilms of S. aureus NCTC8325, with an efficiency that was comparable to or slightly lower than that of lysostaphin (Fig. 6). However, there was no effect on biofilms of S. epidermidis O-47. To our knowledge, no phage lysin has been reported so far to disrupt staphylococcal biofilms.


Figure 6
View larger version (48K):
[in this window]
[in a new window]

  		FIG. 6. Biofilm plate assay of S. aureus NCTC8325. The {phi}11 endolysin lysed biofilms of S. aureus NCTC8325 with an efficiency comparable to that of lysostaphin. In contrast, addition of the {phi}12 endolysin resulted in enhanced staining of the cells. TSB, tryptic soy broth.

Bacterial biofilm formation is part of a survival strategy to resist suboptimal environmental conditions such as limited nutrient availability or lethal concentrations of antibiotics. Antimicrobial agents often show significantly reduced effects on biofilms, which is thought to be due to several biofilm-inherent properties. For example, a low growth rate impedes the action of antibiotics (8, 29), and evidence is emerging that the sessile cells in biofilms live in an altered metabolic state (3, 27). Furthermore, the diffusion velocity of antibiotics is limited within biofilms. A reduced rate of antibiotic penetration leads to a gradually increasing concentration of the antibiotic in the deeper layers of a biofilm, which permits adaptation of the biofilm cells to the antibiotic by stress-induced metabolic and transcriptional changes (17). The {phi}11 endolysin most probably destabilizes the biofilm structure by fast lysis of sessile cells, which are embedded in the extracellular matrix or at the interface of the matrix and surface, whereupon the biofilm is dissolved. Another possibility could be an influence of the intrinsic nature of the biofilm. Neither sodium metaperiodate nor proteinase K treatment could completely eradicate biofilms of S. aureus NCTC8325 grown in tryptic soy broth (data not shown), which suggests that an elevated portion of proteinogenous biofilm is present along with a biofilm mediated by the characteristic polysaccharide (PIA). PIA consists of ß-1,6-linked N-acetylglucosamine residues (11) and is not the target of the {phi}11 murein hydrolase. In contrast to S. aureus NCTC8325, S. epidermidis O-47 biofilms are exclusively polysaccharide mediated. Therefore, different natures of the biofilm matrices tested here could be an explanation for the varying efficacy of the {phi}11 endolysin.

Considering the huge clinical relevance of staphylococcal biofilms in terms of human diseases, the {phi}11 endolysin may constitute a novel strategy to combat S. aureus nosocomial infections that are mediated by biofilm formation on medical devices.


arrow
ACKNOWLEDGMENTS
 
This work was supported by a grant of the Competence Network PathoGenoMik (101.099-1/04-I.1.03) funded by the Bundesministerium für Bildung und Forschung of Germany.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Phone: (49)228-287-1-9103. Fax: (49)228-287-1-4808. E-mail: bierbaum{at}mibi03.meb.uni-bonn.de. Back

{triangledown} Published ahead of print on 3 November 2006. Back


arrow
REFERENCES
 
    1
  1. Baba, T., and O. Schneewind. 1996. Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus. EMBO J. 15:4789-4797.[Medline]
    2. 2
  2. Bateman, A., and N. D. Rawlings. 2003. The CHAP domain: a large family of amidases including GSP amidases and peptidoglycan hydrolases. Trends Biochem. Sci. 28:234-237.[CrossRef][Medline]
    3. 3
  3. Beenken, K. E., P. M. Dunman, F. McAleese, D. Macapagal, E. Murphy, S. J. Projan, J. V. Blevins, and M. S. Smeltzer. 2004. Global gene expression in Staphylococcus aureus biofilms. J. Bacteriol. 186:4665-4684.[Abstract/Free Full Text]
    4. 4
  4. Bierbaum, G., and H. G. Sahl. 1987. Autolytic system of Staphylococcus simulans 22: influence of cationic peptides on activity of N-acetylmuramoyl-L-alanine amidase. J. Bacteriol. 169:5452-5458.[Abstract/Free Full Text]
    5. 5
  5. Costerton, J. W., K. J. Cheng, G. G. Geesey, T. I. Ladd, J. C. Nickel, M. Dasgupta, and T. J. Marrie. 1987. Bacterial biofilms in nature and disease. Annu. Rev. Microbiol. 41:435-464.[CrossRef][Medline]
    6. 6
  6. Delmi, M., P. Vaudaux, D. P. Lew, and H. Vasey. 1994. Role of fibronectin in staphylococcal adhesion to metallic surfaces used as models of orthopaedic devices. J. Orthop. Res. 12:432-438.[CrossRef][Medline]
    7. 7
  7. Donlan, R. M., and J. W. Costerton. 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15:167-193.[Abstract/Free Full Text]
    8. 8
  8. Duguid, I. G., E. Evans, M. R. Brown, and P. Gilbert. 1992. Growth-rate-independent killing by ciprofloxacin of biofilm-derived Staphylococcus epidermidis; evidence for cell-cycle dependency. J. Antimicrob. Chemother. 30:791-802.[Abstract/Free Full Text]
    9. 9
  9. Entenza, J. M., J. M. Loeffler, D. Grandgirard, V. A. Fischetti, and P. Moreillon. 2005. Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae endocarditis in rats. Antimicrob. Agents Chemother. 49:4789-4792.[Abstract/Free Full Text]
    10. 10
  10. Geisel, R., F. J. Schmitz, A. C. Fluit, and H. Labischinski. 2001. Emergence, mechanism, and clinical implications of reduced glycopeptide susceptibility in Staphylococcus aureus. Eur. J. Clin. Microbiol. Infect. Dis. 20:685-697.[CrossRef][Medline]
    11. 11
  11. Götz, F. 2002. Staphylococcus and biofilms. Mol. Microbiol. 43:1367-1378.[CrossRef][Medline]
    12. 12
  12. Gründling, A., and O. Schneewind. 2006. Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus. J. Bacteriol. 188:2463-2472.[Abstract/Free Full Text]
    13. 13
  13. Heilmann, C., C. Gerke, F. Perdreau-Remington, and F. Götz. 1996. Characterization of Tn917 insertion mutants of Staphylococcus epidermidis affected in biofilm formation. Infect. Immun. 64:227-282.
    14. 14
  14. Heilmann, C., G. Thumm, G. S. Chhatwal, J. Hartleib, A. Uekotter, and G. Peters. 2003. Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis. Microbiology 149:2769-2778.[Abstract/Free Full Text]
    15. 15
  15. Heilmann, C., M. Hussain, G. Peters, and F. Götz. 1997. Evidence for autolysin-mediated primary attachment of Staphylococcus epidermidis to a polystyrene surface. Mol. Microbiol. 24:1013-1024.[CrossRef][Medline]
    16. 16
  16. Iandolo, J. J., V. Worrell, K. H. Groicher, Y. Qian, R. Tian, S. Kenton, A. Dorman, H. Ji, S. Lin, P. Loh, S. Qi, H. Zhu, and B. A. Roe. 2002. Comparative analysis of the genomes of the temperate bacteriophages phi 11, phi 12 and phi 13 of Staphylococcus aureus 8325. Gene 289:109-118.[CrossRef][Medline]
    17. 17
  17. Jefferson, K. K., D. A. Goldmann, and G. B. Pier. 2005. Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms. Antimicrob. Agents Chemother. 49:2467-2473.[Abstract/Free Full Text]
    18. 18
  18. Juuti, K. M., B. Sinha, C. Werbick, G. Peters, and P. I. Kuusela. 2004. Reduced adherence and host cell invasion by methicillin-resistant Staphylococcus aureus expressing the surface protein Pls. J. Infect. Dis. 189:1574-1584.[CrossRef][Medline]
    19. 19
  19. Kloos, W. E., and K. H. Schleifer. 1975. Isolation and characterization of staphylococci from human skin. II. Descriptions of four new species: Staphylococcus warneri, Staphylococcus capitis, Staphylococcus hominis, and Staphylococcus simulans. Int. J. Syst. Bacteriol. 25:62-79.[Abstract/Free Full Text]
    20. 20
  20. Loessner, M. J., S. Gaeng, G. Wendlinger, S. K. Maier, and S. Scherer. 1998. The two-component lysis system of Staphylococcus aureus bacteriophage Twort: a large TTG-start holin and an associated amidase endolysin. FEMS Microbiol. Lett. 162:265-274.[CrossRef][Medline]
    21. 21
  21. Low, L. Y., C. Yang, M. Perego, A. Osterman, and R. C. Liddington. 2005. Structure and lytic activity of a Bacillus anthracis prophage endolysin. J. Biol. Chem. 280:35433-35439.[Abstract/Free Full Text]
    22. 22
  22. Lu, J. Z., T. Fujiwara, H. Komatsuzawa, M. Sugai, and J. Sakon. 2006. Cell wall-targeting domain of glycylglycine endopeptidase distinguishes among peptidoglycan cross-bridges. J. Biol. Chem. 281:549-558.[Abstract/Free Full Text]
    23. 23
  23. Mah, T. F., and G. A. O'Toole. 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 9:34-39.[CrossRef][Medline]
    24. 24
  24. Navarre, W. W., H. Ton-That, K. F. Faull, and O. Schneewind. 1999. Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity. J. Biol. Chem. 274:15847-15856.[Abstract/Free Full Text]
    25. 25
  25. O'Flaherty, S., A. Coffey, W. Meaney, G. F. Fitzgerald, and R. P. Ross. 2005. The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus. J. Bacteriol. 187:7161-7164.[Abstract/Free Full Text]
    26. 26
  26. O'Grady, N. P., M. Alexander, E. P. Dellinger, J. L. Gerberding, S. O. Heard, D. G. Maki, H. Masur, R. D. McCormick, L. A. Mermel, M. L. Pearson, I. I. Raad, A. Randolph, R. A. Weinstein, et al. 2002. Guidelines for the prevention of intravascular catheter-related infections. Pediatrics 110:e51.[Abstract/Free Full Text]
    27. 27
  27. Resch, A., R. Rosenstein, C. Nerz, and F. Götz. 2005. Differential gene expression profiling of Staphylococcus aureus cultivated under biofilm and planktonic conditions. Appl. Environ. Microbiol. 71:2663-2676.[Abstract/Free Full Text]
    28. 28
  28. Rupp, M. E., P. D. Fey, C. Heilmann, and F. Götz. 2001. Characterization of the importance of Staphylococcus epidermidis autolysin and polysaccharide intercellular adhesin in the pathogenesis of intravascular catheter-associated infection in a rat model. J. Infect. Dis. 183:1038-1042.[CrossRef][Medline]
    29. 29
  29. Stewart, P. S. 2002. Mechanisms of antibiotic resistance in bacterial biofilms. Int. J. Med. Microbiol. 292:107-113.[CrossRef][Medline]
    30. 30
  30. Studier, F. W., and B. A. Moffatt. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189:113-130.[CrossRef][Medline]
    31. 31
  31. Takano, M., T. Oshida, A. Yasojima, M. Yamada, C. Okagaki, M. Sugai, H. Suginaka, and T. Matsushita. 2000. Modification of autolysis by synthetic peptides derived from the presumptive binding domain of Staphylococcus aureus autolysin. Microbiol. Immunol. 44:463-472.[Medline]
    32. 32
  32. Wolz, C., D. McDevitt, T. J. Foster, and A. L. Cheung. 1996. Influence of agr on fibrinogen binding in Staphylococcus aureus Newman. Infect. Immun. 64:3142-3147.[Abstract]
    33. 33
  33. Wu, J. A., C. Kusuma, J. J. Mond, and J. F. Kokai-Kun. 2003. Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces. Antimicrob. Agents Chemother. 47:3407-3414.[Abstract/Free Full Text]
    34. 34
  34. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119.[CrossRef][Medline]
    35. 35
  35. Yokoi, K. J., N. Kawahigashi, M. Uchida, K. Sugahara, M. Shinohara, K. Kawasaki, S. Nakamura, A. Taketo, and K. Kodaira. 2005. The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage {phi}WMY: cloning, sequencing, expression, and mutational analysis in Escherichia coli. Gene 351:97-108.[CrossRef][Medline]

Applied and Environmental Microbiology, January 2007, p. 347-352, Vol. 73, No. 1
0099-2240/07/$08.00+0     doi:10.1128/AEM.01616-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Horgan, M., O'Flynn, G., Garry, J., Cooney, J., Coffey, A., Fitzgerald, G. F., Ross, R. P., McAuliffe, O. (2009). Phage Lysin LysK Can Be Truncated to Its CHAP Domain and Retain Lytic Activity against Live Antibiotic-Resistant Staphylococci. Appl. Environ. Microbiol. 75: 872-874 [Abstract] [Full Text]  
  • Mayer, M. J., Narbad, A., Gasson, M. J. (2008). Molecular Characterization of a Clostridium difficile Bacteriophage and Its Cloned Biologically Active Endolysin. J. Bacteriol. 190: 6734-6740 [Abstract] [Full Text]  
  • Tallent, S. M., Langston, T. B., Moran, R. G., Christie, G. E. (2007). Transducing Particles of Staphylococcus aureus Pathogenicity Island SaPI1 Are Comprised of Helper Phage-Encoded Proteins. J. Bacteriol. 189: 7520-7524 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sass, P.
Right arrow Articles by Bierbaum, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sass, P.
Right arrow Articles by Bierbaum, G.
Agricola
Right arrow Articles by Sass, P.
Right arrow Articles by Bierbaum, G.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»