Protective effect of probiotics on Salmonella infectivity assessed with combined in vitro gut fermentation-cellular models

Background

The alphaproteobacterium Wolbachia pipientis, the most common endosymbiont in eukaryotes, is found predominantly in insects including many Drosophila species. Although Wolbachia is primarily vertically transmitted, analysis of its genome provides evidence for frequent horizontal transfer, extensive recombination and numerous mobile genetic elements. The genome sequence of Wolbachia in Drosophila simulans Riverside (wRi) is available along with the integrated bacteriophages, enabling a detailed examination of phage genes and the role of these genes in the biology of Wolbachia and its host organisms. Wolbachia is widely known for its ability to modify the reproductive patterns of insects. One particular modification, cytoplasmic incompatibility, has previously been shown to be dependent on Wolbachia density and inversely related to the titer of lytic phage. The wRi genome has four phage regions, two WORiBs, one WORiA and one WORiC.

Results

In this study specific primers were designed to distinguish between these four prophage types in wRi, and quantitative PCR was used to measure the titer of bacteriophages in testes, ovaries, embryos and adult flies. In all tissues tested, WORiA and WORiB were not found to be present in excess of their integrated prophages; WORiC, however, was found to be present extrachromosomally. WORiC is undergoing extrachromosomal replication in wRi. The density of phage particles was found to be consistent in individual larvae in a laboratory population. The WORiC genome is organized in conserved blocks of genes and aligns most closely with other known lytic WO phages, WOVitA and WOCauB.

Conclusions

The results presented here suggest that WORiC is the lytic form of WO in D. simulans, is undergoing extrachromosomal replication in wRi, and belongs to a conserved family of phages in Wolbachia.     

Complete WO Phage Sequences Reveal Their Dynamic Evolutionary Trajectories and Putative Functional Elements Required for Integration into the Wolbachia Genome

Wolbachia endosymbionts are ubiquitously found in diverse insects including many medical and hygienic pests, causing a variety of reproductive phenotypes, such as cytoplasmic incompatibility, and thereby efficiently spreading in host insect populations. Recently, Wolbachia-mediated approaches to pest control and management have been proposed, but the application of these approaches has been hindered by the lack of genetic transformation techniques for symbiotic bacteria. Here, we report the genome and structure of active bacteriophages from a Wolbachia endosymbiont. From the Wolbachia strain wCauB infecting the moth Ephestia kuehniella two closely related WO prophages, WOcauB2 of 43,016 bp with 47 open reading frames (ORFs) and WOcauB3 of 45,078 bp with 46 ORFs, were characterized. In each of the prophage genomes, an integrase gene and an attachment site core sequence were identified, which are putatively involved in integration and excision of the mobile genetic elements. The 3′ region of the prophages encoded genes with sequence motifs related to bacterial virulence and protein-protein interactions, which might represent effector molecules that affect cellular processes and functions of their host bacterium and/or insect. Database searches and phylogenetic analyses revealed that the prophage genes have experienced dynamic evolutionary trajectories. Genes similar to the prophage genes were found across divergent bacterial phyla, highlighting the active and mobile nature of the genetic elements. We suggest that the active WO prophage genomes and their constituent sequence elements would provide a clue to development of a genetic transformation vector for Wolbachia endosymbionts.Members of the genus Wolbachia are endosymbiotic bacteria belonging to the Alphaproteobacteria and infecting a wide range of arthropods, including over 60% of insect species, and some filarial nematodes. They are vertically transmitted through the maternal germ line of their host and are known to distort host reproduction by causing cytoplasmic incompatibility (CI), parthenogenesis, male killing, or feminization. The ability of Wolbachia to cause these reproductive phenotypes is thought to be responsible for their efficient and rapid spread into host populations (5, 21, 35, 51).Recently, Wolbachia-mediated pest control approaches have been proposed. A number of insect pests that have important medical and hygienic consequences, such as tsetse flies and mosquitoes that vector devastating human pathogens including African sleeping disease trypanosomes, malaria plasmodia, dengue viruses, Japanese encephalitis viruses, and others, often also carry Wolbachia infections (8, 24, 25, 34). In theory, if maternally transmitted genetic elements coinherited with a CI-inducing Wolbachia, such as mitochondria, the Wolbachia itself, or other coinfecting endosymbionts, are transformed with a gene of interest (like a gene that confers resistance of the vector insect against the pathogen infection), the genetic trait is expected to be spread and fixed in the host insect population, driven by the symbiont-induced reproductive phenotype (1, 2, 10, 11, 13, 32, 43, 44). The paratransgenesis and Wolbachia-driven population replacement approaches are, although potentially promising in controlling such insect-borne diseases, still at a conceptual stage mainly because no technique has been available for Wolbachia transformation.For genetic transformation of bacteria, mobile genetic elements such as plasmids, bacteriophages, and transposons have been used successfully. For example, pUC plasmids, λ phages, and transposons have been widely utilized for transforming Escherichia coli and other model bacterial species (38). While few plasmids and transposons have been reported from Wolbachia, a family of bacteriophages, called WO phages, has been detected from a diverse array of Wolbachia strains (3, 6, 7, 12, 17, 18, 31, 39, 49). For example, in the genomes of the Wolbachia strains wMel from the fruit fly Drosophila melanogaster and wPip from the mosquito Culex quinquefasciatus, three and five WO prophages are present, respectively (26, 52). Many of the prophages are pseudogenized and inactive while some are active and capable of producing phage particles (4, 7, 15, 17, 30, 40). Such active WO phage elements may provide tools for genetic transformation of Wolbachia endosymbionts.λ phage and many other temperate bacteriophages alternate between lytic phase and lysogenic phase in their life cycles. In the lytic phase, phage particles are produced and released via host cell lysis for infection to new host cells. In the lysogenic phase, the phage genome is integrated into the host genome via a site-specific recombination process, and the integrated phage genome, called prophage, is maintained in the host genome and multiplies together with the host DNA replication (38). Upon infection and lysogenic integration of λ phage, both ends of the linear phage genomic DNA are connected by DNA ligase, and the resultant circular phage genome is inserted into the E. coli genome by site-specific recombination at a region containing a core sequence of an attachment (att) site (28). att sites on the phage genome and the bacterial genome are called attP (phage att site) and attB (bacterial att site), respectively. After integration, attP and attB are located on both ends of the prophage, called attL (left prophage att site) and attR (right prophage att site), respectively. The integration and excision processes are mediated by a site-specific recombinase, called λ integrase, encoded in the phage genome (see Fig. S1 in the supplemental material) (27, 50). Hence, the att site and the integrase are the pivotal functional elements that mediate site-specific integration and excision of λ phage. Considering the structural similarity between λ phage and WO phage (31), identification of the att site and integrase from WO phage is of interest in that these elements could be utilized for delivering foreign genes into the Wolbachia genome.In order to identify a functional att site and integrase of WO phage, the complete genome sequences of active prophage elements producing phage particles should be determined. Here, the Wolbachia strain wCauB derived from the almond moth Cadra cautella was investigated because wCauB was reported to actively produce phage particles, and a partial genome sequence of its WO phage has been determined (15). In the original host insect, C. cautella, wCauB coexists with another Wolbachia strain wCauA, and both cause CI phenotypes and produce phage particles (15, 41). Not to be confounded by the coinfecting Wolbachia strains, we used a transfected line of the Mediterranean flour moth Ephestia kuehniella infected with wCauB only, which was generated by interspecific ooplasm transfer (42). It should be noted that a mass preparation procedure for WO phage particles by centrifugation has been established for the wCauB-infected E. kuehniella (15).In this study, we determined the complete genome sequences of two active WO prophages, named WOcauB2 and WOcauB3, that are capable of producing phage particles and that are located on the genome of the Wolbachia strain wCauB. Furthermore, we identified core sequences of att sites and integrase genes of these WO phages that are putatively involved in integration of the genetic elements into the Wolbachia genome.     

Widespread phages of endosymbionts: Phage WO genomics and the proposed taxonomic classification of Symbioviridae

Wolbachia are the most common obligate, intracellular bacteria in animals. They exist worldwide in arthropod and nematode hosts in which they commonly act as reproductive parasites or mutualists, respectively. Bacteriophage WO, the largest of Wolbachia’s mobile elements, includes reproductive parasitism genes, serves as a hotspot for genetic divergence and genomic rearrangement of the bacterial chromosome, and uniquely encodes a Eukaryotic Association Module with eukaryotic-like genes and an ensemble of putative host interaction genes. Despite WO’s relevance to genome evolution, selfish genetics, and symbiotic applications, relatively little is known about its origin, host range, diversification, and taxonomic classification. Here we analyze the most comprehensive set of 150 Wolbachia and phage WO assemblies to provide a framework for discretely organizing and naming integrated phage WO genomes. We demonstrate that WO is principally in arthropod Wolbachia with relatives in diverse endosymbionts and metagenomes, organized into four variants related by gene synteny, often oriented opposite the putative origin of replication in the Wolbachia chromosome, and the large serine recombinase is an ideal typing tool to distinguish the four variants. We identify a novel, putative lytic cassette and WO’s association with a conserved eleven gene island, termed Undecim Cluster, that is enriched with virulence-like genes. Finally, we evaluate WO-like Islands in the Wolbachia genome and discuss a new model in which Octomom, a notable WO-like Island, arose from a split with WO. Together, these findings establish the first comprehensive Linnaean taxonomic classification of endosymbiont phages, including non-Wolbachia phages from aquatic environments, that includes a new family and two new genera to capture the collective relatedness of these viruses.     

High-Efficiency Thermal Asymmetric Interlaced PCR (hiTAIL-PCR) for Determination of a Highly Degenerated Prophage WO Genome in a Wolbachia Strain Infecting a Fig Wasp Species

Temperate bacteriophage WO is a model system for studying tripartite interactions among viruses, bacteria, and eukaryotes, especially investigations of the genomic stability of obligate intracellular bacteria. Few WO genomes exist because of the difficulty in isolating viral DNA from eukaryotic hosts, and most reports are by-products of Wolbachia sequencing. Only one partial genome of a WO phage has been determined directly from isolated particles. We determine the complete genome sequence of prophage WO (WOSol) in Wolbachia strain wSol, which infects the fig wasp Ceratosolen solmsi (Hymenoptera: Chalcidoidea), by high-efficiency thermal asymmetric interlaced PCR. The genome of WOSol is highly degenerated and disrupted by a large region (14,267 bp) from Wolbachia. Consistent with previous molecular studies of multiple WO genomes, the genome of WOSol appears to have evolved by single nucleotide mutations and recombinations.     

Influence of Wolbachia infection on antimicrobial peptide gene expressions in a cell line of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Wolbachia, a symbiotic bacterium found in a broad range of insects, manipulates host reproduction. In addition to reproductive alterations, Wolbachia may also modify the immune system of host insects to protect them from additional pathogenic infection. We hypothesized that Wolbachia exerts protective effects by activating antimicrobial peptide (AMP) genes. To test this hypothesis, we established immunocompetent cell lines derived from the silkworm, Bombyx mori, which were transinfected with two Wolbachia strains, wKue and wCauB, originating from lepidopteran insects and quantified the expression of four AMP genes, cecropin B, defensin B, attacin, and lebocin 3. The expression was measured in wKue-infected, wCauB-infected, and uninfected cells, before and after treatment with peptidoglycan (PGN) that mimicked a bacterial infection. A two-way ANOVA for each gene showed that both Wolbachia infection and PGN treatment significantly increased the gene expression and their interaction. When treated with PGN, wKue- and wCauB-infected cells showed higher expression of the four AMP genes than those in uninfected cells, suggesting that Wolbachia infection increased the ability of host cells to produce AMPs in response to immune stimulation with PGN. These observations suggest that the two Wolbachia strains have immune priming effects and may protect the host insects from a secondary infection.     

Distribution and Evolution of Bacteriophage WO in Wolbachia, the Endosymbiont Causing Sexual Alterations in Arthropods

Wolbachia are obligatory intracellular and maternally inherited bacteria, known to infect many species of arthropod. In this study, we discovered a bacteriophage-like genetic element in Wolbachia, which was tentatively named bacteriophage WO. The phylogenetic tree based on phage WO genes of several Wolbachia strains was not congruent with that based on chromosomal genes of the same strains, suggesting that phage WO was active and horizontally transmitted among various Wolbachia strains. All the strains of Wolbachia used in this study were infected with phage WO. Although the phage genome contained genes of diverse origins, the average G+C content and codon usage of these genes were quite similar to those of a chromosomal gene of Wolbachia. These results raised the possibility that phage WO has been associated with Wolbachia for a very long time, conferring some benefit to its hosts. The evolution and possible roles of phage WO in various reproductive alterations of insects caused by Wolbachia are discussed. Received: 28 January 2000 / Accepted: 3 August 2000     

Tight Regulation of the intS Gene of the KplE1 Prophage: A New Paradigm for Integrase Gene Regulation

Temperate phages have the ability to maintain their genome in their host, a process called lysogeny. For most, passive replication of the phage genome relies on integration into the host''s chromosome and becoming a prophage. Prophages remain silent in the absence of stress and replicate passively within their host genome. However, when stressful conditions occur, a prophage excises itself and resumes the viral cycle. Integration and excision of phage genomes are mediated by regulated site-specific recombination catalyzed by tyrosine and serine recombinases. In the KplE1 prophage, site-specific recombination is mediated by the IntS integrase and the TorI recombination directionality factor (RDF). We previously described a sub-family of temperate phages that is characterized by an unusual organization of the recombination module. Consequently, the attL recombination region overlaps with the integrase promoter, and the integrase and RDF genes do not share a common activated promoter upon lytic induction as in the lambda prophage. In this study, we show that the intS gene is tightly regulated by its own product as well as by the TorI RDF protein. In silico analysis revealed that overlap of the attL region with the integrase promoter is widely encountered in prophages present in prokaryotic genomes, suggesting a general occurrence of negatively autoregulated integrase genes. The prediction that these integrase genes are negatively autoregulated was biologically assessed by studying the regulation of several integrase genes from two different Escherichia coli strains. Our results suggest that the majority of tRNA-associated integrase genes in prokaryotic genomes could be autoregulated and that this might be correlated with the recombination efficiency as in KplE1. The consequences of this unprecedented regulation for excisive recombination are discussed.     

Genetic elements novel for Corynebacterium diphtheriae: specialized transducing elements and transposons

It was shown in an accompanying paper (Buck and Groman, J. Bacteriol. 148: 131-142, 1981) that γ-tsr-1 phage stocks produced by heat induction of lysogens are a mixture of two phages which differ in the content of their deoxyribonucleic acid (DNA). This difference is evidenced by the appearance of “heterogeneous” (HET) fragments in restriction enzyme digests of γ-tsr-1 phage DNA. It was estimated that 20 to 80% of the phage in these lysates produced HET fragments. The appearance of HET fragments correlated with the appearance of a DNA insertion (DI-1) in the γ phage genome as revealed in heteroduplexes of DNA from γ-tsr-1 and β corynebacteriophages. The HET fragments were seen in DNA from heat-induced lysates, but not in DNA from phage stocks produced by lytic infection. By DNA-DNA hybridization analysis it was shown that a fraction of γ-tsr-1 phages from heat-induced lysates carried an insertion of bacterial DNA in the vegetative phage attachment site (attP), and that this insertion was responsible for the formation of HET fragments. Since the phage produced by this event carried a complete phage genome plus a small segment of bacterial DNA, they were called transducing elements. On the basis of these facts it was concluded that heat-induced γ-tsr-1 prophage was excised at an abnormal site at a very high frequency. Abnormal excision was highly specific, and the change in excision specificity occurred simultaneously with the spontaneous mutation of the phage to heat inducibility. From this and other data it was postulated that a mutation in the immune repressor was reponsible for an alteration in the specificity of the normal excision process. This distinguishes the mechanism of formation of γ-tsr-1 transducing elements from that employed by other phages. A second DNA insertion (DI-2) in the tox (diphtheria toxin) gene of γ-tsr-1 and γ-tsr-2 was also identified as an insertion of bacterial DNA. The DI-2 insertion had a stem-and-loop structure similar to that seen in heteroduplexes visualizing transposons or insertion elements. It seems likely that γ wild-type phage, which is mutant for tox, was originally tox⁺, but that transposition of bacterial DNA into the gene inactivated it.     

Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36–42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13–15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10–33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, Mycobacterium and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.     

Bacteriophage phi297, a new species of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> temperate phages with a mosaic genome: Potential use in phage therapy

The genome of halo-forming temperate Pseudomonas aeruginosa phage phi297 and lytic activity of its virulent mutant were studied. A mosaic structure was revealed for phi297 genome by its complete sequencing. The phi297 genome was partly homologous to the genomes of phages D3 and F116. High lytic activity was assumed for temperate P. aeruginosa bacteriophage phi297 on the basis of morphological features of negative colonies. Virulent mutant phi297vir, which was capable of lysing the wild-type phage bacteria, was isolated. Lytic activity was compared for phi297 and the phages from commercial mixtures of two manufacturers (facilities of Nizhnii Novgorod and Perm’). Phage phi297 caused lysis of the mutant PAO1 bacteria that were resistant to the phages from commercial preparations, but the lytic activity spectrum of phi297 was narrower that the spectra of the commercial phages. The use of nonreverting virulent mutants of certain temperate bacteriophages was proposed for the treatment of P. aeruginosa infections.     

Comparative whole genome analysis of six diagnostic brucellaphages

Whole genome sequencing of six diagnostic brucellaphages, Tbilisi (Tb), Firenze (Fz), Weybridge (Wb), S708, Berkeley (Bk) and R/C, was followed with genomic comparisons including recently described genomes of the Tb phage from Mexico (Tb_M) and Pr phage to elucidate genomic diversity and candidate host range determinants. Comparative whole genome analysis revealed high sequence homogeneity among these brucellaphage genomes and resolved three genetic groups consistent with defined host range phenotypes. Group I was composed of Tb and Fz phages that are predominantly lytic for Brucella abortus and Brucella neotomae; Group II included Bk, R/C, and Pr phages that are lytic mainly for B. abortus, Brucella melitensis and Brucella suis; Group III was composed of Wb and S708 phages that are lytic for B. suis, B. abortus and B. neotomae. We found that the putative phage collar protein is a variable locus with features that may be contributing to the host specificities exhibited by different brucellaphage groups. The presence of several candidate host range determinants is illustrated herein for future dissection of the differential host specificity observed among these phages.     

Excision and duplication of su3+-transducing fragments carried by bacteriophage φ80: II. Red- or rec-dependent excision and duplication

During vegetative growth φ80)sus2psu3⁺ and φ80int3sus2psu3⁺ segregate su3^? progeny phages, which have lost suppressor activity, at high frequency, even in the absence of the host Rec system. DNA molecules of the su3^? segregants were equivalent to φ80 DNA, as determined by heteroduplex analysis. Loss of suppressor activity is ascribed either to unequal intermolecular crossing-over or to excision by internal recombination between two homologous regions of the phage genome which bracket the bacterial segment containing the su3⁺ gene. To investigate the recombination system acting on the segregation of su3^? phages, a fec^?int^? deletion derivative of φ80sus2psu3⁺, φ80Δ4sus2psu3⁺, has been isolated that is stable even after several cycles of growth in the absence of the host Rec system. However, segregation of su3^? phages from φ80Δ4sus2psu3⁺ was observed when it was complemented in vivo with the hybrid phage λatt⁸⁰imm⁸⁰ in the absence of the host Rec system. The Δ4 deletion is 12.4% of the φ80 genome, starting at a distance of 1.6% φ80 unit to the right from the φ80 crossover point, pp′, i.e. located between 54.6% and 67.0% φ80 unit, as measured from the left (0%) termini of the mature φ80 DNA molecules. By locating the regions of homology between the DNAs of λ and φ80 (Fiandt et al., 1971), the region deleted in φ80Δ4sus2psu3⁺ was assigned to the genes of the phage Red system and a part of the int gene. In the presence of the host Rec system, φ80Δ4-sus2psu3⁺ segregates both phages, φ80Δ4sus2 and φ80Δ4sus2p(su3⁺)₂, which were excised or duplicated for su3⁺-transducing fragments. The loss of the duplication in φ80Δ4sus2p(su3⁺)₂ is also promoted by the host Rec system. Either of two generalized recombination systems, viral Red system or host Rec system, can play a role in the production of the excisions and the duplications of transducing fragments.     

Prophage lambda at unusual chromosomal locations: III. The components of the secondary attachment sites

The integration of phage λ occurs by a reciprocal genetic exchange, promoted by the product of phage int gene, at specific sites on the phage and bacterial genomes (att's). Lysogenic bacteria thus contain two att's which bracket the inserted prophage. Genetically, the phage, bacterial and prophage att's differ from each other, indicating that each site has specific elements which segregate during recombination.In hosts that lack the bacterial att, phage integration occurs at about 0.5% the normal frequency. It results from Int-promoted recombination between the phage att and any one of many secondary sites in the bacterial genome. To analyze these sites, we measured Int-promoted recombination at the secondary prophage att's. We found that they differed from the normal prophage att's and from the phage att. The secondary sites, therefore, do not appear to carry any of the specific elements of the phage or bacterial att's.The transducing phage isolated from secondary site lysogens integrate at two loci. In the absence of helper, they insert via homology with the bacterial DNA. Co-infection with helper results in their integration at the normal bacterial att.     

Lysis to Kill: Evaluation of the Lytic Abilities,and Genomics of Nine Bacteriophages Infective for Gordonia spp. and Their Potential Use in Activated Sludge Foam Biocontrol

Nine bacteriophages (phages) infective for members of the genus Gordonia were isolated from wastewater and other natural water environments using standard enrichment techniques. The majority were broad host range phages targeting more than one Gordonia species. When their genomes were sequenced, they all emerged as double stranded DNA Siphoviridae phages, ranging from 17,562 to 103,424 bp in size, and containing between 27 and 127 genes, many of which were detailed for the first time. Many of these phage genomes diverged from the expected modular genome architecture of other characterized Siphoviridae phages and contained unusual lysis gene arrangements. Whole genome sequencing also revealed that infection with lytic phages does not appear to prevent spontaneous prophage induction in Gordonia malaquae lysogen strain BEN700. TEM sample preparation techniques were developed to view both attachment and replication stages of phage infection.     

Genomic analyses of a novel bacteriophage (VB_PmiS-Isfahan) within Siphoviridae family infecting Proteus mirabilis

Proteus mirabilis is one of the most common causes of complicated urinary tract infections (UTI), especially in catheter-associated UTIs. The increased resistance to antibiotics, among P. mirabilis isolates has led us to search for alternative antibacterial agents. In this study, genome of a lytic Proteus phage VB_PmiS-Isfahan, isolated from wastewater, and active against planktonic and biofilms of P. mirabilis, isolated from UTI, was analyzed. Accordingly, the genome was sequenced and its similarity to other phages was assessed by the Mauve and EasyFig softwares. “One Click” was used for phylogenetic tree construction. The complete genome of VB_PmiS-Isfahan was 54,836 bp, dsDNA with a G+C content of 36.09%. Nighty-one open reading frames (ORFs) was deduced, among them, 23 were considered as functional genes, based on the homology to the previously characterized proteins. The BLASTn of VB_PmiS-Isfahan showed low similarity to complete genome of Salmonella phages VB_SenS_Sasha, 9NA, and VB_SenS-Sergei. A comparison of Nucleic acid and amino acid sequence, and phylogenetic analyses indicated that the phage is novel, significantly differs, and is distant from other genera, within Siphoviridae family. No virulence-associated and antibiotic resistance genes were detected. Thus, VB_PmiS-Isfahan phage is suggested as a potential novel candidate for the treatment of diseases, caused by P. mirabilis.     

Characterization of Paenibacillus larvae bacteriophages and their genomic relationships to firmicute bacteriophages

Background

Paenibacillus larvae is a Firmicute bacterium that causes American Foulbrood, a lethal disease in honeybees and is a major source of global agricultural losses. Although P. larvae phages were isolated prior to 2013, no full genome sequences of P. larvae bacteriophages were published or analyzed. This report includes an in-depth analysis of the structure, genomes, and relatedness of P. larvae myoviruses Abouo, Davis, Emery, Jimmer1, Jimmer2, and siphovirus phiIBB_Pl23 to each other and to other known phages.

Results

P. larvae phages Abouo, Davies, Emery, Jimmer1, and Jimmer2 are myoviruses with ~50 kbp genomes. The six P. larvae phages form three distinct groups by dotplot analysis. An annotated linear genome map of these six phages displays important identifiable genes and demonstrates the relationship between phages. Sixty phage assembly or structural protein genes and 133 regulatory or other non-structural protein genes were identifiable among the six P. larvae phages. Jimmer1, Jimmer2, and Davies formed stable lysogens resistant to superinfection by genetically similar phages. The correlation between tape measure protein gene length and phage tail length allowed identification of co-isolated phages Emery and Abouo in electron micrographs. A Phamerator database was assembled with the P. larvae phage genomes and 107 genomes of Firmicute-infecting phages, including 71 Bacillus phages. Phamerator identified conserved domains in 1,501 of 6,181 phamilies (only 24.3%) encoded by genes in the database and revealed that P. larvae phage genomes shared at least one phamily with 72 of the 107 other phages. The phamily relationship of large terminase proteins was used to indicate putative DNA packaging strategies. Analyses from CoreGenes, Phamerator, and electron micrograph measurements indicated Jimmer1, Jimmer2, Abouo and Davies were related to phages phiC2, EJ-1, KC5a, and AQ113, which are small-genome myoviruses that infect Streptococcus, Lactobacillus, and Clostridium, respectively.

Conclusions

This paper represents the first comparison of phage genomes in the Paenibacillus genus and the first organization of P. larvae phages based on sequence and structure. This analysis provides an important contribution to the field of bacteriophage genomics by serving as a foundation on which to build an understanding of the natural predators of P. larvae.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-745) contains supplementary material, which is available to authorized users.     

Core and accessory genome architecture in a group of Pseudomonas aeruginosa Mu-like phages

Background

Bacteriophages that infect the opportunistic pathogen Pseudomonas aeruginosa have been classified into several groups. One of them, which includes temperate phage particles with icosahedral heads and long flexible tails, bears genomes whose architecture and replication mechanism, but not their nucleotide sequences, are like those of coliphage Mu. By comparing the genomic sequences of this group of P. aeruginosa phages one could draw conclusions about their ontogeny and evolution.

Results

Two newly isolated Mu-like phages of P. aeruginosa are described and their genomes sequenced and compared with those available in the public data banks. The genome sequences of the two phages are similar to each other and to those of a group of P. aeruginosa transposable phages. Comparing twelve of these genomes revealed a common genomic architecture in the group. Each phage genome had numerous genes with homologues in all the other genomes and a set of variable genes specific for each genome. The first group, which comprised most of the genes with assigned functions, was named “core genome”, and the second group, containing mostly short ORFs without assigned functions was called “accessory genome”. Like in other phage groups, variable genes are confined to specific regions in the genome.

Conclusion

Based on the known and inferred functions for some of the variable genes of the phages analyzed here, they appear to confer selective advantages for the phage survival under particular host conditions. We speculate that phages have developed a mechanism for horizontally acquiring genes to incorporate them at specific loci in the genome that help phage adaptation to the selective pressures imposed by the host.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1146) contains supplementary material, which is available to authorized users.     

Antirepression System Associated with the Life Cycle Switch in the Temperate Podoviridae Phage SPC32H

Prophages switch from lysogenic to lytic mode in response to the host SOS response. The primary factor that governs this switch is a phage repressor, which is typically a host RecA-dependent autocleavable protein. Here, in an effort to reveal the mechanism underlying the phenotypic differences between the Salmonella temperate phages SPC32H and SPC32N, whose genome sequences differ by only two nucleotides, we identified a new class of Podoviridae phage lytic switch antirepressor that is structurally distinct from the previously reported Sipho- and Myoviridae phage antirepressors. The SPC32H repressor (Rep) is not cleaved by the SOS response but instead is inactivated by a small antirepressor (Ant), the expression of which is negatively controlled by host LexA. A single nucleotide mutation in the consensus sequence of the LexA-binding site, which overlaps with the ant promoter, results in constitutive Ant synthesis and consequently induces SPC32N to enter the lytic cycle. Numerous potential Ant homologues were identified in a variety of putative prophages and temperate Podoviridae phages, indicating that antirepressors may be widespread among temperate phages in the order Caudovirales to mediate a prudent prophage induction.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.