Tritium labeling of thermolysin, elastase, and ribonuclease by exposure to tritium gas at low pressure

The bacterial metalloendoprotease thermolysin, bovine pancreatic ribonuclease, and porcine pancreatic elastase have been tritiated by exposure to subcurie amounts of tritium gas at pressures below 50 mTorr for periods of 1 to 6 h. Thermolysin, ribonuclease, and elastase have been purified to specific radioactivities of 15, 5, and 1 Ci/mol, respectively. Amino acid analyses of the tritiated enzymes revealed higher relative specific radioactivities for His, Pro, and Phe in all three proteins while Val and Ile were among the residues with the lowest relative specific radioactivities. The recovery of enzyme activity was always greater than 95% and the formation of tritiated decomposition products was not observed. This lowpressure gas exposure process requires less tritium gas and less time than the original method of Wilzbach to achieve equal or higher levels of tritium incorporation. In addition, the enzymes were completely active and did not show the presence of highly radioactive byproducts which have been observed in earlier studies of the Wilzbach labeling of proteins.     

Nematode–bacteria mutualism: Selection within the mutualism supersedes selection outside of the mutualism

The coevolution of interacting species can lead to codependent mutualists. Little is known about the effect of selection on partners within verses apart from the association. Here, we determined the effect of selection on bacteria (Xenorhabdus nematophila) both within and apart from its mutualistic partner (a nematode, Steinernema carpocapsae). In nature, the two species cooperatively infect and kill arthropods. We passaged the bacteria either together with (M+), or isolated from (M?), nematodes under two different selection regimes: random selection (S?) and selection for increased virulence against arthropod hosts (S+). We found that the isolated bacteria evolved greater virulence under selection for greater virulence (M?S+) than under random selection (M?S?). In addition, the response to selection in the isolated bacteria (M?S+) caused a breakdown of the mutualism following reintroduction to the nematode. Finally, selection for greater virulence did not alter the evolutionary trajectories of bacteria passaged within the mutualism (M+S+ = M+S?), indicating that selection for the maintenance of the mutualism was stronger than selection for increased virulence. The results show that selection on isolated mutualists can rapidly breakdown beneficial interactions between species, but that selection within a mutualism can supersede external selection, potentially generating codependence over time.     

The identification of residues that control signal peptidase cleavage fidelity and substrate specificity

Signal peptidase, which removes signal peptides from preproteins, has a substrate specificity for small uncharged residues at -1 (P1) and small or larger aliphatic residues at the -3 (P3) position. Structures of the catalytic domain with a 5S-penem inhibitor and a lipopeptide inhibitor reveal candidate residues that make up the S1 and S3 pockets that bind the P1 and P3 specificity residues of the preprotein substrate. We have used site-directed mutagenesis, mass spectrometric analysis, and in vivo and in vitro activity assays as well as molecular modeling to examine the importance of the substrate pocket residues. Generally, we find that the S1 and S3 binding sites can tolerate changes that are expected to increase or decrease the size of the pocket without large effects on activity. One residue that contributes to the high fidelity of cleavage of signal peptidase is the Ile-144 residue. Changes of the Ile-144 residue to cysteine result in cleavage at multiple sites, as determined by mass spectrometry and Edman sequencing analysis. In addition, we find that signal peptidase is able to cleave after phenylalanine at the -1 residue in a double mutant in which both Ile-86 and Ile-144 were changed to an alanine. Also, alteration of the Ile-144 and Ile-86 residues to the corresponding residues found in the homologous Imp1 protease changes the specificity to promote cleavage following a -1 Asn residue. This work shows that Ile-144 and Ile-86 contribute to the signal peptidase substrate specificity and that Ile-144 is important for the accuracy of the cleavage reaction.     

Good vs complementary genes for parasite resistance and the evolution of mate choice

Background  

Female mate choice may be adaptive when males exhibit heritable genetic variation at loci encoding resistance to infectious disease. The Hamilton-Zuk hypothesis predicts that females should assess the genetic quality of males by monitoring traits that indicate health and vigor (condition-dependent choice, or CD). Alternatively, some females may employ a more direct method of screening and select mates based on the dissimilarity of alleles at the major histocompatibility loci (we refer to this as opposites-attract, or OA). Empirical studies suggest that both forms of mate choice exist, but little is known about the potential for natural selection to shape the two strategies in nature.     

Pleistocene glaciation is implicated in the phylogeographical structure of Potamopyrgus antipodarum, a New Zealand snail

Pleistocene glaciation has been identified as an important factor shaping present-day patterns of phylogeographical structure in a diverse array of taxa. The purpose of this study was to use mitochondrial sequence data to address whether Pleistocene glaciation is also a major determinant of phylogeographical patterns in Potamopyrgus antipodarum, a freshwater snail native to New Zealand. We found that haplotypes were separated by no more than 3.7% sequence divergence, and major genetic divisions tended to occur on a north-south axis. These data fit the predictions of the hypothesis that isolation of P. antipodarum in glacial refugia at the northern and southern tip of the South Island of New Zealand during the Pleistocene glaciation underlies the present-day phylogeographical structure. Because sexual P. antipodarum occasionally produce asexual offspring, we also used these data to show that the appearance of asexuality is not phylogeographically constrained. This means that the maintenance of sex in P. antipodarum cannot be wholly due to limited contact between sexual and asexual lineages and must instead be linked to a selective advantage of sexual reproduction.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.     

Spatial variation in infection by digenetic trematodes in a population of freshwater snails (Potamopyrgus antipodarum)

Larval digenetic trematodes commonly castrate their first intermediate hosts, and should therefore impose strong selection on the timing and mode of host reproduction. Here we examine spatial variation in infection by trematodes in the freshwater snail Potamopyrgus antipodarum. Snails were collected at 11 different sites at Lake Alexandrina on the South Island of New Zealand from transects that ran perpendicular to the shore and across several different habitat types (from 0 to 8 m deep). Logistic regression was used to analyze the relationships between the frequency of trematode infection and snail size, habitat type, and transect location. On average, the probability of infection increased 3.3 times with each 1 mm increase in shell length. Prevalence of infection by the most common species of trematode, Microphallus sp., was highest in the shallow-water habitats where its final hosts (waterflow) spend most of their time. Prevalence of infection by another parasite, Telogaster ophistorchis (final host: eels) increased with depth, but because Microphallus was much more common, total infection by all trematodes decreased with depth. The effects of transect location were minor for Telogaster, but there was significant variation in Microphallus prevalence among transects, especially in the shore-bank habitat. Taken together, these results suggest that the risk of infection is spatially variable, but generally higher in shallow-water habitats, which may explain the greater frequency of sexual individuals as well as earlier reproduction among individuals near shore.     

Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail

The small aquatic snail Potamopyrgus antipodarum is an important invading species in Europe, Australia and North America. European populations are generally believed to derive from accidental introductions from New Zealand, probably dating back to the mid-19th century. We have employed mitochondrial DNA sequences to test the proposed New Zealand origin of European Potamopyrgus, and to learn more about its genealogical history. Using a 481-bp region of the 16S ribosomal RNA gene, we identified 17 distinct haplotypes among 65 snails from New Zealand. In marked contrast, only two haplotypes were found across all European samples, which cover a large geographical area. Importantly, these two haplotypes are shared with snails from the North Island of New Zealand. Due to sampling limitations we cannot rule out a South Island origin for one of the haplotypes, but our results clearly demonstrate the New Zealand origin of European populations. The marked divergence among the two European haplotypes implies the successful colonization by two distinct mitochondrial lineages, which is consistent with previous data based on nuclear markers.