Structure and dynamics of the pan-genome of Streptococcus pneumoniae and closely related species

Background

Streptococcus pneumoniae is one of the most important causes of microbial diseases in humans. The genomes of 44 diverse strains of S. pneumoniae were analyzed and compared with strains of non-pathogenic streptococci of the Mitis group.

Results

Despite evidence of extensive recombination, the S. pneumoniae phylogenetic tree revealed six major lineages. With the exception of serotype 1, the tree correlated poorly with capsular serotype, geographical site of isolation and disease outcome. The distribution of dispensable genes - genes present in more than one strain but not in all strains - was consistent with phylogeny, although horizontal gene transfer events attenuated this correlation in the case of ancient lineages. Homologous recombination, involving short stretches of DNA, was the dominant evolutionary process of the core genome of S. pneumoniae. Genetic exchange occurred both within and across the borders of the species, and S. mitis was the main reservoir of genetic diversity of S. pneumoniae. The pan-genome size of S. pneumoniae increased logarithmically with the number of strains and linearly with the number of polymorphic sites of the sampled genomes, suggesting that acquired genes accumulate proportionately to the age of clones. Most genes associated with pathogenicity were shared by all S. pneumoniae strains, but were also present in S. mitis, S. oralis and S. infantis, indicating that these genes are not sufficient to determine virulence.

Conclusions

Genetic exchange with related species sharing the same ecological niche is the main mechanism of evolution of S. pneumoniae. The open pan-genome guarantees the species a quick and economical response to diverse environments.     

Whole genome profiling of spontaneous and chemically induced mutations in Toxoplasma gondii

Background

Next generation sequencing is helping to overcome limitations in organisms less accessible to classical or reverse genetic methods by facilitating whole genome mutational analysis studies. One traditionally intractable group, the Apicomplexa, contains several important pathogenic protozoan parasites, including the Plasmodium species that cause malaria.Here we apply whole genome analysis methods to the relatively accessible model apicomplexan, Toxoplasma gondii, to optimize forward genetic methods for chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) and ethylmethane sulfonate (EMS) at varying dosages.

Results

By comparing three different lab-strains we show that spontaneously generated mutations reflect genome composition, without nucleotide bias. However, the single nucleotide variations (SNVs) are not distributed randomly over the genome; most of these mutations reside either in non-coding sequence or are silent with respect to protein coding. This is in contrast to the random genomic distribution of mutations induced by chemical mutagenesis. Additionally, we report a genome wide transition vs transversion ratio (ti/tv) of 0.91 for spontaneous mutations in Toxoplasma, with a slightly higher rate of 1.20 and 1.06 for variants induced by ENU and EMS respectively. We also show that in the Toxoplasma system, surprisingly, both ENU and EMS have a proclivity for inducing mutations at A/T base pairs (78.6% and 69.6%, respectively).

Conclusions

The number of SNVs between related laboratory strains is relatively low and managed by purifying selection away from changes to amino acid sequence. From an experimental mutagenesis point of view, both ENU (24.7%) and EMS (29.1%) are more likely to generate variation within exons than would naturally accumulate over time in culture (19.1%), demonstrating the utility of these approaches for yielding proportionally greater changes to the amino acid sequence. These results will not only direct the methods of future chemical mutagenesis in Toxoplasma, but also aid in designing forward genetic approaches in less accessible pathogenic protozoa as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-354) contains supplementary material, which is available to authorized users.     

Lack of SOS repair in Streptococcus pneumoniae

Wild-type strains of Streptococcus pneumoniae were non-mutable by UV radiation and thymidine starvation. Moreover, UV-irradiated pneumococcal ω2 phages were not reactivated in an irradiated host. This suggests that, in pneumococcus, there is no efficient inducible repair process similar to the SOS repair described in detail for E. coli. We also report that mutations cannot be induced by a process thought to be linked to competence during transformation with isogenic wild-type DNA either on wild-type strains or in strains in which the hex function of excision and repair of mismatched bases in inactive.     

Intrastrain and interstrain genetic variation within a paralogous gene family in Chlamydia pneumoniae

Background

Chlamydia pneumoniae causes human respiratory diseases and has recently been associated with atherosclerosis. Analysis of the three recently published C. pneumoniae genomes has led to the identification of a new gene family (the Cpn 1054 family) that consists of 11 predicted genes and gene fragments. Each member encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane.

Results

Comparative analysis of this gene family within the published genome sequences provided evidence that multiple levels of genetic variation are evident within this single collection of paralogous genes. Frameshift mutations are found that result in both truncated gene products and pseudogenes that vary among isolates. Several genes in this family contain polycytosine (polyC) tracts either upstream or within the terminal 5' end of the predicted coding sequence. The length of the polyC stretch varies between paralogous genes and within single genes in the three genomes. Sequence analysis of genomic DNA from a collection of 12 C. pneumoniae clinical isolates was used to determine the extent of the variation in the Cpn 1054 gene family.

Conclusions

These studies demonstrate that sequence variability is present both among strains and within strains at several of the loci. In particular, changes in the length of the polyC tract associated with the different Cpn 1054 gene family members are common within each tested C. pneumoniae isolate. The variability identified within this newly described gene family may modulate either phase or antigenic variation and subsequent physiologic diversity within a C. pneumoniae population.     

Significant variation in transformation frequency in Streptococcus pneumoniae

The naturally transformable bacterium Streptococcus pneumoniae is able to take up extracellular DNA and incorporate it into its genome. Maintaining natural transformation within a species requires that the benefits of transformation outweigh its costs. Although much is known about the distribution of natural transformation among bacterial species, little is known about the degree to which transformation frequencies vary within species. Here we find that there is significant variation in transformation frequency between strains of Streptococcus pneumoniae isolated from asymptomatic carriage, and that this variation is not concordant with isolate genetic relatedness. Polymorphism in the signalling system regulating competence is also not causally related to differences in transformation frequency, although this polymorphism does influence the degree of genetic admixture experienced by bacterial strains. These data suggest that bacteria can evolve new transformation frequencies over short evolutionary timescales. This facility may permit cells to balance the potential costs and benefits of transformation by regulating transformation frequency in response to environmental conditions.     

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an ~50 kb ‘plasticity zone’ near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli 0157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C.pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C.pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.     

A Genome-Wide Analysis of Genetic Diversity in Trypanosoma cruzi Intergenic Regions

Background

Trypanosoma cruzi is the causal agent of Chagas Disease. Recently, the genomes of representative strains from two major evolutionary lineages were sequenced, allowing the construction of a detailed genetic diversity map for this important parasite. However this map is focused on coding regions of the genome, leaving a vast space of regulatory regions uncharacterized in terms of their evolutionary conservation and/or divergence.

Methodology

Using data from the hybrid CL Brener and Sylvio X10 genomes (from the TcVI and TcI Discrete Typing Units, respectively), we identified intergenic regions that share a common evolutionary ancestry, and are present in both CL Brener haplotypes (TcII-like and TcIII-like) and in the TcI genome; as well as intergenic regions that were conserved in only two of the three genomes/haplotypes analyzed. The genetic diversity in these regions was characterized in terms of the accumulation of indels and nucleotide changes.

Principal Findings

Based on this analysis we have identified i) a core of highly conserved intergenic regions, which remained essentially unchanged in independently evolving lineages; ii) intergenic regions that show high diversity in spite of still retaining their corresponding upstream and downstream coding sequences; iii) a number of defined sequence motifs that are shared by a number of unrelated intergenic regions. A fraction of indels explains the diversification of some intergenic regions by the expansion/contraction of microsatellite-like repeats.     

Quantifying bacterial evolution in the wild: A birthday problem for Campylobacter lineages

Measuring molecular evolution in bacteria typically requires estimation of the rate at which nucleotide changes accumulate in strains sampled at different times that share a common ancestor. This approach has been useful for dating ecological and evolutionary events that coincide with the emergence of important lineages, such as outbreak strains and obligate human pathogens. However, in multi-host (niche) transmission scenarios, where the pathogen is essentially an opportunistic environmental organism, sampling is often sporadic and rarely reflects the overall population, particularly when concentrated on clinical isolates. This means that approaches that assume recent common ancestry are not applicable. Here we present a new approach to estimate the molecular clock rate in Campylobacter that draws on the popular probability conundrum known as the ‘birthday problem’. Using large genomic datasets and comparative genomic approaches, we use isolate pairs that share recent common ancestry to estimate the rate of nucleotide change for the population. Identifying synonymous and non-synonymous nucleotide changes, both within and outside of recombined regions of the genome, we quantify clock-like diversification to estimate synonymous rates of nucleotide change for the common pathogenic bacteria Campylobacter coli (2.4 x 10⁻⁶ s/s/y) and Campylobacter jejuni (3.4 x 10⁻⁶ s/s/y). Finally, using estimated total rates of nucleotide change, we infer the number of effective lineages within the sample time frame–analogous to a shared birthday–and assess the rate of turnover of lineages in our sample set over short evolutionary timescales. This provides a generalizable approach to calibrating rates in populations of environmental bacteria and shows that multiple lineages are maintained, implying that large-scale clonal sweeps may take hundreds of years or more in these species.     

Complete Genome Sequence of Klebsiella pneumoniae Phage JD001

Klebsiella pneumoniae is a member of the family Enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. The emergence of multidrug-resistant strains of K. pneumoniae has became a public health problem globally. To develop an effective antimicrobial agent, we isolated a bacteriophage, named JD001, from seawater and sequenced its genome. Comparative genome analysis of phage JD001 with other K. pneumoniae bacteriophages revealed that phage JD001 has little similarity to previously published K. pneumoniae phages KP15, KP32, KP34, and phiKO2. Here we announce the complete genome sequence of JD001 and report major findings from the genomic analysis.     

EVOLUTION AND MITOCHONDRIAL DNA IN NEUROSPORA CRASSA

We studied mitochondrial DNA variability in 19 natural Neurospora crassa isolates and one wild-type isolate to examine evolution of these fungi and their mitochondrial DNA (mtDNA). We combined restriction endonuclease analysis of natural isolate mtDNA with DNA-DNA hybridization to cloned EcoR I fragments of a wild-type genome to discriminate between length mutations and site changes due to nucleotide substitution. Most variability was due to length mutations (insertions and deletions); genome size could vary 25% between pairs of isolates. Length-mutation distribution was not random, nor simply explained by the presence of coding versus noncoding regions. Restriction-site changes were few; the estimated amount of nucleotide substitution per nucleotide between the most divergent pair of isolates was 0.78%. Evolutionary relationships among isolates based on both types of mutations were compatible, and suggest that geographically distinct populations of mitochondrial DNA exist in the biological species, N. crassa. In contrast, no such correlation was shown by the previously determined distribution of nuclear heterokaryon incompatibility genes in the same isolates (Mylyk, 1975, 1976).     

Genomic Analysis Reveals the Molecular Basis for Capsule Loss in the Group B Streptococcus Population

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity.     

Whole genome characterization of the mechanisms of daptomycin resistance in clinical and laboratory derived isolates of Staphylococcus aureus

Background

Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.

Methods

Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.

Results

On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.

Conclusion

Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.     

Pherotypes are driving genetic differentiation within Streptococcus pneumoniae

Background  

The boundaries of bacterial species and the mechanisms underlying bacterial speciation are matters of intense debate. Theoretical studies have shown that recombination acts as a strong cohesive force preventing divergence in bacterial populations. Streptococcus pneumoniae populations have the telltale signs of high recombination with competence implicated as the major driving force behind gene exchange. Competence in S. pneumoniae is triggered by a quorum-sensing mechanism controlled by the competence-stimulating peptide pheromone.     

Optimal Strategy for Competence Differentiation in Bacteria

A phylogenetically diverse subset of bacterial species are naturally competent for transformation by DNA. Transformation entails recombination of genes between different lineages, representing a form of bacterial sex that increases standing genetic variation. We first assess whether homologous recombination by transformation is favored by evolution. Using stochastic population genetic computer simulations in which beneficial and deleterious mutations occur at many loci throughout the whole genome, we find that transformation can increase both the rate of adaptive evolution and the equilibrium level of fitness. Secondly, motivated by experimental observations of Bacillus subtilis, we assume that competence additionally entails a weak persister phenotype, i.e., the rates of birth and death are reduced for these cells. Consequently, persisters evolve more slowly than non-persisters. We show via simulation that strains which stochastically switch into and out of the competent phenotype are evolutionarily favored over strains that express only a single phenotype. Our model''s simplicity enables us to derive and numerically solve a system of finite- deterministic equations that describe the evolutionary dynamics. The observed tradeoff between the benefit of recombination and the cost of persistence may explain the previously mysterious observation that only a fractional subpopulation of B. subtilis cells express competence. More generally, this work demonstrates that population genetic forces can give rise to phenotypic diversity even in an unchanging and homogeneous environment.     

The mitochondrial genome of Ifremeria nautilei and the phylogenetic position of the enigmatic deep-sea Abyssochrysoidea (Mollusca: Gastropoda)

The complete nucleotide sequence of the mitochondrial (mt) genome of the deep-sea vent snail Ifremeria nautilei (Gastropoda: Abyssochrysoidea) was determined. The double stranded circular molecule is 15,664 pb in length and encodes for the typical 37 metazoan mitochondrial genes. The gene arrangement of the Ifremeria mt genome is most similar to genome organization of caenogastropods and differs only on the relative position of the trnW gene. The deduced amino acid sequences of the mt protein coding genes of Ifremeria mt genome were aligned with orthologous sequences from representatives of the main lineages of gastropods and phylogenetic relationships were inferred. The reconstructed phylogeny supports that Ifremeria belongs to Caenogastropoda and that it is closely related to hypsogastropod superfamilies. Results were compared with a reconstructed nuclear-based phylogeny. Moreover, a relaxed molecular-clock timetree calibrated with fossils dated the divergence of Abyssochrysoidea in the Late Jurassic–Early Cretaceous indicating a relatively modern colonization of deep-sea environments by these snails.