Assembly and transfer of tripartite integrative and conjugative genetic elements

Integrative and conjugative elements (ICEs) are ubiquitous mobile genetic elements present as “genomic islands” within bacterial chromosomes. Symbiosis islands are ICEs that convert nonsymbiotic mesorhizobia into symbionts of legumes. Here we report the discovery of symbiosis ICEs that exist as three separate chromosomal regions when integrated in their hosts, but through recombination assemble as a single circular ICE for conjugative transfer. Whole-genome comparisons revealed exconjugants derived from nonsymbiotic mesorhizobia received three separate chromosomal regions from the donorMesorhizobium ciceriWSM1271. The three regions were each bordered by two nonhomologous integrase attachment (att) sites, which together comprised three homologous pairs ofattLandattRsites. Sequential recombination between eachattLandattRpair produced correspondingattPandattBsites and joined the three fragments to produce a single circular ICE, ICEMcSym1271. A plasmid carrying the threeattPsites was used to recreate the process of tripartite ICE integration and to confirm the role of integrase genesintS,intM, andintGin this process. Nine additional tripartite ICEs were identified in diverse mesorhizobia and transfer was demonstrated for three of them. The transfer of tripartite ICEs to nonsymbiotic mesorhizobia explains the evolution of competitive but suboptimal N2-fixing strains found in Western Australian soils. The unheralded existence of tripartite ICEs raises the possibility that multipartite elements reside in other organisms, but have been overlooked because of their unusual biology. These discoveries reveal mechanisms by which integrases dramatically manipulate bacterial genomes to allow cotransfer of disparate chromosomal regions.

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A Mating Procedure for Genetic Transfer of Integrative and Conjugative Elements (ICEs) of Streptococci and Enterococci

Methods and Protocols ◽

10.3390/mps4030059 ◽

2021 ◽

Vol 4 (3) ◽

pp. 59

Author(s):

Francesco Iannelli ◽

Francesco Santoro ◽

Valeria Fox ◽

Gianni Pozzi

Keyword(s):

Sequence Data ◽

Bacterial Species ◽

Bacterial Genome ◽

Conjugative Transfer ◽

Bacterial Genomes ◽

Genetic Elements ◽

Integrative And Conjugative Elements ◽

Solid Media ◽

Donor Cells

DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells.

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The coevolution of toxin and antitoxin genes drives the dynamics of bacterial addiction complexes and intragenomic conflict

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2012.0942 ◽

2012 ◽

Vol 279 (1743) ◽

pp. 3706-3715 ◽

Cited By ~ 7

Author(s):

Daniel J. Rankin ◽

Leighton A. Turner ◽

Jack A. Heinemann ◽

Sam P. Brown

Keyword(s):

Theoretical Model ◽

Kin Selection ◽

Population Biology ◽

Bacterial Genomes ◽

Daughter Cells ◽

Intragenomic Conflict ◽

Bacterial Chromosomes ◽

Gene Complexes ◽

Deleterious Genes

Bacterial genomes commonly contain ‘addiction’ gene complexes that code for both a toxin and a corresponding antitoxin. As long as both genes are expressed, cells carrying the complex can remain healthy. However, loss of the complex (including segregational loss in daughter cells) can entail death of the cell. We develop a theoretical model to explore a number of evolutionary puzzles posed by toxin–antitoxin (TA) population biology. We first extend earlier results demonstrating that TA complexes can spread on plasmids, as an adaptation to plasmid competition in spatially structured environments, and highlight the role of kin selection. We then considered the emergence of TA complexes on plasmids from previously unlinked toxin and antitoxin genes. We find that one of these traits must offer at least initially a direct advantage in some but not all environments encountered by the evolving plasmid population. Finally, our study predicts non-transitive ‘rock-paper-scissors’ dynamics to be a feature of intragenomic conflict mediated by TA complexes. Intragenomic conflict could be sufficient to select deleterious genes on chromosomes and helps to explain the previously perplexing observation that many TA genes are found on bacterial chromosomes.

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The clc Element of Pseudomonas sp. Strain B13, a Genomic Island with Various Catabolic Properties

Journal of Bacteriology ◽

10.1128/jb.188.5.1999-2013.2006 ◽

2006 ◽

Vol 188 (5) ◽

pp. 1999-2013 ◽

Cited By ~ 83

Author(s):

Muriel Gaillard ◽

Tatiana Vallaeys ◽

Frank Jörg Vorhölter ◽

Marco Minoia ◽

Christoph Werlen ◽

...

Keyword(s):

Nucleotide Sequence ◽

Open Reading Frames ◽

Genomic Islands ◽

Functional Prediction ◽

Bacterial Genomes ◽

Burkholderia Xenovorans ◽

Integrative And Conjugative Elements ◽

Genomic Regions ◽

Catabolic Plasmids ◽

Reading Frames

ABSTRACT Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

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Type I toxin-antitoxin systems contribute to the maintenance of mobile genetic elements in Clostridioides difficile

Communications Biology ◽

10.1038/s42003-020-01448-5 ◽

2020 ◽

Vol 3 (1) ◽

Author(s):

Johann Peltier ◽

Audrey Hamiot ◽

Julian R. Garneau ◽

Pierre Boudry ◽

Anna Maikova ◽

...

Keyword(s):

Ectopic Expression ◽

Mobile Genetic Elements ◽

Toxin Gene ◽

Type I ◽

Genetic Elements ◽

Clostridioides Difficile ◽

Bacterial Chromosomes ◽

Toxin Protein

AbstractToxin-antitoxin (TA) systems are widespread on mobile genetic elements and in bacterial chromosomes. In type I TA, synthesis of the toxin protein is prevented by the transcription of an antitoxin RNA. The first type I TA were recently identified in the human enteropathogen Clostridioides difficile. Here we report the characterization of five additional type I TA within phiCD630-1 (CD0977.1-RCd11, CD0904.1-RCd13 and CD0956.3-RCd14) and phiCD630-2 (CD2889-RCd12 and CD2907.2-RCd15) prophages of C. difficile strain 630. Toxin genes encode 34 to 47 amino acid peptides and their ectopic expression in C. difficile induces growth arrest that is neutralized by antitoxin RNA co-expression. We show that type I TA located within the phiCD630-1 prophage contribute to its stability and heritability. We have made use of a type I TA toxin gene to generate an efficient mutagenesis tool for this bacterium that allowed investigation of the role of these widespread TA in prophage maintenance.

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Antibiotic Resistance Is Associated with Integrative and Conjugative Elements and Genomic Islands in Naturally Circulating Streptococcus pneumoniae Isolates from Adults in Liverpool, UK

Genes ◽

10.3390/genes11060625 ◽

2020 ◽

Vol 11 (6) ◽

pp. 625

Author(s):

Elissavet Nikolaou ◽

Alasdair T. M. Hubbard ◽

João Botelho ◽

Taylor A. M. Marschall ◽

Daniela M. Ferreira ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Pneumococcal Infection ◽

Mobile Genetic Elements ◽

Genomic Islands ◽

Mortality And Morbidity ◽

Genetic Elements ◽

Integrative And Conjugative Elements ◽

Genetic Context

Pneumonia is the sixth largest cause of death in the UK. It is usually caused by Streptococcus pneumoniae, which healthy individuals can carry in their nose without symptoms of disease. Antimicrobial resistance further increases mortality and morbidity associated with pneumococcal infection, although few studies have analysed resistance in naturally circulating pneumococcal isolates in adult populations. Here, we report on the resistome and associated mobile genetic elements within circulating pneumococcus isolated from adult volunteers enrolled in the experimental human pneumococcal colonisation (EHPC) research program at the Liverpool School of Tropical Medicine, UK. Pneumococcal isolates collected from 30 healthy asymptomatic adults who had volunteered to take part in clinical research were screened for antibiotic susceptibility to erythromycin and tetracycline, and whole-genome sequenced. The genetic context of resistance to one or both antibiotics in four isolates was characterised bioinformatically, and any association of the resistance genes with mobile genetic elements was determined. Tetracycline and macrolide resistance genes [tet(M), erm(B), mef(A), msr(D)] were detected on known Tn916-like integrative and conjugative elements, namely Tn6002 and Tn2010, and tet(32) was found for the first time in S. pneumoniae located on a novel 50 kb genomic island. The widespread use of pneumococcal conjugate vaccines impacts on serotype prevalence and transmission within the community. It is therefore important to continue to monitor antimicrobial resistance (AMR) genes present in both vaccine types and non-vaccine types in response to contemporary antimicrobial therapies and characterise the genetic context of acquired resistance genes to continually optimise antibiotic therapies.

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Conjugative Transfer of the Integrative Conjugative Elements ICESt1 and ICESt3 from Streptococcus thermophilus

Journal of Bacteriology ◽

10.1128/jb.01412-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2764-2775 ◽

Cited By ~ 43

Author(s):

Xavier Bellanger ◽

Adam P. Roberts ◽

Catherine Morel ◽

Frédéric Choulet ◽

Guillaume Pavlovic ◽

...

Keyword(s):

Streptococcus Thermophilus ◽

Genomic Islands ◽

Conjugative Transfer ◽

Integrase Gene ◽

Site Specific ◽

Integrative And Conjugative Elements ◽

Conjugative Transposons ◽

Site Specific Integration ◽

The One ◽

Integrative Conjugative Elements

ABSTRACT Integrative and conjugative elements (ICEs), also called conjugative transposons, are genomic islands that excise, self-transfer by conjugation, and integrate in the genome of the recipient bacterium. The current investigation shows the intraspecies conjugative transfer of the first described ICEs in Streptococcus thermophilus, ICESt1 and ICESt3. Mitomycin C, a DNA-damaging agent, derepresses ICESt3 conjugative transfer almost 25-fold. The ICESt3 host range was determined using various members of the Firmicutes as recipients. Whereas numerous ICESt3 transconjugants of Streptococcus pyogenes and Enterococcus faecalis were recovered, only one transconjugant of Lactococcus lactis was obtained. The newly incoming ICEs, except the one from L. lactis, are site-specifically integrated into the 3′ end of the fda gene and are still able to excise in these transconjugants. Furthermore, ICESt3 was retransferred from E. faecalis to S. thermophilus. Recombinant plasmids carrying different parts of the ICESt1 recombination module were used to show that the integrase gene is required for the site-specific integration and excision of the ICEs, whereas the excisionase gene is required for the site-specific excision only.

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Chromosomal Toxin-Antitoxin Systems May Act as Antiaddiction Modules

Journal of Bacteriology ◽

10.1128/jb.00357-08 ◽

2008 ◽

Vol 190 (13) ◽

pp. 4603-4609 ◽

Cited By ~ 85

Author(s):

Manuel Saavedra De Bast ◽

Natacha Mine ◽

Laurence Van Melderen

Keyword(s):

Physiological Role ◽

Mobile Genetic Elements ◽

Selective Advantage ◽

Vertical Inheritance ◽

Genetic Elements ◽

Daughter Cells ◽

Fitness Advantage ◽

Bacterial Chromosomes ◽

Postsegregational Killing

ABSTRACT Toxin-antitoxin (TA) systems are widespread among bacterial chromosomes and mobile genetic elements. Although in plasmids TA systems have a clear role in their vertical inheritance by selectively killing plasmid-free daughter cells (postsegregational killing or addiction phenomenon), the physiological role of chromosomally encoded ones remains under debate. The assumption that chromosomally encoded TA systems are part of stress response networks and/or programmed cell death machinery has been called into question recently by the observation that none of the five canonical chromosomally encoded TA systems in the Escherichia coli chromosome seem to confer any selective advantage under stressful conditions (V. Tsilibaris, G. Maenhaut-Michel, N. Mine, and L. Van Melderen, J. Bacteriol. 189:6101-6108, 2007). Their prevalence in bacterial chromosomes indicates that they might have been acquired through horizontal gene transfer. Once integrated in chromosomes, they might in turn interfere with their homologues encoded by mobile genetic elements. In this work, we show that the chromosomally encoded Erwinia chrysanthemi ccd (control of cell death) (ccdEch ) system indeed protects the cell against postsegregational killing mediated by its F-plasmid ccd (ccd F) homologue. Moreover, competition experiments have shown that this system confers a fitness advantage under postsegregational conditions mediated by the ccd F system. We propose that ccdEch acts as an antiaddiction module and, more generally, that the integration of TA systems in bacterial chromosomes could drive the evolution of plasmid-encoded ones and select toxins that are no longer recognized by the antiaddiction module.

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Genomic islands and the evolution of livestock-associated Staphylococcus aureus genomes

Bioscience Reports ◽

10.1042/bsr20202287 ◽

2020 ◽

Vol 40 (11) ◽

Author(s):

Relangi Tulasi Rao ◽

Shivani Sharma ◽

Natesan Sivakumar ◽

Kannan Jayakumar

Keyword(s):

Staphylococcus Aureus ◽

Functional Characterization ◽

Vital Role ◽

Genomic Islands ◽

Bacterial Genomes ◽

Future Studies ◽

Niche Adaptation ◽

Major Donors

Abstract Background: Genomic Islands (GIs) are commonly believed to be relics of horizontal transfer and associated with specific metabolic capacities, including virulence of the strain. Horizontal gene transfer (HGT) plays a vital role in the acquisition of GIs and the evolution and adaptation of bacterial genomes. Objective: The present study was designed to predict the GIs and role of HGT in evolution of livestock-associated Staphylococcus aureus (LA-SA). Methods: GIs were predicted with two methods namely, Ensemble algorithm for Genomic Island Detection (EGID) tool, and Seq word Sniffer script. Functional characterization of GI elements was performed with clustering of orthologs. The putative donor predictions of GIs was done with the aid of the pre_GI database. Results: The present study predicted a pan of 46 GIs across the LA-SA genomes. Functional characterization of GI sequences revealed few unique results like the presence of metabolic operons like leuABCD and folPK genes in GIs and showed the importance of GIs in the adaptation to the host niche. The developed framework for GI donor prediction results revealed Rickettsia and Mycoplasma as the major donors of GI elements. Conclusions: The role of GIs during the evolutionary race of LA-SA could be concluded from the present study. Niche adaptation of LA-SA enhanced presumably due to these GIs. Future studies could focus on the evolutionary relationships between Rickettsia and Mycoplasma sp. with S. aureus and also the evolution of Leucine/Isoleucine mosaic operon (leuABCD).

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