Bacterial DNA Uptake Sequences Can Accumulate by Molecular Drive Alone

Uptake signal sequences are DNA motifs that promote DNA uptake by competent bacteria in the family Pasteurellaceae and the genus Neisseria. The genomes of these bacteria contain many copies of their canonical uptake sequence (often >100-fold overrepresentation), so the bias of the uptake machinery causes cells to prefer DNA derived from close relatives over DNA from other sources. However, the molecular and evolutionary forces responsible for the abundance of uptake sequences in these genomes are not well understood, and their presence is not easily explained by any of the current models of the evolution of competence. Here we describe use of a computer simulation model to thoroughly evaluate the simplest explanation for uptake sequences, that they accumulate in genomes by a form of molecular drive generated by biased DNA uptake and evolutionarily neutral (i.e., unselected) recombination. In parallel we used an unbiased search algorithm to characterize genomic uptake sequences and DNA uptake assays to refine the Haemophilus influenzae uptake specificity. These analyses showed that biased uptake and neutral recombination are sufficient to drive uptake sequences to high densities, with the spacings, stabilities, and strong consensuses typical of uptake sequences in real genomes. This result greatly simplifies testing of hypotheses about the benefits of DNA uptake, because it explains how genomes could have passively accumulated sequences matching the bias of their uptake machineries.MANY bacteria are able to take up DNA fragments from their environment, a genetically specified trait called natural competence (Chen and Dubnau 2004; Johnsborg et al. 2007; Maughan et al. 2008). Many other species have homologs of competence genes, suggesting that although they are not competent under laboratory conditions, they may be competent under natural conditions (Claverys and Martin 2003; Kovacs et al. 2009). Such a widespread trait must be beneficial but the evolutionary function of DNA uptake remains controversial. Cells can use the nucleotides released by degradation of both incoming DNA and any strands displaced by its recombination, thus reducing demands on their nucleotide salvage and biosynthesis pathways (Redfield 1993; Palchevskiy and Finkel 2009). Cells may also benefit if recombination of the incoming DNA provides templates for DNA repair or introduces beneficial mutations, but may suffer if recombination introduces damage or harmful mutations (Redfield 1988; Michod et al. 2008).Although most bacteria that have been tested show no obvious preferences for specific DNA sources or sequences, bacteria in the family Pasteurellaceae and the genus Neisseria strongly prefer DNA fragments from close relatives. Two factors are responsible: First, the DNA uptake machineries of these bacteria are strongly biased toward certain short DNA sequence motifs. Second, the genomes of these bacteria contain hundreds of occurrences of the preferred sequences. The Pasteurellacean motif is called the uptake signal sequence (USS); its Neisseria counterpart is called the DNA uptake sequence (DUS). All Neisseria genomes contain the same consensus DUS [core GCCGTCTGAA (Treangen et al. 2008)], but divergence in the Pasteurellaceae has produced two subclades, one of species sharing the canonical Haemophilus influenzae 9-bp USS (Hin-USS core AAGTGCGGT) and the other of species with a variant USS that differs at three core positions (Apl-USS core: ACAAGCGGT) and has a longer flanking consensus (Redfield et al. 2006). Uptake sequence biases are strong but not absolute; for example, replacing the Hin-USS with the Apl-USS reduces H. influenzae DNA uptake only 10-fold (Redfield et al. 2006) and DNA from Escherichia coli is taken up in the absence of competing H. influenzae DNA (Goodgal and Mitchell 1984).Most studies of the distribution and consensus of uptake sequences in genomes have examined only those occurrences that perfectly match the above core DUS and USS sequences. Here we call these perfect matches “core-consensus” (cc) uptake sequences. These cc-uptake sequences occupy ∼1% of their genomes; they are equally frequent in the plus and minus orientations of the genome sequence but are underrepresented in coding sequences, with the noncoding 14% and 20% of their respective genomes containing 35% of cc-USSs and 65% of cc-DUSs (Smith et al. 1995). Although many of these intergenic cc-DUSs and cc-USSs occur in inverted-repeat pairs that function as terminators (Kingsford et al. 2007), most uptake sequences are too far apart or in genes or other locations where termination does not occur. Within coding regions uptake sequences occur more often in weakly conserved genes, in weakly conserved parts of genes, and in reading frames that encode common tripeptides (Findlay and Redfield 2009), all of which are consistent with selection acting mainly to eliminate mutations that improve uptake from genome regions constrained by coding or other functions.Analyses that focus on cc-uptake sequences effectively treat uptake sequences as replicative elements (Smith et al. 1995; Redfield et al. 2006; Ambur et al. 2007; Treangen et al. 2008). However, USS and DUS are known to originate in situ by normal mutational processes, mainly point mutations, and to spread between genomes mainly by homologous recombination (Redfield et al. 2006; Treangen et al. 2008). As they are not replicating elements, why are they up to 1000-fold more common in their genomes than expected for unselected sequences (e.g., H. influenzae, 1471 cc-USS vs. 8 expected by chance; N. gonorrheae, 1892 cc-DUS vs. 2 expected by chance)?The explanation for this abundance must lie with the specificity of the DNA uptake system, because the strong correspondence between the uptake bias and the uptake sequences in the genome is far too improbable to be a coincidence. However, uptake specificity is not easily accommodated by either of the hypothesized functions of DNA uptake. If bacteria take up DNA to get benefits from homologous genetic recombination, the combination of uptake bias and uptake sequences might serve as a mate-choice adaptation that maximizes these benefits by excluding foreign DNAs (Treangen et al. 2008). Although this explanation is appealing, it requires simultaneous evolution of bias in the uptake machinery and of genomic sequences matching this bias. Another problem is that the genomic sequences can be “selected” only after the cell carrying them is dead. On the other hand, if bacteria instead take up DNA as a source of nutrients, all DNAs should be equally useful, so uptake bias and uptake sequences would likely reduce rather than increase this benefit. Although the sequence bias could be explained as a consequence of mechanistic constraints on DNA uptake, this would not account for the high density of the preferred sequences in the genome.However, both hypotheses may be simplified by a process called molecular drive, under which uptake sequences would gradually accumulate over evolutionary time as a direct consequence of biased uptake and recombination (Danner et al. 1980; Bakkali et al. 2004; Bakkali 2007), without any need for natural selection. This drive is proposed to work as follows: First, random mutation continuously creates variation in DNA sequences that affects their probability of uptake, and random cell death allows DNA fragments containing preferred variants to be taken up by other cells. Second, repeated recombination of such preferred DNA sequences with their chromosomal homologs gradually increases their abundance in the genomes of competent cells'' descendants. Thus mutations that create matches to the bias of the uptake machinery are horizontally transmitted to other members of the same species more often than other mutations. Because some recombination may be inevitable even if DNA''s main benefit is nutritional, molecular drive could account for uptake sequence accumulation under both hypotheses, leaving only the biased uptake process to be explained by natural selection for either genetic variation or nutrients.Although drive is plausible, its ability to account for the observed properties of genomic uptake sequences has never been evaluated. To do this, we developed a realistic computer simulation model that includes only the processes thought to generate molecular drive. Below we first use this model to identify the conditions that determine whether uptake sequences will accumulate and then compare the properties of these simulated uptake sequences to those of the uptake sequences in the N. meningitidis and H. influenzae genomes. In parallel we use unbiased motif searches to better characterize genomic uptake sequences and DNA uptake assays to refine the H. influenzae uptake specificity.     

Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.     

A ‘minimum stress inflexion’ in relation to environmental and biotic influences on the dynamics of subtidal encrusting communities?

The sedentary fauna of sublittoral boulders was studied at five sites with very different environmental conditions within Lough Hyne Marine Nature Reserve (51° 30 N, 9° 18 W). The degree of competition was assessed through the construction of competitive hierarchies built up from the results of nearly 3000 interactions between organisms. Communities at all sites were predominantly hierarchically organised with few stand-offs recorded. Density of interactions was correlated with total space occupation. Flow speed at all sites was logged over 24 h and this factor also correlated with space occupation. Degree of disturbance was assessed through the use of marked boulders at each site. Species diversity peaked where all factors were moderate and was lowest at extreme sites. The effects of these environmental variables and biotic factors in determining community composition are discussed, and a working model detailing a Minimum Stress Inflexion is described. This represents an extension of the intermediate disturbance hypothesis in that it is proposed that disturbance in this system may serve to reduce sediment load as opposed to preventing space monopolisation in the classical model.     

Microhabitat preferences of benthic fauna in a woodland stream

Estimates of numbers, biomass, and diversity of benthic macroinvertebrates were made quarterly over a two-year period to investigate microhabitat preferences. Although biomass of most taxa was significantly different among sampling times, physical factors also appeared to be important in determining abundance of many taxa. Optimum depth, velocity, substrate type, and turbulence were determined for major taxa. Optimum conditions for diversity appeared to be 34 cm depth, 60 cm s^?1 velocity, and rubble and boulder substrate type. Habitat preference functions were derived for several taxa based on significant polynomial regressions of biomass on depth, velocity, substrate, and Froude number (turbulence). The relationship between abundance and physical habitat conditions was tested by using the product of the preference factors (range: 0–1) for depth, velocity and substrate type as a measure of habitat suitability (joint preference factor). There were significant correlations between biomass [transformed by log_e (x + 1)] of 10 benthic species and the joint preference factor. The joint preference factors accounted for from 11 to 61% of the variation of biomass of the 10 benthic species. The intercepts of the relationships between biomass of individual species and the joint preference factor were not significantly different from zero for any species. Therefore, the joint preference factors appear to be valid indicators of biomass. The preference functions have utility in habitat assessment studies, specifically with regard to minimum instream flow determinations.     

Regulatory light chain phosphorylation and N-terminal extension increase cross-bridge binding and power output in Drosophila at in vivo myofilament lattice spacing

The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2⁺), truncated N-terminal extension (Dmlc2^Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2^S66A,S67A), and dual mutant (Dmlc2^{Δ2-46; S66A,S67A}). The N-terminal extension truncation and phosphorylation sites disruption mutations decreased oscillatory power output and the frequency of maximum power output in maximally Ca²⁺-activated fibers compressed to near in vivo inter-thick filament spacing, with the phosphorylation sites disruption mutation having a larger affect. The diminished power output parameters with the N-terminal extension truncation and phosphorylation sites disruption mutations were due to the reduction of the number of strongly-bound cross-bridges and rate of myosin force production, with the larger parameter reductions in the phosphorylation sites disruption mutation additionally related to reduced myosin attachment time. The phosphorylation and N-terminal extension-dependent boost in cross-bridge kinetics corroborates previous structural data, which indicate these RLC attributes play a complementary role in moving and orienting myosin heads toward actin target sites, thereby increasing fiber and whole fly power generation.     

Protein osmotic pressure and the state of water in frog myoplasm

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.     

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake.     

Methyl Jasmonate Increases the Tropane Alkaloid Scopolamine and Reduces Natural Herbivory in Brugmansia suaveolens: Is Scopolamine Responsible for Plant Resistance?

The tropane alkaloid (TA) scopolamine is suggested to protect Brugmansia suaveolens (Solanaceae) against herbivorous insects. To test this prediction in a natural environment, scopolamine was induced by methyl jasmonate (MJ) in potted plants which were left 10?days in the field. MJ-treated plants increased their scopolamine concentration in leaves and herbivory decreased. These findings suggest a cause?Ceffect relationship. However, experiments in laboratory showed that scopolamine affect differently the performance of the specialist larvae of the ithomiine butterfly Placidina euryanassa (C. Felder & R. Felder) and the generalist fall armyworm Spodoptera frugiperda (J. E. Smith): the specialist that sequester this TA from B. suaveolens leaves was not negatively affected, but the generalist was. Therefore, scopolamine probably acts only against insects that are not adapted to TAs. Other compounds that are MJ elicited may also play a role in plant resistance against herbivory by generalist and specialist insects, and deserve future investigations.