Restriction/Modification systems and restriction endonucleases are more effective on lactococcal bacteriophages that have emerged recently in the dairy industry

Recently, eight lytic small isometric-headed bacteriophages were isolated from cheese-manufacturing plants throughout North America. The eight phages were different, but all propagated on one strain, Lactococcus lactis NCK203. On the basis of DNA homology, they were classified in the P335 species. Digestion of their genomes in vitro with restriction enzymes resulted in an unusually high number of type II endonuclease sites compared with the more common lytic phages of the 936 (small isometric-headed) and c2 (prolate-headed) species. In vivo, the P335 phages were more sensitive to four distinct lactococcal restriction and modification (R/M) systems than phages belonging to the 936 and c2 species. A significant correlation was found between the number of restriction sites for endonucleases (purified from other bacterial genera) and the relative susceptibility of phages to lactococcal R/M systems. Comparisons among these three phage species indicate that the P335 species may have emerged most recently in the dairy industry.     

Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes

AIMS: To develop PCR assays able to distinguish between groups within lactococcal 936-species bacteriophages, as defined by their different receptor-binding protein (RBP) genes. METHODS AND RESULTS: DNA sequences of RBP genes from 17 lactococcal bacteriophages of the 936-species were compared, and six phage groups were identified. For each phage group a specific primer pair targeting a variable region of the RBP genes was designed. In nine of 20 whey samples, from dairies with recorded phage problems, between one and six phage groups were identified by conventional PCR. The sensitivity and specificity of the method was improved by magnetic capture hybridization (MCH)-PCR using a capture probe targeting an 80-bp highly conserved region just upstream from the RBP gene in all the investigated phages. The MCH-PCR was performed on 100 microl whey samples and the detection limit of the assay was 10(2)-10(3) PFU ml(-1) as opposed to the detection limit of 10(4) PFU ml(-1) for conventional PCR performed on 1-microl whey samples. CONCLUSIONS: In this study, PCR assays have been developed to detect six different types of RBP genes in lactococcal 936-species bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assays have practical applications at cheese plants for detection of 936-species phages with different RBP and thereby potentially with different host ranges. This knowledge will make it possible to improve starter culture rotation systems in the dairy industry.     

Towards the diversification of lactococcal starter and non-starter species in mesophilic dairy culture systems

Lactococcus is one of the earliest identified fermentative bacterial genera and among its member species, the dairy-associated Lactococcus lactis and Lactococcus cremoris are undoubtedly the best studied. These two species are believed to have evolved from plant-associated lactococci and through genome decay and acquisition of plasmids, have adapted to the dairy niche. The past decade has witnessed a surge of activity in novel lactococcal species identification from insect, plant and animal sources. Currently, 22 Lactococcus species are described and in this review, we summarise the genome characteristics of and phylogenetic relationships among these species. Furthermore, we explore the role of mobile elements including plasmids and bacteriophages in the diversification of lactococcal species. The pace of identification of novel lactococcal species suggests that the number of lactococcal species is likely to continue to grow. With additional sequence data for the emerging species, it will be possible to perform pathogenicity/virulence risk evaluations and generate extensive insights into the niche adaptation strategies through which they have evolved.     

Purification and characterization of the lytic activity induced by the prolate-headed bacteriophage P001 in Lactococcus lactis

The lytic activity induced by the lactococcal bacteriophage P001 was isolated from phage lysates of Lactococcus lactis by a four-step purification procedure. Two proteins lytic for L. lactis were identified with molecular weights of 28 kDA and 8 kDa, respectively. The N-terminal amino acid sequences of the two proteins were determined and degenerated oligonucleotide probes corresponding to these sequences were synthesized. DNA hybridization experiments with phage P001-DNA and lactococcal DNA revealed that both proteins were apparently encoded by a single lysin gene located on the phage P001 genome. This was confirmed by alignment of the determined N-terminal amino acid sequences with nucleotide sequences which were deduced from cloned Lactococcus bacteriophage lysin genes.     

Interspecies migration and evolution of bacteriophages of the genus phiKZ: The purpose and criteria of the search for new phiKZ-like bacteriophages

Some properties of bacteriophages with large (200 kb and more) sequenced genomes have been compared. In contrast to other large bacteriophages from different families, bacteriophages active on pseudomonads of various species (phiKZ-like bacterio phages) have some common features, which suggests their phylogenetic relationship and independence of their evolution as a result of migration among bacteria of this family. Among such common features are the absence in the genomes of these phages of sites sensitive to endonuclease PstI, the absence of genes encoding DNA polymerases that are similar to the known enzymes of this type, possible dependence of replication of the phage genome on bacterial DNA polymerase, and a considerably larger average gene size as compared to that for other phages. Criteria are suggested for searching for novel phiKZ-like bacteriophages: the size of a phag e particle, production by bacteria infected with such phages of a large amount of highly viscous mucus. Taking into account the use of these bacteriophages in therapeutic preparations (due to a broad spectrum of lytic activity) and a poor knowledge of a majority of their gene products, it seems necessary to perform a more comprehensive genetic analysis of phages of this genus or their mutants for selecting those adequate for phage therapy.     

The annotated complete DNA sequence of Enterococcus faecalis bacteriophage φEf11 and its comparison with all available phage and predicted prophage genomes

φEf11 is a temperate Siphoviridae bacteriophage isolated by induction from a lysogenic Enterococcus faecalis strain. The φEf11 DNA was completely sequenced and found to be 42,822 bp in length, with a G+C mol% of 34.4%. Genome analysis revealed 65 ORFs, accounting for 92.8% of the DNA content. All except for seven of the ORFs displayed sequence similarities to previously characterized proteins. The genes were arranged in functional modules, organized similar to that of several other phages of low GC Gram-positive bacteria; however, the number and arrangement of lysis-related genes were atypical of these bacteriophages. A 159 bp noncoding region between predicted cI and cro genes is highly similar to the functionally characterized early promoter region of lactococcal temperate phage TP901-1, and possessed a predicted stem-loop structure in between predicted P(L) and P(R) promoters, suggesting a novel mechanism of repression of these two bacteriophages from the λ paradigm. Comparison with all available phage and predicted prophage genomes revealed that the φEf11 genome displays unique features, suggesting that φEf11 may be a novel member of a larger family of temperate prophages that also includes lactococcal phages. Trees based on the blast score ratio grouped this family by tail fiber similarity, suggesting that these trees are useful for identifying phages with similar tail fibers.     

Application of bacteriophages in post-harvest control of human pathogenic and food spoiling bacteria

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.     

Efficacy of a Broad Host Range Lytic Bacteriophage Against <Emphasis Type="Italic">E. coli</Emphasis> Adhered to Urothelium

Persistent urinary tract infections (UTI) are often caused by E. coli adhered to urothelium. This type of cells is generally recognized as very tolerant to antibiotics which renders difficult the treatment of chronic UTI. This study investigates the use of lytic bacteriophages as alternative antimicrobial agents, particularly the interaction of phages with E. coli adhered to urothelium and specifically determines their efficiency against this type of cells. The bacterial adhesion to urothelium was performed varying the bacterial cell concentrations and the period and conditions (static, shaken) of adhesion. Three collection bacteriophages (T1, T4, and phiX174 like phages) were tested against clinical E. coli isolates and only one was selected for further infection experiments. Based on the lytic spectrum against clinical isolates and its ability to infect the highest number of antibiotic resistant strains, the T1-like bacteriophage was selected. This bacteriophage caused nearly a 45% reduction of the bacterial population after 2 h of treatment. This study provides evidence that bacteriophages are effective in controlling suspended and adhered cells and therefore can be a viable alternative to antibiotics to control urothelium- adhered bacteria.     

Genome analysis of the obligately lytic bacteriophage 4268 of Lactococcus lactis provides insight into its adaptable nature

Analysis of the complete nucleotide sequence of the lactococcal phage 4268, which is lytic for the cheese starter Lactococcus lactis DPC4268, is presented. Phage 4268 has a linear genome of 36,596 bp, which is modularly organised and encompasses 49 open reading frames. Putative functions were assigned to approximately 45% of the predicted products of these open reading frames based on sequence similarity with known proteins, N-terminal sequence analysis and identification of conserved domains. Significantly, a segment of the genome has homology to the recently sequenced lysogenic module in lactococcal phage phi31 that contains a lytic switch but no phage integrase or attachment site. This suggests that it is derived from a prophage. A phage 4268-encoded and a host-encoded methylase were found to be highly similar, having only two nucleotide mismatches, suggesting that the phage acquired the methylase gene to protect it from a host endonuclease. Comparative genomic analysis revealed significant homology between phage 4268 and the lactococcal phage BK5-T. The comparative analysis also supported the classification of phage 4268 and other BK5-T-related phage as separate from the proposed P335 species of lactococcal phage.     

Lysozyme expression in Lactococcus lactis

Summary Three lysozyme-encoding genes, one of eukaryotic and two of prokaryotic origin, were expressed in Lactococcus lactis subsp. lactis. Hen egg white lysozyme (HEL) could be detected in L. lactis lysates by Western blotting. No lysozyme activity was observed, however, presumably because of the absence of correctly formed disulphide bonds in the L. lactis product. The functionally related lysozymes of the E. coli bacteriophages T4 and were produced as biologically active proteins in L. lactis. In both cases, the highest expression levels were obtained using configurations in which the bacteriophage lysozyme genes had been translationally coupled to a short open reading frame of lactococcal origin. Both enzymes, like HEL, may prevent the growth of food-spoilage bacteria.     

Genome sequence and global gene expression of Q54, a new phage species linking the 936 and c2 phage species of Lactococcus lactis

The lytic lactococcal phage Q54 was previously isolated from a failed sour cream production. Its complete genomic sequence (26,537 bp) is reported here, and the analysis indicated that it represents a new Lactococcus lactis phage species. A striking feature of phage Q54 is the low level of similarity of its proteome (47 open reading frames) with proteins in databases. A global gene expression study confirmed the presence of two early gene modules in Q54. The unusual configuration of these modules, combined with results of comparative analysis with other lactococcal phage genomes, suggests that one of these modules was acquired through recombination events between c2- and 936-like phages. Proteolytic cleavage and cross-linking of the major capsid protein were demonstrated through structural protein analyses. A programmed translational frameshift between the major tail protein (MTP) and the receptor-binding protein (RBP) was also discovered. A "shifty stop" signal followed by putative secondary structures is likely involved in frameshifting. To our knowledge, this is only the second report of translational frameshifting (+1) in double-stranded DNA bacteriophages and the first case of translational coupling between an MTP and an RBP. Thus, phage Q54 represents a fascinating member of a new species with unusual characteristics that brings new insights into lactococcal phage evolution.     

Phenotypic characterization and genomic analysis of the <Emphasis Type="Italic">Shigella sonnei</Emphasis> bacteriophage SP18

A novel bacteriophage that infects Shigella sonnei was isolated from the Gap River in Korea, and its phenotypic and genomic characteristics were investigated. The virus, called SP18, showed morphology characteristic of the family Myoviridae, and phylogenetic analysis of major capsid gene (gp23) sequences classified it as a T4-like phage. Based on host spectrum analysis, it is lytic to S. sonnei, but not to Shigella flexneri, Shigella boydii or members of the genera Escherichia and Salmonella. Pyrosequencing of the SP18 bacteriophage genome revealed a 170-kb length sequence. In total, 286 ORFs and 3 tRNA genes were identified, and 259 ORFs showed similarity (BLASTP e-value<0.001) to genes of other bacteriophages. The results from comparative genomic analysis indicated that the enterophage JS98, isolated from human stool, is the closest relative of SP18. Based on phylogenetic analysis of gp23 protein-coding sequences, dot plot comparison and BLASTP analysis of genomes, SP18 and JS98 appear to be closely related to T4-even phages. However, several insertions, deletions, and duplications indicate differences between SP18 and JS98. Comparison of duplicated gp24 genes and the soc gene showed that duplication events are responsible for the differentiation and evolution of T4-like bacteriophages.     

Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes

Background  

The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene.     

A new bacteriophage typing scheme forProteus mirabilis andProteus vulgaris strains

The lytic properties of 21 bacteriophages constituting a new typing set forProteus were examined in 507Proteus mirabilis and 29P. vulgaris strains isolated from patients and healthy subjects. Comparison of their morphological, serological, genetic and lytic properties showed that, in theMyoviridae andPodoviridae families, some phages were so closely related that the presence of all of them in the set was redundant. Analysis of the lytic properties revealed that some of the bacteriophages were not active enough to facilitate the differentiation ofProteus strains The size of the final typing set was reduced from 21 to 12 phages but it was suggested that, in order to improve the differentiation capacity of the set, new phages should be included. Second part:Folia Microbiol. 41, 137–140 (1996).     

Phage endolysins are adapted to specific hosts and are evolutionarily dynamic

Endolysins are produced by (bacterio)phages to rapidly degrade the bacterial cell wall and release new viral particles. Despite sharing a common function, endolysins present in phages that infect a specific bacterial species can be highly diverse and vary in types, number, and organization of their catalytic and cell wall binding domains. While much is now known about the biochemistry of phage endolysins, far less is known about the implication of their diversity on phage–host adaptation and evolution. Using CRISPR-Cas9 genome editing, we could genetically exchange a subset of different endolysin genes into distinct lactococcal phage genomes. Regardless of the type and biochemical properties of these endolysins, fitness costs associated to their genetic exchange were marginal if both recipient and donor phages were infecting the same bacterial strain, but gradually increased when taking place between phage that infect different strains or bacterial species. From an evolutionary perspective, we observed that endolysins could be naturally exchanged by homologous recombination between phages coinfecting a same bacterial strain. Furthermore, phage endolysins could adapt to their new phage/host environment by acquiring adaptative mutations. These observations highlight the remarkable ability of phage lytic systems to recombine and adapt and, therefore, explain their large diversity and mosaicism. It also indicates that evolution should be considered to act on functional modules rather than on bacteriophages themselves. Furthermore, the extensive degree of evolvability observed for phage endolysins offers new perspectives for their engineering as antimicrobial agents.

Endolysins are produced by bacteriophages to degrade the host cell wall and release new particles, but the implications of their diversity on phage-host adaptation and evolution is unknown. This study uses CRISPR-Cas9 genome editing to reveal novel insights into bacteriophage endolysin diversity and phage-bacteria interactions as well as into endolysin adaptation towards a new bacterial host.     

Novel Bacteriophages in Enterococcus spp.

Most of the bacteriophages (phages) currently reported in Enterococcus spp. belong to tailed families of bacteriophages Podoviridae, Siphoviridae, and Myoviridae. There is a little information on non-tailed bacteriophages isolated from enterococci. Samples of sewage and piggery effluents were tested on pig and chicken isolates of Enterococcus faecalis, E. faecium and E. gallinarum for lytic phages. In addition, isolates were exposed to mitomycin C to induce lysogenic phages. Bacteriophages that were detected were visualized by electron microscopy. Ten bacteriophages were of isometric shape with long flexible or non-flexible tails, while one had a long head with a long flexible tail; all contained double-stranded DNA molecules. Seven Polyhedral, filamentous, and pleomorphic-shaped phages containing DNA or RNA were also observed. The pleomorphic phages were droplet- or lemon-shaped in morphology. This study is the first report on polyhedral phages in Enterococcus spp. of animal origin and also the first report of filamentous and pleomorphic phages in enterococci.     

The CWPS Rubik’s cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designated cwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A–C). In the present study, the cwps loci of 107 lactococcal strains were compared, revealing the presence of a fourth cwps genotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link between cwps genotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in the cwps cluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galf and phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain.     

Bacteriophage attachment to the S-layer proteins of the mosquito-pathogenic strains ofBacillus sphaericus

The linearly arrayed surface layer proteins found on the mosquito-pathogenic strains ofBacillus sphaericus function as the site of bacteriophage attachment for the ten lytic bacteriophages used in a bacteriophage typing scheme. Attachment to the surface layer proteins was demonstrated by the ability to block bacteriophage binding with antisera and the ability of the purified proteins to neutralize bacteriophage. Bacteriophage-resistant mutants have modified surface proteins that are less able to neutralize bacteriophages than is the protein of the parent strain. No evidence was obtained that sugar residues play a part in bacteriophage attachment. Phage neutralization by surface proteins from strains that do not serve as host to the phage indicates that, although strains in each phage group have a unique surface protein, the proteins do not determine the phage groups.