Structural Basis for Cell-Wall Recognition by Bacteriophage PBC5 Endolysin

1. Download : Download high-res image (208KB)
2. Download : Download full-size image

     

Complete genome sequence of Bacillus cereus bacteriophage PBC1

Bacillus cereus is a ubiquitous, spore-forming bacterium associated with food poisoning cases. To develop an efficient biocontrol agent against B. cereus, we isolated lytic phage PBC1 and sequenced its genome. PBC1 showed a very low degree of homology to previously reported phages, implying that it is novel. Here we report the complete genome sequence of PBC1 and describe major findings from our analysis.     

Transducing Bacteriophage for Bacillus cereus

A phage, designated CP-51, that carries out generalized transduction in Bacillus cereus 569 was isolated from soil. All auxotrophic mutants tested, those requiring tryptophan, histidine, leucine, isoleucine, methionine, or phenylalanine, were transduced to prototrophy. The phage was extremely unstable when stored at 2 to 4 C, but stability was enhanced by storage at higher temperatures. The optimal temperature of those tested for maintenance of plaque-forming units was 15 C.     

Cold Lability of Bacillus cereus Bacteriophage CP-51

Phage CP-51 was rapidly inactivated when stored at the usual refrigerator temperatures (2 to 4 C) and even more rapidly when exposed to 0 C. The loss in viability resulting from exposure to cold appeared to correlate with the increase in number of phage particles having contracted tails. High concentrations (0.01 M) of Mg²⁺, Ca²⁺, or Mn²⁺ stabilized the phage considerably, but even in the presence of these divalent cations, it was much less stable at 0 C than at 15 C.     

Early Biochemical Events Occurring After Infection of Bacillus cereus 569-SP1 with Bacteriophage GSW

After infection of Bacillus cereus 569-SP1 with the 5-hydroxymethyluracil-containing phage GSW, new dTTPase, dUTPase, and dUMP-hydroxymethylase activities appear. No significant changes in activities of other pyrimidine ribonucleoside or 2'-deoxyribonucleoside triphosphate nucleotidohydrolases were detected. dUTP and dUMP inhibit the dTTPase activity, whereas dTTP failed to inhibit dUTPase activity. The K(m) value for the substrate dUTP is 10(-4) M and for dTTP is 4.85 x 10(-4) M. Thymidylate synthetase activity is inhibited only when cells are infected during the late lag or very early log phases of growth; when cells are infected with phage during mid-log, thymidylate synthetase activity is unaffected. The data support the suggestion that, although phage GSW may inhibit an otherwise expected increase in activity of thymidylate synthetase, it fails to affect the already existing activity. The data presented do not allow discrimination as to whether the phage specifies inhibition of de novo synthesis of thymidylate synthetase or the increase in activity of already existing but not fully expressed enzyme.     

Production of Bacteriophage by Temperature-Sensitive Sporulation Mutants of Bacillus cereus T

Five temperature-sensitive sporulation mutants of Bacillus cereus T have been isolated. These mutants are blocked at stage 0 of sporulation at the restrictive temperature (37 C) but are able to sporulate at nearly normal frequencies at the permissive temperature (26 C). A bacteriophage that forms a stable lysogen in the parent strain is induced at increased frequencies in the mutants. This induction is accompanied, in some of the mutants, by a reduction in immunity to the phage. Revertants, selected for their ability to sporulate normally at both temperatures, lose their ability to produce high titers of the phage. In addition to this lytic phage, an apparently defective phage has been found in lysates of the mutants. Strains cured of the plaque-forming phage still carry the defective phage. Comparisons of physical and biological properties of the plaque-forming phage with those of the two Bacillus cereus phages most similar to it have shown that this phage is not identical to either of them. The maximal titer of phage produced in cultures of the parent strain is about 10(3) plaque-forming units (PFU) per ml at both temperatures. The maximal titers of phage produced by the mutant are 4 x 10(9) PFU/ml at 37 C and 7 x 10(8) PFU/ml at 26 C. Both mutant and parent strains release over 90% of the phage they produce after the onset of stationary phase.     

噬菌体内溶素的酶学特性及其应用前景

噬菌体内溶素是噬菌体在入侵宿主菌及侵染后期释放过程中合成的一类酶蛋白,该蛋白质能够破坏宿主细胞壁肽聚糖层。噬菌体编码的内溶素有四种类型:葡糖苷酶、酰胺酶、肽链内切酶和转糖基酶。大部分噬菌体内溶素由于缺少信号肽无法分泌表达,通常需要另外一种噬菌体编码的穴蛋白(holin)破坏细胞膜,然后才能够进入到细胞周间质裂解细菌细胞壁。大部分噬菌体内溶素可以特异地作用于自身宿主菌,同时也可以利用基因工程手段有目的地改造成功能特异的酶蛋白,因此可以用来作为生物制剂预防及控制微生物感染。     

Bacteriophage phi 1 as a gene-cloning vector in Bacillus subtilis

Summary We attempted to use Bacillus subtilis phage 1 as a gene-cloning vector since the 1 genome was found to have few cleavage sites upon digestion with several kinds of restriction endonucleases. A 1 stock supplied by J. Ito (University of Arizona, Tucson, USA) consisted of two phages, 1E1 and 1E2, having one and two EcoRI-cleavage sites in their genomes respectively. From the latter isolate a deletion mutant 1E21 was induced to increase the size range of DNA segments to be cloned. It was demonstrated, by in vitro recombination experiments with phage 11 DNA, that 1E21 can be used for cloning EcoRI fragments of various sizes. We analyzed the DNAs of ten 1 clones isolated from independent transfectants and found that six of them carried 11 DNA fragments inserted at either of the two EcoRI-cleavage sites. Some of the hybrid phage DNAs were found to be cleaved with BamHI and HaeIII endonucleases at the 11 DNA portion, whereas the parental 1E21 DNA was insensitive to any of these enzymes. These hybrid phages would therefore be useful vectors for cloning foreign DNA fragments generated by cleavage with BamHI or HaeIII endonucleases.     

Characterization of Enterococcus faecalis Phage IME-EF1 and Its Endolysin

Enterococcus faecalis is increasingly becoming an important nosocomial infection opportunistic pathogen. E. faecalis can easily obtain drug resistance, making it difficult to be controlled in clinical settings. Using bacteriophage as an alternative treatment to drug-resistant bacteria has been revitalized recently, especially for fighting drug-resistant bacteria. In this research, an E. faecalis bacteriophage named IME-EF1 was isolated from hospital sewage. Whole genomic sequence analysis demonstrated that the isolated IME-EF1 belong to the Siphoviridae family, and has a linear double-stranded DNA genome consisting of 57,081 nucleotides. The IME-EF1 genome has a 40.04% G+C content and contains 98 putative coding sequences. In addition, IME-EF1 has an isometric head with a width of 35 nm to 60 nm and length of 75 nm to 90 nm, as well as morphology resembling a tadpole. IME-EF1 can adsorb to its host cells within 9 min, with an absorbance rate more than 99% and a latent period time of 25 min. The endolysin of IME-EF1 contains a CHAP domain in its N-terminal and has a wider bactericidal spectrum than its parental bacteriophage, including 2 strains of vancomycin-resistant E. faecalis. When administrated intraperitoneally, one dose of IME-EF1 or its endolysin can reduce bacterial count in the blood and protected the mice from a lethal challenge of E. faecalis, with a survival rate of 60% or 80%, respectively. Although bacteriophage could rescue mice from bacterial challenge, to the best of our knowledge, this study further supports the potential function of bacteriophage in dealing with E. faecalis infection in vivo. The results also indicated that the newly isolated bacteriophage IME-EF1 enriched the arsenal library of lytic E. faecalis bacteriophages and presented another choice for phage therapy in the future.     

Antimicrobial activity of a newly identified bacteriocin of Bacillus cereus.

A bacteriocin-producing Bacillus cereus strain was isolated. The bacteriocin, here called cerein, was shown to be active specifically against other B. cereus strains and inactive against all other bacterial species tested. Cerein was detected in the culture supernatants of stationary-phase cells, and its appearance was inhibited by induction of sporulation. The bacterial activity of cerein was insensitive to organic solvents and nonproteolytic enzymes, partially stable to heat, and active over a wide range of pH values. Direct detection of antimicrobial activity on sodium dodecyl sulfate-polyacrylamide gel suggested an apparent molecular mass of about 9 kDa.     

Characterization of a Novel LysM Domain from Lactobacillus fermentum Bacteriophage Endolysin and Its Use as an Anchor To Display Heterologous Proteins on the Surfaces of Lactic Acid Bacteria

The endolysin Lyb5, from Lactobacillus fermentum temperate bacteriophage φPYB5, showed a broad lytic spectrum against Gram-positive as well as Gram-negative bacteria. Sequence analysis revealed that the C terminus of the endolysin Lyb5 (Ly5C) contained three putative lysin motif (LysM) repeat regions, implying that Ly5C was involved in bacterial cell wall binding. To investigate the potential of Ly5C for surface display, green fluorescent protein (GFP) was fused to Ly5C at its N or C terminus and the resulting fusion proteins were expressed in Escherichia coli. After being mixed with various cells in vitro, GFP was successfully displayed on the surfaces of Lactococcus lactis, Lactobacillus casei, Lb. brevis, Lb. plantarum, Lb. fermentum, Lb. delbrueckii, Lb. helveticus, and Streptococcus thermophilus cells. Increases in the fluorescence intensities of chemically pretreated L. lactis and Lb. casei cells compared to those of nonpretreated cells suggested that the peptidoglycan was the binding ligand for Ly5C. Moreover, the pH and concentration of sodium chloride were optimized to enhance the binding capacity of GFP-Ly5C, and high-intensity fluorescence of cells was observed under optimal conditions. All results suggested that Ly5C was a novel anchor for constructing a surface display system for lactic acid bacteria (LAB). To demonstrate the applicability of the Ly5C-mediated surface display system, β-galactosidase (β-Gal) from Paenibacillus sp. strain K1, replacing GFP, was functionally displayed on the surfaces of LAB cells via Ly5C. The success in surface display of GFP and β-Gal opened up the feasibility of employing the cell wall anchor of bacteriophage endolysin for surface display in LAB.Surface display of heterologous proteins or peptides on bacteria is potentially important in several areas of biotechnology, including development of live vaccine delivery systems, diagnostics, whole-cell absorbents, and novel biocatalysts (11). Lactic acid bacteria (LAB) have the status of being generally recognized as safe (GRAS), making them certainly more useful in food and medical applications than other bacterial species. The development of cell surface display systems for LAB has recently become one of the most active research areas. Most of the cell surface display systems for LAB reported thus far have made use of the C terminus of a cell wall-anchoring protein via an LPXTG motif (8, 12, 19, 24). This anchoring mechanism requires processing by a sortase for covalent anchoring of the protein to the cell wall peptidoglycan (15). Various anchoring proteins, such as membrane-spanning protein PgsA (16) and S-layer protein (3), have also been exploited for surface display. However, heterologous proteins have been anchored to the producer cells, and the use of genetically modified organisms is less desirable or at least still being debated. Surface display of heterologous proteins on genetically unmodified Gram-positive bacteria has been successfully carried out using the peptidoglycan binding lysin motif (LysM) domain of the major autolysin AcmA of Lactococcus lactis (1, 2, 4, 18, 28).LysM was first discovered in the lysozyme of Bacillus phage φ29 as a C-terminal repeat composed of 44 amino acids separated by 7 amino acids (6). LysM is a common module found in more than 4,000 proteins of both prokaryotes and eukaryotes (6). Many bacterial proteins containing LysM are peptidoglycan hydrolases, such as p60 (20), Sep (26), LytF (31), AcmA (5), and Mur (7). The best-characterized LysM-containing protein is the N-acetylglucosaminidase AcmA of L. lactis subsp. cremoris MG1363. AcmA is the major autolysin and is required for cell separation and cell lysis during the stationary phase of L. lactis (5). It contains three domains: the N-terminal signal peptide, an active domain, and a C-terminal peptidoglycan anchor (cA) which consists of three LysM repeats (22). Several functional proteins, including malaria parasite surface antigen, β-lactamase, α-amylase, and viral capsid proteins, have been noncovalently bound to cell walls of AcmA-producing and non-AcmA-producing L. lactis as well as several other Gram-positive bacteria via cA (4, 17, 18, 23, 25).Endolysins from bacteriophages are cell wall hydrolases involved in cell lysis to release the progeny particles from the host cells (9, 30). Most endolysins lack a signal peptide and are translocated across the membrane by the aid of the holin protein. This protein typically contains an N-terminal catalytic domain and a C-terminal cell wall binding domain (33). The endolysins Ply118 and Ply500 of a Listeria monocytogenes phage share a unique C-terminal cell wall binding domain which establishes specific recognition of and high-affinity binding to bacterial cell wall carbohydrates (13). The temperate bacteriophage φPYB5, isolated from the Lactobacillus fermentum YB5 strain, has a hexagonal head, noncontractile tails, and several fibers and belongs to Bradley''s group B as defined by the International Committee on Taxonomy of Viruses (32). The sequence of the endolysin gene lyb5 from the genome of φPYB5 has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"EF531306","term_id":"145687952","term_text":"EF531306"}}EF531306, and the gene product has been successfully expressed in Escherichia coli and has shown a broad lytic spectrum (30).Here, we generated a fusion of green fluorescent protein (GFP) to the C terminus of Lyb5 (Ly5C) to construct a surface display system for LAB. The GFP was bound to the surfaces of various LAB cells by the aid of Ly5C. Moreover, by using the system constructed, β-galactosidase (β-Gal) was functionally displayed on the surfaces of LAB cells and retained its activity.     

Rutin-enhanced antibacterial activities of flavonoids against Bacillus cereus and Salmonella enteritidis

The antibacterial activities of flavonoids were found by the paper disk method to be enhanced by combining or mixing them. The combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were much more active than either flavonoid alone. Although rutin did not show activity in itself, the antibacterial activities of quercetin and morin were enhanced in the presence of rutin. The antibacterial activities of flavonoids, in combination with morin and rutin, were evaluated, based on the minimum inhibition concentration (MIC) in a liquid culture, by using Salmonella enteritidis and Bacillus cereus as the test bacteria. The activities of galangin, kaempherol, myricetin and fisetin were each enhanced in the presence of rutin when S. enteritidis was used as the test bacterium. The MIC value for kaempherol was markedly decreased by the addition of rutin. Morin inhibited DNA synthesis, and this effect was promoted by rutin at a concentration of 25 microg/ml.     

Transduction in Bacillus subtilis by Bacteriophage SPP1

Lysates of the virulent bacteriophage SPP1 were shown to be capable of mediating generalized transduction. Suppressible mutants of this bacteriophage (sus) were capable of transduction at a lower multiplicity of infection than virulent SPP1. Linkage analysis demonstrated that bacteriophage SPP1 transduced segments of the genome equal in size to that transferred by SP10. This bacteriophage should be useful in analyzing the regions of the genome where PBS1 appears to give anomalous results.