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Culot A, Abriat G, Furlong KP. High-Performance Genome Annotation for a Safer and Faster-Developing Phage Therapy. Viruses 2025; 17:314. [PMID: 40143245 PMCID: PMC11946116 DOI: 10.3390/v17030314] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/23/2025] [Revised: 02/18/2025] [Accepted: 02/21/2025] [Indexed: 03/28/2025] Open
Abstract
Phage therapy, which uses phages to decrease bacterial load in an ecosystem, introduces a multitude of gene copies (bacterial and phage) into said ecosystem. While it is widely accepted that phages have a significant impact on ecology, the mechanisms underlying their impact are not well understood. It is therefore paramount to understand what is released in the said ecosystem, to avoid alterations with difficult-to-predict-but potentially huge-consequences. An in-depth annotation of therapeutic phage genomes is therefore essential. Currently, the average published phage genome has only 20-30% functionally annotated genes, which represents a hurdle to overcome to deliver safe phage therapy, for both patients and the environment. This study aims to compare the effectiveness of manual versus automated phage genome annotation methods. Twenty-seven phage genomes were annotated using SEA-PHAGE and Rime Bioinformatics protocols. The structural (gene calling) and functional annotation results were compared. The results suggest that during the structural annotation step, the SEA-PHAGE method was able to identify an average of 1.5 more genes per phage (typically a frameshift gene) and 5.3 gene start sites per phage. Despite this difference, the impact on functional annotation appeared to be limited: on average, 1.2 genes per phage had erroneous functions, caused by the structural annotation. Rime Bioinformatics' tool (rTOOLS, v2) performed better at assigning functions, especially where the SEA-PHAGE methods assigned hypothetical proteins: 7.0 genes per phage had a better functional annotation on average, compared to SEA PHAGE's 1.7. The method comparison detailed in this article indicate that (1) manual structural annotation is marginally superior to rTOOLS automated structural annotation; (2) rTOOLS automated functional annotation is superior to manual functional annotation. Previously, the only way to obtain a high-quality annotation was by using manual protocols, such as SEA-PHAGES. In the relatively new field of phage therapy, which requires support to advance, manual work can be problematic due to its high cost. Rime Bioinformatics' rTOOLS software allows for time and money to be saved by providing high-quality genome annotations that are comparable to manual results, enabling a safer and faster-developing phage therapy.
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Affiliation(s)
| | | | - Kieran P. Furlong
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON K1H8M5, Canada
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2
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Kalatzis PG, Mauritzen JJ, Winther-Have CS, Michniewski S, Millard A, Tsertou MI, Katharios P, Middelboe M. Staying below the Radar: Unraveling a New Family of Ubiquitous "Cryptic" Non-Tailed Temperate Vibriophages and Implications for Their Bacterial Hosts. Int J Mol Sci 2023; 24:3937. [PMID: 36835353 PMCID: PMC9966536 DOI: 10.3390/ijms24043937] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2022] [Revised: 02/01/2023] [Accepted: 02/02/2023] [Indexed: 02/18/2023] Open
Abstract
Bacteriophages are the most abundant biological entities in the oceans and play key roles in bacterial activity, diversity and evolution. While extensive research has been conducted on the role of tailed viruses (Class: Caudoviricetes), very little is known about the distribution and functions of the non-tailed viruses (Class: Tectiliviricetes). The recent discovery of the lytic Autolykiviridae family demonstrated the potential importance of this structural lineage, emphasizing the need for further exploration of the role of this group of marine viruses. Here, we report the novel family of temperate phages under the class of Tectiliviricetes, which we propose to name "Asemoviridae" with phage NO16 as a main representative. These phages are widely distributed across geographical regions and isolation sources and found inside the genomes of at least 30 species of Vibrio, in addition to the original V. anguillarum isolation host. Genomic analysis identified dif-like sites, suggesting that NO16 prophages recombine with the bacterial genome based on the XerCD site-specific recombination mechanism. The interactions between the NO16 phage and its V. anguillarum host were linked to cell density and phage-host ratio. High cell density and low phage predation levels were shown to favor the temperate over the lytic lifestyle for NO16 viruses, and their spontaneous induction rate was highly variable between different V. anguillarum lysogenic strains. NO16 prophages coexist with the V. anguillarum host in a mutualistic interaction by rendering fitness properties to the host, such as increased virulence and biofilm formation through lysogenic conversion, likely contributing to their global distribution.
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Affiliation(s)
- Panos G. Kalatzis
- Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Elsinore, Denmark
| | - Jesper Juel Mauritzen
- Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Elsinore, Denmark
| | | | - Slawomir Michniewski
- Department of Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
| | - Andrew Millard
- Department of Genetics and Genome Biology, University of Leicester, University Road, Leicester LE1 7RH, UK
| | - Maria Ioanna Tsertou
- Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Former American Base of Gournes, 71500 Heraklion, Greece
| | - Pantelis Katharios
- Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Former American Base of Gournes, 71500 Heraklion, Greece
| | - Mathias Middelboe
- Marine Biological Section, Department of Biology, University of Copenhagen, 3000 Elsinore, Denmark
- Department of Biology, University of Southern Denmark, 5230 Odense, Denmark
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3
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Mobile Element Integration Reveals a Chromosome Dimer Resolution System in Legionellales. mBio 2022; 13:e0217122. [PMID: 36314797 PMCID: PMC9765430 DOI: 10.1128/mbio.02171-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
In bacteria, the mechanisms used to repair DNA lesions during genome replication include homologous recombination between sister chromosomes. This can lead to the formation of chromosome dimers if an odd number of crossover events occurs. The dimers must be resolved before cell separation to ensure genomic stability and cell viability. Dimer resolution is achieved by the broadly conserved dif/Xer system, which catalyzes one additional crossover event immediately prior to cell separation. While dif/Xer systems have been characterized or predicted in the vast majority of proteobacteria, no homologs to dif or xer have been identified in the order Legionellales. Here, we report the discovery of a distinct single-recombinase dif/Xer system in the intracellular pathogen Legionella pneumophila. The dif site was uncovered by our analysis of Legionella mobile element-1 (LME-1), which harbors a dif site mimic and integrates into the L. pneumophila genome via site-specific recombination. We demonstrate that lpg1867 (here named xerL) encodes a tyrosine recombinase that is necessary and sufficient for catalyzing recombination at the dif site and that deletion of dif or xerL causes filamentation along with extracellular and intracellular growth defects. We show that the dif/XerL system is present throughout Legionellales and that Coxiella burnetii XerL and its cognate dif site can functionally substitute for the native system in L. pneumophila. Finally, we describe an unexpected link between C. burnetii dif/Xer and the maintenance of its virulence plasmids. IMPORTANCE The maintenance of circular chromosomes depends on the ability to resolve aberrant chromosome dimers after they form. In most proteobacteria, broadly conserved Xer recombinases catalyze single crossovers at short, species-specific dif sites located near the replication terminus. Chromosomal dimerization leads to the formation of two copies of dif within the same molecule, leading to rapid site-specific recombination and conversion back into chromosome monomers. The apparent absence of chromosome dimer resolution mechanisms in Legionellales has been a mystery to date. By studying a phage-like mobile genetic element, LME-1, we have identified a previously unknown single-recombinase dif/Xer system that is not only widespread across Legionellales but whose activity is linked to virulence in two important human pathogens.
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Atypical integrative element with strand-biased circularization activity assists interspecies antimicrobial resistance gene transfer from Vibrio alfacsensis. PLoS One 2022; 17:e0271627. [PMID: 35917316 PMCID: PMC9345347 DOI: 10.1371/journal.pone.0271627] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2021] [Accepted: 07/06/2022] [Indexed: 11/19/2022] Open
Abstract
The exchange of antimicrobial resistance (AMR) genes between aquaculture and terrestrial microbial populations has emerged as a serious public health concern. However, the nature of the mobile genetic elements in marine bacteria is poorly documented. To gain insight into the genetic mechanisms underlying AMR gene transfer from marine bacteria, we mated a multidrug-resistant Vibrio alfacsensis strain with an Escherichia coli strain, and then determined the complete genome sequences of the donor and the transconjugant strains. Sequence analysis revealed a conjugative multidrug resistance plasmid in the donor strain, which was integrated into the chromosome of the recipient. The plasmid backbone in the transconjugant chromosome was flanked by two copies of a 7.1 kb unclassifiable integrative element harboring a β-lactamase gene. The 7.1 kb element and the previously reported element Tn6283 share four coding sequences, two of which encode the catalytic R-H-R-Y motif of tyrosine recombinases. Polymerase chain reaction and sequencing experiments revealed that these elements generate a circular copy of one specific strand without leaving an empty site on the donor molecule, in contrast to the movement of integron gene cassettes or ICE/IMEs discovered to date. These elements are termed SEs (strand-biased circularizing integrative elements): SE-6945 (the 7.1 kb element) and SE-6283 (Tn6283). The copy number and location of SE-6945 in the chromosome affected the antibiotic resistance levels of the transconjugants. SEs were identified in the genomes of other Vibrio species. Overall, these results suggest that SEs are involved in the spread of AMR genes among marine bacteria.
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5
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Miele S, Provan JI, Vergne J, Possoz C, Ochsenbein F, Barre FX. The Xer activation factor of TLCΦ expands the possibilities for Xer recombination. Nucleic Acids Res 2022; 50:6368-6383. [PMID: 35657090 PMCID: PMC9226527 DOI: 10.1093/nar/gkac429] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2021] [Revised: 05/03/2022] [Accepted: 06/01/2022] [Indexed: 11/13/2022] Open
Abstract
The chromosome dimer resolution machinery of bacteria is generally composed of two tyrosine recombinases, XerC and XerD. They resolve chromosome dimers by adding a crossover between sister copies of a specific site, dif. The reaction depends on a cell division protein, FtsK, which activates XerD by protein-protein interactions. The toxin-linked cryptic satellite phage (TLCΦ) of Vibrio cholerae, which participates in the emergence of cholera epidemic strains, carries a dif-like attachment site (attP). TLCΦ exploits the Xer machinery to integrate into the dif site of its host chromosomes. The TLCΦ integration reaction escapes the control of FtsK because TLCΦ encodes for its own XerD-activation factor, XafT. Additionally, TLCΦ attP is a poor substrate for XerD binding, in apparent contradiction with the high integration efficiency of the phage. Here, we present a sequencing-based methodology to analyse the integration and excision efficiency of thousands of synthetic mini-TLCΦ plasmids with differing attP sites in vivo. This methodology is applicable to the fine-grained analyses of DNA transactions on a wider scale. In addition, we compared the efficiency with which XafT and the XerD-activation domain of FtsK drive recombination reactions in vitro. Our results suggest that XafT not only activates XerD-catalysis but also helps form and/or stabilize synaptic complexes between imperfect Xer recombination sites.
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Affiliation(s)
- Solange Miele
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - James Iain Provan
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Justine Vergne
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Christophe Possoz
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Françoise Ochsenbein
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - François-Xavier Barre
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
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6
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Kumar A, Das B, Kumar N. Vibrio Pathogenicity Island-1: The Master Determinant of Cholera Pathogenesis. Front Cell Infect Microbiol 2020; 10:561296. [PMID: 33123494 PMCID: PMC7574455 DOI: 10.3389/fcimb.2020.561296] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2020] [Accepted: 09/11/2020] [Indexed: 11/13/2022] Open
Abstract
Cholera is an acute secretory diarrhoeal disease caused by the bacterium Vibrio cholerae. The key determinants of cholera pathogenicity, cholera toxin (CT), and toxin co-regulated pilus (TCP) are part of the genome of two horizontally acquired Mobile Genetic Elements (MGEs), CTXΦ, and Vibrio pathogenicity island 1 (VPI-1), respectively. Besides, V. cholerae genome harbors several others MGEs that provide antimicrobial resistance, metabolic functions, and other fitness traits. VPI-1, one of the most well characterized genomic island (GI), deserved a special attention, because (i) it encodes many of the virulence factors that facilitate development of cholera (ii) it is essential for the acquisition of CTXΦ and production of CT, and (iii) it is crucial for colonization of V. cholerae in the host intestine. Nevertheless, VPI-1 is ubiquitously present in all the epidemic V. cholerae strains. Therefore, to understand the role of MGEs in the evolution of cholera pathogen from a natural aquatic habitat, it is important to understand the VPI-1 encoded functions, their acquisition and possible mode of dissemination. In this review, we have therefore discussed our present understanding of the different functions of VPI-1 those are associated with virulence, important for toxin production and essential for the disease development.
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Affiliation(s)
- Ashok Kumar
- Translational Health Science and Technology Institute, Faridabad, India.,Centre for Doctoral Studies, Advanced Research Centre, Manipal Academy of Higher Education, Manipal, India
| | - Bhabatosh Das
- Translational Health Science and Technology Institute, Faridabad, India.,Centre for Doctoral Studies, Advanced Research Centre, Manipal Academy of Higher Education, Manipal, India
| | - Niraj Kumar
- Translational Health Science and Technology Institute, Faridabad, India.,Centre for Doctoral Studies, Advanced Research Centre, Manipal Academy of Higher Education, Manipal, India
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7
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Molecular insights into the genome dynamics and interactions between core and acquired genomes of Vibrio cholerae. Proc Natl Acad Sci U S A 2020; 117:23762-23773. [PMID: 32873641 DOI: 10.1073/pnas.2006283117] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Bacterial species are hosts to horizontally acquired mobile genetic elements (MGEs), which encode virulence, toxin, antimicrobial resistance, and other metabolic functions. The bipartite genome of Vibrio cholerae harbors sporadic and conserved MGEs that contribute in the disease development and survival of the pathogens. For a comprehensive understanding of dynamics of MGEs in the bacterial genome, we engineered the genome of V. cholerae and examined in vitro and in vivo stability of genomic islands (GIs), integrative conjugative elements (ICEs), and prophages. Recombinant vectors carrying the integration module of these GIs, ICE and CTXΦ, helped us to understand the efficiency of integrations of MGEs in the V. cholerae chromosome. We have deleted more than 250 acquired genes from 6 different loci in the V. cholerae chromosome and showed contribution of CTX prophage in the essentiality of SOS response master regulator LexA, which is otherwise not essential for viability in other bacteria, including Escherichia coli In addition, we observed that the core genome-encoded RecA helps CTXΦ to bypass V. cholerae immunity and allow it to replicate in the host bacterium in the presence of similar prophage in the chromosome. Finally, our proteomics analysis reveals the importance of MGEs in modulating the levels of cellular proteome. This study engineered the genome of V. cholerae to remove all of the GIs, ICEs, and prophages and revealed important interactions between core and acquired genomes.
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8
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Balalovski P, Grainge I. Mobilization of p
dif
modules in
Acinetobacter
: A novel mechanism for antibiotic resistance gene shuffling? Mol Microbiol 2020; 114:699-709. [DOI: 10.1111/mmi.14563] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2020] [Revised: 06/18/2020] [Accepted: 06/18/2020] [Indexed: 02/06/2023]
Affiliation(s)
- Phillip Balalovski
- Biological Sciences School of Environmental and Life Sciences University of Newcastle Callaghan NSW Australia
| | - Ian Grainge
- Biological Sciences School of Environmental and Life Sciences University of Newcastle Callaghan NSW Australia
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9
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Espinosa E, Paly E, Barre FX. High-Resolution Whole-Genome Analysis of Sister-Chromatid Contacts. Mol Cell 2020; 79:857-869.e3. [PMID: 32681820 DOI: 10.1016/j.molcel.2020.06.033] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2019] [Revised: 06/15/2020] [Accepted: 06/22/2020] [Indexed: 12/12/2022]
Abstract
Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.
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Affiliation(s)
- Elena Espinosa
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Université Paris Sud, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - Evelyne Paly
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Université Paris Sud, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
| | - François-Xavier Barre
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Université Paris Sud, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.
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10
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Lin DL, Traglia GM, Baker R, Sherratt DJ, Ramirez MS, Tolmasky ME. Functional Analysis of the Acinetobacter baumannii XerC and XerD Site-Specific Recombinases: Potential Role in Dissemination of Resistance Genes. Antibiotics (Basel) 2020; 9:E405. [PMID: 32668667 PMCID: PMC7399989 DOI: 10.3390/antibiotics9070405] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2020] [Revised: 07/09/2020] [Accepted: 07/11/2020] [Indexed: 12/12/2022] Open
Abstract
Modules composed of a resistance gene flanked by Xer site-specific recombination sites, the vast majority of which were found in Acinetobacter baumannii, are thought to behave as elements that facilitate horizontal dissemination. The A. baumannii xerC and xerD genes were cloned, and the recombinant clones used to complement the cognate Escherichia coli mutants. The complemented strains supported the resolution of plasmid dimers, and, as is the case with E. coli and Klebsiella pneumoniae plasmids, the activity was enhanced when the cells were grown in a low osmolarity growth medium. Binding experiments showed that the partially purified A. baumannii XerC and XerD proteins (XerCAb and XerDAb) bound synthetic Xer site-specific recombination sites, some of them with a nucleotide sequence deduced from existing A. baumannii plasmids. Incubation with suicide substrates resulted in the covalent attachment of DNA to a recombinase, probably XerCAb, indicating that the first step in the recombination reaction took place. The results described show that XerCAb and XerDAb are functional proteins and support the hypothesis that they participate in horizontal dissemination of resistant genes among bacteria.
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Affiliation(s)
- David L. Lin
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA 92831, USA; (D.L.L.); (M.S.R.)
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; (R.B.); (D.J.S.)
| | - German M. Traglia
- Departamento de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, Universidad de la República (UDeLaR), Montevideo 11600, Uruguay;
| | - Rachel Baker
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; (R.B.); (D.J.S.)
| | - David J. Sherratt
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; (R.B.); (D.J.S.)
| | - Maria Soledad Ramirez
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA 92831, USA; (D.L.L.); (M.S.R.)
| | - Marcelo E. Tolmasky
- Center for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA 92831, USA; (D.L.L.); (M.S.R.)
- Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK; (R.B.); (D.J.S.)
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11
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Mauritzen JJ, Castillo D, Tan D, Svenningsen SL, Middelboe M. Beyond Cholera: Characterization of zot-Encoding Filamentous Phages in the Marine Fish Pathogen Vibrio anguillarum. Viruses 2020; 12:v12070730. [PMID: 32640584 PMCID: PMC7412436 DOI: 10.3390/v12070730] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2020] [Revised: 06/29/2020] [Accepted: 07/02/2020] [Indexed: 12/22/2022] Open
Abstract
Zonula occludens toxin (Zot) is a conserved protein in filamentous vibriophages and has been reported as a putative toxin in Vibrio cholerae. Recently, widespread distribution of zot-encoding prophages was found among marine Vibrio species, including environmental isolates. However, little is known about the dynamics of these prophages beyond V. cholerae. In this study, we characterized and quantified the zot-encoding filamentous phage VAIϕ, spontaneously induced from the fish pathogen V. anguillarum. VAIϕ contained 6117 bp encoding 11 ORFs, including ORF8pVAI, exhibiting 27%–73% amino acid identity to Inovirus Zot-like proteins. A qPCR method revealed an average of four VAIϕ genomes per host genome during host exponential growth phase, and PCR demonstrated dissemination of induced VAIϕ to other V. anguillarum strains through re-integration in non-lysogens. VAIϕ integrated into both chromosomes of V. anguillarum by recombination, causing changes in a putative ORF in the phage genome. Phylogenetic analysis of the V. anguillarumInoviridae elements revealed mosaic genome structures related to mainly V. cholerae. Altogether, this study contributes to the understanding of Inovirus infection dynamics and mobilization of zot-like genes beyond human pathogenic vibrios, and discusses their potential role in the evolution of the fish pathogen V. anguillarum.
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Affiliation(s)
- Jesper Juel Mauritzen
- Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark; (J.J.M.); (D.C.)
| | - Daniel Castillo
- Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark; (J.J.M.); (D.C.)
| | - Demeng Tan
- Section for Biomolecular Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200 København N, Denmark; (D.T.); (S.L.S.)
| | - Sine Lo Svenningsen
- Section for Biomolecular Sciences, University of Copenhagen, Ole Maaløes Vej 5, 2200 København N, Denmark; (D.T.); (S.L.S.)
| | - Mathias Middelboe
- Marine Biological Section, University of Copenhagen, Strandpromenaden 5, 3000 Helsingør, Denmark; (J.J.M.); (D.C.)
- Correspondence: ; Tel.: +45-35-32-19-91
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12
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Yeh TY. XerD-dependent integration of a novel filamentous phage Cf2 into the Xanthomonas citri genome. Virology 2020; 548:160-167. [PMID: 32838937 DOI: 10.1016/j.virol.2020.06.010] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2020] [Revised: 06/16/2020] [Accepted: 06/17/2020] [Indexed: 11/27/2022]
Abstract
Filamentous Inoviridae phages integrate into the chromosome of plant pathogens Xanthomonas as prophages, but their diversity and integrative mechanism are not completely understood. A proviral Cf2 sequence of 6454 bases from Xanthomonas citri genome was revived as infectious virions able to lysogenize its host. Unlike other Xanthomonas phages (Cf1c, φLf, Xf109, XacF1), Cf2 phage has RstA/RstB replication protein, and its attP has XerD binding arm and dif central region but lacks XerC binding arm. XerC+/Xf109 and XerD+/Cf2 attPs are in the opposite direction in phage genomes. Moreover, XerCD binding and XerD catalysis for strand exchange are necessary for site-specific integration of XerD+/Cf2 and XerC+/Xf109 attPs. Taken together, these results provide a new insight into the mechanism of XerCD-mediated recombination at XerD + attP.
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Affiliation(s)
- Ting-Yu Yeh
- Agricultural Biotechnology Laboratory, Auxergen Inc., Columbus Center, Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, MD, 21202, USA.
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13
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Shapiro JW, Putonti C. UPΦ phages, a new group of filamentous phages found in several members of Enterobacteriales. Virus Evol 2020; 6:veaa030. [PMID: 32607251 PMCID: PMC7307601 DOI: 10.1093/ve/veaa030] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Filamentous phages establish chronic infections in their bacterial hosts, and new phages are secreted by infected bacteria for multiple generations, typically without causing host death. Often, these viruses integrate in their host's genome by co-opting the host's XerCD recombinase system. In several cases, these viruses also encode genes that increase bacterial virulence in plants and animals. Here, we describe a new filamentous phage, UPϕ901, which we originally found integrated in a clinical isolate of Escherichia coli from urine. UPϕ901 and closely related phages can be found in published genomes of over 200 other bacteria, including strains of Citrobacter koseri, Salmonella enterica, Yersinia enterocolitica, and Klebsiella pneumoniae. Its closest relatives are consistently found in urine or in the blood and feces of patients with urinary tract infections. More distant relatives can be found in isolates from other environments, including sewage, water, soil, and contaminated food. Each of these phages, which we collectively call 'UPϕ viruses', also harbors two or more novel genes of unknown function.
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Affiliation(s)
- Jason W Shapiro
- Department of Biology, Loyola University Chicago, 1032 W Sheridan Rd, Chicago, IL 60660, USA
| | - Catherine Putonti
- Department of Biology, Loyola University Chicago, 1032 W Sheridan Rd, Chicago, IL 60660, USA.,Department of Computer Science, Loyola University Chicago, 1052 W Loyola Ave, Chicago, IL, 60626, USA.,Bioinformatics Program, Loyola University Chicago, 1052 W Loyola Ave, Chicago, IL 60626, USA.,Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, 2160 S First Ave, Maywood, IL 60153, USA
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14
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Salgado-Camargo AD, Castro-Jaimes S, Gutierrez-Rios RM, Lozano LF, Altamirano-Pacheco L, Silva-Sanchez J, Pérez-Oseguera Á, Volkow P, Castillo-Ramírez S, Cevallos MA. Structure and Evolution of Acinetobacter baumannii Plasmids. Front Microbiol 2020; 11:1283. [PMID: 32625185 PMCID: PMC7315799 DOI: 10.3389/fmicb.2020.01283] [Citation(s) in RCA: 59] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2020] [Accepted: 05/20/2020] [Indexed: 11/24/2022] Open
Abstract
Acinetobacter baumannii is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that A. baumannii plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by Bertini et al. (2010). Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the Acinetobacter genus. Moreover, some plasmid lineages are widely distributed along the A. baumannii phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role.
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Affiliation(s)
- Abraham D Salgado-Camargo
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Semiramis Castro-Jaimes
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Rosa-Maria Gutierrez-Rios
- Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Luis F Lozano
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Luis Altamirano-Pacheco
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Jesús Silva-Sanchez
- Grupo de Resistencia Bacteriana, Centro de Investigaciones Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Mexico
| | - Ángeles Pérez-Oseguera
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Patricia Volkow
- Departamento de Infectología, Instituto Nacional de Cancerología, Mexico City, Mexico
| | - Santiago Castillo-Ramírez
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
| | - Miguel A Cevallos
- Programa de Genómica Evolutiva, Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Mexico
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15
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Kirchberger PC, Ochman H. Resurrection of a global, metagenomically defined gokushovirus. eLife 2020; 9:e51599. [PMID: 32101162 PMCID: PMC7062461 DOI: 10.7554/elife.51599] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Accepted: 02/25/2020] [Indexed: 12/16/2022] Open
Abstract
Gokushoviruses are single-stranded, circular DNA bacteriophages found in metagenomic datasets from diverse ecosystems worldwide, including human gut microbiomes. Despite their ubiquity and abundance, little is known about their biology or host range: Isolates are exceedingly rare, known only from three obligate intracellular bacterial genera. By synthesizing circularized phage genomes from prophages embedded in diverse enteric bacteria, we produced gokushoviruses in an experimentally tractable model system, allowing us to investigate their features and biology. We demonstrate that virions can reliably infect and lysogenize hosts by hijacking a conserved chromosome-dimer resolution system. Sequence motifs required for lysogeny are detectable in other metagenomically defined gokushoviruses; however, we show that even partial motifs enable phages to persist cytoplasmically without leading to collapse of their host culture. This ability to employ multiple, disparate survival strategies is likely key to the long-term persistence and global distribution of Gokushovirinae.
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Affiliation(s)
- Paul C Kirchberger
- Department of Integrative Biology University of TexasAustinUnited States
| | - Howard Ochman
- Department of Integrative Biology University of TexasAustinUnited States
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16
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Midonet C, Miele S, Paly E, Guerois R, Barre FX. The TLCΦ satellite phage harbors a Xer recombination activation factor. Proc Natl Acad Sci U S A 2019; 116:18391-18396. [PMID: 31420511 PMCID: PMC6744903 DOI: 10.1073/pnas.1902905116] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens.
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Affiliation(s)
- Caroline Midonet
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Commissariat à l'Energie Atomique et aux Energies Alternatives, CNRS, Université Paris Sud, 91198 Gif sur Yvette, France
| | - Solange Miele
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Commissariat à l'Energie Atomique et aux Energies Alternatives, CNRS, Université Paris Sud, 91198 Gif sur Yvette, France
| | - Evelyne Paly
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Commissariat à l'Energie Atomique et aux Energies Alternatives, CNRS, Université Paris Sud, 91198 Gif sur Yvette, France
| | - Raphaël Guerois
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Commissariat à l'Energie Atomique et aux Energies Alternatives, CNRS, Université Paris Sud, 91198 Gif sur Yvette, France
| | - François-Xavier Barre
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, Commissariat à l'Energie Atomique et aux Energies Alternatives, CNRS, Université Paris Sud, 91198 Gif sur Yvette, France
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17
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Insights into TLCΦ lysogeny: A twist in the mechanism of IMEX integration. Proc Natl Acad Sci U S A 2019; 116:18159-18161. [PMID: 31439815 DOI: 10.1073/pnas.1912633116] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
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18
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CTX phage of Vibrio cholerae: Genomics and applications. Vaccine 2019; 38 Suppl 1:A7-A12. [PMID: 31272871 DOI: 10.1016/j.vaccine.2019.06.034] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 04/22/2019] [Accepted: 06/11/2019] [Indexed: 01/03/2023]
Abstract
The bipartite genome of Vibrio cholerae is divided into two circular non-homologous chromosomes, which harbor several genetic elements like phages, plasmids, transposons, integrative conjugative elements, and pathogenic islands that encode functions responsible for disease development, antimicrobial resistance, and subsistence in hostile environments. These elements are highly heterogeneous, mobile in nature, and encode their own mobility functions or exploit host-encoded enzymes for intra- and inter-cellular movements. The key toxin of V. cholerae responsible for the life-threatening diarrheal disease cholera, the cholera toxin, is coded by part of the genome of a filamentous phage, CTXϕ. The replicative genome of CTXϕ is divided into two distinct modular structures and has adopted a unique strategy for its irreversible integration into the V. cholerae chromosomes. CTXϕ exploits two host-encoded tyrosine recombinases, XerC and XerD, for its integration in the highly conserved dimer resolution site (dif) of V. cholerae chromosomes. CTXϕ can replicate only in the limited number of Vibrio species. In contrast, the phage integration into the bacterial chromosome does not rely on its replication and could integrate to the dif site of large numbers of gram-negative bacteria. Recent pangenomic analysis revealed that like CTXϕ, the bacterial dif site is the integration spot for several other mobile genetic elements such as plasmids and genomic islands. In this review we discuss about current molecular insights into CTXϕ genomics and its replication and integration mechanisms into hosts. Particular emphasis has been given on the exploitation of CTXϕ genomics knowledge in developing genetic tools and designing environmentally safe recombinant live oral cholera vaccine strains.
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19
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Hay ID, Lithgow T. Filamentous phages: masters of a microbial sharing economy. EMBO Rep 2019; 20:e47427. [PMID: 30952693 PMCID: PMC6549030 DOI: 10.15252/embr.201847427] [Citation(s) in RCA: 111] [Impact Index Per Article: 18.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2018] [Revised: 01/30/2019] [Accepted: 03/19/2019] [Indexed: 12/11/2022] Open
Abstract
Bacteriophage ("bacteria eaters") or phage is the collective term for viruses that infect bacteria. While most phages are pathogens that kill their bacterial hosts, the filamentous phages of the sub-class Inoviridae live in cooperative relationships with their bacterial hosts, akin to the principal behaviours found in the modern-day sharing economy: peer-to-peer support, to offset any burden. Filamentous phages impose very little burden on bacteria and offset this by providing service to help build better biofilms, or provision of toxins and other factors that increase virulence, or modified behaviours that provide novel motile activity to their bacterial hosts. Past, present and future biotechnology applications have been built on this phage-host cooperativity, including DNA sequencing technology, tools for genetic engineering and molecular analysis of gene expression and protein production, and phage-display technologies for screening protein-ligand and protein-protein interactions. With the explosion of genome and metagenome sequencing surveys around the world, we are coming to realize that our knowledge of filamentous phage diversity remains at a tip-of-the-iceberg stage, promising that new biology and biotechnology are soon to come.
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Affiliation(s)
- Iain D Hay
- School of Biological Sciences, University of Auckland, Auckland, New Zealand
| | - Trevor Lithgow
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, Vic., Australia
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20
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Pham TD, Nguyen TH, Iwashita H, Takemura T, Morita K, Yamashiro T. Comparative analyses of CTX prophage region of Vibrio cholerae seventh pandemic wave 1 strains isolated in Asia. Microbiol Immunol 2018; 62:635-650. [PMID: 30211956 PMCID: PMC6220881 DOI: 10.1111/1348-0421.12648] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2018] [Revised: 09/02/2018] [Accepted: 09/04/2018] [Indexed: 11/28/2022]
Abstract
Vibrio cholerae O1 causes cholera, and cholera toxin, the principal mediator of massive diarrhea, is encoded by ctxAB in the cholera toxin (CTX) prophage. In this study, the structures of the CTX prophage region of V. cholerae strains isolated during the seventh pandemic wave 1 in Asian countries were determined and compared. Eighteen strains were categorized into eight groups by CTX prophage region‐specific restriction fragment length polymorphism and PCR profiles and the structure of the region of a representative strain from each group was determined by DNA sequencing. Eight representative strains revealed eight distinct CTX prophage regions with various combinations of CTX‐1, RS1 and a novel genomic island on chromosome I. CTX prophage regions carried by the wave 1 strains were diverse in structure. V. cholerae strains with an area specific CTX prophage region are believed to circulate in South‐East Asian countries; additionally, multiple strains with distinct types of CTX prophage region are co‐circulating in the area. Analysis of a phylogenetic tree generated by single nucleotide polymorphism differences across 2483 core genes revealed that V. cholerae strains categorized in the same group based on CTX prophage region structure were segregated in closer clusters. CTX prophage region‐specific recombination events or gain and loss of genomic elements within the region may have occurred at much higher frequencies and contributed to producing a panel of CTX prophage regions with distinct structures among V. cholerae pathogenic strains in lineages with close genetic backgrounds in the early wave 1 period of the seventh cholera pandemic.
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Affiliation(s)
- Tho Duc Pham
- Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
| | - Tuan Hai Nguyen
- Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan
| | - Hanako Iwashita
- Department of Bacteriology, Graduate School of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan
| | - Taichiro Takemura
- Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.,Vietnam Research Station, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
| | - Kouichi Morita
- Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.,Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan
| | - Tetsu Yamashiro
- Department of Bacteriology, Graduate School of Medicine, University of the Ryukyus, Nishihara, Okinawa, Japan
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21
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Dorman MJ, Dorman CJ. Regulatory Hierarchies Controlling Virulence Gene Expression in Shigella flexneri and Vibrio cholerae. Front Microbiol 2018; 9:2686. [PMID: 30473684 PMCID: PMC6237886 DOI: 10.3389/fmicb.2018.02686] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2018] [Accepted: 10/22/2018] [Indexed: 12/13/2022] Open
Abstract
Gram-negative enteropathogenic bacteria use a variety of strategies to cause disease in the human host and gene regulation in some form is typically a part of the strategy. This article will compare the toxin-based infection strategy used by the non-invasive pathogen Vibrio cholerae, the etiological agent in human cholera, with the invasive approach used by Shigella flexneri, the cause of bacillary dysentery. Despite the differences in the mechanisms by which the two pathogens cause disease, they use environmentally-responsive regulatory hierarchies to control the expression of genes that have some features, and even some components, in common. The involvement of AraC-like transcription factors, the integration host factor, the Factor for inversion stimulation, small regulatory RNAs, the RNA chaperone Hfq, horizontal gene transfer, variable DNA topology and the need to overcome the pervasive silencing of transcription by H-NS of horizontally acquired genes are all shared features. A comparison of the regulatory hierarchies in these two pathogens illustrates some striking cross-species similarities and differences among mechanisms coordinating virulence gene expression. S. flexneri, with its low infectious dose, appears to use a strategy that is centered on the individual bacterial cell, whereas V. cholerae, with a community-based, quorum-dependent approach and an infectious dose that is several orders of magnitude higher, seems to rely more on the actions of a bacterial collective.
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Affiliation(s)
- Matthew J Dorman
- Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom
| | - Charles J Dorman
- Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College Dublin, Dublin, Ireland
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22
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Carr CE, Marky LA. Increased Flexibility between Stems of Intramolecular Three-Way Junctions by the Insertion of Bulges. Biophys J 2018; 114:2764-2774. [PMID: 29925014 PMCID: PMC6026347 DOI: 10.1016/j.bpj.2018.05.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2018] [Revised: 04/25/2018] [Accepted: 05/01/2018] [Indexed: 12/20/2022] Open
Abstract
Intramolecular junctions are a ubiquitous structure within DNA and RNA; three-way junctions in particular have high strain around the junction because of the lack of flexibility, preventing the junctions from adopting conformations that would allow for optimal folding. In this work, we used a combination of calorimetric and spectroscopic techniques to study the unfolding of four intramolecular three-way junctions. The control three-way junction, 3H, has the sequence d(GAAATTGCGCT5GCGCGTGCT5GCACAATTTC), which has three arms of different sequences. We studied three other three-way junctions in which one (2HS1H), two (HS12HS1), and three (HS1HS1HS1) cytosine bulges were placed at the junction to allow the arms to adopt a wider range of conformations that may potentially relieve strain. Through calorimetric studies, it was concluded that bulges produce only minor effects on the enthalpic and thermal stability at physiological salt concentrations for 2HS1H and HS1HS1HS1. HS12HS1 displays the strongest effect, with the GTGC stem lacking a defined transition. In addition to unfolding thermodynamics, the differential binding of counterions, water, and protons was determined. It was found that with each bulge, there was a large increase in the binding of counterions; this correlated with a decrease in the immobilization of structural water molecules. The increase in counterion uptake upon folding likely displaces binding of structural water, which is measured by the osmotic stress method, in favor of electrostricted waters. The cytosine bulges do not affect the binding of protons; this finding indicates that the bulges are not forming base-triplet stacks. These results indicate that bulges in junctions do not affect the unfolding profile or the enthalpy of oligonucleotides but do affect the number and amount of molecules immobilized by the junction.
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Affiliation(s)
- Carolyn E Carr
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska
| | - Luis A Marky
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska.
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23
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Carr CE, Marky LA. Effect of GCAA stabilizing loops on three- and four-way intramolecular junctions. Phys Chem Chem Phys 2018; 20:5046-5056. [PMID: 29388988 DOI: 10.1039/c7cp08329g] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
Tetraloops are a common way of changing the melting behavior of a DNA or RNA structure without changing the sequence of the stem. Because of the ubiquitous nature of tetraloops, our goal is to understand the effect a GCAA tetraloop, which belongs to the GNRA family of tetraloops, has on the unfolding thermodynamics of intramolecular junctions. Specifically, we have described the melting behavior of intramolecular three-way and four-way junctions where a T5 loop has been replaced with a GCAA tetraloops in different positions. Their thermodynamic profiles, including ΔnNa+ and ΔnW, were analyzed based on the position of the tetraloop. We obtained between -16.7 and -27.5 kcal mol-1 for all junctions studied. The experimental data indicates the influence of the GCAA tetraloop is primarily dictated by the native unfolding of the junction; if the tetraloop is placed on a stem that unfolds as a single domain when the tetraloop is not present, it will unfold as a single domain when the tetraloop is present but with a higher thermal stability. Conversely, if the tetraloop is placed on a stem which unfolds cooperatively with other stems when the tetraloop is not present, the tetraloop will increase the thermal stability of all the stems in the melting domain. The oligonucleotide structure and not the tetraloop itself affects ion uptake; three-way junctions do not gain an increase in ion uptake, but four-way junctions do. This is not the case for water immobilization, where the position of the tetraloop dictates the amount of water immobilized.
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Affiliation(s)
- Carolyn E Carr
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA.
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24
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Carr CE, Marky LA. Investigation of the Melting Behavior of DNA Three-Way Junctions in the Closed and Open States. Biophys J 2017; 113:529-539. [PMID: 28793208 DOI: 10.1016/j.bpj.2017.06.024] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2017] [Revised: 06/09/2017] [Accepted: 06/14/2017] [Indexed: 10/19/2022] Open
Abstract
Intramolecular three-way junctions are commonly found in both DNA and RNA. These structures are functionally relevant in ribozymes, riboswitches, rRNA, and during replication. In this work, we present a thermodynamic description of the unfolding of DNA intramolecular three-way junctions. We used a combination of spectroscopic and calorimetric techniques to investigate the folding/unfolding thermodynamics of two three-way junctions with a closed (Closed-J) or open (Open-J) junction and their appropriate control stem-loop motifs (GAAATT-Hp, CTATC-Hp, and Dumbbell). The overall results show that both junctions are stable over a wide range of salt concentrations. However, Open-J is more stable due to a higher enthalpy contribution from the formation of a higher number of basepair stacks whereas Closed-J has a defined structure and retains the basepair stacking of all three stems. The comparison of the experimental results of Closed-J and Open-J with those of their component stem-loop motifs allowed us to be more specific about their cooperative unfolding. For instance, Closed-J sacrifices thermal stability of the Dumbbell structure to maintain an overall folded state. At higher salt concentration, the simultaneous unfolding of the above domains is lost, resulting in the unfolding of the three separate stems. In contrast, the junction of Open-J in low salt retains the thermal and enthalpic stability of the Dumbbell structure although sacrificing stability of the CTATC stem. The relative stability of Dumbbell is the primary reason for the higher ΔG°(5), or free energy, value seen for Open-J at low salt. Higher salt not only maintains thermal stability of the Dumbbell structure in Open-J but causes the CTATC stem to fully fold.
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Affiliation(s)
- Carolyn E Carr
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska
| | - Luis A Marky
- Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Nebraska Medical Center, Omaha, Nebraska.
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25
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Lean SS, Yeo CC. Small, Enigmatic Plasmids of the Nosocomial Pathogen, Acinetobacter baumannii: Good, Bad, Who Knows? Front Microbiol 2017; 8:1547. [PMID: 28861061 PMCID: PMC5559437 DOI: 10.3389/fmicb.2017.01547] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2017] [Accepted: 07/31/2017] [Indexed: 12/16/2022] Open
Abstract
Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has become a serious healthcare concern within a span of two decades due to its ability to rapidly acquire resistance to all classes of antimicrobial compounds. One of the key features of the A. baumannii genome is an open pan genome with a plethora of plasmids, transposons, integrons, and genomic islands, all of which play important roles in the evolution and success of this clinical pathogen, particularly in the acquisition of multidrug resistance determinants. An interesting genetic feature seen in majority of A. baumannii genomes analyzed is the presence of small plasmids that usually ranged from 2 to 10 kb in size, some of which harbor antibiotic resistance genes and homologs of plasmid mobilization genes. These plasmids are often overlooked when compared to their larger, conjugative counterparts that harbor multiple antibiotic resistance genes and transposable elements. In this mini-review, we will examine our current knowledge of these small A. baumannii plasmids and look into their genetic diversity and phylogenetic relationships. Some of these plasmids, such as the Rep-3 superfamily group and the pRAY-type, which has no recognizable replicase genes, are quite widespread among diverse A. baumannii clinical isolates worldwide, hinting at their usefulness to the lifestyle of this pathogen. Other small plasmids especially those from the Rep-1 superfamily are truly enigmatic, encoding only hypothetical proteins of unknown function, leading to the question of whether these small plasmids are “good” or “bad” to their host A. baumannii.
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Affiliation(s)
- Soo Sum Lean
- Saw Swee Hock School of Public Health, National University of SingaporeSingapore, Singapore
| | - Chew Chieng Yeo
- Faculty of Medicine, Biomedical Research Centre, Universiti Sultan Zainal AbidinKuala Terengganu, Malaysia
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26
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Abstract
One of the major mechanisms driving the evolution of all organisms is genomic rearrangement. In hyperthermophilic Archaea of the order Thermococcales, large chromosomal inversions occur so frequently that even closely related genomes are difficult to align. Clearly not resulting from the native homologous recombination machinery, the causative agent of these inversions has remained elusive. We present a model in which genomic inversions are catalyzed by the integrase enzyme encoded by a family of mobile genetic elements. We characterized the integrase from Thermococcus nautili plasmid pTN3 and showed that besides canonical site-specific reactions, it catalyzes low sequence specificity recombination reactions with the same outcome as homologous recombination events on DNA segments as short as 104bp both in vitro and in vivo, in contrast to other known tyrosine recombinases. Through serial culturing, we showed that the integrase-mediated divergence of T. nautili strains occurs at an astonishing rate, with at least four large-scale genomic inversions appearing within 60 generations. Our results and the ubiquitous distribution of pTN3-like integrated elements suggest that a major mechanism of evolution of an entire order of Archaea results from the activity of a selfish mobile genetic element. Mobile elements (MEs) such as viruses, plasmids and transposons infect most living organisms and often encode recombinases promoting their insertion into cellular genomes. These insertions alter the genome of their host according to two main mechanisms. First, MEs provide new functions to the cell by integrating their own genetic information into the DNA of the host, at one or more locations. Secondly, cellular homologous recombination will act upon multiple integrated copies and produce a variety of large-scale chromosomal rearrangements. If such modifications are advantageous, they will spread into the population by natural selection. Typically, enzymes involved in cellular homologous recombination and the integration of MEs are distinct. We describe here a novel plasmid-encoded archaeal integrase which in addition to site-specific recombination can catalyze low sequence specificity recombination reactions akin to homologous recombination.
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27
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Castillo F, Benmohamed A, Szatmari G. Xer Site Specific Recombination: Double and Single Recombinase Systems. Front Microbiol 2017; 8:453. [PMID: 28373867 PMCID: PMC5357621 DOI: 10.3389/fmicb.2017.00453] [Citation(s) in RCA: 66] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2016] [Accepted: 03/03/2017] [Indexed: 12/20/2022] Open
Abstract
The separation and segregation of newly replicated bacterial chromosomes can be constrained by the formation of circular chromosome dimers caused by crossing over during homologous recombination events. In Escherichia coli and most bacteria, dimers are resolved to monomers by site-specific recombination, a process performed by two Chromosomally Encoded tyrosine Recombinases (XerC and XerD). XerCD recombinases act at a 28 bp recombination site dif, which is located at the replication terminus region of the chromosome. The septal protein FtsK controls the initiation of the dimer resolution reaction, so that recombination occurs at the right time (immediately prior to cell division) and at the right place (cell division septum). XerCD and FtsK have been detected in nearly all sequenced eubacterial genomes including Proteobacteria, Archaea, and Firmicutes. However, in Streptococci and Lactococci, an alternative system has been found, composed of a single recombinase (XerS) genetically linked to an atypical 31 bp recombination site (difSL). A similar recombination system has also been found in 𝜀-proteobacteria such as Campylobacter and Helicobacter, where a single recombinase (XerH) acts at a resolution site called difH. Most Archaea contain a recombinase called XerA that acts on a highly conserved 28 bp sequence dif, which appears to act independently of FtsK. Additionally, several mobile elements have been found to exploit the dif/Xer system to integrate their genomes into the host chromosome in Vibrio cholerae, Neisseria gonorrhoeae, and Enterobacter cloacae. This review highlights the versatility of dif/Xer recombinase systems in prokaryotes and summarizes our current understanding of homologs of dif/Xer machineries.
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Affiliation(s)
- Fabio Castillo
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, MontréalQC, Canada
| | | | - George Szatmari
- Département de Microbiologie, Infectiologie et Immunologie, Université de Montréal, MontréalQC, Canada
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Yeh TY. Complete nucleotide sequence of a new filamentous phage, Xf109, which integrates its genome into the chromosomal DNA of Xanthomonas oryzae. Arch Virol 2016; 162:567-572. [PMID: 27743252 DOI: 10.1007/s00705-016-3105-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2016] [Accepted: 10/02/2016] [Indexed: 12/13/2022]
Abstract
Unlike Ff-like coliphages, certain filamentous Inoviridae phages integrate their genomes into the host chromosome and enter a prophage state in their infectious cycle. This lysogenic life cycle was first reported for Xanthomonas citri Cf phage. However, except for the X. citri phages Cf and XacF1, complete genome sequence information about lysogenic Xanthomonas phages is not available to date. A proviral sequence of Xf109 phage was identified in the genome of Xanthomonas oryzae, the rice bacterial blight pathogen, and revived as infectious virions to lysogenize its host de novo. The genome of Xf109 phage is 7190 nucleotides in size and contains 12 predicted open reading frames in an organization similar to that of the Cf phage genome. Seven of the Xf109 proteins show significant sequence similarity to Cf and XacF1 phage proteins, while its ORF4 shares 92 % identity with the major coat protein of X. phage oryzae Xf. Integration of Xf109 phage DNA into the host genome is site-specific, and the attP/attB sequence contains the dif core sequence 5'-TATACATTATGCGAA-3', which is identical to that of Cf, XacF1, and Xanthomonas campestris phage ϕLf. To my knowledge, this is the first complete genome sequence of a filamentous bacteriophage that infects X. oryzae.
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Affiliation(s)
- Ting Y Yeh
- Agricultural Biotechnology Laboratory, Plant Health Division, Auxergen Inc., 1100 Wicomico Street, Suite 700, Baltimore, MD, 21230, USA. .,Atom Health Corporation, Hsinchu Biomedical Science Park, 2F, No. 6-1, Section 2, Shengyi Road, Zhubei, Hsinchu, 30261, Taiwan.
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Nivina A, Escudero JA, Vit C, Mazel D, Loot C. Efficiency of integron cassette insertion in correct orientation is ensured by the interplay of the three unpaired features of attC recombination sites. Nucleic Acids Res 2016; 44:7792-803. [PMID: 27496283 PMCID: PMC5027507 DOI: 10.1093/nar/gkw646] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Revised: 07/05/2016] [Accepted: 07/10/2016] [Indexed: 01/29/2023] Open
Abstract
The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of cassettes carrying protein-coding sequences, and is responsible for the emergence and rise of multiresistance in Gram-negative bacteria. The functionality of this system depends on the insertion of promoterless cassettes in correct orientation, allowing their expression from the promoter located upstream of the cassette array. Correct orientation is ensured by strand selectivity of integron integrases for the bottom strand of cassette recombination sites (attC), recombined in form of folded single-stranded hairpins. Here, we investigated the basis of such strand selectivity by comparing recombination of wild-type and mutated attC sites with different lengths, sequences and structures. We show that all three unpaired structural features that distinguish the bottom and top strands contribute to strand selectivity. The localization of Extra-Helical Bases (EHBs) directly favors integrase binding to the bottom strand. The Unpaired Central Spacer (UCS) and the Variable Terminal Structure (VTS) influence strand selectivity indirectly, probably through the stabilization of the bottom strand and the resulting synapse due to the nucleotide skew between the two strands. These results underscore the importance of the single-stranded nature of the attC site that allows such tight control over integron cassette orientation.
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Affiliation(s)
- Aleksandra Nivina
- Institut Pasteur, Bacterial Genome Plasticity Unit, 75724 Paris, France CNRS UMR3525, 75724 Paris, France Paris Descartes, Sorbonne Paris Cité, Paris, France
| | - José Antonio Escudero
- Institut Pasteur, Bacterial Genome Plasticity Unit, 75724 Paris, France CNRS UMR3525, 75724 Paris, France
| | - Claire Vit
- Institut Pasteur, Bacterial Genome Plasticity Unit, 75724 Paris, France CNRS UMR3525, 75724 Paris, France
| | - Didier Mazel
- Institut Pasteur, Bacterial Genome Plasticity Unit, 75724 Paris, France CNRS UMR3525, 75724 Paris, France
| | - Céline Loot
- Institut Pasteur, Bacterial Genome Plasticity Unit, 75724 Paris, France CNRS UMR3525, 75724 Paris, France
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FtsK translocation permits discrimination between an endogenous and an imported Xer/dif recombination complex. Proc Natl Acad Sci U S A 2016; 113:7882-7. [PMID: 27317749 DOI: 10.1073/pnas.1523178113] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
In bacteria, the FtsK/Xer/dif (chromosome dimer resolution site) system is essential for faithful vertical genetic transmission, ensuring the resolution of chromosome dimers during their segregation to daughter cells. This system is also targeted by mobile genetic elements that integrate into chromosomal dif sites. A central question is thus how Xer/dif recombination is tuned to both act in chromosome segregation and stably maintain mobile elements. To explore this question, we focused on pathogenic Neisseria species harboring a genomic island in their dif sites. We show that the FtsK DNA translocase acts differentially at the recombination sites flanking the genomic island. It stops at one Xer/dif complex, activating recombination, but it does not stop on the other site, thus dismantling it. FtsK translocation thus permits cis discrimination between an endogenous and an imported Xer/dif recombination complex.
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Abstract
The integron is a powerful system which, by capturing, stockpiling, and rearranging new functions carried by gene encoding cassettes, confers upon bacteria a rapid adaptation capability in changing environments. Chromosomally located integrons (CI) have been identified in a large number of environmental Gram-negative bacteria. Integron evolutionary history suggests that these sedentary CIs acquired mobility among bacterial species through their association with transposable elements and conjugative plasmids. As a result of massive antibiotic use, these so-called mobile integrons are now widespread in clinically relevant bacteria and are considered to be the principal agent in the emergence and rise of antibiotic multiresistance in Gram-negative bacteria. Cassette rearrangements are catalyzed by the integron integrase, a site-specific tyrosine recombinase. Central to these reactions is the single-stranded DNA nature of one of the recombination partners, the attC site. This makes the integron a unique recombination system. This review describes the current knowledge on this atypical recombination mechanism, its implications in the reactions involving the different types of sites, attC and attI, and focuses on the tight regulation exerted by the host on integron activity through the control of attC site folding. Furthermore, cassette and integrase expression are also highly controlled by host regulatory networks and the bacterial stress (SOS) response. These intimate connections to the host make the integron a genetically stable and efficient system, granting the bacteria a low cost, highly adaptive evolution potential "on demand".
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Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation. Nat Commun 2016; 7:10937. [PMID: 26961432 PMCID: PMC4792948 DOI: 10.1038/ncomms10937] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2015] [Accepted: 02/03/2016] [Indexed: 12/28/2022] Open
Abstract
Tyrosine (Y)-recombinases have evolved to deliver mechanistically different reactions on a variety of substrates, but these evolutionary transitions are poorly understood. Among them, integron integrases are hybrid systems recombining single- and double-stranded DNA partners. These reactions are asymmetric and need a replicative resolution pathway, an exception to the canonical second strand exchange model of Y-recombinases. Integron integrases possess a specific domain for this specialized pathway. Here we show that despite this, integrases are still capable of efficiently operating the ancestral second strand exchange in symmetrical reactions between double-stranded substrates. During these reactions, both strands are reactive and Holliday junction resolution can follow either pathway. A novel deep-sequencing approach allows mapping of the crossover point for the second strand exchange. The persistence of the ancestral activity in integrases illustrates their robustness and shows that innovation towards new recombination substrates and resolution pathways was a smooth evolutionary process. The integron integrases have evolved to perform recombination of single and double stranded DNA. Here the authors show that the ancestral pathway is still functional at double stranded sites, revealing the evolution towards the modern resolution pathway.
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Martínez E, Campos-Gómez J, Barre FX. CTXϕ: Exploring new alternatives in host factor-mediated filamentous phage replications. BACTERIOPHAGE 2016; 6:e1128512. [PMID: 27607139 DOI: 10.1080/21597081.2015.1128512] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/15/2015] [Accepted: 11/24/2015] [Indexed: 10/22/2022]
Abstract
For a long time Ff phages from Escherichia coli provided the majority of the knowledge about the rolling circle replication mechanism of filamentous phages. Host factors involved in coliphages replication have been fully identified. Based on these studies, the function of Rep protein as the accessory helicase directly implicated in filamentous phage replication was considered a paradigm. We recently reported that the replication of some filamentous phages from Vibrio cholerae, including the cholera toxin phage CTXϕ, depended on the accessory helicase UvrD instead of Rep. We also identified HU protein as one of the host factors involved in CTXϕ and VGJϕ replication. The requirement of UvrD and HU for rolling circle replication was previously reported in some family of plasmids but had no precedent in filamentous phages. Here, we enrich the discussion of our results and present new preliminary data highlighting remarkable divergence in the lifestyle of filamentous phages.
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Affiliation(s)
- Eriel Martínez
- Southern Research Institute, Department of Biochemistry and Molecular Biology, Drug Discovery Division, Birmingham, AL, USA; Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Univ. Paris Sud, Gif sur Yvette, France
| | - Javier Campos-Gómez
- Southern Research Institute, Department of Biochemistry and Molecular Biology, Drug Discovery Division , Birmingham, AL, USA
| | - François-Xavier Barre
- Institute for Integrative Biology of the Cell (I2BC), Université Paris-Saclay, CEA, CNRS, Univ. Paris Sud , Gif sur Yvette, France
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Xer Site-Specific Recombination: Promoting Vertical and Horizontal Transmission of Genetic Information. Microbiol Spectr 2016; 2. [PMID: 26104463 DOI: 10.1128/microbiolspec.mdna3-0056-2014] [Citation(s) in RCA: 48] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Two related tyrosine recombinases, XerC and XerD, are encoded in the genome of most bacteria where they serve to resolve dimers of circular chromosomes by the addition of a crossover at a specific site, dif. From a structural and biochemical point of view they belong to the Cre resolvase family of tyrosine recombinases. Correspondingly, they are exploited for the resolution of multimers of numerous plasmids. In addition, they are exploited by mobile DNA elements to integrate into the genome of their host. Exploitation of Xer is likely to be advantageous to mobile elements because the conservation of the Xer recombinases and of the sequence of their chromosomal target should permit a quite easy extension of their host range. However, it requires means to overcome the cellular mechanisms that normally restrict recombination to dif sites harbored by a chromosome dimer and, in the case of integrative mobile elements, to convert dedicated tyrosine resolvases into integrases.
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Abstract
One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes.
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Effect of LexA on Chromosomal Integration of CTXϕ in Vibrio cholerae. J Bacteriol 2015; 198:268-75. [PMID: 26503849 DOI: 10.1128/jb.00674-15] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2015] [Accepted: 10/12/2015] [Indexed: 12/26/2022] Open
Abstract
UNLABELLED The genesis of toxigenic Vibrio cholerae involves acquisition of CTXϕ, a single-stranded DNA (ssDNA) filamentous phage that encodes cholera toxin (CT). The phage exploits host-encoded tyrosine recombinases (XerC and XerD) for chromosomal integration and lysogenic conversion. The replicative genome of CTXϕ produces ssDNA by rolling-circle replication, which may be used either for virion production or for integration into host chromosome. Fine-tuning of different ssDNA binding protein (Ssb) levels in the host cell is crucial for cellular functioning and important for CTXϕ integration. In this study, we mutated the master regulator gene of SOS induction, lexA, of V. cholerae because of its known role in controlling levels of Ssb proteins in other bacteria. CTXϕ integration decreased in cells with a ΔlexA mutation and increased in cells with an SOS-noninducing mutation, lexA (Ind(-)). We also observed that overexpression of host-encoded Ssb (VC0397) decreased integration of CTXϕ. We propose that LexA helps CTXϕ integration, possibly by fine-tuning levels of host- and phage-encoded Ssbs. IMPORTANCE Cholera toxin is the principal virulence factor responsible for the acute diarrheal disease cholera. CT is encoded in the genome of a lysogenic filamentous phage, CTXϕ. Vibrio cholerae has a bipartite genome and harbors single or multiple copies of CTXϕ prophage in one or both chromosomes. Two host-encoded tyrosine recombinases (XerC and XerD) recognize the folded ssDNA genome of CTXϕ and catalyze its integration at the dimer resolution site of either one or both chromosomes. Fine-tuning of ssDNA binding proteins in host cells is crucial for CTXϕ integration. We engineered the V. cholerae genome and created several reporter strains carrying ΔlexA or lexA (Ind(-)) alleles. Using the reporter strains, the importance of LexA control of Ssb expression in the integration efficiency of CTXϕ was demonstrated.
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Abstract
Horizontal gene transfer plays a major role in microbial evolution, allowing microbes to acquire new genes and phenotypes. Integrative and conjugative elements (ICEs, a.k.a. conjugative transposons) are modular mobile genetic elements integrated into a host genome and are passively propagated during chromosomal replication and cell division. Induction of ICE gene expression leads to excision, production of the conserved conjugation machinery (a type IV secretion system), and the potential to transfer DNA to appropriate recipients. ICEs typically contain cargo genes that are not usually related to the ICE life cycle and that confer phenotypes to host cells. We summarize the life cycle and discovery of ICEs, some of the regulatory mechanisms, and how the types of cargo have influenced our view of ICEs. We discuss how ICEs can acquire new cargo genes and describe challenges to the field and various perspectives on ICE biology.
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Affiliation(s)
- Christopher M Johnson
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; ,
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Wright LD, Johnson CM, Grossman AD. Identification of a Single Strand Origin of Replication in the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis. PLoS Genet 2015; 11:e1005556. [PMID: 26440206 PMCID: PMC4595007 DOI: 10.1371/journal.pgen.1005556] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2015] [Accepted: 09/07/2015] [Indexed: 11/24/2022] Open
Abstract
We identified a functional single strand origin of replication (sso) in the integrative and conjugative element ICEBs1 of Bacillus subtilis. Integrative and conjugative elements (ICEs, also known as conjugative transposons) are DNA elements typically found integrated into a bacterial chromosome where they are transmitted to daughter cells by chromosomal replication and cell division. Under certain conditions, ICEs become activated and excise from the host chromosome and can transfer to neighboring cells via the element-encoded conjugation machinery. Activated ICEBs1 undergoes autonomous rolling circle replication that is needed for the maintenance of the excised element in growing and dividing cells. Rolling circle replication, used by many plasmids and phages, generates single-stranded DNA (ssDNA). In many cases, the presence of an sso enhances the conversion of the ssDNA to double-stranded DNA (dsDNA) by enabling priming of synthesis of the second DNA strand. We initially identified sso1 in ICEBs1 based on sequence similarity to the sso of an RCR plasmid. Several functional assays confirmed Sso activity. Genetic analyses indicated that ICEBs1 uses sso1 and at least one other region for second strand DNA synthesis. We found that Sso activity was important for two key aspects of the ICEBs1 lifecycle: 1) maintenance of the plasmid form of ICEBs1 in cells after excision from the chromosome, and 2) stable acquisition of ICEBs1 following transfer to a new host. We identified sequences similar to known plasmid sso's in several other ICEs. Together, our results indicate that many other ICEs contain at least one single strand origin of replication, that these ICEs likely undergo autonomous replication, and that replication contributes to the stability and spread of these elements. Mobile genetic elements facilitate movement of genes, including those conferring antibiotic resistance and other traits, between bacteria. Integrative and conjugative elements (ICEs) are a large family of mobile genetic elements that are typically found integrated in the chromosome of their host bacterium. Under certain conditions (e.g., DNA damage, high cell density, stationary phase) an ICE excises from the host chromosome to form a circle. A linear single strand of ICE DNA can be transferred to an appropriate recipient through the ICE-encoded conjugation machinery. In addition, following excision from the chromosome, at least some (perhaps most) ICEs undergo autonomous rolling circle replication, a mechanism used by many plasmids and phages. Rolling circle replication generates single-stranded DNA (ssDNA). We found that ICEBs1, from Bacillus subtilis, contains at least two regions that enable conversion of ssDNA to double-stranded DNA. At least one of these regions functions as an sso (single strand origin of replication). ICEBs1 Sso activity was important for the ability of transferred ICEBs1 to be acquired by recipients and for the ability of ICEBs1 to replicate autonomously after excising from its host’s chromosome. We identified putative sso's in several other ICEs, indicating that Sso activity is likely important for the replication, stability and spread of these elements.
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Affiliation(s)
- Laurel D. Wright
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
| | - Christopher M. Johnson
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
| | - Alan D. Grossman
- Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
- * E-mail:
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Hellard E, Fouchet D, Vavre F, Pontier D. Parasite-Parasite Interactions in the Wild: How To Detect Them? Trends Parasitol 2015; 31:640-652. [PMID: 26440785 DOI: 10.1016/j.pt.2015.07.005] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2014] [Revised: 07/06/2015] [Accepted: 07/31/2015] [Indexed: 01/26/2023]
Abstract
Inter-specific interactions between parasites impact on parasite intra-host dynamics, host health, and disease management. Identifying and understanding interaction mechanisms in the wild is crucial for wildlife disease management. It is however complex because several scales are interlaced. Parasite-parasite interactions are likely to occur via mechanisms at the within-host level, but also at upper levels (host population and community). Furthermore, interactions occurring at one level of organization spread to upper levels through cascade effects. Even if cascade effects are important confounding factors, we argue that we can also benefit from them because upper scales often provide a way to survey a wider range of parasites at lower cost. New protocols and theoretical studies (especially across scales) are necessary to take advantage of this opportunity.
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Affiliation(s)
- Eléonore Hellard
- Laboratoire de Biométrie et Biologie Evolutive, Université de Lyon, Université Lyon I, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5558, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne, France; Percy FitzPatrick Institute, DST-NRF Centre of Excellence, University of Cape Town, Private Bag X3, Rondebosch 7701, South Africa.
| | - David Fouchet
- Laboratoire de Biométrie et Biologie Evolutive, Université de Lyon, Université Lyon I, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5558, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne, France; LabEx Ecofect, Ecoevolutionary Dynamics of Infectious Diseases, University of Lyon, France
| | - Fabrice Vavre
- Laboratoire de Biométrie et Biologie Evolutive, Université de Lyon, Université Lyon I, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5558, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne, France; LabEx Ecofect, Ecoevolutionary Dynamics of Infectious Diseases, University of Lyon, France
| | - Dominique Pontier
- Laboratoire de Biométrie et Biologie Evolutive, Université de Lyon, Université Lyon I, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche 5558, 43 Boulevard du 11 Novembre 1918, 69622, Villeurbanne, France; LabEx Ecofect, Ecoevolutionary Dynamics of Infectious Diseases, University of Lyon, France
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Martínez E, Paly E, Barre FX. CTXφ Replication Depends on the Histone-Like HU Protein and the UvrD Helicase. PLoS Genet 2015; 11:e1005256. [PMID: 25992634 PMCID: PMC4439123 DOI: 10.1371/journal.pgen.1005256] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Accepted: 04/29/2015] [Indexed: 02/06/2023] Open
Abstract
The Vibrio cholerae bacterium is the agent of cholera. The capacity to produce the cholera toxin, which is responsible for the deadly diarrhea associated with cholera epidemics, is encoded in the genome of a filamentous phage, CTXφ. Rolling-circle replication (RCR) is central to the life cycle of CTXφ because amplification of the phage genome permits its efficient integration into the genome and its packaging into new viral particles. A single phage-encoded HUH endonuclease initiates RCR of the proto-typical filamentous phages of enterobacteriaceae by introducing a nick at a specific position of the double stranded DNA form of the phage genome. The rest of the process is driven by host factors that are either essential or crucial for the replication of the host genome, such as the Rep SF1 helicase. In contrast, we show here that the histone-like HU protein of V. cholerae is necessary for the introduction of a nick by the HUH endonuclease of CTXφ. We further show that CTXφ RCR depends on a SF1 helicase normally implicated in DNA repair, UvrD, rather than Rep. In addition to CTXφ, we show that VGJφ, a representative member of a second family of vibrio integrative filamentous phages, requires UvrD and HU for RCR while TLCφ, a satellite phage, depends on Rep and is independent from HU. One of the major strategies to prevent Cholera epidemics is the development of oral vaccines based on live attenuated Vibrio cholerae strains. The most promising vaccine strains have been obtained by deletion of the cholera toxin genes, which are harboured in the genome of an integrated phage, CTXϕ. However, they can re-acquire the cholera toxin genes when re-infected by CTXϕ or by hybrid phages between CTXϕ and other vibrio phages, which raised safety concerns about their use. Here, we developed a screening strategy to identify non-essential host factors implicated in CTXϕ replication. We show that the histone-like HU protein and the UvrD helicase are both absolutely required for its replication. We further show that they are essential for the replication of VGJϕ, a representative member of a family of phages that can form hybrids with CTXϕ. Accordingly, we demonstrate that the disruption of the two subunits of HU and/or of UvrD prevents infection of the V. cholerae by CTXϕ and VGJϕ. In addition, we show that it limits CTXϕ horizontal transmission. Taken together, these results indicate that HU- and/or UvrD- cells are promising candidates for the development of safer live attenuated cholera vaccine.
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Affiliation(s)
- Eriel Martínez
- Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Université Paris Sud, Gif sur Yvette, France
| | - Evelyne Paly
- Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Université Paris Sud, Gif sur Yvette, France
| | - François-Xavier Barre
- Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Université Paris Sud, Gif sur Yvette, France
- * E-mail:
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Ilyina TS. Filamentous bacteriophages and their role in the virulence and evolution of pathogenic bacteria. MOLECULAR GENETICS MICROBIOLOGY AND VIROLOGY 2015. [DOI: 10.3103/s0891416815010036] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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Krupovic M, Forterre P. Single-stranded DNA viruses employ a variety of mechanisms for integration into host genomes. Ann N Y Acad Sci 2015; 1341:41-53. [PMID: 25675979 DOI: 10.1111/nyas.12675] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Single-stranded DNA (ssDNA) viruses are widespread in the environment and include economically, medically, and ecologically important pathogens. Recently, it has been discovered that ssDNA virus genomes are also prevalent in the chromosomes of their bacterial, archaeal, and eukaryotic hosts. Sequences originating from viruses of the families Parvoviridae, Circoviridae, and Geminiviridae are particularly widespread in the genomes of eukaryotes, where they are often fossilized as endogenous viral elements. ssDNA viruses have evolved diverse mechanisms to invade cellular genomes, and these principally vary between viruses infecting bacteria/archaea and eukaryotes. Filamentous bacteriophages (Inoviridae) use at least three major mechanisms of integration. Some of these phages encode integrases of serine or tyrosine recombinase superfamilies, while others utilize DDE transposases of the IS3, IS30, or IS110/IS492 families, whereas some inoviruses, and possibly certain members of the Microviridae, hijack the host XerCD recombination machinery. By contrast, eukaryotic viruses for integration rely on the endonuclease activity of their rolling-circle replication-initiation proteins, mimicking the mechanisms used by some bacterial transposons. Certain bacterial and eukaryotic ssDNA viruses have embraced a transposon-like means of propagation, with occasionally dramatic effects on host genome evolution. Here, we review the diversity of experimentally verified and hypothetical mechanisms of genome integration employed by ssDNA viruses, and consider the evolutionary implications of these processes, particularly in the emergence of novel virus groups.
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Affiliation(s)
- Mart Krupovic
- Institut Pasteur, Unité Biologie Moléculaire du Gène chez les Extrêmophiles, Paris, France
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Derbise A, Carniel E. YpfΦ: a filamentous phage acquired by Yersinia pestis. Front Microbiol 2014; 5:701. [PMID: 25566217 PMCID: PMC4266046 DOI: 10.3389/fmicb.2014.00701] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2014] [Accepted: 11/26/2014] [Indexed: 11/13/2022] Open
Abstract
Yersinia pestis, the plague bacillus, has an exceptional pathogenicity for humans. The plague bacillus emerged very recently (≈3,000 years ago) from the enteropathogen Y. pseudotuberculosis. Early after its emergence, Y. pestis became infected by a filamentous phage named YpfΦ. During the microevolution of the plague bacillus, the phage remained in the various lineages as an unstable extrachromosomal element. However, in the sub branch that caused the third plague pandemic, YpfΦ integrated itself into the bacterial chromosome to become a stable prophage. The genome of this phage has the same genetic organization as that of other filamentous phages such as the Vibrio cholerae CTXΦ phage, and shares high sequence identity with the CUS-1 filamentous phage of a high-virulence Escherichia coli K1 clone. In addition to genes involved in phage physiology, YpfΦ carries at each extremity of its genome two open reading frames with no predicted functions. This filamentous phage confers some selective properties to Y. pestis during the infectious process, which may explain why it was conserved duringY. pestis microevolution, despite its instability as an extrachromosomal element in most branches.
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Affiliation(s)
- Anne Derbise
- Yersinia Research Unit, Department of Microbiology, Institut Pasteur Paris, France
| | - Elisabeth Carniel
- Yersinia Research Unit, Department of Microbiology, Institut Pasteur Paris, France
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Das B. Mechanistic insights into filamentous phage integration in Vibrio cholerae. Front Microbiol 2014; 5:650. [PMID: 25506341 PMCID: PMC4246890 DOI: 10.3389/fmicb.2014.00650] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2014] [Accepted: 11/10/2014] [Indexed: 02/03/2023] Open
Abstract
Vibrio cholerae, the etiological agent of acute diarrhoeal disease cholera, harbors large numbers of lysogenic filamentous phages, contribute significantly to the host pathogenesis and provide fitness factors to the pathogen that help the bacterium to survive in natural environment. Most of the vibriophage genomes are not equipped with integrase and thus exploit two host-encoded tyrosine recombinases, XerC and XerD, for lysogenic conversion. Integration is site-specific and it occurs at dimer resolution site (dif) of either one or both chromosomes of V. cholerae. Each dif sequence contains two recombinase-binding sequences flanking a central region. The integration follows a sequential strand exchanges between dif and attP sites within a DNA-protein complex consisting of one pair of each recombinase and two DNA fragments. During entire process of recombination, both the DNA components and recombinases of the synaptic complex keep transiently interconnected. Within the context of synaptic complex, both of the actuated enzymes mediate cleavage of phosphodiester bonds. First cleavage generates a phosphotyrosyl-linked recombinase-DNA complex at the recombinase binding sequence and free 5′-hydroxyl end at the first base of the central region. Following the cleavage, the exposed bases with 5′-hydroxyl ends of the central region of dif and attP sites melt from their complementary strands and react with the recombinase-DNA phosphotyrosyl linkage of their recombining partner. Subsequent ligation between dif and attP strands requires complementary base pair interactions at the site of phosphodiester bond formation. Integration mechanism is mostly influenced by the compatibility of dif and attP sequences. dif sites are highly conserved across bacterial phyla. Different phage genomes have different attP sequences; therefore they rely on different mechanisms for integration. Here, I review our current understanding of integration mechanisms used by the vibriophages.
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Affiliation(s)
- Bhabatosh Das
- Centre for Human Microbial Ecology, Translational Health Science and Technology Institute Gurgaon, India
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XerD-mediated FtsK-independent integration of TLCϕ into the Vibrio cholerae genome. Proc Natl Acad Sci U S A 2014; 111:16848-53. [PMID: 25385643 DOI: 10.1073/pnas.1404047111] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
As in most bacteria, topological problems arising from the circularity of the two Vibrio cholerae chromosomes, chrI and chrII, are resolved by the addition of a crossover at a specific site of each chromosome, dif, by two tyrosine recombinases, XerC and XerD. The reaction is under the control of a cell division protein, FtsK, which activates the formation of a Holliday Junction (HJ) intermediate by XerD catalysis that is resolved into product by XerC catalysis. Many plasmids and phages exploit Xer recombination for dimer resolution and for integration, respectively. In all cases so far described, they rely on an alternative recombination pathway in which XerC catalyzes the formation of a HJ independently of FtsK. This is notably the case for CTXϕ, the cholera toxin phage. Here, we show that in contrast, integration of TLCϕ, a toxin-linked cryptic satellite phage that is almost always found integrated at the chrI dif site before CTXϕ, depends on the formation of a HJ by XerD catalysis, which is then resolved by XerC catalysis. The reaction nevertheless escapes the normal cellular control exerted by FtsK on XerD. In addition, we show that the same reaction promotes the excision of TLCϕ, along with any CTXϕ copy present between dif and its left attachment site, providing a plausible mechanism for how chrI CTXϕ copies can be eliminated, as occurred in the second wave of the current cholera pandemic.
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Abstract
Bacteriophage genomes found in a range of bacterial pathogens encode a diverse array of virulence factors ranging from superantigens or pore forming lysins to numerous exotoxins. Recent studies have uncovered an entirely new class of bacterial virulence factors, called effector proteins or effector toxins, which are encoded within phage genomes that reside among several pathovars of Escherichia coli and Salmonella enterica. These effector proteins have multiple domains resulting in proteins that can be multifunctional. The effector proteins encoded within phage genomes are translocated directly from the bacterial cytosol into their eukaryotic target cells by specialized bacterial type three secretion systems (T3SSs). In this review, we will give an overview of the different types of effector proteins encoded within phage genomes and examine their roles in bacterial pathogenesis.
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Affiliation(s)
- E Fidelma Boyd
- Department of Biological Sciences; University of Delaware; Newark, DE USA
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Plasmid-Mediated Antibiotic Resistance and Virulence in Gram-negatives: the Klebsiella pneumoniae Paradigm. Microbiol Spectr 2014; 2:1-15. [PMID: 25705573 DOI: 10.1128/microbiolspec.plas-0016-2013] [Citation(s) in RCA: 91] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Plasmids harbor genes coding for specific functions including virulence factors and antibiotic resistance that permit bacteria to survive the hostile environment found in the host and resist treatment. Together with other genetic elements such as integrons and transposons, and using a variety of mechanisms, plasmids participate in the dissemination of these traits resulting in the virtual elimination of barriers among different kinds of bacteria. In this article we review the current information about physiology and role in virulence and antibiotic resistance of plasmids from the gram-negative opportunistic pathogen Klebsiella pneumoniae. This bacterium has acquired multidrug resistance and is the causative agent of serious communityand hospital-acquired infections. It is also included in the recently defined ESKAPE group of bacteria that cause most of US hospital infections.
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A novel, broad-range, CTXΦ-derived stable integrative expression vector for functional studies. J Bacteriol 2014; 196:4071-80. [PMID: 25225263 DOI: 10.1128/jb.01966-14] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
CTXΦ, a filamentous vibriophage encoding cholera toxin, uses a unique strategy for its lysogeny. The single-stranded phage genome forms intramolecular base-pairing interactions between two inversely oriented XerC and XerD binding sites (XBS) and generates a functional phage attachment site, attP(+), for integration. The attP(+) structure is recognized by the host-encoded tyrosine recombinases XerC and XerD (XerCD), which enables irreversible integration of CTXΦ into the chromosome dimer resolution site (dif) of Vibrio cholerae. The dif site and the XerCD recombinases are widely conserved in bacteria. We took advantage of these conserved attributes to develop a broad-host-range integrative expression vector that could irreversibly integrate into the host chromosome using XerCD recombinases without altering the function of any known open reading frame (ORF). In this study, we engineered two different arabinose-inducible expression vectors, pBD62 and pBD66, using XBS of CTXΦ. pBD62 replicates conditionally and integrates efficiently into the dif of the bacterial chromosome by site-specific recombination using host-encoded XerCD recombinases. The expression level of the gene of interest could be controlled through the PBAD promoter by modulating the functions of the vector-encoded transcriptional factor AraC. We validated the irreversible integration of pBD62 into a wide range of pathogenic and nonpathogenic bacteria, such as V. cholerae, Vibrio fluvialis, Vibrio parahaemolyticus, Escherichia coli, Salmonella enterica, and Klebsiella pneumoniae. Gene expression from the PBAD promoter of integrated vectors was confirmed in V. cholerae using the well-studied reporter genes mCherry, eGFP, and lacZ.
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Ahmad AA, Askora A, Kawasaki T, Fujie M, Yamada T. The filamentous phage XacF1 causes loss of virulence in Xanthomonas axonopodis pv. citri, the causative agent of citrus canker disease. Front Microbiol 2014; 5:321. [PMID: 25071734 PMCID: PMC4076744 DOI: 10.3389/fmicb.2014.00321] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2014] [Accepted: 06/11/2014] [Indexed: 12/15/2022] Open
Abstract
In this study, filamentous phage XacF1, which can infect Xanthomonas axonopodis pv. citri (Xac) strains, was isolated and characterized. Electron microscopy showed that XacF1 is a member of the family Inoviridae and is about 600 nm long. The genome of XacF1 is 7325 nucleotides in size, containing 13 predicted open reading frames (ORFs), some of which showed significant homology to Ff-like phage proteins such as ORF1 (pII), ORF2 (pV), ORF6 (pIII), and ORF8 (pVI). XacF1 showed a relatively wide host range, infecting seven out of 11 strains tested in this study. Frequently, XacF1 was found to be integrated into the genome of Xac strains. This integration occurred at the host dif site (attB) and was mediated by the host XerC/D recombination system. The attP sequence was identical to that of Xanthomonas phage Cf1c. Interestingly, infection by XacF1 phage caused several physiological changes to the bacterial host cells, including lower levels of extracellular polysaccharide production, reduced motility, slower growth rate, and a dramatic reduction in virulence. In particular, the reduction in virulence suggested possible utilization of XacF1 as a biological control agent against citrus canker disease.
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Affiliation(s)
- Abdelmonim Ali Ahmad
- Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima UniversityHigashi-Hiroshima, Japan
| | - Ahmed Askora
- Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima UniversityHigashi-Hiroshima, Japan
- Department of Microbiology, Faculty of Science, Zagazig UniversityZagazig, Sharkia, Egypt
| | - Takeru Kawasaki
- Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima UniversityHigashi-Hiroshima, Japan
| | - Makoto Fujie
- Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima UniversityHigashi-Hiroshima, Japan
| | - Takashi Yamada
- Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima UniversityHigashi-Hiroshima, Japan
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RS1 satellite phage promotes diversity of toxigenic Vibrio cholerae by driving CTX prophage loss and elimination of lysogenic immunity. Infect Immun 2014; 82:3636-43. [PMID: 24935981 DOI: 10.1128/iai.01699-14] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
In El Tor biotype strains of toxigenic Vibrio cholerae, the CTXϕ prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1ϕ particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTXϕ repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTXϕ lysogeny. We found that superinfection of toxigenic El Tor strains with RS1ϕ, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies using recA deletion mutants and a cloned rstC gene showed that the excision event was recA dependent and that introduction of additional copies of the cloned rstC gene instead of infection with RS1ϕ was sufficient to enhance CTXϕ elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTXϕ lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies of rstC, the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTXϕ or by chitin-induced transformation. These results provide new insights into the role of RS1ϕ in V. cholerae evolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti.
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