Spontaneous Resistance of Erwinia amylovora Against Bacteriophage Y2 Affects Infectivity of Multiple Phages.

Broad application of antibiotics gave rise to increasing numbers of antibiotic resistant bacteria. Therefore, effective alternatives are currently investigated. Bacteriophages, natural predators of bacteria, could work as such an alternative. Although phages can be highly effective at eliminating specific bacteria, phage resistance can be observed after application. The nature of this resistance, however, can differ depending on the phage. Exposing Erwinia amylovora CFBP 1430, the causative agent of fire blight, to the different phages Bue1, L1, S2, S6, or M7 led to transient resistance. The bacteria reversed to a phage sensitive state after the phage was eliminated. When wild type bacteria were incubated with Y2, permanently resistant colonies (1430 Y2R ) formed spontaneously. In addition, 1430 Y2R revealed cross-resistance against other phages (Bue1) or lowered the efficiency of plating (L1, S2, and S6). Pull down experiments revealed that Y2 is no longer able to bind to the mutant suggesting mutation or masking of the Y2 receptor. Other phages tested were still able to bind to 1430 Y2R . Bue1 was observed to still adsorb to the mutant, but no host lysis was found. These findings indicated that, in addition to the alterations of the Y2 receptor, the 1430 Y2R mutant might block phage attack at different stage of infection. Whole genome sequencing of 1430 Y2R revealed a deletion in the gene with the locus tag EAMY_2231. The gene, which encodes a putative galactosyltransferase, was truncated due to the resulting frameshift. The mutant 1430 Y2R was monitored for potential defects or fitness loss. Weaker growth was observed in LB medium compared to the wild type but not in minimal medium. Strain 1430 Y2R was still highly virulent in blossoms even though amylovoran production was observed to be reduced. Additionally, LPS structures were analyzed and were clearly shown to be altered in the mutant. Complementation of the truncated EAMY_2231 in trans restored the wild type phenotype. The truncation of EAMY_2231 can therefore be associated with manifold modifications in 1430 Y2R , which can affect different phages simultaneously.

Bacteriophage S6 requires bacterial cellulose for Erwinia amylovora infection.

Bacteriophages are highly selective in targeting bacteria. This selectivity relies on the specific adsorption of phages to the host cell surface. In this study, a Tn5 transposon mutant library of Erwinia amylovora, the causative agent of fire blight, was screened to identify bacterial receptors required for infection by the podovirus S6. Phage S6 was unable to infect mutants with defects in the bacterial cellulose synthase operon (bcs). The Bcs complex produces and secretes bacterial cellulose, an extracellular polysaccharide associated with bacterial biofilms. Deletion of the bcs operon or associated genes (bcsA, bcsC and bcsZ) verified the crucial role of bacterial cellulose for S6 infection. Application of the cellulose binding dye Congo Red blocked infection by S6. We demonstrate that infective S6 virions degraded cellulose and that Gp95, a phage-encoded cellulase, is involved to catalyse the reaction. In planta S6 did not significantly inhibit fire blight symptom development. Moreover, deletion of bcs genes in E. amylovora did not affect bacterial virulence in blossom infections, indicating that sole application of cellulose targeting phages is less appropriate to biologically control E. amylovora. The interplay between cellulose synthesis, host cell infection and maintenance of the host cell population is discussed.

Isolation of a Novel Lytic Bacteriophage against a Nosocomial Methicillin-Resistant Staphylococcus aureus Belonging to ST45.

Methicillin-resistant Staphylococcus aureus (MRSA) can cause a wide range of infections from mild to life-threatening conditions. Its enhanced antibiotic resistance often leads to therapeutic failures and therefore alternative eradication methods must be considered. Potential candidates to control MRSA infections are bacteriophages and their lytic enzymes, lysins. In this study, we isolated a bacteriophage against a nosocomial MRSA strain belonging to the ST45 epidemiologic group. The phage belonging to Caudovirales, Siphoviridae, showed a narrow host range and stable lytic activity without the emergence of resistant MRSA clones. Phylogenetic analysis showed that the newly isolated Staphylococcus phage R4 belongs to the Triavirus genus in Siphoviridae family. Genetic analysis of the 45 kb sequence of R4 revealed 69 ORFs. No remnants of mobile genetic elements and traces of truncated genes were observed. We have localized the lysin (N-acetylmuramoyl-L-alanine amidase) gene of the new phage that was amplified, cloned, expressed, and purified. Its activity was verified by zymogram analysis. Our findings could potentially be used to develop specific anti-MRSA bacteriophage- and phage lysin-based therapeutic strategies against major clonal lineages and serotypes.

A major-capsid-protein-based multiplex PCR assay for rapid identification of selected virulent bacteriophage types.

Bacteriophages represent a promising alternative for controlling pathogenic bacteria. They are ubiquitous in the environment, and their isolation is usually simple and fast. However, not every phage is suitable for biocontrol applications. It must be virulent (i.e., strictly lytic), non-transducing, and safe. We have developed a method for identifying selected types of virulent phages at an early stage of the isolation process to simplify the search for suitable candidates. Using the major capsid protein (MCP) as a phylogenetic marker, we designed degenerate primers for the identification of Felix O1-, GJ1-, N4-, SP6-, T4-, T7-, and Vi1-like phages in multiplex PCR setups with single phage plaques as templates. Performance of the MCP PCR assay was evaluated with a set of 26 well-characterized phages. Neither false-positive nor false-negative results were obtained. In addition, 154 phages from enrichment cultures from various environmental samples were subjected to MCP PCR analysis. Eight of them, specific for Salmonella enterica, Escherichia coli, or Erwinia amylovora, belonged to one of the selected phage types. Their PCR-based identification was successfully confirmed by pulsed-field gel electrophoresis of the phage genomes, electron microscopy, and sequencing of the amplified mcp gene fragment. The MCP PCR assay was shown to be a simple method for preliminary assignment of new phages to a certain group and thus to identify candidates for biocontrol immediately after their isolation. Given that sufficient sequence data are available, this method can be extended to any phage group of interest.

Complete Genome Sequences of Erwinia amylovora Phages vB_EamP-S2 and vB_EamM-Bue1.

Phages vB_EamP-S2 (S2) and vB_EamM-Bue1 (Bue1) infect the plant pathogen Erwinia amylovora. S2 has a genome size of 45,495 bp and belongs to the genus SP6virus. The genome size of Bue1, related to Salmonella phage Vil, is 164,037 bp. Both phages possess a depolymerase enzyme, a frequent feature of E. amylovora phages.

Erwinia amylovora phage vB_EamM_Y3 represents another lineage of hairy Myoviridae.

To date, a small number of jumbo myoviruses have been reported to possess atypical whisker-like structures along the surface of their contractile tails. Erwinia amylovora phage vB_EamM_Y3 is another example. It possesses a genome of 261,365 kbp with 333 predicted ORFs. Using a combination of BLASTP, Interproscan and HHpred, about 21% of its putative proteins could be assigned functions involved in nucleotide metabolism, DNA replication, virion structure and cell wall degradation. The phage was found to have a signal-arrest-release (SAR) endolysin (Y3_301) possessing a soluble lytic transglycosylase domain. Like other SAR endolysins, inducible expression of Y3_301 caused Escherichia coli lysis, which is dependent on the presence of an N-terminal signal sequence. Phylogenetic analysis showed that its closest relatives are other jumbo phages including Pseudomonas aeruginosa phage PaBG and P. putida phage Lu11, sharing 105 and 87 homologous proteins respectively. Like these phages, Y3 also shares a distant relationship to Ralstonia solanacearum phage ΦRSL1 (sharing 55 homologous proteins). As these phages are unrelated to the Rak2-like group of hairy phages, Y3 along with Lu11 represent a second lineage of hairy myoviruses.

Engineering of Bacteriophages Y2::dpoL1-C and Y2::luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora.

Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting members of the Rosaceae Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range E. amylovora virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of E. amylovora virus L1 (dpoL1-C) or a bacterial luxAB fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein orf68, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::dpoL1-C demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of E. amylovora to colonize the surface of detached flowers. The reporter phage Y2::luxAB transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both in vitro and in plant material.IMPORTANCE Fire blight, caused by Erwinia amylovora, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce E. amylovora on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of E. amylovora cells.

Erwinia gerundensis sp. nov., a cosmopolitan epiphyte originally isolated from pome fruit trees.

A survey to obtain potential antagonists of pome fruit tree diseases yielded two yellow epiphytic bacterial isolates morphologically similar to Pantoea agglomerans, but showing no biocontrol activity. Whole-cell MALDI-TOF mass spectrometry and analysis of 16S rRNA gene and gyrB sequences suggested the possibility of a novel species with a phylogenetic position in either the genus Pantoea or the genus Erwinia. Multi-locus sequence analysis (MLSA) placed the two strains in the genus Erwinia and supported their classification as a novel species. The strains showed general phenotypic characteristics typical of this genus and results of DNA-DNA hybridizations confirmed that they represent a single novel species. Both strains showed a DNA G+C content, as determined by HPLC, of 54.5 mol% and could be discriminated from phylogenetically related species of the genus Erwinia by their ability to utilize potassium gluconate, potassium 2-ketogluconate, maltose, melibiose and raffinose. Whole-genome sequencing of strain EM595T revealed the presence of a chromosomal carotenoid biosynthesis gene cluster similar to those found in species of the genera Cronobacter and Pantoea that explains the pigmentation of the strain, which is atypical for the genus Erwinia. Additional strains belonging to the same species were recovered from different plant hosts in three different continents, revealing the cosmopolitan nature of this epiphyte. The name Erwinia gerundensis sp. nov. is proposed, with EM595T ( = LMG 28990T = CCOS 903T) as the designated type strain.

Fe2+ chelator proferrorosamine A: a gene cluster of Erwinia rhapontici P45 involved in its synthesis and its impact on growth of Erwinia amylovora CFBP1430.

Proferrorosamine A (proFRA) is an iron (Fe2+) chelator produced by the opportunistic plant pathogen Erwinia rhapontici P45. To identify genes involved in proFRA synthesis, transposon mutagenesis was performed. The identified 9.3 kb gene cluster, comprising seven genes, designated rosA-rosG, encodes proteins that are involved in proFRA synthesis. Based on gene homologies, a biosynthetic pathway model for proFRA is proposed. To obtain a better understanding of the effect of proFRA on non-proFRA producing bacteria, E. rhapontici P45 was co-cultured with Erwinia amylovora CFBP1430, a fire-blight-causing plant pathogen. E. rhapontici P45, but not corresponding proFRA-negative mutants, led to a pink coloration of E. amylovora CFBP1430 colonies on King's B agar, indicating accumulation of the proFRA-iron complex ferrorosamine, and growth inhibition in vitro. By saturating proFRA-containing extracts with Fe2+, the inhibitory effect was neutralized, suggesting that the iron-chelating capability of proFRA is responsible for the growth inhibition of E. amylovora CFBP1430.

Protection of Erwinia amylovora bacteriophage Y2 from UV-induced damage by natural compounds.

Bacteriophages have regained much attention as biocontrol agents against bacterial pathogens. However, with respect to stability, phages are biomolecules and are therefore sensitive to a number of environmental influences. UV-irradiation can readily inactivate phage infectivity, which impedes their potential application in the plant phyllosphere. Therefore, phages for control of Erwinia amylovora, the causative agent of fire blight, need to be protected from UV-damage by adequate measures. We investigated the protective effect of different light-absorbing substances on phage particles exposed to UV-light. For this, natural extracts from carrot, red pepper, and beetroot, casein and soy peptone in solution, and purified substances such as astaxanthin, aromatic amino acids, and Tween 80 were prepared and tested as natural sunscreens for phage. All compounds were found to significantly increase half-life of UV-irradiated phage particles and they did not negatively affect phage viability or infectivity. Altogether, a range of readily available, natural substances are suitable as UV-protectants to prevent phage particles from UV-light damage.

The tail-associated depolymerase of Erwinia amylovora phage L1 mediates host cell adsorption and enzymatic capsule removal, which can enhance infection by other phage.

The depolymerase enzyme (DpoL1) encoded by the T7-like phage L1 efficiently degrades amylovoran, an important virulence factor and major component of the extracellular polysaccharide (EPS) of its host, the plant pathogen Erwinia amylovora. Mass spectrometry analysis of hydrolysed EPS revealed that DpoL1 cleaves the galactose-containing backbone of amylovoran. The enzyme is most active at pH 6 and 50°C, and features a modular architecture. Removal of 180 N-terminal amino acids was shown not to affect enzyme activity. The C-terminus harbours the hydrolase activity, while the N-terminal domain links the enzyme to the phage particle. Electron microscopy demonstrated that DpoL1-specific antibodies cross-link phage particles at their tails, either lateral or frontal, and immunogold staining confirmed that DpoL1 is located at the tail spikes. Exposure of high-level EPS-producing Er. amylovora strain CFBP1430 to recombinant DpoL1 dramatically increased sensitivity to the Dpo-negative phage Y2, which was not the case for EPS-negative mutants or low-level EPS-producing Er. amylovora. Our findings indicate that enhanced phage susceptibility is based on enzymatic removal of the EPS capsule, normally a physical barrier to Y2 infection, and that use of DpoL1 together with the broad host range, virulent phage Y2 represents an attractive combination for biocontrol of fire blight.

Inducible Clostridium perfringens bacteriophages ΦS9 and ΦS63: Different genome structures and a fully functional sigK intervening element.

Two inducible temperate bacteriophages ΦS9 and ΦS63 from Clostridium perfringens were sequenced and analyzed. Isometric heads and long non-contractile tails classify ΦS9 and ΦS63 in the Siphoviridae family, and their genomes consist of 39,457 bp (ΦS9) and 33,609 bp (ΦS63) linear dsDNA, respectively. ΦS63 has 3'-overlapping cohesive genome ends, whereas ΦS9 is the first Clostridium phage featuring an experimentally proven terminally redundant and circularly permuted genome. A total of 50 and 43 coding sequences were predicted for ΦS9 and ΦS63, respectively, organized into 6 distinct lifestyle-associated modules typical for temperate Siphoviruses. Putative functions could be assigned to 26 gene products of ΦS9, and to 25 of ΦS63. The ΦS9 attB attachment and insertion site is located in a non-coding region upstream of a putative phosphorylase gene. Interestingly, ΦS63 integrates into the 3' part of sigK in C. perfringens, and represents the first functional skin-element-like phage described for this genus. With respect to possible effects of lysogeny, we did not obtain evidence that ΦS9 may influence sporulation of a lysogenized host. In contrast, interruption of sigK, a sporulation associated gene in various bacteria, by the ΦS63 prophage insertion is more likely to affect sporulation of its carrier.

Novel virulent and broad-host-range Erwinia amylovora bacteriophages reveal a high degree of mosaicism and a relationship to Enterobacteriaceae phages.

A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.

Comparative genome analysis of Listeria bacteriophages reveals extensive mosaicism, programmed translational frameshifting, and a novel prophage insertion site.

The genomes of six Listeria bacteriophages were sequenced and analyzed. Phages A006, A500, B025, P35, and P40 are members of the Siphoviridae and contain double-stranded DNA genomes of between 35.6 kb and 42.7 kb. Phage B054 is a unique myovirus and features a 48.2-kb genome. Phage B025 features 3' overlapping single-stranded genome ends, whereas the other viruses contain collections of terminally redundant, circularly permuted DNA molecules. Phages P35 and P40 have a broad host range and lack lysogeny functions, correlating with their virulent lifestyle. Phages A500, A006, and B025 integrate into bacterial tRNA genes, whereas B054 targets the 3' end of translation elongation factor gene tsf. This is the first reported case of phage integration into such an evolutionarily conserved genetic element. Peptide fingerprinting of viral proteins revealed that both A118 and A500 utilize +1 and -1 programmed translational frameshifting for generating major capsid and tail shaft proteins with C termini of different lengths. In both cases, the unusual +1 frameshift at the 3' ends of the tsh coding sequences is induced by overlapping proline codons and cis-acting shifty stops. Although Listeria phage genomes feature a conserved organization, they also show extensive mosaicism within the genome building blocks. Of particular interest is B025, which harbors a collection of modules and sequences with relatedness not only to other Listeria phages but also to viruses infecting other members of the Firmicutes. In conclusion, our results yield insights into the composition and diversity of Listeria phages and provide new information on their function, genome adaptation, and evolution.