Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport

Bolocam Survey for 1.1 mm Dust Continuum Emission in the c2d Legacy Clouds. II. Ophiuchus

We present a large-scale millimeter continuum map of the Ophiuchus molecular cloud. Nearly 11 square degrees, including all of the area in the cloud with visual extinction more than 3 magnitudes, was mapped at 1.1 mm with Bolocam on the Caltech Submillimeter Observatory (CSO). By design, the map also covers the region mapped in the infrared with the Spitzer Space Telescope. We detect 44 definite sources, and a few likely sources are also seen along a filament in the eastern streamer. The map indicates that dense cores in Ophiuchus are very clustered and often found in filaments within the cloud. Most sources are round, as measured at the half power point, but elongated when measured at lower contour levels, suggesting spherical sources lying within filaments. The masses, for an assumed dust temperature of 10 K, range from 0.24 to 3.9 solar masses, with a mean value of 0.96 solar masses. The total mass in distinct cores is 42 solar masses, 0.5 to 2% of the total cloud mass, and the total mass above 4 sigma is about 80 solar masses. The mean densities in the cores are quite high, with an average of 1.6 x 10^6 per cc, suggesting short free-fall times. The core mass distribution can be fitted with a power law with slope of 2.1 plus or minus 0.3 for M>0.5 solar masses, similar to that found in other regions, but slightly shallower than that of some determinations of the local IMF. In agreement with previous studies, our survey shows that dense cores account for a very small fraction of the cloud volume and total mass. They are nearly all confined to regions with visual extinction at least 9 mag, a lower threshold than found previously.Comment: 47 pages, 16 figures, accepted for Ap

The Dust Emissivity Spectral Index in the Starless Core TMC-1C

In this paper we present a dust emission map of the starless core TMC-1C taken at 2100 microns. Along with maps at 160, 450, 850 and 1200 microns, we study the dust emissivity spectral index from the (sub)millimeter spectral energy distribution, and find that it is close to the typically assumed value of beta = 2. We also map the dust temperature and column density in TMC-1C, and find that at the position of the dust peak (A_V ~ 50), the line-of-sight-averaged temperature is ~7 K. Employing simple Monte Carlo modeling, we show that the data are consistent with a constant value for the emissivity spectral index over the whole map of TMC-1C.Comment: 11 pages, including 5 pages of figures. Accepted to Ap

FusorSV: an algorithm for optimally combining data from multiple structural variation detection methods.

Comprehensive and accurate identification of structural variations (SVs) from next generation sequencing data remains a major challenge. We develop FusorSV, which uses a data mining approach to assess performance and merge callsets from an ensemble of SV-calling algorithms. It includes a fusion model built using analysis of 27 deep-coverage human genomes from the 1000 Genomes Project. We identify 843 novel SV calls that were not reported by the 1000 Genomes Project for these 27 samples. Experimental validation of a subset of these calls yields a validation rate of 86.7%. FusorSV is available at https://github.com/TheJacksonLaboratory/SVE . Genome Biol 2018 Mar 20; 19(1):38

The Twin-Arginine Translocation Pathway in α-Proteobacteria Is Functionally Preserved Irrespective of Genomic and Regulatory Divergence

The twin-arginine translocation (Tat) pathway exports fully folded proteins out of the cytoplasm of Gram-negative and Gram-positive bacteria. Although much progress has been made in unraveling the molecular mechanism and biochemical characterization of the Tat system, little is known concerning its functionality and biological role to confer adaptive skills, symbiosis or pathogenesis in the α-proteobacteria class. A comparative genomic analysis in the α-proteobacteria class confirmed the presence of tatA, tatB, and tatC genes in almost all genomes, but significant variations in gene synteny and rearrangements were found in the order Rickettsiales with respect to the typically described operon organization. Transcription of tat genes was confirmed for Anaplasma marginale str. St. Maries and Brucella abortus 2308, two α-proteobacteria with full and partial intracellular lifestyles, respectively. The tat genes of A. marginale are scattered throughout the genome, in contrast to the more generalized operon organization. Particularly, tatA showed an approximately 20-fold increase in mRNA levels relative to tatB and tatC. We showed Tat functionality in B. abortus 2308 for the first time, and confirmed conservation of functionality in A. marginale. We present the first experimental description of the Tat system in the Anaplasmataceae and Brucellaceae families. In particular, in A. marginale Tat functionality is conserved despite operon splitting as a consequence of genome rearrangements. Further studies will be required to understand how the proper stoichiometry of the Tat protein complex and its biological role are achieved. In addition, the predicted substrates might be the evidence of role of the Tat translocation system in the transition process from a free-living to a parasitic lifestyle in these α-proteobacteria

A holin and an endopeptidase are essential for chitinolytic protein secretion in <i>Serratia marcescens</i>

Pathogenic bacteria adapt to their environment and manipulate the biochemistry of hosts by secretion of effector molecules. Serratia marcescens is an opportunistic pathogen associated with healthcare-acquired infections and is a prolific secretor of proteins, including three chitinases (ChiA, ChiB, and ChiC) and a chitin binding protein (Cbp21). In this work, genetic, biochemical, and proteomic approaches identified genes that were required for secretion of all three chitinases and Cbp21. A genetic screen identified a holin-like protein (ChiW) and a putative l-alanyl-d-glutamate endopeptidase (ChiX), and subsequent biochemical analyses established that both were required for nonlytic secretion of the entire chitinolytic machinery, with chitinase secretion being blocked at a late stage in the mutants. In addition, live-cell imaging experiments demonstrated bimodal and coordinated expression of chiX and chiA and revealed that cells expressing chiA remained viable. It is proposed that ChiW and ChiX operate in tandem as components of a protein secretion system used by gram-negative bacteria