Multilocus sequence typing (MLST) of Entamoeba histolytica identifies kerp2 as a genetic marker associated with disease outcomes

Amoebiasis caused by protozoan parasite Entamoeba histolytica has diverse infection outcomes. The relationship between parasite genotypes and outcome of amoebic infection is still a paradox and needs to be explored. Genome information of infecting strains from endemic areas throughout the world is essential to explore this relation. Comparative genetics between E. histolytica populations from different disease outcomes have been studied to identify potential genetic markers having single nucleotide polymorphisms (SNPs) significantly associated with specific clinical outcome. Coding and non-coding regions have significantly different rates of polymorphism. Non-synonymous base substitutions were significantly more frequent than synonymous within coding loci. Both synonymous and non-synonymous SNPs within lysine- and glutamic acid rich protein 2 (kerp2) locus were significantly associated with disease outcomes. An incomplete linkage disequilibrium (LD) value with potential recombination events and significant population differentiation (F_ST) value have also been identified at kerp2 locus within the study population. Presence of disease specific SNPs, potential recombination events, and significant F_ST value at kerp2 locus indicate that kerp2 gene and its gene product are under constant selection pressure exerted by host on parasite and could also be a potential determinant of disease outcome of E. histolytica infection. Furthermore, E. histolytica isolated from asymptomatic carriers are phylogenetically closer to those causing liver abscess in human and exhibit potential inter-population recombination among them. Individuals with persistent asymptomatic E. histolytica infection may be under high risk of developing amoebic liver abscess formation in future and detailed investigation of asymptomatic individuals from endemic areas should be always required.     

Molecular analysis of intragenic recombination at the tryptophan synthetase locus in Neurospora crassa

Fifteen different classically generated and mapped mutations at the tryptophan synthetase locus in Neurospora crassa have been characterized to the level of the primary sequence of the gene. This sequence analysis has demonstrated that intragenic recombination is accurate to order mutations within one open reading frame. While classic genetic analysis correctly ordered the mutations, the position of mutations characterized by gene sequence analysis was more accurate. A leaky mutation was found to have a wild-type primary sequence. The presence of unique polymorphisms in the primary sequence of the trp-3 gene from strain 861 confirms that it has a unique history relative to the other strains studied. Most strains that were previously shown to be immunologically nonreactive with antibody preparations raised against tryptophan synthetase protein were shown to have nonsense mutations. This work defines 14 alleles of the N. crassa trp-3 gene.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Complex Population Structure and Virulence Differences among Serotype 2 Streptococcus suis Strains Belonging to Sequence Type 28

Streptococcus suis is a major swine pathogen and a zoonotic agent. Serotype 2 strains are the most frequently associated with disease. However, not all serotype 2 lineages are considered virulent. Indeed, sequence type (ST) 28 serotype 2 S. suis strains have been described as a homogeneous group of low virulence. However, ST28 strains are often isolated from diseased swine in some countries, and at least four human ST28 cases have been reported. Here, we used whole-genome sequencing and animal infection models to test the hypothesis that the ST28 lineage comprises strains of different genetic backgrounds and different virulence. We used 50 S. suis ST28 strains isolated in Canada, the United States and Japan from diseased pigs, and one ST28 strain from a human case isolated in Thailand. We report a complex population structure among the 51 ST28 strains. Diversity resulted from variable gene content, recombination events and numerous genome-wide polymorphisms not attributable to recombination. Phylogenetic analysis using core genome single-nucleotide polymorphisms revealed four discrete clades with strong geographic structure, and a fifth clade formed by US, Thai and Japanese strains. When tested in experimental animal models, strains from this latter clade were significantly more virulent than a Canadian ST28 reference strain, and a closely related Canadian strain. Our results highlight the limitations of MLST for both phylogenetic analysis and virulence prediction and raise concerns about the possible emergence of ST28 strains in human clinical cases.     

Chromosomal Rearrangements between Serotype A and D Strains in Cryptococcus neoformans

Cryptococcus neoformans is a major human pathogenic fungus that can cause meningoencephalitis in immunocompromised hosts. It contains two divergent varieties, var. grubii (serotype A) and var. neoformans (serotype D), as well as hybrids (serotype AD) between these two varieties. In this study, we investigated the extent of chromosomal rearrangements between the two varieties, estimated the effects of chromosomal rearrangements on recombination frequencies, and surveyed the potential polymorphisms of the rearrangements among natural strains of the three serotypes. Through the analyses of two sequenced genomes from strains H99 (representing var. grubii) and JEC21 (representing var. neoformans), we revealed a total of 32 unambiguous chromosome rearrangements, including five translocations, nine simple inversions, and 18 complex rearrangements. Our analyses identified that overall, rearranged regions had recombination frequencies about half of those around syntenic regions. Using a direct PCR screening strategy, we examined the potential polymorphisms of 11 rearrangements among 64 natural C. neoformans strains from five countries. We found no polymorphism within var. neoformans and very limited polymorphism within var. grubii. However, strains of serotype AD showed significant polymorphism, consistent with their hybrid origins coupled with differential loss of heterozygosity. We discuss the implications of these results on the genome structure, ecology, and evolution of C. neoformans.     

Multiple Interactions of a DNA-Binding Protein IN VIVO. III. Phage T4 Gene-32 Mutations Differentially Affect Insertion-Type Recombination and Membrane Properties

We have investigated the in vivo roles of T4 gene- 32 protein in recombination. We have studied the effects of gene- 32 mutations under conditions that allow normal DNA replication and are permissive for progeny production. Under these conditions, certain gene- 32 mutations specifically reduce insertion-type (short-interval) recombination but none affect crossover-type (long-interval) recombination (see Figure 5). Heterozygote frequencies in all gene-32 mutants are similar to or higher than in a gene-32⁺ background and are not correlated with recombination deficiencies. "Recombination-deficient" alleles are dominant or codominant over the "recombination-proficient" gene-32 mutation tsL171. This explains apparent discrepancies between a gene-32 map deduced from two-factor crosses and the map derived from three-factor crosses.

We have also found that the "recombination proficient" mutation tsL171 and its homoalleles suppress the characteristic plaque morphology of rII mutants. Under restrictive conditions, tsL171 is partially suppressed by rII mutations, which allow the use of host ligase in recombination.

Our present and previous results are discussed in terms of current recombination models. We conclude that gene-32 protein functions in recombination by forming a complex with DNA, with recombination enzymes and with membrane components. Since gene-32 protein interacts with many components of this recombination complex, gene-32 mutations may differentially affect various recombination steps.

     

Recombination and Its Impact on the Genome of the Haplodiploid Parasitoid Wasp Nasonia

Homologous meiotic recombination occurs in most sexually reproducing organisms, yet its evolutionary advantages are elusive. Previous research explored recombination in the honeybee, a eusocial hymenopteran with an exceptionally high genome-wide recombination rate. A comparable study in a non-social member of the Hymenoptera that would disentangle the impact of sociality from Hymenoptera-specific features such as haplodiploidy on the evolution of the high genome-wide recombination rate in social Hymenoptera is missing. Utilizing single-nucleotide polymorphisms (SNPs) between two Nasonia parasitoid wasp genomes, we developed a SNP genotyping microarray to infer a high-density linkage map for Nasonia. The map comprises 1,255 markers with an average distance of 0.3 cM. The mapped markers enabled us to arrange 265 scaffolds of the Nasonia genome assembly 1.0 on the linkage map, representing 63.6% of the assembled N. vitripennis genome. We estimated a genome-wide recombination rate of 1.4–1.5 cM/Mb for Nasonia, which is less than one tenth of the rate reported for the honeybee. The local recombination rate in Nasonia is positively correlated with the distance to the center of the linkage groups, GC content, and the proportion of simple repeats. In contrast to the honeybee genome, gene density in the parasitoid wasp genome is positively associated with the recombination rate; regions of low recombination are characterized by fewer genes with larger introns and by a greater distance between genes. Finally, we found that genes in regions of the genome with a low recombination frequency tend to have a higher ratio of non-synonymous to synonymous substitutions, likely due to the accumulation of slightly deleterious non-synonymous substitutions. These findings are consistent with the hypothesis that recombination reduces interference between linked sites and thereby facilitates adaptive evolution and the purging of deleterious mutations. Our results imply that the genomes of haplodiploid and of diploid higher eukaryotes do not differ systematically in their recombination rates and associated parameters.     

Attachment site mutants of bacteriophage lambda

Insertion and excision of the chromosome of phage λ occurs by recombination at special regions of the phage and bacterial chromosomes known as attachment sites (alt's). We have isolated att mutants which display reduced recombination frequencies. The mutations are cis-dominant, trans-recessive, and can be crossed into a phage, bacterial or prophage att. These results suggest that the att's, although different over-all, include the same DNA sequence as part of their structure, and that the mutations reside in these sequences. Crosses between mutant and wild-type att's occasionally yield heterozygotes. This result suggests that recombination of the att's generates complementary single-strands via staggered nicks in these common sequences. Recombinant att's are then formed by the interannealing of single-strands of different att's followed by ligation.     

Comparative Genomic and Phylogenetic Approaches to Characterize the Role of Genetic Recombination in Mycobacterial Evolution

The genus Mycobacterium encompasses over one hundred named species of environmental and pathogenic organisms, including the causative agents of devastating human diseases such as tuberculosis and leprosy. The success of these human pathogens is due in part to their ability to rapidly adapt to their changing environment and host. Recombination is the fastest way for bacterial genomes to acquire genetic material, but conflicting results about the extent of recombination in the genus Mycobacterium have been reported. We examined a data set comprising 18 distinct strains from 13 named species for evidence of recombination. Genomic regions common to all strains (accounting for 10% to 22% of the full genomes of all examined species) were aligned and concatenated in the chromosomal order of one mycobacterial reference species. The concatenated sequence was screened for evidence of recombination using a variety of statistical methods, with each proposed event evaluated by comparing maximum-likelihood phylogenies of the recombinant section with the non-recombinant portion of the dataset. Incongruent phylogenies were identified by comparing the site-wise log-likelihoods of each tree using multiple tests. We also used a phylogenomic approach to identify genes that may have been acquired through horizontal transfer from non-mycobacterial sources. The most frequent associated lineages (and potential gene transfer partners) in the Mycobacterium lineage-restricted gene trees are other members of suborder Corynebacterinae, but more-distant partners were identified as well. In two examined cases of potentially frequent and habitat-directed transfer (M. abscessus to Segniliparus and M. smegmatis to Streptomyces), observed sequence distances were small and consistent with a hypothesis of transfer, while in a third case (M. vanbaalenii to Streptomyces) distances were larger. The analyses described here indicate that whereas evidence of recombination in core regions within the genus is relatively sparse, the acquisition of genes from non-mycobacterial lineages is a significant feature of mycobacterial evolution.     

Tandem chromosomal duplications in Salmonella typhimurium: fusion of histidine genes to novel promoters.

Salmonella strains harboring tandem chromosomal duplications have been identified following selection for expression of a histidine biosynthetic gene whose promoter is deleted. In such strains, tandem duplications fuse the selected his gene to “foreign” regulatory elements, thereby allowing gene expression. Selection is made for hisD⁺ activity in deletion strain hisOG203. Among the revertants, strains harboring tandem chromosomal duplications have been identified by a number of their properties. (1) Their HisD⁺ phenotype is genetically unstable. (2) Such instability is dependent on recombination (recA) activity. (3) Genetic tests demonstrate that these strains are merodiploid for large regions (up to 25%) of the Salmonella genome. (4) Recipient strains that inherit the HisD⁺ phenotype of these duplication-carrying revertants also inherit the donor's merodiploid state. (5) In certain revertants the functional hisD⁺ gene and the sequence which promotes merodiploid transductant formation are linked to chromosomal markers located far from the normal his region.Previous reports have concluded that the instability of strains isolated by this selection is due to translocation of the hisD⁺ gene to an extrachromosomal element (the pi-histidine factor). We believe that in all strains we have tested (33 independent isolates) instability can better be accounted for as due to tandem duplication events which permit expression of hisD. At least two mechanisms are responsible for duplication formation. One mechanism is dependent on recombination function and generates identical revertants having a duplication of 16% of the chromosome. A second mechanism operates independently of recombination activity; individual duplications produced by this process have variable endpoints.     

The role of recombination in the formation of circular oligomers of the lambda plasmid

The λdv1 plasmid forms an extensive oligomeric series of circular DNA molecules in recombination-proficient (recsu⁺) Escherichia coli. These rec⁺ [λdv1]⁺ strains can be typed into the following four classes according to which member of the oligomeric series is most frequent: monomer, dimer, trimer, and tetramer strains. Each of these strains forms a set of circular λdv1 DNA molecules in which most members belong to the series l, 2l, 3l, 4l, where l is the length of the most frequent circular DNA that characterizes the strain—i.e. l equals the length of the most frequent oligomer in the respective strain. In a given strain, the frequency of a molecular species decreases as its length becomes a larger multiple of l. For example, the dimer strains produce dimers, tetramers, hexamers, octomers, etc., in decreasing frequencies, which reach the limits of detection at about the hexadecamer.When recA^? mutations that are absolutely defective for host recombination are introduced into each of these four strains, l retains the same values as in the parent rec⁺ strain, but oligomers larger than 2l are not formed, and the frequency of the 2l oligomer is much reduced. The introduction of recB^? or recC^? mutations, which are only partially defective for host recombination, produces a much smaller perturbation of the rec⁺ distributions, and $\text{rec}{}^{\mathrm{+}}\text{recA}{}^{\mathrm{?}}$ merodiploids exhibit the rec⁺ phenotype with respect to both oligomerization and host recombination.The effects of rec^? mutations on the distribution of λdv1 oligomers and the nature of the oligomeric series produced in rec⁺ cells all indicate that an intermolecular reciprocal recombination between two circular λdv1 DNAs is the principal reaction responsible for oligomerization. It is suggested that the small residual oligomerization that yields 2l oligomers in recA^?cells results from aberrant segregation of the DNA strands at the termination of the replication of l-sized molecules.The inactivation of recA, but not of recB or C, also results in a marked reduction in the frequency of spontaneous curing which in recA⁺ [λdv1⁺]hosts leads to the segregation of [λdv^?]cells. However, spontaneous curing does not appear to be dependent upon the recombination reactions that yield the [λdv 1⁺]oligomers, since the frequency of oligomerization in recA⁺ hosts decreases with increasing l, whereas the frequency of curing increases with increasing l.     

Phylogeny of Mycobacterium tuberculosis Beijing strains constructed from polymorphisms in genes involved in DNA replication, recombination and repair

Background

The Beijing family is a successful group of M. tuberculosis strains, often associated with drug resistance and widely distributed throughout the world. Polymorphic genetic markers have been used to type particular M. tuberculosis strains. We recently identified a group of polymorphic DNA repair replication and recombination (3R) genes. It was shown that evolution of M. tuberculosis complex strains can be studied using 3R SNPs and a high-resolution tool for strain discrimination was developed. Here we investigated the genetic diversity and propose a phylogeny for Beijing strains by analyzing polymorphisms in 3R genes.

Methodology/Principal Findings

A group of 3R genes was sequenced in a collection of Beijing strains from different geographic origins. Sequence analysis and comparison with the ones of non-Beijing strains identified several SNPs. These SNPs were used to type a larger collection of Beijing strains and allowed identification of 26 different sequence types for which a phylogeny was constructed. Phylogenetic relationships established by sequence types were in agreement with evolutionary pathways suggested by other genetic markers, such as Large Sequence Polymorphisms (LSPs). A recent Beijing genotype (Bmyc10), which included 60% of strains from distinct parts of the world, appeared to be predominant.

Conclusions/Significance

We found SNPs in 3R genes associated with the Beijing family, which enabled discrimination of different groups and the proposal of a phylogeny. The Beijing family can be divided into different groups characterized by particular genetic polymorphisms that may reflect pathogenic features. These SNPs are new, potential genetic markers that may contribute to better understand the success of the Beijing family.     

Analysis of the Mycoplasma genitalium MgpB Adhesin to Predict Membrane Topology,Investigate Antibody Accessibility,Characterize Amino Acid Diversity,and Identify Functional and Immunogenic Epitopes

Mycoplasma genitalium is a sexually transmitted pathogen and is associated with reproductive tract disease that can be chronic in nature despite the induction of a strong antibody response. Persistent infection exacerbates the likelihood of transmission, increases the risk of ascension to the upper tract, and suggests that M. genitalium may possess immune evasion mechanism(s). Antibodies from infected patients predominantly target the MgpB adhesin, which is encoded by a gene that recombines with homologous donor sequences, thereby generating sequence variation within and among strains. We have previously characterized mgpB heterogeneity over the course of persistent infection and have correlated the induction of variant-specific antibodies with the loss of that particular variant from the infected host. In the current study, we examined the membrane topology, antibody accessibility, distribution of amino acid diversity, and the location of functional and antigenic epitopes within the MgpB adhesin. Our results indicate that MgpB contains a single transmembrane domain, that the majority of the protein is surface exposed and antibody accessible, and that the attachment domain is located within the extracellular C-terminus. Not unexpectedly, amino acid diversity was concentrated within and around the three previously defined variable regions (B, EF, and G) of MgpB; while nonsynonymous mutations were twice as frequent as synonymous mutations in regions B and G, region EF had equal numbers of nonsynonymous and synonymous mutations. Interestingly, antibodies produced during persistent infection reacted predominantly with the conserved C-terminus and variable region B. In contrast, infection-induced antibodies reacted poorly with the N-terminus, variable regions EF and G, and intervening conserved regions despite the presence of predicted B cell epitopes. Overall, this study provides an important foundation to define how different segments of the MgpB adhesin contribute to functionality, variability, and immunogenicity during persistent M. genitalium infection.     

Comparative Genomics of Wolbachia and the Bacterial Species Concept

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.     

Are Protein Domains Modules of Lateral Genetic Transfer?

Background

In prokaryotes and some eukaryotes, genetic material can be transferred laterally among unrelated lineages and recombined into new host genomes, providing metabolic and physiological novelty. Although the process is usually framed in terms of gene sharing (e.g. lateral gene transfer, LGT), there is little reason to imagine that the units of transfer and recombination correspond to entire, intact genes. Proteins often consist of one or more spatially compact structural regions (domains) which may fold autonomously and which, singly or in combination, confer the protein''s specific functions. As LGT is frequent in strongly selective environments and natural selection is based on function, we hypothesized that domains might also serve as modules of genetic transfer, i.e. that regions of DNA that are transferred and recombined between lineages might encode intact structural domains of proteins.

Methodology/Principal Findings

We selected 1,462 orthologous gene sets representing 144 prokaryotic genomes, and applied a rigorous two-stage approach to identify recombination breakpoints within these sequences. Recombination breakpoints are very significantly over-represented in gene sets within which protein domain-encoding regions have been annotated. Within these gene sets, breakpoints significantly avoid the domain-encoding regions (domons), except where these regions constitute most of the sequence length. Recombination breakpoints that fall within longer domons are distributed uniformly at random, but those that fall within shorter domons may show a slight tendency to avoid the domon midpoint. As we find no evidence for differential selection against nucleotide substitutions following the recombination event, any bias against disruption of domains must be a consequence of the recombination event per se.

Conclusions/Significance

This is the first systematic study relating the units of LGT to structural features at the protein level. Many genes have been interrupted by recombination following inter-lineage genetic transfer, during which the regions within these genes that encode protein domains have not been preferentially preserved intact. Protein domains are units of function, but domons are not modules of transfer and recombination. Our results demonstrate that LGT can remodel even the most functionally conservative modules within genomes.     

Size Variation of the Nonrecombining Region on the Mating-Type Chromosomes in the Fungal Podospora anserina Species Complex

Sex chromosomes often carry large nonrecombining regions that can extend progressively over time, generating evolutionary strata of sequence divergence. However, some sex chromosomes display an incomplete suppression of recombination. Large genomic regions without recombination and evolutionary strata have also been documented around fungal mating-type loci, but have been studied in only a few fungal systems. In the model fungus Podospora anserina (Ascomycota, Sordariomycetes), the reference S strain lacks recombination across a 0.8-Mb region around the mating-type locus. The lack of recombination in this region ensures that nuclei of opposite mating types are packaged into a single ascospore (pseudohomothallic lifecycle). We found evidence for a lack of recombination around the mating-type locus in the genomes of ten P. anserina strains and six closely related pseudohomothallic Podospora species. Importantly, the size of the nonrecombining region differed between strains and species, as indicated by the heterozygosity levels around the mating-type locus and experimental selfing. The nonrecombining region is probably labile and polymorphic, differing in size and precise location within and between species, resulting in occasional, but infrequent, recombination at a given base pair. This view is also supported by the low divergence between mating types, and the lack of strong linkage disequilibrium, chromosomal rearrangements, transspecific polymorphism and genomic degeneration. We found a pattern suggestive of evolutionary strata in P. pseudocomata. The observed heterozygosity levels indicate low but nonnull outcrossing rates in nature in these pseudohomothallic fungi. This study adds to our understanding of mating-type chromosome evolution and its relationship to mating systems.     

Evaluation of the efficiency and utility of recombinant enzyme-free seamless DNA cloning methods

Simple and low-cost recombinant enzyme-free seamless DNA cloning methods have recently become available. In vivo Escherichia coli cloning (iVEC) can directly transform a mixture of insert and vector DNA fragments into E. coli, which are ligated by endogenous homologous recombination activity in the cells. Seamless ligation cloning extract (SLiCE) cloning uses the endogenous recombination activity of E. coli cellular extracts in vitro to ligate insert and vector DNA fragments. An evaluation of the efficiency and utility of these methods is important in deciding the adoption of a seamless cloning method as a useful tool. In this study, both seamless cloning methods incorporated inserting DNA fragments into linearized DNA vectors through short (15–39 bp) end homology regions. However, colony formation was 30–60-fold higher with SLiCE cloning in end homology regions between 15 and 29 bp than with the iVEC method using DH5α competent cells. E. coli AQ3625 strains, which harbor a sbcA gene mutation that activates the RecE homologous recombination pathway, can be used to efficiently ligate insert and vector DNA fragments with short-end homology regions in vivo. Using AQ3625 competent cells in the iVEC method improved the rate of colony formation, but the efficiency and accuracy of SLiCE cloning were still higher. In addition, the efficiency of seamless cloning methods depends on the intrinsic competency of E. coli cells. The competency of chemically competent AQ3625 cells was lower than that of competent DH5α cells, in all cases of chemically competent cell preparations using the three different methods. Moreover, SLiCE cloning permits the use of both homemade and commercially available competent cells because it can use general E. coli recA^? strains such as DH5α as host cells for transformation. Therefore, between the two methods, SLiCE cloning provides both higher efficiency and better utility than the iVEC method for seamless DNA plasmid engineering.     

Molecular Insights Into the Evolutionary Pathway of Vibrio cholerae O1 Atypical El Tor Variants

Pandemic V. cholerae strains in the O1 serogroup have 2 biotypes: classical and El Tor. The classical biotype strains of the sixth pandemic, which encode the classical type cholera toxin (CT), have been replaced by El Tor biotype strains of the seventh pandemic. The prototype El Tor strains that produce biotype-specific cholera toxin are being replaced by atypical El Tor variants that harbor classical cholera toxin. Atypical El Tor strains are categorized into 2 groups, Wave 2 and Wave 3 strains, based on genomic variations and the CTX phage that they harbor. Whole-genome analysis of V. cholerae strains in the seventh cholera pandemic has demonstrated gradual changes in the genome of prototype and atypical El Tor strains, indicating that atypical strains arose from the prototype strains by replacing the CTX phages. We examined the molecular mechanisms that effected the emergence of El Tor strains with classical cholera toxin-carrying phage. We isolated an intermediary V. cholerae strain that carried two different CTX phages that encode El Tor and classical cholera toxin, respectively. We show here that the intermediary strain can be converted into various Wave 2 strains and can act as the source of the novel mosaic CTX phages. These results imply that the Wave 2 and Wave 3 strains may have been generated from such intermediary strains in nature. Prototype El Tor strains can become Wave 3 strains by excision of CTX-1 and re-equipping with the new CTX phages. Our data suggest that inter-chromosomal recombination between 2 types of CTX phages is possible when a host bacterial cell is infected by multiple CTX phages. Our study also provides molecular insights into population changes in V. cholerae in the absence of significant changes to the genome but by replacement of the CTX prophage that they harbor.