Essential Roles for Mycobacterium tuberculosis Rel beyond the Production of (p)ppGpp

In Mycobacterium tuberculosis, the stringent response to amino acid starvation is mediated by the M. tuberculosis Rel (Rel(Mtb)) enzyme, which transfers a pyrophosphate from ATP to GDP or GTP to synthesize ppGpp and pppGpp, respectively. (p)ppGpp then influences numerous metabolic processes. Rel(Mtb) also encodes a second, distinct catalytic domain that hydrolyzes (p)ppGpp into pyrophosphate and GDP or GTP. Rel(Mtb) is required for chronic M. tuberculosis infection in mice; however, it is unknown which catalytic activity of Rel(Mtb) mediates pathogenesis and whether (p)ppGpp itself is necessary. In order to individually investigate the roles of (p)ppGpp synthesis and hydrolysis during M. tuberculosis pathogenesis, we generated Rel(Mtb) point mutants that were either synthetase dead (Rel(Mtb)(H344Y)) or hydrolase dead (Rel(Mtb)(H80A)). M. tuberculosis strains expressing the synthetase-dead Rel(Mtb)(H344Y) mutant did not persist in mice, demonstrating that the Rel(Mtb) (p)ppGpp synthetase activity is required for maintaining bacterial titers during chronic infection. Deletion of a second predicted (p)ppGpp synthetase had no effect on pathogenesis, demonstrating that Rel(Mtb) was the major contributor to (p)ppGpp production during infection. Interestingly, expression of an allele encoding the hydrolase-dead Rel(Mtb) mutant, Rel(Mtb)(H80A), that is incapable of hydrolyzing (p)ppGpp but still able to synthesize (p)ppGpp decreased the growth rate of M. tuberculosis and changed the colony morphology of the bacteria. In addition, Rel(Mtb)(H80A) expression during acute or chronic M. tuberculosis infection in mice was lethal to the infecting bacteria. These findings highlight a distinct role for Rel(Mtb)-mediated (p)ppGpp hydrolysis that is essential for M. tuberculosis pathogenesis

The California-Kepler Survey. II. Precise Physical Properties of 2025 Kepler Planets and Their Host Stars

We present stellar and planetary properties for 1305 Kepler Objects of Interest (KOIs) hosting 2025 planet candidates observed as part of the California-Kepler Survey. We combine spectroscopic constraints, presented in Paper I, with stellar interior modeling to estimate stellar masses, radii, and ages. Stellar radii are typically constrained to 11%, compared to 40% when only photometric constraints are used. Stellar masses are constrained to 4%, and ages are constrained to 30%. We verify the integrity of the stellar parameters through comparisons with asteroseismic studies and Gaia parallaxes. We also recompute planetary radii for 2025 planet candidates. Because knowledge of planetary radii is often limited by uncertainties in stellar size, we improve the uncertainties in planet radii from typically 42% to 12%. We also leverage improved knowledge of stellar effective temperature to recompute incident stellar fluxes for the planets, now precise to 21%, compared to a factor of two when derived from photometry.Comment: 13 pages, 4 figures, 4 tables, accepted for publication in AJ; full versions of tables 3 and 4 are include

The California-Kepler Survey. I. High Resolution Spectroscopy of 1305 Stars Hosting Kepler Transiting Planets

The California-Kepler Survey (CKS) is an observational program to improve our knowledge of the properties of stars found to host transiting planets by NASA's Kepler Mission. The improvement stems from new high-resolution optical spectra obtained using HIRES at the W. M. Keck Observatory. The CKS stellar sample comprises 1305 stars classified as Kepler Objects of Interest, hosting a total of 2075 transiting planets. The primary sample is magnitude-limited (Kp < 14.2) and contains 960 stars with 1385 planets. The sample was extended to include some fainter stars that host multiple planets, ultra short period planets, or habitable zone planets. The spectroscopic parameters were determined with two different codes, one based on template matching and the other on direct spectral synthesis using radiative transfer. We demonstrate a precision of 60 K in effective temperature, 0.10 dex in surface gravity, 0.04 dex in [Fe/H], and 1.0 km/s in projected rotational velocity. In this paper we describe the CKS project and present a uniform catalog of spectroscopic parameters. Subsequent papers in this series present catalogs of derived stellar properties such as mass, radius and age; revised planet properties; and statistical explorations of the ensemble. CKS is the largest survey to determine the properties of Kepler stars using a uniform set of high-resolution, high signal-to-noise ratio spectra. The HIRES spectra are available to the community for independent analyses.Comment: 20 pages, 19 figures, accepted for publication in AJ; a full version of Table 5 is included as tab_cks.csv and tab_cks.te

Inclusion of Host Quality Data Improves Predictions of Herbivore Phenology

Understanding the correspondence between ambient temperature and insect development is necessary to forecast insect phenology under novel environments. In the face of climate change, both conservation and pest control efforts require accurate phenological predictions. Here, we compare a suite of degree-day models to assess their ability to predict the phenology of a common, oligophagous butterfly, the silver-spotted skipper, Epargyreus clarus (Cramer) (Lepidoptera: Hesperiidae). To estimate model parameters, we used development time of eggs and larvae reared in the laboratory at six constant temperatures ranging from 8 to 38 °C and on two host plants of contrasting quality (kudzu and wisteria). We employed three approaches to determine the base temperature to calculate degree days: linear regression, modified reduced major axis regression, and application of a generic base temperature value of 10 °C, which is commonly used in the absence of laboratory data. To calculate the number of degree days required to complete a developmental stage, we used data from caterpillars feeding on high- and low-quality hosts, both in the field and in the laboratory. To test model accuracy, we predicted development time of seven generations of larvae reared in the field on the same host plants across 3 years (2014–2016). To compare performance among models, we regressed predicted vs. observed development time, and found that r2 values were significantly larger when accounting for host plant quality. The accuracy of development time predictions varied across the season, with estimates of the first two generations being more accurate than estimates of the third generation, when ambient temperatures dropped outside the range in which development rate and temperature have a linear relationship. Overall, we show that accounting for variation in host plant quality when calculating development time in the field is more important than the choice of the base temperature for calculating degree days

The California-Kepler Survey. III. A Gap in the Radius Distribution of Small Planets

The size of a planet is an observable property directly connected to the physics of its formation and evolution. We used precise radius measurements from the California-Kepler Survey (CKS) to study the size distribution of 2025 $\textit{Kepler}$ planets in fine detail. We detect a factor of $\geq$2 deficit in the occurrence rate distribution at 1.5-2.0 R$_{\oplus}$. This gap splits the population of close-in ($P$ < 100 d) small planets into two size regimes: R$_P$ < 1.5 R$_{\oplus}$ and R$_P$ = 2.0-3.0 R$_{\oplus}$, with few planets in between. Planets in these two regimes have nearly the same intrinsic frequency based on occurrence measurements that account for planet detection efficiencies. The paucity of planets between 1.5 and 2.0 R$_{\oplus}$ supports the emerging picture that close-in planets smaller than Neptune are composed of rocky cores measuring 1.5 R$_{\oplus}$ or smaller with varying amounts of low-density gas that determine their total sizes.Comment: Paper III in the California-Kepler Survey series, accepted to the Astronomical Journa

Effects of increasing the affinity of CarD for RNA polymerase on Mycobacterium tuberculosis growth, rRNA transcription, and virulence

CarD is an essential RNA polymerase (RNAP) interacting protein in Mycobacterium tuberculosis that stimulates formation of RNAP-promoter open complexes. CarD plays a complex role in M. tuberculosis growth and virulence that is not fully understood. Therefore, to gain further insight into the role of CarD in M. tuberculosis growth and virulence, we determined the effect of increasing the affinity of CarD for RNAP. Using site-directed mutagenesis guided by crystal structures of CarD bound to RNAP, we identified amino acid substitutions that increase the affinity of CarD for RNAP. Using these substitutions, we show that increasing the affinity of CarD for RNAP increases the stability of the CarD protein in M. tuberculosis. In addition, we show that increasing the affinity of CarD for RNAP increases the growth rate in M. tuberculosis without affecting 16S rRNA levels. We further show that increasing the affinity of CarD for RNAP reduces M. tuberculosis virulence in a mouse model of infection despite the improved growth rate in vitro. Our findings suggest that the CarD-RNAP interaction protects CarD from proteolytic degradation in M. tuberculosis, establish that growth rate and rRNA levels can be uncoupled in M. tuberculosis and demonstrate that the strength of the CarD-RNAP interaction has been finely tuned to optimize virulence. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis, remains a major global health problem. In order to develop new strategies to battle this pathogen, we must gain a better understanding of the molecular processes involved in its survival and pathogenesis. We have previously identified CarD as an essential transcriptional regulator in mycobacteria. In this study, we detail the effects of increasing the affinity of CarD for RNAP on transcriptional regulation, CarD protein stability, and virulence. These studies expand our understanding of the global transcription regulator CarD, provide insight into how CarD activity is regulated, and broaden our understanding of prokaryotic transcription

The California-Kepler Survey V. Peas in a Pod: Planets in a Kepler Multi-planet System are Similar in Size and Regularly Spaced

We have established precise planet radii, semimajor axes, incident stellar fluxes, and stellar masses for 909 planets in 355 multi-planet systems discovered by Kepler. In this sample, we find that planets within a single multi-planet system have correlated sizes: each planet is more likely to be the size of its neighbor than a size drawn at random from the distribution of observed planet sizes. In systems with three or more planets, the planets tend to have a regular spacing: the orbital period ratios of adjacent pairs of planets are correlated. Furthermore, the orbital period ratios are smaller in systems with smaller planets, suggesting that the patterns in planet sizes and spacing are linked through formation and/or subsequent orbital dynamics. Yet, we find that essentially no planets have orbital period ratios smaller than $1.2$, regardless of planet size. Using empirical mass-radius relationships, we estimate the mutual Hill separations of planet pairs. We find that $93\%$ of the planet pairs are at least 10 mutual Hill radii apart, and that a spacing of $\sim20$ mutual Hill radii is most common. We also find that when comparing planet sizes, the outer planet is larger in $65 \pm 0.4\%$ of cases, and the typical ratio of the outer to inner planet size is positively correlated with the temperature difference between the planets. This could be the result of photo-evaporation.Comment: Published in The Astronomical Journal. 15 pages, 17 figure

Interaction of CarD with RNA polymerase mediates Mycobacterium tuberculosis viability, rifampin resistance, and pathogenesis

Mycobacterium tuberculosis infection continues to cause substantial human suffering. New chemotherapeutic strategies, which require insight into the pathways essential for M. tuberculosis pathogenesis, are imperative. We previously reported that depletion of the CarD protein in mycobacteria compromises viability, resistance to oxidative stress and fluoroquinolones, and pathogenesis. CarD associates with the RNA polymerase (RNAP), but it has been unknown which of the diverse functions of CarD are mediated through the RNAP; this question must be answered to understand the CarD mechanism of action. Herein, we describe the interaction between the M. tuberculosis CarD and the RNAP β subunit and identify point mutations that weaken this interaction. The characterization of mycobacterial strains with attenuated CarD/RNAP β interactions demonstrates that the CarD/RNAP β association is required for viability and resistance to oxidative stress but not for fluoroquinolone resistance. Weakening the CarD/RNAP β interaction also increases the sensitivity of mycobacteria to rifampin and streptomycin. Surprisingly, depletion of the CarD protein did not affect sensitivity to rifampin. These findings define the CarD/RNAP interaction as a new target for chemotherapeutic intervention that could also improve the efficacy of rifampin treatment of tuberculosis. In addition, our data demonstrate that weakening the CarD/RNAP β interaction does not completely phenocopy the depletion of CarD and support the existence of functions for CarD independent of direct RNAP binding

Acute Ethanol Administration Rapidly Increases Phosphorylation of Conventional Protein Kinase C in Specific Mammalian Brain Regions in Vivo

Background Protein kinase C (PKC) is a family of isoenzymes that regulate a variety of functions in the central nervous system including neurotransmitter release, ion channel activity, and cell differentiation. Growing evidence suggests that specific isoforms of PKC influence a variety of behavioral, biochemical, and physiological effects of ethanol in mammals. The purpose of this study was to determine whether acute ethanol exposure alters phosphorylation of conventional PKC isoforms at a threonine 674 (p-cPKC) site in the hydrophobic domain of the kinase, which is required for its catalytic activity. Methods Male rats were administered a dose range of ethanol (0, 0.5, 1, or 2 g/kg, intragastric) and brain tissue was removed 10 minutes later for evaluation of changes in p-cPKC expression using immunohistochemistry and Western blot methods. Results Immunohistochemical data show that the highest dose of ethanol (2 g/kg) rapidly increases p-cPKC immunoreactivity specifically in the nucleus accumbens (core and shell), lateral septum, and hippocampus (CA3 and dentate gyrus). Western blot analysis further showed that ethanol (2 g/kg) increased p-cPKC expression in the P2 membrane fraction of tissue from the nucleus accumbens and hippocampus. Although p-cPKC was expressed in numerous other brain regions, including the caudate nucleus, amygdala, and cortex, no changes were observed in response to acute ethanol. Total PKC? immunoreactivity was surveyed throughout the brain and showed no change following acute ethanol injection