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1.
David M. Donovan Shengli Dong Wes Garrett Genevive M. Rousseau Sylvain Moineau David G. Pritchard 《Applied microbiology》2006,72(4):2988-2996
The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63°C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections. 相似文献
2.
LambdaSa2 prophage endolysin requires Cpl-7-binding domains and amidase-5 domain for antimicrobial lysis of streptococci 总被引:1,自引:0,他引:1
Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of multidrug-resistant streptococci. Thus, there is a need for new antimicrobials. Bacteriophage endolysins (peptidoglycan hydrolases) comprise one group of new antimicrobials that are reportedly refractory to resistance development. The LambdaSa2 prophage endolysin gene was recently isolated from a Group B streptococcal genome, expressed on an Escherichia coli plasmid, and shown by homology screening and biochemical analysis to harbor an amidase-5 (endopeptidase) domain, an amidase-4 (glycosidase) domain, and two Cpl-7 cell wall-binding domains. In this study, turbidity reduction and plate lysis assays indicate that this hydrolase shows strong lytic activity toward Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus equi, GES, and GGS. Deletion analysis indicates that the N-terminal endopeptidase domain with both Cpl-7 domains can lyse with a higher specific activity than the full-length protein (against some strains). This dual Cpl-7 domain truncated version also shows weak lytic activity against methicillin-resistant Staphylococcus aureus (MRSA) and the coagulase negative staphylococci, Staphylococcus xylosus. The truncated constructs harboring the glycosidase domain are virtually inactive, showing only minimal activity on plate lysis assays. 相似文献
3.
The Staphylococcus aureus bacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and an SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis pathogens, Staphylococcus aureus and coagulase-negative staphylococci (Staphylococcus chronogenes, Staphylococcus epidermidis, Staphylococcus hyicus, Staphylococcus simulans, Staphylococcus warneri and Staphylococcus xylosus), making it a strong candidate protein antimicrobial. This lytic activity is maintained at the pH (6.7), and the "free" calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins containing only the endopeptidase domain also lyse staphylococci in the absence of the SH3b-binding domain. 相似文献
4.
Schmelcher M Powell AM Becker SC Camp MJ Donovan DM 《Applied and environmental microbiology》2012,78(7):2297-2305
Staphylococci cause bovine mastitis, with Staphylococcus aureus being responsible for the majority of the mastitis-based losses to the dairy industry (up to $2 billion/annum). Treatment is primarily with antibiotics, which are often ineffective and potentially contribute to resistance development. Bacteriophage endolysins (peptidoglycan hydrolases) present a promising source of alternative antimicrobials. Here we evaluated two fusion proteins consisting of the streptococcal λSA2 endolysin endopeptidase domain fused to staphylococcal cell wall binding domains from either lysostaphin (λSA2-E-Lyso-SH3b) or the staphylococcal phage K endolysin, LysK (λSA2-E-LysK-SH3b). We demonstrate killing of 16 different S. aureus mastitis isolates, including penicillin-resistant strains, by both constructs. At 100 μg/ml in processed cow milk, λSA2-E-Lyso-SH3b and λSA2-E-LysK-SH3b reduced the S. aureus bacterial load by 3 and 1 log units within 3 h, respectively, compared to a buffer control. In contrast to λSA2-E-Lyso-SH3b, however, λSA2-E-LysK-SH3b permitted regrowth of the pathogen after 1 h. In a mouse model of mastitis, infusion of 25 μg of λSA2-E-Lyso-SH3b or λSA2-E-LysK-SH3b into mammary glands reduced S. aureus CFU by 0.63 or 0.81 log units, compared to >2 log for lysostaphin. Both chimeras were synergistic with lysostaphin against S. aureus in plate lysis checkerboard assays. When tested in combination in mice, λSA2-E-LysK-SH3b and lysostaphin (12.5 μg each/gland) caused a 3.36-log decrease in CFU. Furthermore, most protein treatments reduced gland wet weights and intramammary tumor necrosis factor alpha (TNF-α) concentrations, which serve as indicators of inflammation. Overall, our animal model results demonstrate the potential of fusion peptidoglycan hydrolases as antimicrobials for the treatment of S. aureus-induced mastitis. 相似文献
5.
Rodríguez-Rubio L Martínez B Rodríguez A Donovan DM García P 《Applied and environmental microbiology》2012,78(7):2241-2248
Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections. 相似文献
6.
Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice 总被引:23,自引:0,他引:23
Kerr DE Plaut K Bramley AJ Williamson CM Lax AJ Moore K Wells KD Wall RJ 《Nature biotechnology》2001,19(1):66-70
Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle. 相似文献
7.
Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria
Rubén Pérez Pulido Maria José Grande Burgos Rosario Lucas López 《Critical reviews in biotechnology》2016,36(5):851-861
Bacteriophages have attracted great attention for application in food biopreservation. Lytic bacteriophages specific for human pathogenic bacteria can be isolated from natural sources such as animal feces or industrial wastes where the target bacteria inhabit. Lytic bacteriophages have been tested in different food systems for inactivation of main food-borne pathogens including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, Salmonella enterica, Shigella spp., Campylobacter jejuni and Cronobacter sakazkii, and also for control of spoilage bacteria. Application of lytic bacteriophages could selectively control host populations of concern without interfering with the remaining food microbiota. Bacteriophages could also be applied for inactivation of bacteria attached to food contact surfaces or grown as biofilms. Bacteriophages may receive a generally recognized as safe status based on their lack of toxicity and other detrimental effects to human health. Phage preparations specific for L. monocytogenes, E. coli O157:H7 and S. enterica serotypes have been commercialized and approved for application in foods or as part of surface decontamination protocols. Phage endolysins have a broader host specificity compared to lytic bacteriophages. Cloned endolysins could be used as natural preservatives, singly or in combination with other antimicrobials such as bacteriocins. 相似文献
8.
Rodríguez-Rubio L Gutiérrez D Martínez B Rodríguez A García P 《Applied and environmental microbiology》2012,78(9):3469-3472
Bacteriophage endolysins have an interesting potential as antimicrobials. The endolysin LysH5, encoded by Staphylococcus aureus phage vB_SauS-phi-IPLA88, was expressed and secreted in Lactococcus lactis using the signal peptide of bacteriocin lactococcin 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of extracellular active endolysin was detected in culture supernatants, but most of the protein (up to 323 U/mg) remained in the cell extracts. 相似文献
9.
Rodríguez-Rubio L Gutiérrez D Martínez B Rodríguez A Götz F García P 《Applied and environmental microbiology》2012,78(17):6369-6371
Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections. 相似文献
10.
Bacteriophage endolysins are peptidoglycan hydrolases employed by the virus to lyse the host at the end of its multiplication phase. They have found many uses in biotechnology; not only as antimicrobials, but also for the development of novel diagnostic tools for rapid detection of pathogenic bacteria. These enzymes generally show a modular organization, consisting of N‐terminal enzymatically active domains (EADs) and C‐terminal cell wall‐binding domains (CBDs) which specifically target the enzymes to their substrate in the bacterial cell envelope. In this work, we used individual functional modules of Listeria phage endolysins to create fusion proteins with novel and optimized properties for labelling and lysis of Listeria cells. Chimaeras consisting of individual EAD and CBD modules from PlyPSA and Ply118 endolysins with different binding specificity and catalytic activity showed swapped properties. EAD118–CBDPSA fusion proteins exhibited up to threefold higher lytic activity than the parental endolysins. Recombineering different CBD domains targeting various Listeria cell surfaces into novel heterologous tandem proteins provided them with extended recognition and binding properties, as demonstrated by fluorescent GFP‐tagged CBD fusions. It was also possible to combine the binding specificities of different single CBDs in heterologous tandem CBD constructs such as CBD500‐P35 and CBDP35‐500, which were then able to recognize the majority of Listeria strains. Duplication of CBD500 increased the equilibrium cell wall binding affinity by approximately 50‐fold, and the enzyme featuring tandem CBD modules showed increased activity at higher ionic strength. Our results demonstrate that modular engineering of endolysins is a powerful approach for the rational design and optimization of desired functional properties of these proteins. 相似文献
11.
Use of bacteriophages as biocontrol agents is a promising tool for controlling pathogenic bacteria including antibiotic-resistant bacteria. Not only bacteriophages but also endolysins, the peptidoglycan hydrolyzing enzymes encoded by bacteriophages, have high potential for applications as biocontrol agents against food-borne pathogens. In this study, a putative endolysin gene was identified in the genome of the bacteriophage BPS13, which infects Bacillus cereus. In silico analysis of this endolysin, designated LysBPS13, showed that it consists of an N-terminal catalytic domain (PGRP domain) and a C-terminal cell wall binding domain (SH3_5 domain). Further characterization of the purified LysBPS13 revealed that this endolysin is an N-acetylmuramyl-l-alanine amidase, the activity of which was not influenced by addition of EDTA. In addition, LysBPS13 demonstrated remarkable thermostability in the presence of glycerol, and it retained its lytic activity even after incubation at 100 °C for 30 min. Taken together, these results indicate that LysBPS13 can be considered a favorable candidate for a new antimicrobial agent to control B. cereus. 相似文献
12.
Engineering Disease Resistant Cattle 总被引:12,自引:0,他引:12
Mastitis is a disease of the mammary gland caused by pathogens that find their way into the lumen of the gland through the
teat canal. Mammary gland infections cost the US dairy industry approximately $2 billion dollars annually and have a similar
impact in Europe. In the absence of effective treatments or breeding strategies to enhance mastitis resistance, we have created
transgenic dairy cows that express lysostaphin in their mammary epithelium and secrete the antimicrobial peptide into milk.
Staphylococcus aureus, a major mastitis pathogen, is exquisitely sensitive to lysostaphin. The transgenic cattle resist S. aureus mammary gland challenges, and their milk kills the bacteria, in a dose dependent manner. This first step in protecting cattle
against mastitis will be followed by introduction of other genes to deal with potential resistance issues and other mastitis
causing organisms. Care will be taken to avoid altering milk’s nutritional and manufacturing properties. Multi-cistronic constructs
may be required to achieve our goals as will other strategies possibly involving RNAi and gene targeting technology. This
work demonstrates the possibility of using transgenic technology to address disease problems in agriculturally important species.
The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged. 相似文献
13.
Pritchard DG Dong S Kirk MC Cartee RT Baker JR 《Applied and environmental microbiology》2007,73(22):7150-7154
Putative N-acetylmuramyl-l-alanine amidase genes from LambdaSa1 and LambdaSa2 prophages of Streptococcus agalactiae were cloned and expressed in Escherichia coli. The purified enzymes lysed the cell walls of Streptococcus agalactiae, Streptococcus pneumoniae, and Staphylococcus aureus. The peptidoglycan digestion products in the cell wall lysates were not consistent with amidase activity. Instead, the structure of the muropeptide digestion fragments indicated that both the LambdaSa1 and LambdaSa2 lysins exhibited gamma-d-glutaminyl-l-lysine endopeptidase activity. The endopeptidase cleavage specificity of the lysins was confirmed using a synthetic peptide substrate corresponding to a portion of the stem peptide and cross bridge of Streptococcus agalactiae peptidoglycan. The LambdaSa2 lysin also displayed beta-d-N-acetylglucosaminidase activity. 相似文献
14.
Myllys V. Honkanen-Buzalski T. Huovinen P. Sandholm M. Nurmi E. 《Acta veterinaria Scandinavica》1994,35(4):363-369
The results of mastitis bacteriology made by the National Veterinary and Food Research Institute in Finland during the past 50 years (1.15 million samples) are viewed in relation to simultaneous changes in dairy cow management. Although intensive preventive measures have been applied for decades, the prevalence of bovine mastitis has not decreased. Instead, pathogenic bacteria are becoming progressively less susceptible to the available therapy. In part this must be due to the emergence of antibiotic resistant strains of bacteria, but it seems that the bacterial spectrum has also changed. The incidence of Streptococcus agalactiae (group B streptococci) has decreased, while in contrast, the incidence of staphylococci, initially Staphylococcus aureus and later coagulase-negative staphylococci, has increased. Results suggest that external pressure, like changes in animal husbandry, including antimicrobial treatments and introduction of modern milking machines, act as selective forces on the bacterial species which cause bovine mastitis. 相似文献
15.
Phage lytic enzymes (enzybiotics) have gained attention as prospective tools to eradicate Gram-positive pathogens resistant to antibiotics. Attempts to purify the P16 endolysin of Staphylococcus aureus phage P68 were unsuccessful owing to the poor solubility of the protein. To overcome this limitation, we constructed a chimeric endolysin (P16-17) comprised of the inferred N-terminal d-alanyl-glycyl endopeptidase domain and the C-terminal cell wall targeting domain of the S. aureus phage P16 endolysin and the P17 minor coat protein, respectively. The domain swapping approach and the applied purification procedure resulted in soluble P16-17 protein, which exhibited antimicrobial activity towards S. aureus. In addition, P16-17 augmented the antimicrobial efficacy of the antibiotic gentamicin. This synergistic effect could be useful to reduce the effective dose of aminoglycoside antibiotics. 相似文献
16.
Borysowski J Weber-Dabrowska B Górski A 《Experimental biology and medicine (Maywood, N.J.)》2006,231(4):366-377
Endolysins are double-stranded DNA bacteriophage-encoded peptidoglycan hydrolases produced in phage-infected bacterial cells toward the end of the lytic cycle. They reach the peptidoglycan through membrane lesions formed by holins and cleave it, thus, inducing lysis of the bacterial cell and enabling progeny virions to be released. Endolysins are also capable of degrading peptidoglycan when applied externally (as purified recombinant proteins) to the bacterial cell wall, which also results in a rapid lysis of the bacterial cell. The unique ability of endolysins to rapidly cleave peptidoglycan in a generally species-specific manner renders them promising potential antibacterial agents. Originally developed with a view to killing bacteria colonizing mucous membranes (with the first report published in 2001), endolysins also hold promise for the treatment of systemic infections. As potential antibacterials, endolysins possess several important features, for instance, a novel mode of action, a narrow antibacterial spectrum, activity against bacteria regardless of their antibiotic sensitivity, and a low probability of developing resistance. However, there is only one report directly comparing the activity of an endolysin with that of an antibiotic, and no general conclusions can be drawn regarding whether lysins are more effective than traditional antibiotics. The results of the first preclinical studies indicate that the most apparent potential problems associated with endolysin therapy (e.g., their immunogenicity, the release of proinflammatory components during bacteriolysis, or the development of resistance), in fact, may not seriously hinder their use. However, all data regarding the safety and therapeutic effectiveness of endolysins obtained from preclinical studies must be ultimately verified by clinical trials. This review discusses the prophylactic and therapeutic applications of endolysins, especially with respect to their potential use in human medicine. Additionally, we outline current knowledge regarding the structure and natural function of the enzymes in phage biology, including the most recent findings. 相似文献
17.
Stephen C. Becker Shengli Dong John R. Baker Juli Foster-Frey David G. Pritchard & David M. Donovan 《FEMS microbiology letters》2009,294(1):52-60
LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d -alanine of the stem peptide and glycine of the cross-bridge peptide, and N -acetylmuramoyl- l -alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK. 相似文献
18.
In vitro efficacy of the novel peptide CECT7121 against bacteria isolated from mastitic dairy cattle
Aims: To evaluate the in vitro bactericidal activity of the novel antimicrobial peptide (AP) CECT7121 against Gram-positive bacteria from mastitic dairy cattle.
Methods and Results: A total of 15 Staphylococcus aureus , 10 Streptococcus dysgalactiae , 7 Strep. uberis , 1 Strep. agalactiae strains were isolated from 33 different mastitic dairy cattle, sourced from two dairies in Tandil-Argentina. Isolates from each of the bacterial species screened which developed the lowest inhibition zones in response to the peptide, were further evaluated in a series of time-killing curve studies. No survivors were detected in whole strains (from the three Streptococcal species isolated) within 120 min of incubation in presence of the peptide. The Staph. aureus isolates were less sensitive but, nevertheless, a drop in viable counts to below the detection limit was achieved for all the test strains by the final postincubation sampling point at 180 min.
Conclusions: The study demonstrated the in vitro efficacy of the AP-CECT7121 against a variety strains of Gram positives isolated from mastitic dairy cattle.
Significance and Impact of the Study: There is urgent global interest in the development of natural alternatives for the control and prevention of mastitis. Confirmation of the in vitro activity of the novel AP-CECT7121 against Gram-positive isolates encourages further research. 相似文献
Methods and Results: A total of 15 Staphylococcus aureus , 10 Streptococcus dysgalactiae , 7 Strep. uberis , 1 Strep. agalactiae strains were isolated from 33 different mastitic dairy cattle, sourced from two dairies in Tandil-Argentina. Isolates from each of the bacterial species screened which developed the lowest inhibition zones in response to the peptide, were further evaluated in a series of time-killing curve studies. No survivors were detected in whole strains (from the three Streptococcal species isolated) within 120 min of incubation in presence of the peptide. The Staph. aureus isolates were less sensitive but, nevertheless, a drop in viable counts to below the detection limit was achieved for all the test strains by the final postincubation sampling point at 180 min.
Conclusions: The study demonstrated the in vitro efficacy of the AP-CECT7121 against a variety strains of Gram positives isolated from mastitic dairy cattle.
Significance and Impact of the Study: There is urgent global interest in the development of natural alternatives for the control and prevention of mastitis. Confirmation of the in vitro activity of the novel AP-CECT7121 against Gram-positive isolates encourages further research. 相似文献
19.
The extent of subclinical mastitis in three breeds of cattle, Kankrej, Gir, and Crossbred, was performed at cattle farms in Anand town of Gujarat State, India. The prevalence of subclinical mastitis in crossbred cattle was higher compared to local breed of cattle. Causative agents identified using 16S rDNA polymerase chain reaction (PCR)-based molecular method were Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Bacillus megaterium. In vitro antibacterial activity of ethyl acetate extract of plant Terminalia chebula (Combretaceae) was checked by agar well diffusion method against four isolated and molecularly identified microorganisms. Ethyl acetate extract shows antimicrobial activity with varying magnitudes against all identified isolates. Among the three different concentrations, 500?µg/mL conc. of extract is as effective as that of standard amoxicillin. In vitro results support the use of plant extract from T. chebula as an alternative to antibiotics therapy against bovine subclinical mastitis. 相似文献
20.
Briers Y Volckaert G Cornelissen A Lagaert S Michiels CW Hertveldt K Lavigne R 《Molecular microbiology》2007,65(5):1334-1344
Pseudomonas aeruginosa bacteriophage endolysins KZ144 (phage phiKZ) and EL188 (phage EL) are highly lytic peptidoglycan hydrolases (210 000 and 390 000 units mg(-1)), active on a broad range of outer membrane-permeabilized Gram-negative species. Site-directed mutagenesis indicates E115 (KZ144) and E155 (EL188) as their respective essential catalytic residues. Remarkably, both endolysins have a modular structure consisting of an N-terminal substrate-binding domain and a predicted C-terminal catalytic module, a property previously only demonstrated in endolysins originating from phages infecting Gram-positives and only in an inverse arrangement. Both binding domains contain conserved repeat sequences, consistent with those of some peptidoglycan hydrolases of Gram-positive bacteria. Fusions of these domains with green fluorescent protein immediately label all outer membrane-permeabilized Gram-negative bacteria tested, isolated P. aeruginosa peptidoglycan and N-acetylated Bacillus subtilis peptidoglycan, demonstrating the broad range of peptidoglycan-binding capacity by these domains. Specifically, A1 chemotype peptidoglycan and fully N-acetylated glucosamine units are essential for binding. Both KZ144 and EL188 appear to be a natural chimeric enzyme, originating from a recombination of a cell wall-binding domain encoded by a Bacillus or Clostridium species and a catalytic domain of an unknown ancestor. 相似文献