Evolutionary history of bacteriophages with double-stranded DNA genomes

<p>Abstract</p> <p>Background</p> <p>Reconstruction of evolutionary history of bacteriophages is a difficult problem because of fast sequence drift and lack of omnipresent genes in phage genomes. Moreover, losses and recombinational exchanges of genes are so pervasive in phages that the plausibility of phylogenetic inference in phage kingdom has been questioned.</p> <p>Results</p> <p>We compiled the profiles of presence and absence of 803 orthologous genes in 158 completely sequenced phages with double-stranded DNA genomes and used these gene content vectors to infer the evolutionary history of phages. There were 18 well-supported clades, mostly corresponding to accepted genera, but in some cases appearing to define new taxonomic groups. Conflicts between this phylogeny and trees constructed from sequence alignments of phage proteins were exploited to infer 294 specific acts of intergenome gene transfer.</p> <p>Conclusion</p> <p>A notoriously reticulate evolutionary history of fast-evolving phages can be reconstructed in considerable detail by quantitative comparative genomics.</p> <p>Open peer review</p> <p>This article was reviewed by Eugene Koonin, Nicholas Galtier and Martijn Huynen.</p

Genome classification by gene distribution: An overlapping subspace clustering approach

<p>Abstract</p> <p>Background</p> <p>Genomes of lower organisms have been observed with a large amount of horizontal gene transfers, which cause difficulties in their evolutionary study. Bacteriophage genomes are a typical example. One recent approach that addresses this problem is the unsupervised clustering of genomes based on gene order and genome position, which helps to reveal species relationships that may not be apparent from traditional phylogenetic methods.</p> <p>Results</p> <p>We propose the use of an overlapping subspace clustering algorithm for such genome classification problems. The advantage of subspace clustering over traditional clustering is that it can associate clusters with gene arrangement patterns, preserving genomic information in the clusters produced. Additionally, overlapping capability is desirable for the discovery of multiple conserved patterns within a single genome, such as those acquired from different species via horizontal gene transfers. The proposed method involves a novel strategy to vectorize genomes based on their gene distribution. A number of existing subspace clustering and biclustering algorithms were evaluated to identify the best framework upon which to develop our algorithm; we extended a generic subspace clustering algorithm called HARP to incorporate overlapping capability. The proposed algorithm was assessed and applied on bacteriophage genomes. The phage grouping results are consistent overall with the Phage Proteomic Tree and showed common genomic characteristics among the TP901-like, Sfi21-like and sk1-like phage groups. Among 441 phage genomes, we identified four significantly conserved distribution patterns structured by the terminase, portal, integrase, holin and lysin genes. We also observed a subgroup of Sfi21-like phages comprising a distinctive divergent genome organization and identified nine new phage members to the Sfi21-like genus: <it>Staphylococcus </it>71, phiPVL108, <it>Listeria </it>A118, 2389, <it>Lactobacillus phi </it>AT3, A2, <it>Clostridium </it>phi3626, <it>Geobacillus </it>GBSV1, and <it>Listeria monocytogenes </it>PSA.</p> <p>Conclusion</p> <p>The method described in this paper can assist evolutionary study through objectively classifying genomes based on their resemblance in gene order, gene content and gene positions. The method is suitable for application to genomes with high genetic exchange and various conserved gene arrangement, as demonstrated through our application on phages.</p

Examining recombination and intra-genomic conflict dynamics in the evolution of anti-microbial resistant bacteria

The spread of antimicrobial resistance (AMR) among pathogenic bacterial species threatens to undercut much of the progress made in treating infectious diseases. AMR genes can disseminate between and within populations via horizontal gene transfer (HGT). Selfish mobile genetic elements (MGEs) can encode resistance and spread between host cells. Homologous recombination can alter the core genes of pathogens with resistant donors via HGT too. MGEs may be cured from host genomes through transformation. Hence, MGEs may be able to avoid deletion by disrupting transformation. This work aims to understand how the dynamics of these processes affect the epidemiology of AMR pathogens. To understand these dynamics, I co-developed a new version of the popular recombination detection tool Gubbins. Through simulation studies, I find this new version to be both accurate in reconstructing the relationships between isolates, and efficient in terms of its use of computational resources. I then apply Gubbins to both AMR lineages and species-wide datasets of the pathogen Streptococcus pneumoniae. I find that recombination frequently occurs around core genes involved in both drug resistance and the host immune response. Additionally, an MGE was able to successfully spread within a population by disrupting the transformation machinery, preventing its loss from the host. Finally, I investigate two recent examples of MGEs disrupting transformation in the gram-negative species Acinetobacter baumannii and Legionella pneumophila. I find that while these insertions may decrease the efficiency of transformations within cells, the observed recombination rates largely reflect the selection pressures on isolates. With MGEs only partially able to inhibit these observable transformation events. These results show how selection pressures from clinical interventions shape pathogen genomes through diverse, often interspecies, recombination events. The spread of MGEs can also be favoured by both these selection pressures, and their ability to disrupt host cell machinery.Open Acces

Convergent evolution of PICIs-mediated phage interference

Staphylococcal pathogenicity islands (SaPIs), the prototype members of the family of phage inducible chromosomal islands (PICIs), are extremely mobile phage satellites, which are transferred between bacterial hosts after their induction by a helper phage. The intimate relationship between SaPIs and their helper phages is one of the most studied examples of virus satellite interactions in prokaryotic cells. SaPIs encode and disseminate virulence and fitness factors, representing a driving force for bacterial adaptation and pathogenesis. Many SaPIs encode a conserved morphogenetic operon, including a core set of genes whose function allows them to parasitize and exploit the phage life cycle. One of the central mechanisms of this molecular piracy is the specific packaging of the SaPI genomes into reduced sized capsid structures derived from phage proteins. Pac phages were classically thought to be the only phages involved in the mobilisation of phage-mediated virulence genes, including the transfer of SaPIs within related and non-related bacteria. This study presents the involvement of S. aureus cos phages in the intra- and intergeneric transfer of cos SaPIs for the first time. A novel example of molecular parasitism is shown, by which this newly characterised group of cos SaPIs uses two distinct and complementary mechanisms to take over the helper phage packaging machinery for their own reproduction. SaPIbov5, the prototype of the cos SaPIs, does not encode the characteristic morphogenetic operon found in pac SaPIs. However, cos SaPIs features both pac and cos phage cleavage sequences in their genome, ensuring SaPI packaging in small- and full-sized phage particles, depending on the helper phage. Moreover, cos-site packaging in S. aureus was shown to require the activity of a phage HNH nuclease. The HNH protein functions together with the large terminase subunit, triggering cleavage and melting of the cos-site sequence. In addition, a novel piracy strategy, severely interfering with the helper phage reproduction, was identified in cos SaPIs and characterised. This mechanism of piracy depends on the cos SaPI-encoded ccm gene, which encodes a capsid protein involved in the formation of small phage particles, modifying the assembling process via a scaffolding mechanism. This strategy resembles the ones described for pac SaPIs and represents a remarkable example of convergent evolution. A further convergent mechanism of capsid size-reduction was identified and characterised for the Enterococcus faecalis EfCIV583 pathogenicity island, another member of the PICI family. In this case, the self-encoded CpmE conducts this molecular piracy through a putative scaffolding function. Similar to cos SaPIs, EfCIV583 carries the helper phage cleavage sequence in its genome enabling its mobilisation by the phage terminase complex. The results presented in this thesis show how two examples of non-related members of the PICI family follow the same evolutionary convergent strategy to interfere with their helper phage. These findings could indicate that the described strategies might be widespread among PICIs and implicate a significant impact of PICIs mediated-virulence gene transfer in bacterial evolution and the emergence of pathogenic bacteria

Fleas of fleas: The potential role of bacteriophages in Salmonella diversity and pathogenicity.

Non-typhoidal salmonellosis is an important foodborne and zoonotic infection, that causes significant global public health concern. Diverse serovars are multidrug-resistant and encode several virulence indicators, however, little is known on the role prophages play in driving these characteristics. Here, we extracted prophages from 75 Salmonella genomes, which represent the 15 most important serovars in the United Kingdom. We analysed the genomes of the intact prophages for the presence of virulence factors which were associated with; diversity, evolution and pathogenicity of Salmonella and to establish their genomic relationships. We identified 615 prophage elements from the Salmonella genomes, from which 195 prophages are intact, 332 being incomplete while 88 are questionable. The average prophage carriage was found to be more prevalent in S. Heidelberg, S. Inverness and S. Newport (10.2-11.6 prophages/strain), compared to S. Infantis, S. Stanley, S. Typhimurium and S. Virchow (8.2-9 prophages/strain) and S. Agona, S. Braenderup, S. Bovismorbificans, S. Choleraesuis, S. Dublin, and S. Java (6-7.8 prophages/strain), and S. Javiana and S. Enteritidis (5.8 prophages/strain). Cumulatively, 2760 virulence factors were detected from the intact prophages and associated with cellular functionality being linked to effector delivery/secretion system (73%), adherence (22%), magnesium uptake (2.7%), resistance to antimicrobial peptides (0.94%), stress/survival (0.4%), exotoxins (0.32%) and antivirulence (0.18%). Close and distant clusters were formed among the prophage genomes suggesting different lineages and associations with bacteriophages of other Enterobacteriaceae. We show that diverse repertoire of Salmonella prophages are associated with numerous virulence factors, and may contribute to diversity, pathogenicity and success of specific serovars

Characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting Shiga toxin encoding bacteriophage

Shigatoxigenic Escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of Shiga toxin-encoding lambdoid bacteriophages. At least some of these Stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. Here, increasing levels of Stx phage Φ24B integrase expression in multiple lysogen cultures are demonstrated along with apparently negligible repression of integrase expression by the cognate CI repressor. The Φ24B int transcription start site and promoter region were identified and found to differ from in silico predictions. The unidirectional activity of this integrase was determined in an in situ, inducible tri-partite reaction. This indicated that Φ24B must encode a novel directionality factor that is controlling excision events during prophage induction. This excisionase was subsequently identified and characterized through complementation experiments. In addition, the previous proposal that a putative antirepressor was responsible for the lack of immunity to superinfection through inactivation of CI has been revisited and a new hypothesis involving the role of this protein in promoting efficient induction of the Φ24B prophage is proposed

Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons

Background: Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. Results: Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. Conclusions: Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain

Exploring the Non-vertical Component of Bacterial Evolution Using Network Structures

The central tree metaphor has been challenged over the last couple of decades with the observation of incongruent trees derived largely from protein-coding genes in prokaryotic genomes. There are an increasing number of evolutionary processes and entities that confuse and confound the traditional understanding of evolution. As a result, these processes and entities are very often omitted from phylogenetic studies altogether. In this thesis I attempt to uncover the importance of non-tree like evolution. I discuss the types of genes that do not adhere to vertical patterns of inheritance such as fusion genes and mobile genetic elements. Furthermore I explore the alternative of using network structures in describing the evolutionary history of bacteria. This thesis recounts two key uses of networks for revealing the less commonly noted aspects of bacterial evolution. Firstly I present each stage in the development of a new method for identifying fusions of unrelated genes from conception of the idea, through the implementation to its application to data. Secondly I use networks of gene sharing to elucidate patterns of divergence among a group of closely related bacteria that would have once formed a single species cloud. These studies reveal an abundance of the types of genes that contradict traditional tree-thinking and support the notion that a strictly vertical view of evolution is inadequate when describing bacterial relationships