Quantifying bacterial evolution in the wild: A birthday problem for Campylobacter lineages

Measuring molecular evolution in bacteria typically requires estimation of the rate at which nucleotide changes accumulate in strains sampled at different times that share a common ancestor. This approach has been useful for dating ecological and evolutionary events that coincide with the emergence of important lineages, such as outbreak strains and obligate human pathogens. However, in multi-host (niche) transmission scenarios, where the pathogen is essentially an opportunistic environmental organism, sampling is often sporadic and rarely reflects the overall population, particularly when concentrated on clinical isolates. This means that approaches that assume recent common ancestry are not applicable. Here we present a new approach to estimate the molecular clock rate in Campylobacter that draws on the popular probability conundrum known as the ‘birthday problem’. Using large genomic datasets and comparative genomic approaches, we use isolate pairs that share recent common ancestry to estimate the rate of nucleotide change for the population. Identifying synonymous and non-synonymous nucleotide changes, both within and outside of recombined regions of the genome, we quantify clock-like diversification to estimate synonymous rates of nucleotide change for the common pathogenic bacteria Campylobacter coli (2.4 x 10⁻⁶ s/s/y) and Campylobacter jejuni (3.4 x 10⁻⁶ s/s/y). Finally, using estimated total rates of nucleotide change, we infer the number of effective lineages within the sample time frame–analogous to a shared birthday–and assess the rate of turnover of lineages in our sample set over short evolutionary timescales. This provides a generalizable approach to calibrating rates in populations of environmental bacteria and shows that multiple lineages are maintained, implying that large-scale clonal sweeps may take hundreds of years or more in these species.     

IQGAP1 is necessary for pulmonary vascular barrier protection in murine acute lung injury and pneumonia

We recently reported that integrin α(v)β(3) is necessary for vascular barrier protection in mouse models of acute lung injury and peritonitis. Here, we used mass spectrometric sequencing of integrin complexes to isolate the novel β(3)-integrin binding partner IQGAP1. Like integrin β(3), IQGAP1 localized to the endothelial cell-cell junction after sphingosine-1-phosphate (S1P) treatment, and IQGAP1 knockdown prevented cortical actin formation and barrier enhancement in response to S1P. Furthermore, knockdown of IQGAP1 prevented localization of integrin α(v)β(3) to the cell-cell junction. Similar to β(3)-null animals, IQGAP1-null mice had increased pulmonary vascular leak compared with wild-type controls 3 days after intratracheal LPS. In an Escherichia coli pneumonia model, IQGAP1 knockout mice had increased lung weights, lung water, and lung extravascular plasma equivalents of (125)I-labeled albumin compared with wild-type controls. Taken together, these experiments indicate that IQGAP1 is necessary for S1P-mediated vascular barrier protection during acute lung injury and is required for junctional localization of the barrier-protective integrin α(v)β(3).     

Patterns of population genetic structure and diversity across bumble bee communities in the Pacific Northwest

Patterns of genetic structure and diversity are largely mediated by a species’ ecological niche and sensitivity to climate variation. Some species with narrow ecological niches have been found to exhibit increased population differentiation, limited gene flow across populations, and reduced population genetic diversity. In this study, we examine patterns of population genetic structure and diversity of four bumble bee species that are broadly sympatric, but do not necessarily inhabit the same ecological niche in the Pacific Northwest of the United States. Testing for the effect of isolation by geographic distance (IBD) with linearized F _st and D _est found that Bombus sylvicola and B. mixtus exhibited significant IBD across populations. In contrast, both B. melanopygus and B. flavifrons, two species that are distributed across a broad elevation gradient, exhibited no IBD, a result further corroborated by Bayesian a priori population assignment tests. Furthermore, we discovered that B. sylvicola populations distributed on the Olympic Peninsula have significantly less average allelic diversity than populations distributed in the Cascade Mountains. Our results suggest that populations distributed in the Olympic Mountains represent a distinct genetic cluster relative to the Cascade Mountains, with B. sylvicola and B. mixtus likely experiencing the greatest degree of population genetic differentiation relative to B. flavifrons and B. melanopygus. While bumble bees are known to co-exist across a diversity of habitats, our results demonstrate that underlying population genetic structure and diversity may not necessarily be similar across species, and are largely governed by their respective niches.     

Evaluation of temperature gradient gel electrophoresis for the analysis of prey DNA within the guts of invertebrate predators

The utility of temperature gradient gel electrophoresis (TGGE) as a means of analysing the gut contents of predators was evaluated. Generalist predators consume multiple prey species and a species-specific primer approach may not always be a practical means of analysing predator responses to prey diversity in complex and biodiverse ecosystems. General invertebrate primers were used to amplify the gut contents of predators, generating banding patterns that identified component prey remains. There was no evidence of dominance of the polymerase chain reaction (PCR) by predator DNA. When applied to field samples of the carabid predator Pterostichus melanarius (Illiger) nine banding patterns were detected, including one for aphids. To further distinguish between species, group-specific primers were designed to separate species of earthworm and aphid. TGGE of the earthworm PCR products generated banding patterns that varied with haplotype in some species. Aphid and earthworm DNA could be detected in the guts of carabids for up to 24 h using TGGE. In P. melanarius, with low numbers of prey per insect gut (mean<3), interpretation of banding patterns proved to be tractable. Potential problems of interpretation of TGGE gels caused by multiple prey bands, cryptic bands, haplotype variation, taxonomic uncertainties (especially with regard to earthworms), secondary predation, scavenging and presence of parasites and parasitoids in the prey or the predators, are discussed. The results suggest that PCR, using combinations of general invertebrate and group-specific primers followed by TGGE, provides a potentially useful approach to the analysis of multiple uncharacterized prey in predators.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

Dosage-mortality and time-mortality studies of a granulosis virus in a laboratory strain of the codling moth,Laspeyresia pomonella

Different doses of a granulosis virus were administered to first- and fifth-instar larvae of the codling moth Laspeyresia pomonella. Virus was very pathogenic for both larval instars. The LD₅₀ values for first- and fifth-instar larvae were 5 and 49 capsules/larva, respectively. However, fifth-instar larvae were much more variable in their response to virus than first-instar larvae. Using probit methods it was calculated that 1 capsule could cause death in about 25% of both larval instars but 1578 capsules were required to cause 70% mortality of fifth-instar larvae as compared to 12 capsules for first-instar larvae. This is the first report of a decided difference in variability of response to virus by two larval instars of the same species. A bimodal response by both larval instars was observed in time-mortality studies. Apparently, about 20% of the larvae were very resistant to virus infections.     

Potassium stimulated calcium dependent release of immunoreactive somatostatin from incubated rat hypothalamus

S omatostatin (somatotropin release inhibiting factor, SRIF) is present in the median eminence of the hypothalamus in high concentration (K ronheim et al., 1976), is visualized in nerve endings (H ökfelt et al., 1974) and has been found to be concentrated in the synaptosome fraction of hypothalamic homogenates (E pelbaum et al., 1977; B erelowitz et al., 1978), suggesting a true neurosecretory role. To further explore this possibility we have studied the release of immunoreactive SRIF from the incubated rat hypothalamus (B radbury et al., 1974: R otsztein et al., 1977), basally and in response to depolarising concentrations of potassium, and have assessed the calcium dependence of this release.     

The effect of pH and hypertonic KC1 on the collection of the cyclic AMP-protein complex from rat skeletal muscle and erythrocyte extracts.

The effect of pH on the extent of binding of cyclic AMP was evaluated by membrane filtration, charcoal exclusion and Sephadex G-25 chromatography. The amount of binding activity found in hemolysates of rat erythrocytes and 105,000 × g supernatants of rat thigh muscle homogenates varies appreciably with pH and method of measurement. Measurements of binding activity in a muscle extract by exclusion from Sephadex G-25 indicated the presence of two pH optima, one at pH 4.5 and the other at pH 7.4 or higher. The filtration method gave higher values than the charcoal method at pH 4.5 while the reverse was true at pH 7.4. With the erythrocyte preparation no binding was evident above pH 5.0 by either procedure except in the presence of 0.8 M KCl. Hypertonic KCl raised the pH of optimum binding to 5–5.5 for both tissues as indicated by both the filtration and charcoal methods. It is apparent from these results that the determination of cyclic AMP binding proteins from various tissues requires that more attention be paid to the role of ionic strength, pH and the mode of collection of the bound complex.     

Phylogenetic relationships of honey bees (Hymenoptera:Apinae:Apini) inferred from nuclear and mitochondrial DNA sequence data

Two different genomic regions (ND2 mitochondrial gene and EF1-alpha intron) were PCR amplified, cloned and sequenced for the ten known honey bee species collected within their natural range distribution. DNA sequences were analyzed using parsimony, distance and maximum likelihood methods to investigate phylogenetic relationships within Apis. The phylogenetic analyses strongly supported the basic topology recoverable from morphometric analysis, grouping the honey bees into three major clusters: giant bees (A. dorsata, A. binghami, and A. laboriosa), dwarf bees (A. andreniformis and A. florea), and cavity-nesting bees (A. mellifera, A. cerana, A. koschevnikovi, A. nuluensis, and A. nigrocincta). However, the clade of Asian cavity-nesting bees included paraphyletic taxa. Exemplars of Apis cerana collected from divergent portions of its range were less related to each other than were sympatric A. cerana, A. nuluensis, and A. nigrocincta taxa. Nucleotide sequence divergence between allopatrically distributed western (A. mellifera) and eastern (A. cerana, A. koschevnikovi, A. nigrocincta, and A. nuluensis) cavity-nesting species, around 18% for the mitochondrial gene and 10-15% for the nuclear intron, suggested an earlier divergence for these groups than previously estimated from morphometric and behavioral studies. This latter finding neccessitates reevaluation of the hypothesized origin of extant European, African, and west Asian Apis mellifera. Sequence divergence between A. laboriosa and A. dorsata was consistent with behavioral data and supports the species status of A. laboriosa.