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1.  Comparative and Evolutionary Analysis of the Bacterial Homologous Recombination Systems  
PLoS Genetics  2005;1(2):e15.
Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes.
Genomes evolve mostly by modifications involving large pieces of genetic material (DNA). Exchanges of chromosome pieces between different organisms as well as intragenomic movements of DNA regions are the result of a process named homologous recombination. The central actor of this process, the RecA protein, is amazingly conserved from bacteria to human. In addition to its role in the generation of genetic variability, homologous recombination is also the guardian of genome integrity, as it acts to repair DNA damage. RecA-catalyzed DNA exchange (synapse) is facilitated by the action of presynaptic enzymes and completed by postsynaptic enzymes (resolvases). In addition, some enzymes counteract RecA. Here, the researchers assess the diversity of recombination proteins among 117 different bacterial species. They find that resolvases are nearly as ubiquitous and as well conserved at the sequence level as RecA. This suggests that the large majority of bacterial genomes are capable of homologous recombination. Presynaptic systems are less ubiquitous, and there is no obvious correlation between their presence and experimental data on the frequency of recombination. However, there is a significant pattern of co-occurrence between these systems and antirecombinant proteins.
PMCID: PMC1193525  PMID: 16132081
2.  The Interplay between Entamoeba and Enteropathogenic Bacteria Modulates Epithelial Cell Damage 
Mixed intestinal infections with Entamoeba histolytica, Entamoeba dispar and bacteria with exacerbated manifestations of disease are common in regions where amoebiasis is endemic. However, amoeba–bacteria interactions remain largely unexamined.
Trophozoites of E. histolytica and E. dispar were co-cultured with enteropathogenic bacteria strains Escherichia coli (EPEC), Shigella dysenteriae and a commensal Escherichia coli. Amoebae that phagocytosed bacteria were tested for a cytopathic effect on epithelial cell monolayers. Cysteine proteinase activity, adhesion and cell surface concentration of Gal/GalNAc lectin were analyzed in amoebae showing increased virulence. Structural and functional changes and induction of IL-8 expression were determined in epithelial cells before and after exposure to bacteria. Chemotaxis of amoebae and neutrophils to human IL-8 and conditioned culture media from epithelial cells exposed to bacteria was quantified.
Principal Findings
E. histolytica digested phagocytosed bacteria, although S. dysenteriae retained 70% viability after ingestion. Phagocytosis of pathogenic bacteria augmented the cytopathic effect of E. histolytica and increased expression of Gal/GalNAc lectin on the amoebic surface and increased cysteine proteinase activity. E. dispar remained avirulent. Adhesion of amoebae and damage to cells exposed to bacteria were increased. Additional increases were observed if amoebae had phagocytosed bacteria. Co-culture of epithelial cells with enteropathogenic bacteria disrupted monolayer permeability and induced expression of IL-8. Media from these co-cultures and human recombinant IL-8 were similarly chemotactic for neutrophils and E. histolytica.
Epithelial monolayers exposed to enteropathogenic bacteria become more susceptible to E. histolytica damage. At the same time, phagocytosis of pathogenic bacteria by amoebae further increased epithelial cell damage.
The in vitro system presented here provides evidence that the Entamoeba/enteropathogenic bacteria interplay modulates epithelial cell responses to the pathogens. In mixed intestinal infections, where such interactions are possible, they could influence the outcome of disease. The results offer insights to continue research on this phenomenon.
Author Summary
In amoebiasis, a human disease that is a serious health problem in many developing countries, efforts have been made to identify responsible factors for the tissue damage inflicted by the parasite Entamoeba histolytica. This amoeba lives in the lumen of the colon without causing damage to the intestinal mucosa, but under unknown circumstances becomes invasive, destroying the intestinal tissue. Bacteria in the intestinal flora have been proposed as inducers of higher amoebic virulence, but the causes or mechanisms responsible for the induction are still undetermined. Mixed intestinal infections with Entamoeba histolytica and enteropathogenic bacteria, showing exacerbated manifestations of disease, are common in endemic countries. We implemented an experimental system to study amoebic virulence in the presence of pathogenic bacteria and its consequences on epithelial cells. Results showed that amoebae that ingested enteropathogenic bacteria became more virulent, causing more damage to epithelial cells. Bacteria induced release of inflammatory proteins by the epithelial cells that attracted amoebae, facilitating amoebic contact to the epithelial cells and higher damage. Our results, although a first approach to this complex problem, provide insights into amoebic infections, as interplay with other pathogens apparently influences the intestinal environment, the behavior of cells involved and the manifestations of the disease.
PMCID: PMC2447883  PMID: 18648517
3.  Oligonucleotide Recombination in Gram-Negative Bacteria 
Molecular Microbiology  2009;75(1):138-148.
This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any additional phage encoded functions. Oligo recombination was tested in four genera of Gram-negative bacteria and in all cases evidence for recombination was apparent. The experiments presented here were designed with an eye towards learning to use oligo recombination in order to bootstrap identification and development of phage encoded recombination systems for recombineering in a wide range of bacteria. The results show that oligo concentration and sequence have the greatest influence on recombination frequency, while oligo length was less important. Apart from the utility of oligo recombination, these findings also provide insights regarding the details of recombination mediated by phage-encoded functions. Establishing that oligos can recombine with bacterial genomes provides a link to similar observations of oligo recombination in archaea and eukaryotes suggesting the possibility that this process is evolutionary conserved.
PMCID: PMC3404488  PMID: 19943907
Recombineering; Pseudomonas; Homologous Recombination; Oligonucleotide; Gene Conversion
4.  Purifying selection in mitochondria, free-living and obligate intracellular proteobacteria 
The effectiveness of elimination of slightly deleterious mutations depends mainly on drift and recombination frequency. Here we analyze the influence of these two factors on the strength of the purifying selection in mitochondrial and proteobacterial orthologous genes taking into account the differences in the organism lifestyles.
(I) We found that the probability of fixation of nonsynonymous substitutions (Kn/Ks) in mitochondria is significantly lower compared to obligate intracellular bacteria and even marginally significantly lower compared to free-living bacteria. The comparison of bacteria of different lifestyles demonstrates more effective elimination of slightly deleterious mutations in (II) free-living bacteria as compared to obligate intracellular species and in (III) obligate intracellular parasites as compared to obligate intracellular symbionts. (IV) Finally, we observed that the level of the purifying selection (i.e. 1-Kn/Ks) increases with the density of mobile elements in bacterial genomes.
This study shows that the comparison of patterns of molecular evolution of orthologous genes between ecologically different groups of organisms allow to elucidate the genetic consequences of their various lifestyles. Comparing the strength of the purifying selection among proteobacteria with different lifestyles we obtained results, which are in concordance with theoretical expectations: (II) low effective population size and level of recombination in obligate intracellular proteobacteria lead to less effective elimination of mutations compared to free-living relatives; (III) rare horizontal transmissions, i.e. effectively zero recombination level in symbiotic obligate intracellular bacteria leads to less effective purifying selection than in parasitic obligate intracellular bacteria; (IV) the increased frequency of recombination in bacterial genomes with high mobile element density leads to a more effective elimination of slightly deleterious mutations. At the same time, (I) more effective purifying selection in relatively small populations of nonrecombining mitochondria as compared to large populations of recombining proteobacteria was unexpected. We hypothesize that additional features such as the high number of protein-protein interactions or female germ-cell atresia increase evolutionary constraints and maintain the effective purifying selection in mitochondria, but more work is needed to definitely establish these additional features.
PMCID: PMC1803777  PMID: 17295908
5.  The Population and Evolutionary Dynamics of Homologous Gene Recombination in Bacteria 
PLoS Genetics  2009;5(8):e1000601.
In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination—broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT)—plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR) to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1) the contribution of HGR to the rate of adaptive evolution in these populations and (2) the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1) HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2) once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent invasion of non-recombining populations, even when recombination engenders a modest fitness cost; and (3) because of the density- and frequency-dependent nature of HGR in bacteria, this capacity to increase rates of adaptive evolution is not sufficient as a selective force to provide a recombining population a selective advantage when it is rare. Under realistic conditions, homologous gene recombination will increase the rate of adaptive evolution in bacterial populations and, once established, selection for higher rates of evolution will promote the maintenance of bacteria-encoded mechanisms for HGR. On the other hand, increasing rates of adaptive evolution by HGR is unlikely to be the sole or even a dominant selective pressure responsible for the original evolution of transformation.
Author Summary
For many species of bacteria, recombination in the form of the acquisition and expression of genes and genetic elements acquired from other bacteria, eukaryotes, and archaea, HGT is an important source of variation for adaptive evolution. Not so clear is the contribution of recombination of homologous genes to adaptive evolution and as a selective pressure for the evolution and maintenance of HGT. Using computer simulations, we explore the role of HGR to adaptive evolution and selection for the evolution and maintenance of HGT. We demonstrate that under realistic conditions by shuffling genes within a bacterial population, HGR will increase its rate of adaptive evolution. Once established, this capacity to increase the rate of adaptive evolution can serve as a selective force for the maintenance of HGT. On the other hand, HGR cannot provide an advantage to a population when its density is low or when the recombining population is rare relative to non-recombining competitors. Thus, we postulate that it is unlikely that the only bacteria—rather than plasmid (or phage)—determined mechanism of HGR, transformation, evolved in response to selection for higher rates of evolution by gene shuffling.
PMCID: PMC2717328  PMID: 19680442
6.  Key Role for Clumping Factor B in Staphylococcus aureus Nasal Colonization of Humans 
PLoS Medicine  2008;5(1):e17.
Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor.
Methods and Findings
We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB− mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 ± 1 d versus 7 ± 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not.
The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.
Heiman Wertheim and colleagues investigate the role ofStaphylococcus aureus clumping factor B, a cell wall protein, in bacterial adherence to epithelial cells and persistent colonization of human nostrils.
Editors' Summary
Staphylococcus aureus are common bacteria that normally live on the skin. They also colonize the nostrils of about one in five adults permanently and another one in three adults intermittently. Although these bacteria usually coexist peacefully with their human carriers, they can cause minor infections such as pimples and boils if they enter the skin through a cut or a sore. They can also cause potentially life-threatening infections such as blood poisoning and pneumonia. These serious, invasive infections are often “autoinfections.” That is, they are caused by strains of S. aureus that are present in the patient's nose before they become ill. Minor S. aureus infections can be treated without antibiotics—by draining a boil, for example. Invasive infections are usually treated with antibiotics such as flucloxacillin.
Why Was This Study Done?
There is no effective vaccine against S. aureus infections and these bacteria are becoming increasingly resistant to flucloxacillin, methicillin, and other antibiotics. Worryingly, although methicillin-resistant S. aureus (MRSA) infections occur most frequently among people in health-care facilities who have weakened immune systems, community-acquired MRSA infections among otherwise healthy people are increasingly common. Consequently, new ways to avoid S. aureus infections are urgently needed. Because persistent nasal carriers of S. aureus have an increased risk of infection, one strategy might be to prevent nasal colonization with S. aureus. How these bacteria colonize the nose is poorly understood, but is likely to involve interactions between molecules expressed on the surface of the bacteria and molecules expressed on the surface of the cells lining the nostrils. In this study, the researchers use a new human nasal colonization assay to investigate the involvement of a bacterial surface protein called clumping factor B (ClfB) in the survival of S. aureus in the human nose. ClfB binds to cytokeratin 10, a protein expressed by cells lining the human nose, and has been implicated in the colonization of mouse noses by S. aureus.
What Did the Researchers Do and Find?
The researchers introduced a strain of S. aureus that made ClfB and an otherwise identical, mutant strain that lacked ClfB into the nostrils of healthy human volunteers and measured how long the two strains survived. For safety reasons, the S. aureus strains used in this study have an additional defect that makes them less likely to colonize and persist in the human nose than the strains found in natural S. aureus carriers. Although both strains grew equally well in the laboratory, the mutant strain was eliminated from human noses much quicker than the strain that made ClfB. Mutant bacteria lacking ClfB were cleared from the nostrils of all the volunteers within two weeks, whereas the bacteria that made ClfB were still present in some of the volunteers four weeks after their introduction. When the researchers investigated how well the two strains stuck to a layer of human cytokeratin 10 in a plastic dish, they found that the bacteria that made ClfB stuck to the human protein but the mutant bacteria did not. Furthermore, the strain with ClfB stuck particularly well to cytokeratin 10 when the bacteria had been grown in conditions where nutrients were limiting, a situation that mimics bacterial growth in the human nose.
What Do These Findings Mean?
These findings show that ClfB is an important factor in the establishment of human nasal colonization by S. aureus and suggest that ClfB might be a target for S. aureus decolonization strategies. Furthermore, although ClfB is clearly important in human nasal colonization by S. aureus, it is likely that additional bacterial factors will also be involved in this process. The human nasal colonization model used in this study may be useful in the identification of these additional factors and also as a test bed for potential S. aureus decolonization strategies.
Additional Information.
Please access these Web sites via the online version of this summary at
The MedlinePlus encyclopedia has a page on Staphylococcus aureus and MRSA (in English and Spanish)
The US Centers for Disease Control and Prevention provides information on community-associated MRSA (in English and Spanish)
The UK National Health Service's health website (NHS Direct) provides information about staphylococcal infections and about MRSA
The UK Health Protection Agency provides information about Staphylococcus aureus
PMCID: PMC2194749  PMID: 18198942
7.  Live bacterial vaccines – a review and identification of potential hazards 
The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development.
PMCID: PMC1538998  PMID: 16796731
8.  Diversity and recombination in Wolbachia and Cardinium from Bryobia spider mites 
BMC Microbiology  2012;12(Suppl 1):S13.
Wolbachia and Cardinium are endosymbiotic bacteria infecting many arthropods and manipulating host reproduction. Although these bacteria are maternally transmitted, incongruencies between phylogenies of host and parasite suggest an additional role for occasional horizontal transmission. Consistent with this view is the strong evidence for recombination in Wolbachia, although it is less clear to what extent recombination drives diversification within single host species and genera. Furthermore, little is known concerning the population structures of other insect endosymbionts which co-infect with Wolbachia, such as Cardinium. Here, we explore Wolbachia and Cardinium strain diversity within nine spider mite species (Tetranychidae) from 38 populations, and quantify the contribution of recombination compared to point mutation in generating Wolbachia diversity.
We found a high level of genetic diversity for Wolbachia, with 36 unique strains detected (64 investigated mite individuals). Sequence data from four Wolbachia genes suggest that new alleles are 7.5 to 11 times more likely to be generated by recombination than point mutation. Consistent with previous reports on more diverse host samples, our data did not reveal evidence for co-evolution of Wolbachia with its host. Cardinium was less frequently found in the mites, but also showed a high level of diversity, with eight unique strains detected in 15 individuals on the basis of only two genes. A lack of congruence among host and Cardinium phylogenies was observed.
We found a high rate of recombination for Wolbachia strains obtained from host species of the spider mite family Tetranychidae, comparable to rates found for horizontally transmitted bacteria. This suggests frequent horizontal transmission of Wolbachia and/or frequent horizontal transfer of single genes. Our findings strengthens earlier reports of recombination for Wolbachia, and shows that high recombination rates are also present on strains from a restrictive host range. Cardinium was found co-infecting several spider mite species, and phylogenetic comparisons suggest also horizontal transmission of Cardinium among hosts.
PMCID: PMC3287510  PMID: 22375894
9.  Role of commensal and probiotic bacteria in human health: a focus on inflammatory bowel disease 
The human gut is one of the most complex ecosystems, composed of 1013-1014 microorganisms which play an important role in human health. In addition, some food products contain live bacteria which transit through our gastrointestinal tract and could exert beneficial effects on our health (known as probiotic effect). Among the numerous proposed health benefits attributed to commensal and probiotic bacteria, their capacity to interact with the host immune system is now well demonstrated. Currently, the use of recombinant lactic acid bacteria to deliver compounds of health interest is gaining importance as an extension of the probiotic concept. This review summarizes some of the recent findings and perspectives in the study of the crosstalk of both commensal and probiotic bacteria with the human host as well as the latest studies in recombinant commensal and probiotic bacteria. Our aim is to highlight the potential roles of recombinant bacteria in this ecosystem.
PMCID: PMC3726476  PMID: 23876056
Bacteria-host crosstalk; Dysbiosis; Genetically modified microorganisms
10.  The Recombination Genes addAB Are Not Restricted to Gram-Positive Bacteria: Genetic Analysis of the Recombination Initiation Enzymes RecF and AddAB in Rhizobium etli 
Journal of Bacteriology  2004;186(23):7905-7913.
Single-strand gaps (SSGs) and double-strand breaks (DSBs) are the major initiation sites for recombination. In bacteria, the SSGs are repaired by RecFOR, while the DSBs are processed by RecBCD in gram-negative bacteria and AddAB in gram-positive bacteria. Unexpectedly, instead of recBCD genes, the addAB genes were found in members of the α-proteobacteria group (gram negative). Taking Rhizobium etli as a model, the role of recF and addAB genes in homologous recombination and repair of damaged DNA was evaluated. Inactivation of either recF or addA provoked strong sensitivity to UV radiation and mitomycin C, while an additive effect was observed in the recF-addA mutant. The DSBs generated by nalidixic acid caused low viability only in the addA mutant. The recombination frequency of large and small plasmids was reduced in the recF mutant (24- and 36-fold, respectively), whereas a slight decrease (threefold) in the addA mutant was observed. Moreover, an additive effect (47- and 90-fold, respectively) was observed in the double mutant, but it was not as dramatic as that in a recA mutant. Interestingly, the frequency of deletion and Campbell-type recombination was slightly affected in either single or double mutants. These results suggest that another pathway exists that allows plasmid and Campbell-type recombination in the absence of recF and addA genes.
PMCID: PMC529079  PMID: 15547262
11.  An investigation of horizontal transfer of feed introduced DNA to the aerobic microbiota of the gastrointestinal tract of rats 
BMC Research Notes  2012;5:170.
Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes.
Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates.
The analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 108 cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.
PMCID: PMC3364145  PMID: 22463741
12.  Homologous Recombination as a Replication Fork Escort: Fork-Protection and Recovery 
Biomolecules  2012;3(1):39-71.
Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.
PMCID: PMC4030885  PMID: 24970156
homologous recombination; DNA replication; DNA repair; fork-restart; fork-repair; fork stabilization
13.  Microevolution of Helicobacter pylori during Prolonged Infection of Single Hosts and within Families 
PLoS Genetics  2010;6(7):e1001036.
Our understanding of basic evolutionary processes in bacteria is still very limited. For example, multiple recent dating estimates are based on a universal inter-species molecular clock rate, but that rate was calibrated using estimates of geological dates that are no longer accepted. We therefore estimated the short-term rates of mutation and recombination in Helicobacter pylori by sequencing an average of 39,300 bp in 78 gene fragments from 97 isolates. These isolates included 34 pairs of sequential samples, which were sampled at intervals of 0.25 to 10.2 years. They also included single isolates from 29 individuals (average age: 45 years) from 10 families. The accumulation of sequence diversity increased with time of separation in a clock-like manner in the sequential isolates. We used Approximate Bayesian Computation to estimate the rates of mutation, recombination, mean length of recombination tracts, and average diversity in those tracts. The estimates indicate that the short-term mutation rate is 1.4×10−6 (serial isolates) to 4.5×10−6 (family isolates) per nucleotide per year and that three times as many substitutions are introduced by recombination as by mutation. The long-term mutation rate over millennia is 5–17-fold lower, partly due to the removal of non-synonymous mutations due to purifying selection. Comparisons with the recent literature show that short-term mutation rates vary dramatically in different bacterial species and can span a range of several orders of magnitude.
Author Summary
Mutation rates in bacteria have generally been considered to be much slower than in viruses. This is partly because estimates of long-term mutation rates for the evolution of distinct species have been inappropriately used for dating divergence within species. Furthermore, the most commonly used long-term mutation rate is based on geological dates that are no longer accepted. In addition, only few short-term mutation rates have been calculated within bacterial species, and these differ with the species by several orders of magnitude. Here, we provide robust estimates for short-term mutation and recombination rates within Helicobacter pylori, a bacterium that commonly infects the human gastric mucosa, based on serial isolates from long-term infections and on differences between isolates from multiple family members. These short-term mutation rates are 5–17-fold faster than long-term mutation rates in H. pylori that have been calibrated by parallel ancient migrations of humans. Short-term mutation rates in bacteria, including those for H. pylori, can be quite fast, partially overlapping with those for viruses. Future calculations of ages of bacterial species will need to account for dramatic differences in mutation rate between species and for dramatic differences between short- and long-term mutation rates.
PMCID: PMC2908706  PMID: 20661309
14.  Comparative Genomics of Wolbachia and the Bacterial Species Concept 
PLoS Genetics  2013;9(4):e1003381.
The importance of host-specialization to speciation processes in obligate host-associated bacteria is well known, as is also the ability of recombination to generate cohesion in bacterial populations. However, whether divergent strains of highly recombining intracellular bacteria, such as Wolbachia, can maintain their genetic distinctness when infecting the same host is not known. We first developed a protocol for the genome sequencing of uncultivable endosymbionts. Using this method, we have sequenced the complete genomes of the Wolbachia strains wHa and wNo, which occur as natural double infections in Drosophila simulans populations on the Seychelles and in New Caledonia. Taxonomically, wHa belong to supergroup A and wNo to supergroup B. A comparative genomics study including additional strains supported the supergroup classification scheme and revealed 24 and 33 group-specific genes, putatively involved in host-adaptation processes. Recombination frequencies were high for strains of the same supergroup despite different host-preference patterns, leading to genomic cohesion. The inferred recombination fragments for strains of different supergroups were of short sizes, and the genomes of the co-infecting Wolbachia strains wHa and wNo were not more similar to each other and did not share more genes than other A- and B-group strains that infect different hosts. We conclude that Wolbachia strains of supergroup A and B represent genetically distinct clades, and that strains of different supergroups can co-exist in the same arthropod host without converging into the same species. This suggests that the supergroups are irreversibly separated and that barriers other than host-specialization are able to maintain distinct clades in recombining endosymbiont populations. Acquiring a good knowledge of the barriers to genetic exchange in Wolbachia will advance our understanding of how endosymbiont communities are constructed from vertically and horizontally transmitted genes.
Author Summary
Speciation in sexual organisms is defined as the inability of two populations to get viable offspring. Speciation in asexual, obligate endosymbionts is thought to be an indirect consequence of host-specialization. An important question is if divergent endosymbionts would start blending if the host barrier isolating them were removed. Here, we have studied Wolbachia, an abundant group of bacteria in the insect world. Wolbachia is classified into supergroups based on multi-locus sequence typing. We have sequenced the genomes from the Wolbachia strains wNo and wHa. These are particularly interesting since they belong to different supergroups yet co-occur as a double-infection in natural populations of Drosophila simulans. A comparative genomics study showed that wHa and wNo contain no uniquely shared genes. Instead, each strain shares unique gene functions with members of the same supergroup that infect other hosts. This unexpected finding suggests an alternative means of ecological speciation, indicating that speciation is not restricted to host-specialization but rather that related endosymbionts can coexist as separate species in the same host. Our study sheds light on the genomic divergence between different partners inhabiting the intracellular niche of the same host organism.
PMCID: PMC3616963  PMID: 23593012
15.  Rhizosphere Competitiveness of Trichloroethylene-Degrading, Poplar-Colonizing Recombinant Bacteria 
Applied and Environmental Microbiology  2000;66(11):4673-4678.
Indigenous bacteria from poplar tree (Populus canadensis var. eugenei ‘Imperial Carolina’) and southern California shrub rhizospheres, as well as two tree-colonizing Rhizobium strains (ATCC 10320 and ATCC 35645), were engineered to express constitutively and stably toluene o-monooxygenase (TOM) from Burkholderia cepacia G4 by integrating the tom locus into the chromosome. The poplar and Rhizobium recombinant bacteria degraded trichloroethylene at a rate of 0.8 to 2.1 nmol/min/mg of protein and were competitive against the unengineered hosts in wheat and barley rhizospheres for 1 month (colonization occurred at a level of 1.0 × 105 to 23 × 105 CFU/cm of root). In addition, six of these recombinants colonized poplar roots stably and competitively with populations as large as 79% ± 12% of all rhizosphere bacteria after 28 days (0.2 × 105 to 31 × 105 CFU/cm of root). Furthermore, five of the most competitive poplar recombinants (e.g., Pb3-1 and Pb5-1, which were identified as Pseudomonas sp. strain PsK recombinants) retained the ability to express TOM for 29 days as 100% ± 0% of the recombinants detected in the poplar rhizosphere expressed TOM constitutively.
PMCID: PMC92365  PMID: 11055909
16.  Induction of Protective Cellular Immunity against Mycobacterium tuberculosis by Recombinant Attenuated Self-Destructing Listeria monocytogenes Strains Harboring Eukaryotic Expression Plasmids for Antigen 85 Complex and MPB/MPT51  
Infection and Immunity  2004;72(4):2014-2021.
We report here the induction of specific protective cellular immunity against Mycobacterium tuberculosis by the employment of vaccination with recombinant attenuated Listeria monocytogenes strains. We constructed self-destructing attenuated L. monocytogenes Δ2 strains carrying eukaryotic expression plasmids for the antigen 85 complex (Ag85A and Ag85B) and for MPB/MPT51 (mycobacterial protein secreted by M. bovis BCG/mycobacterial protein secreted by M. tuberculosis) molecules. Infection of these recombinant bacteria allowed expression of the genes in the J774A.1 murine macrophage cell line. Intraperitoneal vaccination of C57BL/6 mice with these recombinant bacteria was capable of inducing purified protein derivative-specific cellular immune responses, such as foot pad reactions, proliferative responses of splenocytes, and gamma interferon production from splenocytes, suggesting the efficacy of vaccination against mycobacterial infection by use of these recombinant L. monocytogenes strains. Furthermore, intravenous vaccination with recombinant bacteria carrying expression plasmids for Ag85A, Ag85B, or MPB/MPT51 in BALB/c mice elicited significant protective responses, comparable to those evoked by a live Mycobacterium bovis BCG vaccine. Notably, this is the first report to show that MPB/MPT51 is a major protective antigen in addition to Ag85A and Ag85B, which have been reported to be major mycobacterial protective antigens.
PMCID: PMC375201  PMID: 15039321
17.  The Unconventional Xer Recombination Machinery of Streptococci/Lactococci 
PLoS Genetics  2007;3(7):e117.
Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (dif) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in E. coli is highly conserved among prokaryotes. We show here that Streptococci and Lactococci carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (difSL) associated with a dedicated tyrosine recombinase (XerS). In contrast to the E. coli Xer system, only a single recombinase is required to recombine difSL, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when difSL sites are located on chromosome dimers. Moreover, the XerS/difSL recombination requires the streptococcal protein FtsKSL, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of Lactococcus lactis. Acquisition of the XerS recombination module can be considered as a landmark of the separation of Streptococci/Lactococci from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.
Author Summary
In bacteria, genetic information is mainly carried by a single circular chromosome. The replication of this circular molecule sometimes leads to the formation of a chromosome dimer unable to segregate in the daughter cells during the division process. In the bacterial model E. coli, chromosome dimers are resolved in monomers by site-specific recombination: two recombinases (XerC and XerD) cooperatively catalyze the recombination at a chromosomal site (dif), located at the junction of the two replication arms. This recombination is intimately coupled to cell division by the control of the septal protein FtsK. Xer recombination machinery as described in E. coli appears highly conserved among bacterial species. We show by comparative genomics and genetic studies that Streptococci use an alternative Xer recombination system, renamed XerS/difSL, which is composed of a single recombinase phylogenetically unrelated to XerCD proteins and a noncanonical dif site. We also demonstrate that the streptococcal FtsK protein localizes at the division septum and operates the XerS/difSL recombination. This is the first identification of an alternative Xer recombination system in prokaryotes to out knowledge, which might indicate that other unusual chromosome dimer resolution systems could exist in bacterial phyla where a canonical dif site is not detected.
PMCID: PMC1914069  PMID: 17630835
18.  Holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories 
Nucleic Acids Research  2000;28(18):3417-3432.
Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII–colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage λ exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and Aquifex. Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including McrA and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the λ exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.
PMCID: PMC110722  PMID: 10982859
19.  Could DNA uptake be a side effect of bacterial adhesion and twitching motility? 
Archives of Microbiology  2013;195(4):279-289.
DNA acquisition promotes the spread of resistance to antibiotics and virulence among bacteria. It is also linked to several natural phenomena including recombination, genome dynamics, adaptation and speciation. Horizontal DNA transfer between bacteria occurs via conjugation, transduction or competence for natural transformation by DNA uptake. Among these, competence is the only mechanism of transformation initiated and entirely controlled by the chromosome of the recipient bacteria. While the molecular mechanisms allowing the uptake of extracellular DNA are increasingly characterized, the function of competence for natural transformation by DNA uptake, the selective advantage maintaining it and the reasons why bacteria take up DNA in the first place are still debated. In this synthesis, I review some of the literature and discuss the four hypotheses on how and why do bacteria take up DNA. I argue that DNA uptake by bacteria is an accidental by-product of bacterial adhesion and twitching motility. Adhesion and motility are generally increased in stressful conditions, which may explain why bacteria increase DNA uptake in these conditions. In addition to its fundamental scientific relevance, the new hypothesis suggested here has significant clinical implications and finds further support from the fact that antibiotics sometimes fail to eliminate the targeted bacterium while inevitably causing stress to others. The widespread misuse of antibiotics may thus not only be selecting for resistant strains, but may also be causing bacteria to take up more DNA with the consequent increase in the chances of acquiring drug resistance and virulence—a scenario in full concordance with the previously reported induction of competence genes by antibiotics in Streptococcus pneumoniae and Legionella pneumophila.
PMCID: PMC3597990  PMID: 23381940
Competence; DNA uptake; Transformation; Horizontal gene transfer; Type IV pilus; Twitching motility; Social gliding; Swarming; Bacteria
20.  Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase 
DNA repair  2008;7(10):1624-1635.
Thymidylate deprivation brings about “thymineless death” in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.
PMCID: PMC2586641  PMID: 18603020
21.  ruvA Mutants That Resolve Holliday Junctions but Do Not Reverse Replication Forks 
PLoS Genetics  2008;4(3):e1000012.
RuvAB and RuvABC complexes catalyze branch migration and resolution of Holliday junctions (HJs) respectively. In addition to their action in the last steps of homologous recombination, they process HJs made by replication fork reversal, a reaction which occurs at inactivated replication forks by the annealing of blocked leading and lagging strand ends. RuvAB was recently proposed to bind replication forks and directly catalyze their conversion into HJs. We report here the isolation and characterization of two separation-of-function ruvA mutants that resolve HJs, based on their capacity to promote conjugational recombination and recombinational repair of UV and mitomycin C lesions, but have lost the capacity to reverse forks. In vivo and in vitro evidence indicate that the ruvA mutations affect DNA binding and the stimulation of RuvB helicase activity. This work shows that RuvA's actions at forks and at HJs can be genetically separated, and that RuvA mutants compromised for fork reversal remain fully capable of homologous recombination.
Author Summary
DNA replication is the process by which DNA strands are copied to ensure the transmission of the genetic material to daughter cells. Chromosome replication is not a continuous process but is subjected to accidental arrests, owing to the encounter of obstacles or to the dysfunctioning of a replication protein. In bacteria, inactivated replication forks restart but they are most often remodeled before restarting. Interestingly, enzymes involved in homologous recombination, the process that rearranges chromosomes, are also involved in fork-remodeling reactions. The subject of the present study is RuvAB, a highly conserved bacterial complex used as the model enzyme for resolution of recombination intermediates, which we found to also act at blocked forks. We describe here the isolation and characterization of ruvA mutants that have specifically lost the capability to act at inactivated replication forks, although they remain fully capable of homologous recombination. The existence of such ruvA mutants, their properties and those of the purified RuvA mutant proteins, indicate that the action of RuvAB at replication forks is more demanding that its action at recombination intermediates, but have nevertheless been preserved during evolution.
PMCID: PMC2265524  PMID: 18369438
22.  SoxAX Binding Protein, a Novel Component of the Thiosulfate-Oxidizing Multienzyme System in the Green Sulfur Bacterium Chlorobium tepidum▿  
Journal of Bacteriology  2008;190(18):6097-6110.
From the photosynthetic green sulfur bacterium Chlorobium tepidum (pro synon. Chlorobaculum tepidum), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome c554. These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome c551, composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in Escherichia coli cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome c554 reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose sox gene cluster sequences have been deposited so far and ranging over the Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae, etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.
PMCID: PMC2546802  PMID: 18641134
23.  Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia. 
Journal of Clinical Investigation  1995;95(1):142-150.
Streptococcus pneumoniae is one of the most common etiologic agents of community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S. pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+) bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after infection. The presence of pneumolysin also contributed to the development of bacteremia, which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects of pneumolysin on lung injury and on the ability of pneumococci to evade local lung defenses was confirmed by addition of purified recombinant pneumolysin to inocula of PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels comparable to those seen with the PLY(+) strain. We further demonstrated the contributions of both the cytolytic and the complement-activating properties of pneumolysin on enhanced bacterial growth in murine lungs using genetically modified pneumolysin congeners and genetically complement-deficient mice. Thus, pneumolysin facilitates intraalveolar replication of pneumococci, penetration of bacteria from alveoli into the interstitium of the lung, and dissemination of pneumococci into the bloodstream during experimental pneumonia. Moreover, both the cytotoxic and the complement-activating activities of pneumolysin may contribute independently to the acute pulmonary injury and the high rates of bacteremia which characterize pneumococcal pneumonia.
PMCID: PMC295392  PMID: 7814608
24.  Productive Homologous and Non-homologous Recombination of Hepatitis C Virus in Cell Culture 
PLoS Pathogens  2013;9(3):e1003228.
Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13–36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5′ end to the NS2–NS3 region followed by JFH1 sequence from Core to the 3′ end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients.
Author Summary
Genetic recombination is the alternative joining of nucleic acids leading to novel combinations of genetic information. While DNA recombination in cells is of importance for evolution and adaptive immunity, RNA recombination often has only transient effects. However, RNA viruses are rapidly evolving and recombination can be an important evolutionary step in addition to mutations introduced by the viral polymerase. Recombination can allow escape from the host immune system and from antiviral treatment, and recombination of live attenuated viral vaccines has led to re-emergence of disease. Hepatitis C virus (HCV) is an important human pathogen that chronically infects more than 130 million worldwide and leads to serious liver disease. For HCV, naturally occurring recombinants are rare but clinically important. HCV recombination constitutes a challenge to antiviral treatment and can potentially provide an escape mechanism for the virus. In this study, we established an assay for HCV RNA recombination and characterized the emerging homologous and non-homologous recombinant viruses. Interestingly, recombination did not depend on viral replication, occurred most efficiently between isolates of the same genotype and did not occur with strong site-specificity. Better diagnosis of clinically important recombinants and an increased knowledge on viral recombination could strengthen antiviral and vaccine development.
PMCID: PMC3610614  PMID: 23555245
25.  Impact of homologous and non-homologous recombination in the genomic evolution of Escherichia coli 
BMC Genomics  2012;13:256.
Escherichia coli is an important species of bacteria that can live as a harmless inhabitant of the guts of many animals, as a pathogen causing life-threatening conditions or freely in the non-host environment. This diversity of lifestyles has made it a particular focus of interest for studies of genetic variation, mainly with the aim to understand how a commensal can become a deadly pathogen. Many whole genomes of E. coli have been fully sequenced in the past few years, which offer helpful data to help understand how this important species evolved.
We compared 27 whole genomes encompassing four phylogroups of Escherichia coli (A, B1, B2 and E). From the core-genome we established the clonal relationships between the isolates as well as the role played by homologous recombination during their evolution from a common ancestor. We found strong evidence for sexual isolation between three lineages (A+B1, B2, E), which could be explained by the ecological structuring of E. coli and may represent on-going speciation. We identified three hotspots of homologous recombination, one of which had not been previously described and contains the aroC gene, involved in the essential shikimate metabolic pathway. We also described the role played by non-homologous recombination in the pan-genome, and showed that this process was highly heterogeneous. Our analyses revealed in particular that the genomes of three enterohaemorrhagic (EHEC) strains within phylogroup B1 have converged from originally separate backgrounds as a result of both homologous and non-homologous recombination.
Recombination is an important force shaping the genomic evolution and diversification of E. coli, both by replacing fragments of genes with an homologous sequence and also by introducing new genes. In this study, several non-random patterns of these events were identified which correlated with important changes in the lifestyle of the bacteria, and therefore provide additional evidence to explain the relationship between genomic variation and ecological adaptation.
PMCID: PMC3505186  PMID: 22712577

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