Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle. IMPORTANCE: Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms

The Cosmic Ultraviolet Baryon Survey (CUBS) I. Overview and the diverse environments of Lyman limit systems at z<1

We present initial results from the Cosmic Ultraviolet Baryon Survey (CUBS). CUBS is designed to map diffuse baryonic structures at redshift z<~1 using absorption-line spectroscopy of 15 UV-bright QSOs with matching deep galaxy survey data. CUBS QSOs are selected based on their NUV brightness to avoid biases against the presence of intervening Lyman Limit Systems (LLSs) at zabs~ 17.2 over a total redshift survey pathlength of dz=9.3, and a number density of n(z)=0.43 (-0.18, +0.26). Considering all absorbers with log N(HI)/cm^-2 > 16.5 leads to n(z)=1.08 (-0.25, +0.31) at z<1. All LLSs exhibit a multi-component structure and associated metal transitions from multiple ionization states such as CII, CIII, MgII, SiII, SiIII, and OVI absorption. Differential chemical enrichment levels as well as ionization states are directly observed across individual components in three LLSs. We present deep galaxy survey data obtained using the VLT-MUSE integral field spectrograph and the Magellan Telescopes, reaching sensitivities necessary for detecting galaxies fainter than 0.1L* at d<~300 physical kpc (pkpc) in all five fields. A diverse range of galaxy properties is seen around these LLSs, from a low-mass dwarf galaxy pair, a co-rotating gaseous halo/disk, a star-forming galaxy, a massive quiescent galaxy, to a galaxy group. The closest galaxies have projected distances ranging from d=15 to 72 pkpc and intrinsic luminosities from ~0.01L* to ~3L*. Our study shows that LLSs originate in a variety of galaxy environments and trace gaseous structures with a broad range of metallicities.Comment: 26 pages, 14 figures, MNRAS in pres

The HIPASS Catalogue - II. Completeness, Reliability, and Parameter Accuracy

The HI Parkes All Sky Survey (HIPASS) is a blind extragalactic HI 21-cm emission line survey covering the whole southern sky from declination -90 to +25. The HIPASS catalogue (HICAT), containing 4315 HI-selected galaxies from the region south of declination +2, is presented in Meyer et al. (2004a, Paper I). This paper describes in detail the completeness and reliability of HICAT, which are calculated from the recovery rate of synthetic sources and follow-up observations, respectively. HICAT is found to be 99 per cent complete at a peak flux of 84 mJy and an integrated flux of 9.4 Jy km/s. The overall reliability is 95 per cent, but rises to 99 per cent for sources with peak fluxes >58 mJy or integrated flux > 8.2 Jy km/s. Expressions are derived for the uncertainties on the most important HICAT parameters: peak flux, integrated flux, velocity width, and recessional velocity. The errors on HICAT parameters are dominated by the noise in the HIPASS data, rather than by the parametrization procedure.Comment: Accepted for publication in MNRAS. 12 pages, 11 figures. Paper with higher resolution figures can be downloaded from http://hipass.aus-vo.or

The Northern HIPASS catalogue - Data presentation, completeness and reliability measures

The Northern HIPASS catalogue (NHICAT) is the northern extension of the HIPASS catalogue, HICAT (Meyer et al. 2004). This extension adds the sky area between the declination range of +2 deg < dec. < +25.5 deg to HICAT's declination range of -90 deg < dec. < +2 deg. HIPASS is a blind HI survey using the Parkes Radio Telescope covering 71% of the sky (including this northern extension) and a heliocentric velocity range of -1,280 km/s to 12,700 km/s . The entire Virgo Cluster region has been observed in the Northern HIPASS. The galaxy catalogue, NHICAT, contains 1002 sources with v_hel > 300 km/s . Sources with -300 km/s < v_hel < 300 km/s were excluded to avoid contamination by Galactic emission. In total, the entire HIPASS survey has found 5317 galaxies identified purely by their HI content. The full galaxy catalogue is publicly-available at .Comment: 12 pages, accepted for publication by MNRA

Extreme Feedback and the Epoch of Reionization: Clues in the Local Universe

The source responsible for reionizing the universe at z > 6 remains uncertain. While an energetically adequate population of star-forming galaxies may be in place, it is unknown whether a large enough fraction of their ionizing radiation can escape into the intergalactic medium. Attempts to measure this escape-fraction in intensely star-forming galaxies at lower redshifts have largely yielded upper limits. In this paper we present new HST COS and archival FUSE far-UV spectroscopy of a sample of eleven Lyman Break Analogs (LBAs), a rare population of local galaxies that strongly resemble the high-z Lyman Break galaxies. We combine these data with SDSS optical spectra and Spitzer photometry. We also analyze archival FUSE observations of fifteen typical UV-bright local starbursts. We find evidence of small covering factors for optically-thick neutral gas in 3 cases. This is based on two independent pieces of evidence: a significant residual intensity in the cores of the strongest interstellar absorption-lines tracing neutral gas and a small ratio of extinction-corrected H-alpha to UV plus far-IR luminosities. These objects represent three of the four LBAs that contain a young, very compact (~100pc), and highly massive (~10^9 Mo) dominant central object (DCO). These three objects also differ from the other galaxies in showing a significant amount of blueshifted Ly-alpha emission, which may be related to the low covering factor of neutral gas. All four LBAs with DCOs in our sample show extremely high velocity outflows of interstellar gas, with line centroids blueshifted by about 700km/s and maximum outflow velocities reaching at least 1500km/s. We show that these properties are consistent with an outflow driven by a powerful starburst that is exceptionally compact. We speculate that such extreme feedback may be required to enable the escape of ionizing radiation from star forming galaxies.Comment: Accepted for publication in ApJ. 14 pages, 8 figures, 2 table

Genetic Dissection of Acute Ethanol Responsive Gene Networks in Prefrontal Cortex: Functional and Mechanistic Implications

Background Individual differences in initial sensitivity to ethanol are strongly related to the heritable risk of alcoholism in humans. To elucidate key molecular networks that modulate ethanol sensitivity we performed the first systems genetics analysis of ethanol-responsive gene expression in brain regions of the mesocorticolimbic reward circuit (prefrontal cortex, nucleus accumbens, and ventral midbrain) across a highly diverse family of 27 isogenic mouse strains (BXD panel) before and after treatment with ethanol. Results Acute ethanol altered the expression of ~2,750 genes in one or more regions and 400 transcripts were jointly modulated in all three. Ethanol-responsive gene networks were extracted with a powerful graph theoretical method that efficiently summarized ethanol\u27s effects. These networks correlated with acute behavioral responses to ethanol and other drugs of abuse. As predicted, networks were heavily populated by genes controlling synaptic transmission and neuroplasticity. Several of the most densely interconnected network hubs, including Kcnma1 and Gsk3β, are known to influence behavioral or physiological responses to ethanol, validating our overall approach. Other major hub genes like Grm3, Pten and Nrg3 represent novel targets of ethanol effects. Networks were under strong genetic control by variants that we mapped to a small number of chromosomal loci. Using a novel combination of genetic, bioinformatic and network-based approaches, we identified high priority cis-regulatory candidate genes, including Scn1b,Gria1, Sncb and Nell2. Conclusions The ethanol-responsive gene networks identified here represent a previously uncharacterized intermediate phenotype between DNA variation and ethanol sensitivity in mice. Networks involved in synaptic transmission were strongly regulated by ethanol and could contribute to behavioral plasticity seen with chronic ethanol. Our novel finding that hub genes and a small number of loci exert major influence over the ethanol response of gene networks could have important implications for future studies regarding the mechanisms and treatment of alcohol use disorders

The first myriapod genome sequence reveals conservative arthropod gene content and genome organisation in the centipede Strigamia maritima.

Myriapods (e.g., centipedes and millipedes) display a simple homonomous body plan relative to other arthropods. All members of the class are terrestrial, but they attained terrestriality independently of insects. Myriapoda is the only arthropod class not represented by a sequenced genome. We present an analysis of the genome of the centipede Strigamia maritima. It retains a compact genome that has undergone less gene loss and shuffling than previously sequenced arthropods, and many orthologues of genes conserved from the bilaterian ancestor that have been lost in insects. Our analysis locates many genes in conserved macro-synteny contexts, and many small-scale examples of gene clustering. We describe several examples where S. maritima shows different solutions from insects to similar problems. The insect olfactory receptor gene family is absent from S. maritima, and olfaction in air is likely effected by expansion of other receptor gene families. For some genes S. maritima has evolved paralogues to generate coding sequence diversity, where insects use alternate splicing. This is most striking for the Dscam gene, which in Drosophila generates more than 100,000 alternate splice forms, but in S. maritima is encoded by over 100 paralogues. We see an intriguing linkage between the absence of any known photosensory proteins in a blind organism and the additional absence of canonical circadian clock genes. The phylogenetic position of myriapods allows us to identify where in arthropod phylogeny several particular molecular mechanisms and traits emerged. For example, we conclude that juvenile hormone signalling evolved with the emergence of the exoskeleton in the arthropods and that RR-1 containing cuticle proteins evolved in the lineage leading to Mandibulata. We also identify when various gene expansions and losses occurred. The genome of S. maritima offers us a unique glimpse into the ancestral arthropod genome, while also displaying many adaptations to its specific life history.This work was supported by the following grants: NHGRIU54HG003273 to R.A.G; EU Marie Curie ITN #215781 “Evonet” to M.A.; a Wellcome Trust Value in People (VIP) award to C.B. and Wellcome Trust graduate studentship WT089615MA to J.E.G; Marine rhythms of Life” of the University of Vienna, an FWF (http://www.fwf.ac.at/) START award (#AY0041321) and HFSP (http://www.hfsp.org/) research grant (#RGY0082/2010) to KT-­‐R; MFPL Vienna International PostDoctoral Program for Molecular Life Sciences (funded by Austrian Ministry of Science and Research and City of Vienna, Cultural Department -­‐Science and Research to T.K; Direct Grant (4053034) of the Chinese University of Hong Kong to J.H.L.H.; NHGRI HG004164 to G.M.; Danish Research Agency (FNU), Carlsberg Foundation, and Lundbeck Foundation to C.J.P.G.; U.S. National Institutes of Health R01AI55624 to J.H.W.; Royal Society University Research fellowship to F.M.J.; P.D.E. was supported by the BBSRC via the Babraham Institute;This is the final version of the article. It first appeared from PLOS via http://dx.doi.org/10.1371/journal.pbio.100200