Identification of Group A Streptococcus Genes Directly Regulated by CsrRS and Novel Intermediate Regulators

Adaptation of group A Streptococcus (GAS) to its human host is mediated by two-component systems that transduce external stimuli to regulate bacterial physiology. Among such systems, CsrRS (also known as CovRS) is the most extensively characterized for its role in regulating ∼10% of the GAS genome, including several virulence genes. Here, we show that extracellular magnesium and the human antimicrobial peptide LL-37 have opposing effects on the phosphorylation of the response regulator CsrR by the receptor kinase CsrS. Genetic inactivation of CsrS phosphatase or kinase activity, respectively, had similar but more pronounced effects on CsrR phosphorylation compared to growth in magnesium or LL-37. These changes in CsrR phosphorylation were correlated with the repression or activation of CsrR-regulated genes as assessed by NanoString analysis. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) revealed CsrR occupancy at CsrRS-regulated promoters and lower-affinity associations at many other locations on the GAS chromosome. Because ChIP-seq did not detect CsrR occupancy at promoters associated with some CsrR-regulated genes, we investigated whether these genes might be controlled indirectly by intermediate regulators whose expression is modulated by CsrR. Transcriptional profiling of mutant strains deficient in the expression of either of two previously uncharacterized transcription regulators in the CsrR regulon indicated that one or both proteins participated in the regulation of 22 of the 42 CsrR-regulated promoters for which no CsrR association was detected by ChIP-seq. Taken together, these results illuminate CsrRS-mediated regulation of GAS gene expression through modulation of CsrR phosphorylation, CsrR association with regulated promoters, and the control of intermediate transcription regulators. Importance: Group A Streptococcus (GAS) is an important public health threat as a cause of sore throat, skin infections, life-threatening invasive infections, and the postinfectious complications of acute rheumatic fever, a leading cause of acquired heart disease. This work characterizes CsrRS, a GAS system for the detection of environmental signals that enables adaptation of the bacteria for survival in the human throat by regulating the production of products that allow the bacteria to resist clearance by the human immune system. CsrRS consists of two proteins: CsrS, which is on the bacterial surface to detect specific stimuli, and CsrR, which receives signals from CsrS and, in response, represses or activates the expression of genes coding for proteins that enhance bacterial survival. Some of the genes regulated by CsrR encode proteins that are themselves regulators of gene expression, thereby creating a regulatory cascade

Low Luminosity States of the Black Hole Candidate GX~339--4. II. Timing Analysis

Here we present timing analysis of a set of eight Rossi X-ray Timing Explorer (RXTE) observations of the black hole candidate GX 339-4 that were taken during its hard/low state. On long time scales, the RXTE All Sky Monitor data reveal evidence of a 240 day periodicity, comparable to timescales expected from warped, precessing accretion disks. On short timescales all observations save one show evidence of a persistent f approximately equal to 0.3 Hz QPO. The broad band (10^{-3}-10^2 Hz) power appears to be dominated by two independent processes that can be modeled as very broad Lorentzians with Q approximately less than 1. The coherence function between soft and hard photon variability shows that if these are truly independent processes, then they are individually coherent, but they are incoherent with one another. This is evidenced by the fact that the coherence function between the hard and soft variability is near unity between 0.005-10 Hz but shows evidence of a dip at f approximately equal to 1 Hz. This is the region of overlap between the broad Lorentzian fits to the PSD. Similar to Cyg X-1, the coherence also drops dramatically at frequencies approximately greater than 10 Hz. Also similar to Cyg X-1, the hard photon variability is seen to lag the soft photon variability with the lag time increasing with decreasing Fourier frequency. The magnitude of this time lag appears to be positively correlated with the flux of GX 339-4. We discuss all of these observations in light of current theoretical models of both black hole spectra and temporal variability.Comment: To Appear in the AStrophysical Journa

Improved transformation efficiency of group A Streptococcus by inactivation of a type I restriction modification system

Streptococcus pyogenes or group A Streptococcus (GAS) is a leading cause of bacterial pharyngitis, skin and soft tissue infections, life-threatening invasive infections, and the post-infectious autoimmune syndromes of acute rheumatic fever and post-streptococcal glomerulonephritis. Genetic manipulation of this important pathogen is complicated by resistance of the organism to genetic transformation. Very low transformation efficiency is attributed to recognition and degradation of introduced foreign DNA by a type I restriction-modification system encoded by the hsdRSM locus. DNA sequence analysis of this locus in ten GAS strains that had been previously transformed with an unrelated plasmid revealed that six of the ten harbored a spontaneous mutation in hsdR, S, or M. The mutations were all different, and at least five of the six were predicted to result in loss of function of the respective hsd gene product. The unexpected occurrence of such mutations in previously transformed isolates suggested that the process of transformation selects for spontaneous inactivating mutations in the Hsd system. We investigated the possibility of exploiting the increased transformability of hsd mutants by constructing a deletion mutation in hsdM in GAS strain 854, a clinical isolate representative of the globally dominant M1T1 clonal group. Mutant strain 854ΔhsdM exhibited a 5-fold increase in electrotransformation efficiency compared to the wild type parent strain and no obvious change in growth or off-target gene expression. We conclude that genetic transformation of GAS selects for spontaneous mutants in the hsdRSM restriction modification system. We propose that use of a defined hsdM mutant as a parent strain for genetic manipulation of GAS will enhance transformation efficiency and reduce the likelihood of selecting spontaneous hsd mutants with uncharacterized genotypes

The Escape of Ionizing Photons from OB Associations in Disk Galaxies: Radiation Transfer Through Superbubbles

By solving the time-dependent radiation transfer problem of stellar radiation through evolving superbubbles within a smoothly varying HI distribution, we estimate the fraction of ionizing photons emitted by OB associations that escapes the HI disk of our Galaxy into the halo and intergalactic medium (IGM). We consider both coeval star-formation and a Gaussian star-formation history with a time spread sigma_t = 2 Myr. We consider both a uniform H I distribution and a two-phase (cloud/intercloud) model, with a negligible filling factor of hot gas. We find that the shells of the expanding superbubbles quickly trap or attenuate the ionizing flux, so that most of the escaping radiation escapes shortly after the formation of the superbubble. For the coeval star-formation history, the total fraction of Lyman Continuum photons that escape both sides of the disk in the solar vicinity is f_esc approx 0.15 +/- 0.05. For the Gaussian star formation history, f_esc approx 0.06 +/- 0.03, a value roughly a factor of two lower than the results of Dove & Shull (1994), where superbubbles were not considered. For a local production rate of ionizing photons Psi_LyC = 4.95 X 10^7 cm^{-2} s^{-1}, the flux escaping the disk is Phi_LyC approx (1.5-3.0) X 10^6 cm^{-2} s^{-1} for coeval and Gaussian star formation, comparable to the flux required to sustain the Reynolds layer.Comment: Revised version (expanded), accepted for publication by ApJ, 38 pages, 8 figures, aasms4.sty and aabib.sty files include

Selectively bred oysters can alter their biomineralization pathways, promoting resilience to environmental acidification

Commercial shellfish aquaculture is vulnerable to the impacts of ocean acidification driven by increasing carbon dioxide (CO2) absorption by the ocean as well as to coastal acidification driven by land run off and rising sea level. These drivers of environmental acidification have deleterious effects on biomineralization. We investigated shell biomineralization of selectively bred and wild‐type families of the Sydney rock oyster Saccostrea glomerata in a study of oysters being farmed in estuaries at aquaculture leases differing in environmental acidification. The contrasting estuarine pH regimes enabled us to determine the mechanisms of shell growth and the vulnerability of this species to contemporary environmental acidification. Determination of the source of carbon, the mechanism of carbon uptake and use of carbon in biomineral formation are key to understanding the vulnerability of shellfish aquaculture to contemporary and future environmental acidification. We, therefore, characterized the crystallography and carbon uptake in the shells of S. glomerata, resident in habitats subjected to coastal acidification, using high‐resolution electron backscatter diffraction and carbon isotope analyses (as δ13C). We show that oyster families selectively bred for fast growth and families selected for disease resistance can alter their mechanisms of calcite crystal biomineralization, promoting resilience to acidification. The responses of S. glomerata to acidification in their estuarine habitat provide key insights into mechanisms of mollusc shell growth under future climate change conditions. Importantly, we show that selective breeding in oysters is likely to be an important global mitigation strategy for sustainable shellfish aquaculture to withstand future climate‐driven change to habitat acidification

The Effect of Galactic Properties on the Escape Fraction of Ionizing Photons

The escape fraction, fesc, of ionizing photons from early galaxies is a crucial parameter for determining whether the observed galaxies at z > 6 are able to reionize the high-redshift intergalactic medium. Previous attempts to measure fesc have found a wide range of values, varying from less than 0.01 to nearly 1. Rather than finding a single value of fesc, we clarify through modeling how internal properties of galaxies affect fesc through the density and distribution of neutral hydrogen within the galaxy, along with the rate of ionizing photons production. We find that the escape fraction depends sensitively on the covering factor of clumps, along with the density of the clumped and interclump medium. One must therefore be cautious when dealing with an inhomogeneous medium. Fewer, high-density clumps lead to a greater escape fraction than more numerous low-density clumps. When more ionizing photons are produced in a starburst, fesc increases, as photons escape more readily from the gas layers. Large variations in the predicted escape fraction, caused by differences in the hydrogen distribution, may explain the large observed differences in fesc among galaxies. Values of fesc must also be consistent with the reionization history. High-mass galaxies alone are unable to reionize the universe, because fesc > 1 would be required. Small galaxies are needed to achieve reionization, with greater mean escape fraction in the past.Comment: 27 pages, 8 figures. Accepted to ApJ. v2: Improvements based on referee's comment

Regulated Expression of Chromobox Homolog 5 Revealed in Tumors of ApcMin/+ ROSA11 Gene Trap Mice

The gene-trap lacZ reporter insertion, ROSA11, in the Cbx5 mouse gene illuminates the regulatory complexity of this locus in ApcMin/+ mice. The insertion site of the β-Geo gene-trap element lies in the 24-kb intron proximal to the coding region of Cbx5. Transcript analysis indicates that two promoters for Cbx5 flank this insertion site. Heterozygotes for the insertion express lacZ widely in fetal tissues but show limited expression in adult tissues. In the intestine, strong expression is limited to proliferative zones of crypts and tumors. Homozygotes for ROSA11, found at a lower than Mendelian frequency, express reduced levels of the coding region transcript in normal tissues, using a downstream promoter. Analysis via real-time polymerase chain reaction indicates that the upstream promoter is the dominant promoter in normal epithelium and tumors. Bioinformatic analysis of the Cbx5 locus indicates that WNT and its target transcription factor MYC can establish a feedback loop that may play a role in regulating the self-renewal of the normal intestinal epithelium and its tumors