Recurrent adenylation domain replacement in the microcystin synthetase gene cluster

Background  

Microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. Microcystins are synthesized on large NRPS-PKS enzyme complexes. Many structural variants of microcystins are produced simulatenously. A recombination event between the first module of mcyB (mcyB1) and mcyC in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus Microcystis.     

Specific enrichment of nonribosomal peptide synthetase module by an affinity probe for adenylation domains

We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5′-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated l-Phe-AMS (l-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates l-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes.     

Diversity of and selection acting on cylindrospermopsin cyrB gene adenylation domain sequences in Florida

Aphanizomenon ovalisporum is the only confirmed cylindrospermopsin producer identified in the United States to date. On the other hand, Cylindrospermopsis raciborskii is a prominent feature of many lakes in Florida and other regions of the United States. To see the variation in cylindrospermopsin cyrB gene adenylation domain sequences and possibly discover new cylindrospermopsin producers, we collected water samples for a 3-year period from 17 different systems in Florida. Positive amplicons were cloned and sequenced, revealing that approximately 92% of sequences were A. ovalisporum-like (>99% identity). Interestingly, 6% of sequences were very similar (>99% identity) to cyrB sequences of C. raciborskii from Australia and of Aphanizomenon sp. from Germany. Neutrality tests suggest that A. ovalisporum-like cyrB adenylation domain sequences are under purifying selection, with abundant low-frequency polymorphisms within the population. On the other hand, when compared between species by codon-based methods, amino acids of CyrB also seem to be under purifying selection, in accordance with the one proposed amino acid thought to be activated by the CyrB adenylation domain.     

Prediction of the substrate for nonribosomal peptide synthetase (NRPS) adenylation domains by virtual screening

Nonribosomal peptide synthetases (NRPSs) synthesize a diverse array of bioactive small peptides, many of which are used in medicine. There is considerable interest in predicting NRPS substrate specificity in order to facilitate investigation of the many “cryptic” NRPS genes that have not been linked to any known product. However, the current sequence similarity‐based methods are unable to produce reliable predictions when there is a lack of prior specificity data, which is a particular problem for fungal NRPSs. We conducted virtual screening on the specificity‐determining domain of NRPSs, the adenylation domain, and found that virtual screening using experimentally determined structures results in good enrichment of the cognate substrate. Our results indicate that the conformation of the adenylation domain and in particular the conformation of a key conserved aromatic residue is important in determining the success of the virtual screening. When homology models of NRPS adenylation domains of known specificity, rather than experimentally determined structures, were built and used for virtual screening, good enrichment of the cognate substrate was also achieved in many cases. However, the accuracy of the models was key to the reliability of the predictions and there was a large variation in the results when different models of the same domain were used. This virtual screening approach is promising and is able to produce enrichment of the cognate substrates in many cases, but improvements in building and assessing homology models are required before the approach can be reliably applied to these models. Proteins 2015; 83:2052–2066. © 2015 Wiley Periodicals, Inc.     

Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains

DltA, the D-alanine:D-alanyl carrier protein ligase responsible for the initial step of lipoteichoic acid D-alanylation in Gram-positive bacteria, belongs to the adenylation domain superfamily, which also includes acetyl-CoA synthetase and the adenylation domains of non-ribosomal synthetases. The two-step reaction catalyzed by these enzymes (substrate adenylation followed by transfer to the reactive thiol group of CoA or the phosphopantheinyl prosthetic group of peptidyl carrier proteins) has been suggested to proceed via large scale rearrangements of structural domains within the enzyme. The structures of DltA reported here reveal the determinants for D-Ala substrate specificity and confirm that the peptidyl carrier protein-activating domains are able to adopt multiple conformational states, in this case corresponding to the thiolation reaction. Comparisons of available structures allow us to propose a mechanism whereby small perturbations of finely balanced metastable structural states would be able to direct an ordered formation of non-ribosomal synthetase products.     

Evidence for purifying selection acting on silent sites in BRCA1

In mammals, it is usually assumed that selection cannot be strong enough to act on nucleotide mutations that do not cause a change at the protein level (i.e. 'silent' or 'synonymous' mutations). Here we report the results of a molecular evolutionary analysis of BRCA1. We find a repeatable pronounced peak in the ratio of nonsynonymous to synonymous substitutions between codons 200-300. Unusually, this peak is caused by a plummet in the silent-site rate of evolution. The most parsimonious interpretation of these data is that purifying selection is acting on silent sites.     

Evidence for directional selection acting on pheromone-binding proteins in the genus Choristoneura

Patterns of nucleotide variation consistent with the action of natural selection have been discovered at a number of different gene loci. Here, pheromone-binding proteins (PBPs) are examined to determine if selection has acted to fix amino acid changes in PBPs in lineages in which pheromone changes have occurred. PBPs from five different species of moths in the genus Choristoneura were sequenced, along with the PBP of Argyrotaenia velutinana, which serves as an outgroup. Three independent major pheromone changes are represented within this group of five Choristoneura species. Two different lineages show evidence for selection based on polymorphism and divergence comparisons and comparisons of rates of replacement evolution to silent and noncoding evolution. Along one of these lineages, leading to Choristoneura fumiferana, there has been a change to an aldehyde pheromone from an acetate pheromone. The second branch does not appear to be associated with a major pheromone change. Other branches in the tree show a trend toward greater replacement fixation than expected under neutrality. This trend could reflect undetected selective events within this group of PBPs. Selection appears to have acted to fix amino acid changes in the PBP of moths from the genus Choristoneura, but it is not clear that this selection is due to pheromone changes between species.     

Evidence for positive selection on Mycobacterium tuberculosis within patients

Background

While the pathogenesis and epidemiology of tuberculosis are well studied, relatively little is known about the evolution of the infectious agent Mycobacterium tuberculosis, especially at the within-host level. The insertion sequence IS6110 is a genetic marker that is widely used to track the transmission of tuberculosis between individuals. This and other markers may also facilitate our understanding of the disease within patients.

Results

This article presents three lines of evidence supporting the action of positive selection on M. tuberculosis within patients. The arguments are based on a comparison between empirical findings from molecular epidemiology, and population genetic models of evolution. Under the hypothesis of neutrality of genotypes, 1) the mutation rate of the marker IS6110 is unusually high, 2) the time it takes for substitutions to occur within patients is too short, and 3) the amount of polymorphism within patients is too low.

Conclusions

Empirical observations are explained by the action of positive selection during infection, or alternatively by very low effective population sizes. I discuss the possible roles of antibiotic treatment, the host immune system and extrapulmonary dissemination in creating opportunities for positive selection.

     

Application of Bayesian network including Microcystis morphospecies for microcystin risk assessment in three cyanobacterial bloom-plagued lakes,China

Microcystis spp., which occur as colonies of different sizes under natural conditions, have expanded in temperate and tropical freshwater ecosystems and caused seriously environmental and ecological problems. In the current study, a Bayesian network (BN) framework was developed to access the probability of microcystins (MCs) risk in large shallow eutrophic lakes in China, namely, Taihu Lake, Chaohu Lake, and Dianchi Lake. By means of a knowledge-supported way, physicochemical factors, Microcystis morphospecies, and MCs were integrated into different network structures. The sensitive analysis illustrated that Microcystis aeruginosa biomass was overall the best predictor of MCs risk, and its high biomass relied on the combined condition that water temperature exceeded 24 °C and total phosphorus was above 0.2 mg/L. Simulated scenarios suggested that the probability of hazardous MCs (≥1.0 μg/L) was higher under interactive effect of temperature increase and nutrients (nitrogen and phosphorus) imbalance than that of warming alone. Likewise, data-driven model development using a naïve Bayes classifier and equal frequency discretization resulted in a substantial technical performance (CCI = 0.83, K = 0.60), but the performance significantly decreased when model excluded species-specific biomasses from input variables (CCI = 0.76, K = 0.40). The BN framework provided a useful screening tool to evaluate cyanotoxin in three studied lakes in China, and it can also be used in other lakes suffering from cyanobacterial blooms dominated by Microcystis.     

Evidence for positive selection on the floral scent gene isoeugenol-O-methyltransferase

Isoeugenol-O-methyltransferase (IEMT) is an enzyme involved in the production of the floral volatile compounds methyl eugenol and methyl isoeugenol in Clarkia breweri (Onagraceae). IEMT likely evolved by gene duplication from caffeic acid-O-methyltransferase followed by amino acid divergence, leading to the acquisition of its novel function. To investigate the selective context under which IEMT evolved, maximum likelihood methods that estimate variable d(N)/d(S) ratios among lineages, among sites, and among a combination of both lineages and sites were utilized. Statistically significant support was obtained for a hypothesis of positive selection driving the evolution of IEMT since its origin. Subsequent Bayesian analyses identified several sites in IEMT that have experienced positive selection. Most of these positions are in the active site of IEMT and have been shown by site-directed mutagenesis to have large effects on substrate specificity. Although the selective agent is unknown, the adaptive evolution of this gene may have resulted in increased effectiveness of pollinator attraction or herbivore repellence.     

Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in Microcystis strains

Toxic Microcystis strains often produce several isoforms of the cyclic hepatotoxin microcystin, and more than 65 isoforms are known. This has been attributed to relaxed substrate specificity of the adenylation domain. Our results show that in addition to this, variability is also caused by genetic variation in the microcystin synthetase genes. Genetic characterization of a region of the adenylation domain in module mcyB1 resulted in identification of two groups of genetic variants in closely related Microcystis strains. Sequence analyses suggested that the genetic variation is due to recombination events between mcyB1 and the corresponding domains in mcyC. Each variant could be correlated to a particular microcystin isoform profile, as identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Among the Microcystis species studied, we found 11 strains containing different variants of the mcyABC gene cluster and 7 strains lacking the genes. Furthermore, there is no concordance between the phylogenies generated with mcyB1, 16S ribosomal DNA, and DNA fingerprinting. Collectively, these results suggest that recombination between imperfect repeats, gene loss, and horizontal gene transfer can explain the distribution and variation within the mcyABC operon.     

Evidence for positive selection on the leptin gene in Cetacea and Pinnipedia

The leptin gene has received intensive attention and scientific investigation for its importance in energy homeostasis and reproductive regulation in mammals. Furthermore, study of the leptin gene is of crucial importance for public health, particularly for its role in obesity, as well as for other numerous physiological roles that it plays in mammals. In the present work, we report the identification of novel leptin genes in 4 species of Cetacea, and a comparison with 55 publicly available leptin sequences from mammalian genome assemblies and previous studies. Our study provides evidence for positive selection in the suborder Odontoceti (toothed whales) of the Cetacea and the family Phocidae (earless seals) of the Pinnipedia. We also detected positive selection in several leptin gene residues in these two lineages. To test whether leptin and its receptor evolved in a coordinated manner, we analyzed 24 leptin receptor gene (LPR) sequences from available mammalian genome assemblies and other published data. Unlike the case of leptin, our analyses did not find evidence of positive selection for LPR across the Cetacea and Pinnipedia lineages. In line with this, positively selected sites identified in the leptin genes of these two lineages were located outside of leptin receptor binding sites, which at least partially explains why co-evolution of leptin and its receptor was not observed in the present study. Our study provides interesting insights into current understanding of the evolution of mammalian leptin genes in response to selective pressures from life in an aquatic environment, and leads to a hypothesis that new tissue specificity or novel physiologic functions of leptin genes may have arisen in both odontocetes and phocids. Additional data from other species encompassing varying life histories and functional tests of the adaptive role of the amino acid changes identified in this study will help determine the factors that promote the adaptive evolution of the leptin genes in marine mammals.     

Evidence for positive selection on Drosophila melanogaster seminal fluid protease homologs

Proteins present in the seminal fluid of Drosophila melanogaster (accessory gland proteins Acps) contribute to female postmating behavioral changes, sperm storage, sperm competition, and immunity. Consequently, male-female coevolution and host-pathogen interactions are thought to underlie the rapid, adaptive evolution that characterizes several Acp-encoding genes. We propose that seminal fluid proteases are likely targets of selection due to their demonstrated or potential roles in between-sex interactions and immune processes. We use within- and between-species sequence data for 5 predicted protease-encoding Acp loci to test this hypothesis. Our polymorphism-based analyses find evidence for positive selection at 2 genes, both of which encode predicted serine protease homologs. One of these genes, CG6069, also shows evidence for consistent selection on a subset of codons over a deeper evolutionary time scale. The second gene, CG9997, was previously shown to be essential for normal sperm usage, suggesting that sexual selection may underlie its history of adaptation.     

Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

Background  

CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions.     

Evidence for positive selection in the TLR9 gene of teleosts

Toll-like receptors (TLRs) have been identified as key sensors of invading microbes by identifying pathogen-associated molecular patterns and activating innate immune responses. Whereas purifying selection has been suggested in mammalian TLR9, evolutionary features of TLR9 in teleosts have not been investigated in detail. We therefore analysed TLR9 DNA sequences of eight teleost species, including zebrafish (Danio rerio), Japanese flounder (Paralichthys olivaceus), pufferfish (Takifugu rubripes), and five seabreams. Eleven sites subjected to positive selection were identified using the codon-substitution models of PAML 3.15. Ten of these 11 sites were found to be associated with leucine-rich repeats (LRRs). Seven of these 10 positively selected sites were associated with the convex surface of the LRR solenoids, leading to variations of the structures of the LRRs possibly by the introduction of flexibility into the LRR solenoids. The positive selection of LRRs in TLR9 may indicate the adaptation of teleosts to different oligodeoxynucleotides present in different bacterial species.     

Nonribosomal peptide synthetase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC

Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.     

In vivo selection of lethal mutations reveals two functional domains in arginyl-tRNA synthetase

Using random mutagenesis and a genetic screening in yeast, we isolated 26 mutations that inactivate Saccharomyces cerevisiae arginyl-tRNA synthetase (ArgRS). The mutations were identified and the kinetic parameters of the corresponding proteins were tested after purification of the expression products in Escherichia coli. The effects were interpreted in the light of the crystal structure of ArgRS. Eighteen functional residues were found around the arginine-binding pocket and eight others in the carboxy-terminal domain of the enzyme. Mutations of these residues all act by strongly impairing the rates of tRNA charging and arginine activation. Thus, ArgRS and tRNA(Arg) can be considered as a kind of ribonucleoprotein, where the tRNA, before being charged, is acting as a cofactor that activates the enzyme. Furthermore, by using different tRNA(Arg) isoacceptors and heterologous tRNA(Asp), we highlighted the crucial role of several residues of the carboxy-terminal domain in tRNA recognition and discrimination.     

Evidence of positive selection on a class I ADH locus

The alcohol dehydrogenase (ADH) family of enzymes catalyzes the reversible oxidation of alcohol to acetaldehyde. Seven ADH genes exist in a segment of ~370 kb on 4q21. Products of the three class I ADH genes that share 95% sequence identity are believed to play the major role in the first step of ethanol metabolism. Because the common belief that selection has operated at the ADH1B*47His allele in East Asian populations lacks direct biological or statistical evidence, we used genomic data to test the hypothesis. Data consisted of 54 single-nucleotide polymorphisms (SNPs) across the ADH clusters in a global sampling of 42 populations. Both the F(st) statistic and the long-range haplotype (LRH) test provided positive evidence of selection in several East Asian populations. The ADH1B Arg47His functional polymorphism has the highest F(st) of the 54 SNPs in the ADH cluster, and it is significantly above the mean F(st) of 382 presumably neutral sites tested on the same 42 population samples. The LRH test that uses cores including that site and extending on both sides also gives significant evidence of positive selection in some East Asian populations for a specific haplotype carrying the ADH1B*47His allele. Interestingly, this haplotype is present at a high frequency in only some East Asian populations, whereas the specific allele also exists in other East Asian populations and in the Near East and Europe but does not show evidence of selection with use of the LRH test. Although the ADH1B*47His allele conveys a well-confirmed protection against alcoholism, that modern phenotypic manifestation does not easily translate into a positive selective force, and the nature of that selective force, in the past and/or currently, remains speculative.