The implications of satellite DNA instability on cellular function and evolution.

Abundant tandemly repeated satellite DNA is present in most eukaryotic genomes. Previous limitations including a pervasive view that it was uninteresting junk DNA, combined with challenges in studying it, are starting to dissolve - and recent studies have found important functions for satellite DNAs. The observed rapid evolution and implied instability of satellite DNA now has important significance for their functions and maintenance within the genome. In this review, we discuss the processes that lead to satellite DNA copy number instability, and the importance of mechanisms to manage the potential negative effects of instability. Satellite DNA is vulnerable to challenges during replication and repair, since it forms difficult-to-process secondary structures and its homology within tandem arrays can result in various types of recombination. Satellite DNA instability may be managed by DNA or chromatin-binding proteins ensuring proper nuclear localization and repair, or by proteins that process aberrant structures that satellite DNAs tend to form. We also discuss the pattern of satellite DNA mutations from recent mutation accumulation (MA) studies that have tracked changes in satellite DNA for up to 1000 generations with minimal selection. Finally, we highlight examples of satellite evolution from studies that have characterized satellites across millions of years of Drosophila fruit fly evolution, and discuss possible ways that selection might act on the satellite DNA composition.

Longitudinal Evolution of the Pseudomonas-Derived Cephalosporinase (PDC) Structure and Activity in a Cystic Fibrosis Patient Treated with ß-Lactams.

Traditional studies on the evolution of antibiotic resistance development use approaches that can range from laboratory-based experimental studies, to epidemiological surveillance, to sequencing of clinical isolates. However, evolutionary trajectories also depend on the environment in which selection takes place, compelling the need to more deeply investigate the impact of environmental complexities and their dynamics over time. Herein, we explored the within-patient adaptive long-term evolution of a Pseudomonas aeruginosa hypermutator lineage in the airways of a cystic fibrosis (CF) patient by performing a chronological tracking of mutations that occurred in different subpopulations; our results demonstrated parallel evolution events in the chromosomally encoded class C ß-lactamase (blaPDC). These multiple mutations within blaPDC shaped diverse coexisting alleles, whose frequency dynamics responded to the changing antibiotic selective pressures for more than 26 years of chronic infection. Importantly, the combination of the cumulative mutations in blaPDC provided structural and functional protein changes that resulted in a continuous enhancement of its catalytic efficiency and high level of cephalosporin resistance. This evolution was linked to the persistent treatment with ceftazidime, which we demonstrated selected for variants with robust catalytic activity against this expanded-spectrum cephalosporin. A "gain of function" of collateral resistance toward ceftolozane, a more recently introduced cephalosporin that was not prescribed to this patient, was also observed, and the biochemical basis of this cross-resistance phenomenon was elucidated. This work unveils the evolutionary trajectories paved by bacteria toward a multidrug-resistant phenotype, driven by decades of antibiotic treatment in the natural CF environmental setting. IMPORTANCE Antibiotics are becoming increasingly ineffective to treat bacterial infections. It has been consequently predicted that infectious diseases will become the biggest challenge to human health in the near future. Pseudomonas aeruginosa is considered a paradigm in antimicrobial resistance as it exploits intrinsic and acquired resistance mechanisms to resist virtually all antibiotics known. AmpC ß-lactamase is the main mechanism driving resistance in this notorious pathogen to ß-lactams, one of the most widely used classes of antibiotics for cystic fibrosis infections. Here, we focus on the ß-lactamase gene as a model resistance determinant and unveil the trajectory P. aeruginosa undertakes on the path toward a multidrug-resistant phenotype during the course of two and a half decades of chronic infection in the airways of a cystic fibrosis patient. Integrating genetic and biochemical studies in the natural environment where evolution occurs, we provide a unique perspective on this challenging landscape, addressing fundamental molecular mechanisms of resistance.

Jump-Chain Simulation of Markov Substitution Processes Over Phylogenies.

We draw attention to an under-appreciated simulation method for generating artificial data in a phylogenetic context. The approach, which we refer to as jump-chain simulation, can invoke rich models of molecular evolution having intractable likelihood functions. As an example, we simulate data under a context-dependent model allowing for CpG hypermutability and show how such a feature can mislead common codon models used for detecting positive selection. We discuss more generally how this method can serve to elucidate the ways by which currently used models for inference are susceptible to violations of their underlying assumptions. Finally, we show how the method could serve as an inference engine in the Approximate Bayesian Computation framework.

Localized pmrB hypermutation drives the evolution of colistin heteroresistance.

Colistin has emerged as an important last line of defense for the treatment of infections caused by antibiotic-resistant gram-negative pathogens, but colistin resistance remains poorly understood. Here, we investigate the responses of ≈1,000 populations of a multi-drug-resistant (MDR) strain of P. aeruginosa to a high dose of colistin. Colistin exposure causes rapid cell death, but some populations eventually recover due to the growth of sub-populations of heteroresistant cells. Heteroresistance is unstable, and resistance is rapidly lost under culture in colistin-free medium. The evolution of heteroresistance is primarily driven by selection for heteroresistance at two hotspot sites in the PmrAB regulatory system. Localized hypermutation of pmrB generates colistin resistance at 103-104 times the background resistance mutation rate (≈2 × 10-5 per cell division). PmrAB provides resistance to antimicrobial peptides that are involved in host immunity, suggesting that this pathogen may have evolved a highly mutable pmrB as an adaptation to host immunity.

Adaptive antimicrobial resistance, a description of microbial variants, and their relevance to periprosthetic joint infection.

Periprosthetic joint infection (PJI) is a difficult complication requiring a comprehensive eradication protocol. Cure rates have essentially stalled in the last two decades, using methods of antimicrobial cement joint spacers and parenteral antimicrobial agents. Functional spacers with higher-dose antimicrobial-loaded cement and antimicrobial-loaded calcium sulphate beads have emphasized local antimicrobial delivery on the premise that high-dose local antimicrobial delivery will enhance eradication. However, with increasing antimicrobial pressures, microbiota have responded with adaptive mechanisms beyond traditional antimicrobial resistance genes. In this review we describe adaptive resistance mechanisms that are relevant to the treatment of PJI. Some mechanisms are well known, but others are new. The objective of this review is to inform clinicians of the known adaptive resistance mechanisms of microbes relevant to PJI. We also discuss the implications of these adaptive mechanisms in the future treatment of PJI. Cite this article: Bone Joint J 2022;104-B(5):575-580.

A look towards the clonal origin of metastatic pulmonary carcinosarcoma: Report of a patient with an unexpected long-term survival.

INTRODUCTION: Pulmonary carcinosarcoma is a rare histological subtype of non-small cell lung cancer, defined by the combination of epithelial and mesenchimal elements. Prognosis is usually dismal, with a median survival of about 6 months. The use of immunotherapy by blockade of PD1/PD-L1 immune checkpoint signaling has been shown to improve patients' survival. However, local aggressiveness and distant metastases are frequent. Spread to the gastrointestinal tract is seldom reported. The genetic landscape of the disease has only recently begun to emerge, pointing at TP53, KRAS, EGFR and MET as the most common mutated genes. CASE DESCRIPTION: We describe the case of a metastatic patient with 37 months overall survival, treated by an aggressive multimodal approach combining surgery, chemotherapy, radiotherapy and immunotherapy. To shed new light on the molecular basis for sarcomatoid component in lung carcinoma, we performed next generation sequencing analysis of the squamous and sarcomatoid component by the two sites. We demonstrated a clonal origin and hypermutability of the sarcomatous elements that may account for the good response to immunotherapy. Moreover, we identified some mutations involving TP53 and EGFR genes, targetable by already available drugs. CONCLUSIONS: We depicted a model of how a squamous cell carcinoma can differentiate during its natural history into sub-clonal populations with different features and may ultimately result in a neoplasm (i.e. pulmonary carcinosarcoma) showing clonal heterogeneity. Our data might contribute to a better understanding of the pathogenesis and molecular mechanisms of this rare tumor and open new ways for a more tailored approach.

Investigation of an Alternative Marker for Hypermutability Evaluation in Different Tumors.

A growing number of studies have shown immunotherapy to be a promising treatment strategy for several types of cancer. Short tandem repeats (STRs) have been proven to be alternative markers for the evaluation of hypermutability in gastrointestinal (GI) cancers. However, the status of STRs and microsatellite instability (MSI) in other tumors have not yet been investigated. To further compare STR and MSI alterations in different tumors, a total of 407 paired DNAs were analyzed from the following eight tumor types: breast cancer (BC), hepatocellular cancer (HCC), pancreatic cancer (PC), colorectal cancer (CRC), gastric cancer (GC), lung cancer (LC), esophageal cancer (EC), and renal cell cancer (RCC). The STR alteration frequencies varied in different tumors as expected. Interestingly, none of the patients possessed MSI-low (MSI-L) or MSI-high (MSI-H), except for the GI patients. The highest STR alteration was detected in EC (77.78%), followed by CRC (69.77%), HCC (63.33%), GC (54.55%), LC (48.00%), RCC (40.91%), BC (36.11%), and PC (25.71%). The potential cutoff for hypermutability was predicted using the published objective response rate (ORR), and the cutoff of LC and HCC was the same as that of GI cancers (26.32%). The cutoffs of 31.58% and 10.53% should be selected for BC and RCC, respectively. In summary, we compared MSI and STR status in eight tumor types, and predicted the potential threshold for hypermutability of BC, HCC, CRC, GC, LC, EC, and RCC.

Resistência aos antimicrobianos e diversidade genética de variantes fenotípicas de Pseudomonas aeruginosa não sensíveis aos carbapenêmicos recuperadas de pacientes com fibrose cística com infecção pulmonar crônica: uma análise temporal / Antimicrobial resistance and genetic diversity of phenotypic variants of carbapenem non susceptible Pseudomonas aeruginosa recovered from patients with cystic fibrosis with chronic lung infection: a temporal analysis

Pseudomonas aeruginosa, bactéria ubíqua e versátil, pode se comportar como um patógeno oportunista, com ampla capacidade adaptativa, por múltiplos fatores de virulência e resistência. Como agente patogênico nas infecções pulmonares em pacientes com fibrose cística (FC), é motivo de prognóstico ruim, aumento de hospitalizações e altas taxas de morbimortalidade, sendo quase impossível a sua erradicação, ao evoluírem para a cronicidade. Globalmente, é notável o aumento nos índices de amostras não sensíveis aos carbapenêmicos e a múltiplos antimicrobianos, essenciais à terapêutica. Assim, avaliamos temporalmente a susceptibilidade aos antimicrobianos e a presença de amostras hipermutáveis (HPM) em P. aeruginosa de diferentes morfotipos, não sensíveis aos carbapenêmicos (PANSC), obtidas de pacientes FC com infecção pulmonar crônica, acompanhados em dois centros de referência no Rio de Janeiro. De 2007 a 2016, a análise retrospectiva, através dos resultados obtidos no teste de disco-difusão (TDD), permitiu selecionar amostras de PANSC incluídas neste trabalho. Usando os resultados obtidos no TDD, foi definida a susceptibilidade a outros antimicrobianos, bem como os fenótipos de resistência, multi-(MDR), extensivo-(XDR) e pandroga resistentes (PDR). Adicionalmente, determinou-se a concentração inibitória mínima (CIM) para imipenem (IPM), meropenem (MEM), doripenem (DOR) e polimixina (POL). Através de teste fenotípico, foi calculada a frequência de mutação espontânea e as amostras hipermutáveis foram caracterizadas. O sequenciamento de genoma total (SGT) foi realizado em seis amostras de diferentes morfotipos, incluindo uma variante fenotípica rara, a small colony variant (SCV). Essas amostras foram recuperadas em dois episódios de exacerbação do paciente. Foram investigadas a clonalidade, resistência a antimicrobianos e virulência. Das 143 amostras, de 18 pacientes (9 pediátricos e 9 adultos), os resultados do TDD apontaram taxas de não susceptibilidade superiores a 44% para gentamicina, amicacina, tobramicina e ciprofloxacina, e maiores de 30 % para POL. Pela determinação da CIM, quase a totalidade (96%) das amostras foram não sensíveis a IMP, seguidos de 56% para MEM e 44% para DOR. Analisando-se a distribuição dos valores da CIM50 e CIM90 nos dois grupos de pacientes, os valores para IMP foram maiores entre as amostras dos pacientes pediátricos, equivalendo a 32 µg/mL e 64 µg/mL, respectivamente. Cerca de 25%, 37% e 6% eram MDR, XDR e PDR, respectivamente. Aproximadamente 12% eram HPM, e mais da metade destas foram XDR. Após o SGT, as seis amostras, recuperadas do caso clínico foram classificadas em um novo sequence type (ST2744), com a presença de genes de resistência adquiridos blaPAO, blaOXA-50, aph(3')-Iib, fosA, catB7 e crpP, apresentando mutações em genes codificadores de porinas e bombas de efluxo. Entretanto, não foram observados marcadores genéticos clássicos exclusivos para os fenótipos SCV e HPM. Este é o primeiro relato de P. aeruginosa SCV na FC, no Brasil. A vigilância epidemiológica de P. aeruginosa é crucial para a conduta terapêutica, bem como para o sucesso da resposta do paciente e erradicação da infecção pulmonar, justificando o uso de técnicas fenotípicas e moleculares na detecção dos mecanismos de resistência e virulência desse microrganismo na FC.

Pseudomonas aeruginosa, a ubiquitous and versatile bacterium, can behave as an opportunistic pathogen, with strong adaptive capacity, due to multiple virulence and resistance factors. As a pulmonary infection pathogen in patients with cystic fibrosis (CF), it is related with poor prognosis, increased hospitalizations and high rates of morbidity and mortality, and the eradication is almost impossible, especially after chronicity. The increase rates of isolates non-susceptible to carbapenem and multiple antimicrobials, essentials to therapy, have been observed worldwide. Therefore, we assessed the antimicrobial susceptibility and the presence of hypermutability (HPM) in non-susceptible to carbapenem P. aeruginosa (PANSC) isolates from different morphotypes, obtained from CF patients with chronic pulmonary infection, followed at two reference centers in Rio de Janeiro. Using the results obtained by disk-diffusion test (DDT) between 2007 to 2016, we select 143 PANSC and susceptibility to other antimicrobials was defined, as well as the resistance phenotypes, multi- (MDR), extensive- (XDR) and pandrug resistant (PDR). Additionally, the minimum inhibitory concentration (MIC) for imipenem (IPM), meropenem (MEM), doripenem (DOR) and polymyxin (POL) was determined. Hypermutable isolates were characterized by determination of mutation frequency. Whole genome sequencing (WGS) was performed in six morphotypes isolates, including the small colony variant (SCV), a rare variant phenotype. These isolates were recovered in two exacerbation episodes. Clonality, antimicrobial resistance and virulence were investigated. Of the total (143 isolates) isolated from 18 patients (9 pediatric and 9 adults), non-susceptibility rates above than 44% for gentamicin, amikacin, tobramycin and ciprofloxacin, and more than 30% for POL were observed. Almost all (96%) of the isolates were non-susceptible to IPM by MIC determination, followed by 56% for MEM and 44% for DOR. MIC50 (32 µg/mL) and MIC90 (64 µg/mL) rates for IPM were higher among pediatric patient isolates and 25%, 37% and 6% were MDR, XDR and PDR, respectively. 12% of all isolates were classified as HPM and more than half were categorized as XDR. Using WGS, the six isolates recovered from the clinical case, were identified as a new sequence type (ST2744). Acquired resistance genes blaPAO, blaOXA-50, aph (3')-Iib, fosA, catB7 and crpP and mutations in encoding genes for porins and efflux pumps, was annotated. None exclusive classic genetic markers related to SCV and HPM phenotypes were not observed. This is the first Brazilian report of P. aeruginosa SCV in CF. Our results highlight the importance of epidemiological surveillance in P. aeruginosa. The application of phenotypic and molecular techniques to investigate resistance and virulence mechanisms, can contribute to therapeutic success in CF.

Hypermutator Pseudomonas aeruginosa Exploits Multiple Genetic Pathways To Develop Multidrug Resistance during Long-Term Infections in the Airways of Cystic Fibrosis Patients.

Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI, encoding the ß-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying ß-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence.

Detecting genetic hypermutability of gastrointestinal tumor by using a forensic STR kit.

Growing evidence suggests that somatic hypermutational status and programmed cell death-1 overexpression are potential predictive biomarkers indicating treatment benefits from immunotherapy using immune checkpoint inhibitors. However, biomarker-matched trials are still limited, and many of the genomic alterations remain difficult to target. To isolate the potential somatic hypermutational tumor from microsatellite instability low/microsatellite stability (MSI-L/MSS) cases, we employed two commercial kits to determine MSI and forensic short tandem repeat (STR) alternations in 250 gastrointestinal (GI) tumors. Three types of forensic STR alternations, namely, allelic loss, Aadd, and Anew, were identified. 62.4% (156/250) of the patients with GI exhibited STR alternation, including 100% (15/15) and 60% (141/235) of the microsatellite high instability and MSI-L/MSS cases, respectively. 30% (75/250) of the patients exhibited STR instability with more than 26.32% (26.32%-84.21%) STR alternation. The cutoff with 26.32% of the STR alternations covered all 15 MSI cases and suggested that it might be a potential threshold. Given the similar mechanism of the mutations of MSI and forensic STR, the widely used forensic identifier STR kit might provide potential usage for identifying hypermutational status in GI cancers.

Mutator genomes decay, despite sustained fitness gains, in a long-term experiment with bacteria.

Understanding the extreme variation among bacterial genomes remains an unsolved challenge in evolutionary biology, despite long-standing debate about the relative importance of natural selection, mutation, and random drift. A potentially important confounding factor is the variation in mutation rates between lineages and over evolutionary history, which has been documented in several species. Mutation accumulation experiments have shown that hypermutability can erode genomes over short timescales. These results, however, were obtained under conditions of extremely weak selection, casting doubt on their general relevance. Here, we circumvent this limitation by analyzing genomes from mutator populations that arose during a long-term experiment with Escherichia coli, in which populations have been adaptively evolving for >50,000 generations. We develop an analytical framework to quantify the relative contributions of mutation and selection in shaping genomic characteristics, and we validate it using genomes evolved under regimes of high mutation rates with weak selection (mutation accumulation experiments) and low mutation rates with strong selection (natural isolates). Our results show that, despite sustained adaptive evolution in the long-term experiment, the signature of selection is much weaker than that of mutational biases in mutator genomes. This finding suggests that relatively brief periods of hypermutability can play an outsized role in shaping extant bacterial genomes. Overall, these results highlight the importance of genomic draft, in which strong linkage limits the ability of selection to purge deleterious mutations. These insights are also relevant to other biological systems evolving under strong linkage and high mutation rates, including viruses and cancer cells.

Is Mutation Random or Targeted?: No Evidence for Hypermutability in Snail Toxin Genes.

Ever since Luria and Delbruck, the notion that mutation is random with respect to fitness has been foundational to modern biology. However, various studies have claimed striking exceptions to this rule. One influential case involves toxin-encoding genes in snails of the genus Conus, termed conotoxins, a large gene family that undergoes rapid diversification of their protein-coding sequences by positive selection. Previous reconstructions of the sequence evolution of conotoxin genes claimed striking patterns: (1) elevated synonymous change, interpreted as being due to targeted "hypermutation" in this region; (2) elevated transversion-to-transition ratios, interpreted as reflective of the particular mechanism of hypermutation; and (3) much lower rates of synonymous change in the codons encoding several highly conserved cysteine residues, interpreted as strong position-specific codon bias. This work has spawned a variety of studies on the potential mechanisms of hypermutation and on causes for cysteine codon bias, and has inspired hypermutation hypotheses for various other fast-evolving genes. Here, I show that all three findings are likely to be artifacts of statistical reconstruction. First, by simulating nonsynonymous change I show that high rates of dN can lead to overestimation of dS. Second, I show that there is no evidence for any of these three patterns in comparisons of closely related conotoxin sequences, suggesting that the reported findings are due to breakdown of statistical methods at high levels of sequence divergence. The current findings suggest that mutation and codon bias in conotoxin genes may not be atypical, and that random mutation and selection can explain the evolution of even these exceptional loci.

Intrapopulation variability in mutator prevalence among urinary tract infection isolates of Escherichia coli.

Bacteria with elevated mutation rates represent a risk factor for treatment failure and are often found with high frequency in clinical isolates from different sources. How this frequency reflects the among-population and within-population proportion of hypermutators is unknown, despite its importance to the choice of antibiotic therapies that minimize the likelihood of resistance development. Here we screened for hypermutators among the urine of 80 patients with urinary tract infections, at an unprecedented resolution of 24 isolates per sample. We found hypermutators in four patients (5%), at frequencies ranging from 4.2% to 62.5%. Molecular characterization revealed alterations in the oxidized guanine (GO) and methly-directed mistmatch repair (MMR) systems as the genetic basis of hypermutability. These observations suggest that mutators may be present in more patients than previously anticipated, at frequencies that are difficult to detect but still sufficient to impact on adaptation to antibiotics or the host environment.

Extreme dNTP pool changes and hypermutability in dcd ndk strains.

Cells lacking deoxycytidine deaminase (DCD) have been shown to have imbalances in the normal dNTP pools that lead to multiple phenotypes, including increased mutagenesis, increased sensitivity to oxidizing agents, and to a number of antibiotics. In particular, there is an increased dCTP pool, often accompanied by a decreased dTTP pool. In the work presented here, we show that double mutants of Escherichia coli lacking both DCD and NDK (nucleoside diphosphate kinase) have even more extreme imbalances of dNTPs than mutants lacking only one or the other of these enzymes. In particular, the dCTP pool rises to very high levels, exceeding even the cellular ATP level by several-fold. This increased level of dCTP, coupled with more modest changes in other dNTPs, results in exceptionally high mutation levels. The high mutation levels are attenuated by the addition of thymidine. The results corroborate the critical importance of controlling DNA precursor levels for promoting genome stability. We also show that the addition of certain exogenous nucleosides can influence replication errors in DCD-proficient strains that are deficient in mismatch repair.

Sustained fitness gains and variability in fitness trajectories in the long-term evolution experiment with Escherichia coli.

Many populations live in environments subject to frequent biotic and abiotic changes. Nonetheless, it is interesting to ask whether an evolving population's mean fitness can increase indefinitely, and potentially without any limit, even in a constant environment. A recent study showed that fitness trajectories of Escherichia coli populations over 50 000 generations were better described by a power-law model than by a hyperbolic model. According to the power-law model, the rate of fitness gain declines over time but fitness has no upper limit, whereas the hyperbolic model implies a hard limit. Here, we examine whether the previously estimated power-law model predicts the fitness trajectory for an additional 10 000 generations. To that end, we conducted more than 1100 new competitive fitness assays. Consistent with the previous study, the power-law model fits the new data better than the hyperbolic model. We also analysed the variability in fitness among populations, finding subtle, but significant, heterogeneity in mean fitness. Some, but not all, of this variation reflects differences in mutation rate that evolved over time. Taken together, our results imply that both adaptation and divergence can continue indefinitely--or at least for a long time--even in a constant environment.

Mini-review: Strategies for Variation and Evolution of Bacterial Antigens.

Across the eubacteria, antigenic variation has emerged as a strategy to evade host immunity. However, phenotypic variation in some of these antigens also allows the bacteria to exploit variable host niches as well. The specific mechanisms are not shared-derived characters although there is considerable convergent evolution and numerous commonalities reflecting considerations of natural selection and biochemical restraints. Unlike in viruses, mechanisms of antigenic variation in most bacteria involve larger DNA movement such as gene conversion or DNA rearrangement, although some antigens vary due to point mutations or modified transcriptional regulation. The convergent evolution that promotes antigenic variation integrates various evolutionary forces: these include mutations underlying variant production; drift which could remove alleles especially early in infection or during life history phases in arthropod vectors (when the bacterial population size goes through a bottleneck); selection not only for any particular variant but also for the mechanism for the production of variants (i.e., selection for mutability); and overcoming negative selection against variant production. This review highlights the complexities of drivers of antigenic variation, in particular extending evaluation beyond the commonly cited theory of immune evasion. A deeper understanding of the diversity of purpose and mechanisms of antigenic variation in bacteria will contribute to greater insight into bacterial pathogenesis, ecology and coevolution with hosts.

Characterization of Staphylococcus aureus small colony variant strains isolated from Italian patients attending a regional cystic fibrosis care centre.

Small colony variant (SCV) Staphylococcus aureus are a subpopulation of auxotroph, slow-growing strains causing persisting and relapsing infections in cystic fibrosis (CF) patients. Twenty-eight SCV and 29 normal S. aureus strains were isolated from 42 out of 222 Italian CF patients. The isolates were characterized for: susceptibility to antibiotics, methicillin-resistance (MR), Panton Valentine leukocidin, auxotrophy, hypermutability and biofilm formation. Clonal identity of SCV and normal strains was determined by pulsed-field gel electrophoresis. We found that 27 out of 28 SCVs were thymidine-dependent. Furthermore, in contrast to normal phenotype, SCVs were characterized by antibiotic resistance. We also found that 39.3% SCV vs 20.7% normal strains were strong mutators. Moreover, SCVs showed a higher capability to form biofilm compared to normal strains (100% vs 59%). Importantly, we found evidence of clonal spread of SCV strain among CF patients. Using molecular typing, we found that five patients shared the same type A and five out of seven MR-SCV belonged to the same clone (Clone C). The particular virulence and spreading ability of MR-SCV observed highlights the importance of accurate identification and susceptibility testing of these strains. It is important to adopt the optimal approach to treat patients and to prevent cross-infection in CF centres.

Evolutionary consequences of DNA methylation on the GC content in vertebrate genomes.

The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution, mainly three mechanisms have been proposed: selection for GC content, mutation bias, and GC-biased gene conversion. At present, the mechanism of GC-biased gene conversion, i.e., short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis, we investigated one mammalian (human) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm and made use of a publicly available whole-genome methylation map of human sperm. Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one that neglects and one that incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ by approximately 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated.

The apparent enhancement of CpG transversions in primate lineage is a consequence of multiple replacements.

We claim that the apparently enhanced CpG transversions in the form CpG to CpC/GpG or to ApG/CpT are caused by the hypermutable CpG to CpA/TpG transition. The nucleotide replacement counts obtained from the human/chimpanzee/gorilla/orangutan sequence alignments representing the replacements due to the evolutionary species divergence and the results of 1000 genomes project that provide us with the differences due to the intraspecies diversification were analyzed to estimate the ratio of CpG versus non-CpG transversion probabilities. The trinucleotide replacement counts were extracted from the regions that are free of functional constraints. The CpG transversion probabilities based upon the genomic comparisons were found to exceed more than twice the non-CpG transversions. The diversity data emerging from 14 population groups were partitioned in five classes as a function of the parameter quantifying the spread of the polymorphic allele among the group of individuals. The results based upon the human polymorphism exhibit a trend where CpG over non-CpG transversion probability ratio is less and less exceeding unity as the values of the derived allele frequency (DAF) of snps are diminishing. A computer simulation of a simplified model indicates that the phenomenon of the apparent enhancement of CpG transversions can have its source in the interference of the entropic effects with the maximum likelihood methodologies.