Enhanced plasmid stability through post-segregational killing of plasmid-free cells

Summary In order to develop an extremely stable, inducible host/vector system, the following genetic manipulations were made: a recA mutation was introduced into the chromosome of the host strain, the plasmid selectable marker was changed from ampicillin to kanamycin, and the parB stability locus of plasmid R1 was added to the plasmid. The stability of the new vector, pTKW106, was increased such that the fraction of plasmid-bearing cells present during chemostat fermentations under selective pressure increased from 1.75% to 100% when plasmid protein production was fully induced. At this level of induction, -galactosidase represents 10% of the total cell protein. In addition, the frequency of generation of plasmid-free cells was shown to decrease from 1.0 per generation to less than 10^–11 with full promoter induction under non-selective conditions.     

Field evidence for apparent competition mediated via the shared parasites of two gamebird species

Although apparent competition mediated via shared parasites is potentially an important force influencing community structure, there is limited evidence to demonstrate its occurrence in the field. Here we show that the intensity of infection by the caecal nematode Heterakis gallinarum picked up by naive grey partridges, both maintained in pens and released on six gamebird estates in the UK, is significantly correlated with the intensity of infection recorded in the previous year from pheasants on those estates. Furthermore, the worm burdens picked up appeared to be sufficient to negatively influence host condition. These results provide evidence that infection from pheasants determines the worm burdens of partridges in the field, supporting the hypothesis that parasite-mediated apparent competition with the pheasant may be a factor influencing the decline and subsequent recovery of wild grey partridges.     

The effect of the location of the proteic post-segregational stability system within the replicon of plasmid pTF-FC2 on the fine regulation of plasmid replication

The broad host-range IncQ-2 family plasmid, pTF-FC2, is a mobilizable, medium copy number plasmid that lacks an active partitioning system. Plasmid stability is enhanced by a toxin–antitoxin (TA) system known as pas (plasmid addiction system) that is located within the replicon between the repB (primase) and the repA (helicase) and repC (DNA-binding) genes. The discovery of a closely related IncQ-2 plasmid, pRAS3, with a completely different TA system located between the repB and repAC genes raised the question of whether the location of pas within the replicon had an effect on the plasmid in addition to its ability to act as a TA system. In this work we demonstrate that the presence of the strongly expressed, autoregulated pas operon within the replicon resulted in an increase in the expression of the downstream repAC genes when autoregulation was relieved. While deletion of the pas module did not affect the average plasmid copy number, a pas-containing plasmid exhibited increased stability compared with a pas deletion plasmid even when the TA system was neutralized. It is proposed that the location of a strongly expressed, autoregulated operon within the replicon results in a rapid, but transient, expression of the repAC genes that enables the plasmid to rapidly restore its normal copy number should it fall below a threshold.     

The form of sexual selection arising from male-male competition depends on the presence of females in the social environment

Sexual selection arises from social interactions, and if social environments vary so too should sexual selection. For example, male-male competition often occurs either in the presence or in the absence of females, and such changes in the social environment could affect the form and strength of sexual selection. Here we examine how the presence of a female influences selection arising from male-male competition in a leaf-footed cactus bug, Narnia femorata, which has a resource defence mating system. Males compete for territories on cacti because females lay eggs on the cactus plants. Females are not always present when this competition first occurs; however, the presence or absence of the female matters. We found that both the form and strength of selection on male traits, those traits that influenced success in intrasexual competition, depended on the social context. When a female was not present, male size and the area of the sexually dimorphic hind legs was only marginally important to winning a contest. However, males with larger overall size and leg area were more likely to win in the presence of a female. There was also positive quadratic selection on these traits when a female was present with both the largest and the smallest males winning. The implication is unexpected alternative strategies when females are present. Our results support the notion that sexual selection should be studied under all relevant social contexts.     

Experimental evidence for accelerated adaptation to desiccation through sexual selection on males

The impact of sexual selection on the adaptive process remains unclear. On the one hand, sexual selection might hinder adaptation by favouring costly traits and preferences that reduce nonsexual fitness. On the other hand, condition dependence of success in sexual selection may accelerate adaptation. Here, we used replicate populations of Drosophila melanogaster to artificially select on male desiccation resistance while manipulating the opportunity for precopulatory sexual selection in a factorial design. Following five generations of artificial selection, we measured the desiccation resistance of males and females to test whether the addition of sexual selection accelerated adaptation. We found a significant interaction between the effects of natural selection and sexual selection: desiccation resistance was highest in populations where sexual selection was allowed to operate. Despite only selecting on males, we also found a correlated response in females. These results provide empirical support for the idea that sexual selection can accelerate the rate of adaptation.     

Regions on plasmid pCU1 required for the killing of Klebsiella pneumoniae.

Plasmid pCU1 was Kik+ (promotes killing of Klebsiella pneumoniae). All Tn5 insertions within the tra region of pCU1 were Kik-. Two other regions, kikA and kikB, were needed. They may be separated on different plasmids, but both must be mobilized into Klebsiella pneumoniae. Establishment of one kik region in K. pneumoniae followed by receipt of the second did not lead to killing. Kik was therefore intracellular and required concerted and transient action of both regions.     

Dynamics of multiple infection and within-host competition by the anther-smut pathogen

Infection of one host by multiple pathogen genotypes represents an important area of pathogen ecology and evolution that lacks a broad empirical foundation. Multiple infection of Silene latifolia by Microbotryum violaceum was studied under field and greenhouse conditions using the natural polymorphism for mating-type bias as a marker. Field transmission resulted in frequent multiple infection, and each stem of the host was infected independently. Within-host diversity of infections equaled that of nearby inoculum sources by the end of the growing season. The number of diseased stems per plant was positively correlated with multiple infection and with overwintering mortality. As a result, multiply infected plants were largely purged from the population, and there was lower within-host pathogen diversity in the second season. However, among plants with a given number of diseased stems, multiply infected plants had a lower risk of overwintering mortality. Following simultaneous and sequential inoculation, strong competitive exclusion was demonstrated, and the first infection had a significant advantage. Dynamics of multiple infection initially included components of coinfection models for virulence evolution and then components of superinfection models after systemic colonization. Furthermore, there was evidence for an advantage of genotypes with mating-type bias, which may contribute to maintenance of this polymorphism in natural populations.     

Conjugal transfer from Streptococcus lactis ME2 of plasmids encoding phage resistance, nisin resistance and lactose-fermenting ability: evidence for a high-frequency conjugative plasmid responsible for abortive infection of virulent bacteriophage

Streptococcus lactis ME2 exhibits at least three mechanisms which confer resistance to virulent bacteriophage. These include plasmid-induced interference with phage adsorption, host-controlled restriction and modification activities, and a heat-sensitive mechanism which suppresses development of virulent phage. Conjugal mating experiments were done with S. lactis ME2 to determine if phage-defence mechanisms present in this strain could be mobilized, associated with plasmid DNA elements and phenotypically characterized in transconjugants. Agar-surface matings of S. lactis ME2 with S. lactis LM0230 demonstrated that lactose-fermenting ability (Lac+) was transferred in a conjugation-like process at frequencies of 10(-6) per donor cell and was associated with a 40 MDal plasmid designated pTR1040. Resistance to nisin (Nisr) was acquired or lost simultaneously with Lac+, indicating that pTR1040 carried determinants for both phenotypes. Lac+ Nisr transconjugants that carried a 30 MDal plasmid (pTR2030) exhibited a heat-sensitive phage-defence mechanism (Hsp+) which limited the burst size and plaque size of phage c2 without altering the efficiency of plaquing (e.o.p.) or the level of adsorption. The ability of phage c2 to initiate plaquing at an e.o.p. of 1.0 indicated that DNA injection and early viral gene expression are not affected in the Hsp+ transconjugants. We suggest, therefore, that the Hsp+ phenotype may result from plasmid-induced abortive infection of phage dependent on the presence of pTR2030. Hsp+ transconjugants carrying pTR2030 also promoted high-frequency conjugal transfer of Lac+ Nisr associated with pTR1040 (greater than 10(-1) per donor cell). It was concluded that Hsp+ and determinants for conjugal transfer ability (Tra+) are located on pTR2030.     

Babesia bovis: Proteins of virulent and avirulent parasites passaged through ticks and splenectomized or intact calves

Passage of the avirulent vaccine (K) strain of Babesia bovis (K_A) through either Boophilus microplus ticks, intact calves, or intact calves and then ticks, resulted in two distinct protein and protein antigen profiles as analyzed by two-dimensional gel electrophoresis of biosynthetically labeled proteins and immunoprecipitates. Different degrees of expression of two major acidic antigens of K_A designated Ka₁ (M_r 47,500) and Ka₂ (M_r 43,000) were observed. Ka₁ was apparently lost following passage of K_AB. bovis through intact calves but was strongly represented in the parasite population following a single tick passage. In contrast, passage through ticks of the virulent K_VB. bovis (from which K_A was derived by passage in splenectomized calves) did not lead to strong representation of the Ka₁ protein although there was increased representation of another major acidic protein antigen, designated Kv (M_r 35,000). These data suggest that the previously recognized reversion to a strain-dependent basal antigenic type in the tick vector depends also on intrastrain characteristics such as virulence and strain heterogeneity. The data suggest that K_A is a more heterogeneous population than K_V although cloned isolates are required to establish this point. Comparable syringe passage of another strain of B. bovis, designated C strain, through splenectomized calves resulted in less marked differences between the putative C_A and C_VB. bovis. This may explain the less stable avirulence of C_A compared to K_AB. bovis. Various selection pressures must act, in either the tick or the vertebrate host, on subpopulations in heterogeneous isolates to produce the changes described in protein antigen profiles of B. bovis. The possible relevance of changes in representation of proteins to biological characteristics of B. bovis (such as virulence and tick transmissibility) is discussed.     

Establishing a blueprint for CED-3-dependent killing through identification of multiple substrates for this protease

Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that approximately 3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts.     

Resource competition and community response to fertilization: the outcome depends on spatial strategies

Decreases in plant species richness and shifts in community structure following fertilization are usually attributed to increasing light limitation. However, there is increasing evidence that light limitation alone does not account for all of the observed effects of fertilization on plant communities. We present a model of competition for a single, spatially heterogeneous resource that shows fertility-mediated changes in community structure without light competition. This model predicts that in a low-productivity spatially heterogeneous habitat, species that interact with the resource environment over small spatial scales may exclude species that experience the environment at larger spatial scales, even when the latter species are better resource competitors in a uniform environment (have a lower R*). Increasing overall habitat fertility under these conditions minimizes the effects of spatial heterogeneity on the species that forage at a larger spatial scale, resulting in changes in species dominance and the potential for species coexistence. This analysis suggests that considering differences in the spatial scales at which species interact with environmental heterogeneity may help explain observed changes in community structure following fertilization.     

Optimal killing for obligate killers: the evolution of life histories and virulence of semelparous parasites.

Many viral, bacterial and protozoan parasites of invertebrates first propagate inside their host without releasing any transmission stages and then kill their host to release all transmission stages at once. Life history and the evolution of virulence of these obligately killing parasites are modelled, assuming that within-host growth is density dependent. We find that the parasite should kill the host when its per capita growth rate falls to the level of the host mortality rate. The parasite should kill its host later when the carrying capacity, K, is higher, but should kill it earlier when the parasite-independent host mortality increases or when the parasite has a higher birth rate. When K(t), for parasite growth, is not constant over the duration of an infection, but increases with time, the parasite should kill the host around the stage when the growth rate of the carrying capacity decelerates strongly. In case that K(t) relates to host body size, this deceleration in growth is around host maturation.     

Response to joint selection on germination and flowering phenology depends on the direction of selection

Flowering and germination time are components of phenology, a complex phenotype that incorporates a number of traits. In natural populations, selection is likely to occur on multiple components of phenology at once. However, we have little knowledge of how joint selection on several phenological traits influences evolutionary response. We conducted one generation of artificial selection for all combinations of early and late germination and flowering on replicated lines within two independent base populations in the herb Campanula americana. We then measured response to selection and realized heritability for each trait. Response to selection and heritability were greater for flowering time than germination time, indicating greater evolutionary potential of this trait. Selection for earlier phenology, both flowering and germination, did not depend on the direction of selection on the other trait, whereas response to selection to delay germination and flowering was greater when selection on the other trait was in the opposite direction (e.g., early germination and late flowering), indicating a negative genetic correlation between the traits. Therefore, the extent to which correlations shaped response to selection depended on the direction of selection. Furthermore, the genetic correlation between timing of germination and flowering varies across the trait distributions. The negative correlation between germination and flowering time found when selecting for delayed phenology follows theoretical predictions of constraint for traits that jointly determine life history schedule. In contrast, the lack of constraint found when selecting for an accelerated phenology suggests a reduction of the covariance due to strong selection favoring earlier flowering and a shorter life cycle. This genetic architecture, in turn, will facilitate further evolution of the early phenology often favored in warm climates.     

Melittin-induced bilayer leakage depends on lipid material properties: evidence for toroidal pores

The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the "barrel stave" model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the "toroidal pore" melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid headgroups. Here we test these models by measuring both the free energy of melittin transfer (DeltaG degrees ) and melittin-induced leakage as a function of bilayer elastic (material) properties that determine the energetics of bilayer bending, including the area compressibility modulus (K(a)), bilayer bending modulus (k(c)), and monolayer spontaneous curvature (R(o)). The addition of cholesterol to phosphatidylcholine (PC) bilayers, which increases K(a) and k(c), decreases both DeltaG degrees and the melittin-induced vesicle leakage. In contrast, the addition to PC bilayers of molecules with either positive R(o), such as lysoPC, or negative R(o), such as dioleoylglycerol, has little effect on DeltaG degrees , but produces large changes in melittin-induced leakage, from 86% for 8:2 PC/lysoPC to 18% for 8:2 PC/dioleoylglycerol. We observe linear relationships between melittin-induced leakage and both K(a) and 1/R(o)(2). However, in contrast to what would be expected for a barrel stave model, there is no correlation between observed leakage and bilayer hydrocarbon thickness. All of these results demonstrate the importance of bilayer material properties on melittin-induced leakage and indicate that the melittin-induced pores are defects in the bilayer lined in part by lipid molecules.     

Age-specific reproductive success: evidence for the selection hypothesis

Age-specific reproductive success has been demonstrated in many species. Three hypotheses have been raised to explain this general phenomenon: the experience hypothesis based on age-specific reproductive experience, the effort hypothesis based on age-specific reproductive effort, and the selection hypothesis based on progressive disappearance of phenotypes due to variation in individual productivity and survival. We used data from a long-term study of Leach's storm-petrels (Oceanodroma leucorhoa) to present a single test of mutually exclusive predictions about the relationship between early breeding success and longevity. There should be no correlation between early breeding success and longevity under the experience hypothesis, a negative correlation under the effort hypothesis, and a positive correlation under the selection hypothesis. We found a significant (P < 0.0001) positive relationship between success in the first two breeding attempts and longevity in this population of long-lived seabirds, strongly suggesting that low-productivity parents were also less likely to survive early breeding. These data provide some of the strongest support to date for the selection hypothesis.     

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (K_d) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.