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.     

Structural and spectropotentiometric analysis of Blastochloris viridis heterodimer mutant reaction center

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 × 100 × 100 μm, belonged to space group P4₃2₁2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 Å resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C₃₈₀, revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.     

Further considerations on the statistical mechanics of biological associations

In this continuation of a previous report it is shown how the Volterra population dynamics, which underlies the statistical theory, can be based on a variational principle; how the dynamics can be generalized as regards both the behavior of total populations and migration phenomena; and how many directly observable data, such as amplitudes and frequencies of oscillation of a population, fit into the statistical theory and can test it. Such a test is carried out in some detail using the fox-catch data of Elton, with a clear indication that the theory is capable of comprehending the major statistical properties of population-time curves. A final section sketches an extension of the theory to cover secular variations of external conditions such as temperature of the environment.     

Diffusional mobility of parvalbumin in spiny dendrites of cerebellar Purkinje neurons quantified by fluorescence recovery after photobleaching

Ca(2+)-binding proteins (CaBPs) represent key factors for the modulation of cellular Ca(2+) dynamics. Especially in thin extensions of nerve cells, Ca(2+) binding and buffered diffusion of Ca(2+) by CaBPs is assumed to effectively control the spatio-temporal extend of Ca(2+) signals. However, no quantitative data about the mobility of specific CaBPs in the neuronal cytosol are available. We quantified the diffusion of the endogenous CaPB parvalbumin (PV) in spiny dendrites of cerebellar Purkinje neurons with two-photon fluorescence recovery after photobleaching. Fluorescently labeled PV diffused readily between spines and dendrites with a median time constant of 49 ms (37-61 ms, interquartile range). Based on published data on spine geometry, this value corresponds to an apparent diffusion coefficient of 43 microm(2) s(-1) (34-56 microm(2) s(-1)). The absence of large or immobile binding partners for PV was confirmed in PV null-mutant mice. Our data validate the common but so far unproven assumption that PV is highly mobile in neurons and will facilitate simulations of neuronal Ca(2+) buffering. Our experimental approach represents a versatile tool for quantifying the mobility of proteins in neuronal dendrites.     

Nuclear double-fluorescent reporter for in vivo and ex vivo analyses of biological transitions in mouse nuclei

Cre-responsive dual-fluorescent alleles allow in situ marking of cell lineages or genetically modified cells. Here we report a dual-fluorescent allele, ROSA ^nT-nG , which directs nuclear accumulation of tdTomato in Cre-naïve lineages. Cre converts the allele to ROSA ^nG , which drives nuclear EGFP accumulation. Conditions were established for analyzing marked nuclei by flow cytometry on the basis of red–green fluorescence and ploidy, with a particular focus on liver nuclei. Hydrodynamic delivery of a Cre-expression plasmid was used to time-stamp arbitrary hepatocytes for lineage tracing. The distinct green fluorescence of nuclei from Cre-exposed lineages facilitated analyses of ploidy transitions within clones. To assess developmental transitions in liver nuclei, ROSA ^nT-nG was combined with the hepatocyte-specific AlbCre transgene, facilitating discrimination between hepatocyte and nonhepatocyte nuclei. Nuclei extracted from postnatal day 2 (P2) livers were 41 % green and 59 % red and reached a stable level of 84 % green by P22. Until P20, green nuclei were >98 % diploid (2N); at P40 they were ~56 % 2N, 43 % 4N, and <1 % 8N; and by P70 they reached a stable distribution of ~46 % 2N, 45 % 4N, and 9 % 8N. In conclusion, ROSA ^nT-nG will facilitate in vivo and ex vivo studies on liver and will likely be valuable for studies on tissues like muscle, kidney, or brain in which cells are refractory to whole-cell flow cytometry, or like trophectoderm derivatives or cancers in which cells undergo ploidy transitions.     

Thalamic circuitry and thalamocortical synchrony

The corticothalamic system has an important role in synchronizing the activities of thalamic and cortical neurons. Numerically, its synapses dominate the inputs to relay cells and to the gamma-amino butyric acid (GABA)ergic cells of the reticular nucleus (RTN). The capacity of relay neurons to operate in different voltage-dependent functional modes determines that the inputs from the cortex have the capacity directly to excite the relay cells, or indirectly to inhibit them via the RTN, serving to synchronize high- or low-frequency oscillatory activity respectively in the thalamocorticothalamic network. Differences in the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subunit composition of receptors at synapses formed by branches of the same corticothalamic axon in the RTN and dorsal thalamus are an important element in the capacity of the cortex to synchronize low-frequency oscillations in the network. Interactions of focused corticothalamic axons arising from layer VI cortical cells and diffuse corticothalamic axons arising from layer V cortical cells, with the specifically projecting core relay cells and diffusely projecting matrix cells of the dorsal thalamus, form a substrate for synchronization of widespread populations of cortical and thalamic cells during high-frequency oscillations that underlie discrete conscious events.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.