Layla Lakes, Saudi Arabia: The World-Wide Largest Lacustrine Gypsum Tufas

Throughout the center of Saudi Arabia the anhydrite upper Jurassic Heethformation can be followed N to S. Locally it is punctured by hypogene karst sinkholes. The most prominent are the former Layla Lakes at 22.17°N 46.70°E. The lakes (17 originally) have been drained in the late 1980’s, revealing 19 sinkholes, some of them composites of several subsidence centers. The largest is 1.1 km long, 0.4 km wide and about 40 m deep. Others are less than 10 m across and rather recent. The bottom of the former lakes and the flats around them are composed of thick layers of fine-grained lake chalks. The most striking feature of these sinkholes is the several meters thick tufa covering the vertical walls of the sinkholes. It formed sub-aqueous and is entirely composed of gypsum. Morphologically the tufa displays thick bulbous forms at the bottom changing to conical forms at middle depthto gour-, gutter-, or shovel-like forms near to the former lake surface. The mineralogy and morphology of this tufa appear to be singular world-wide

Bacillus spores as building blocks for stimuli-responsive materials and nanogenerators

Engineering and Applied Science

A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis

BACKGROUND: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. METHODOLOGY/PRINCIPAL FINDINGS: The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. CONCLUSIONS/SIGNIFICANCE: The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes

A Distance-Weighted Interaction Map Reveals a Previously Uncharacterized Layer of the Bacillus subtilis Spore Coat

SummaryBacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all Bacillus spores and may play critical functions in the environment

Gene Clusters Located on Two Large Plasmids Determine Spore Crystal Association (SCA) in Bacillus thuringiensis Subsp. finitimus Strain YBT-020

Crystals in Bacillus thuringiensis are usually formed in the mother cell compartment during sporulation and are separated from the spores after mother cell lysis. In a few strains, crystals are produced inside the exosporium and are associated with the spores after sporulation. This special phenotype, named ‘spore crystal association’ (SCA), typically occurs in B. thuringiensis subsp. finitimus. Our aim was to identify genes determining the SCA phenotype in B. thuringiensis subsp. finitimus strain YBT-020. Plasmid conjugation experiments indicated that the SCA phenotype in this strain was tightly linked with two large plasmids (pBMB26 and pBMB28). A shuttle bacterial artificial chromosome (BAC) library of strain YBT-020 was constructed. Six fragments from BAC clones were screened from this library and discovered to cover the full length of pBMB26; four others were found to cover pBMB28. Using fragment complementation testing, two fragments, each of approximately 35 kb and located on pBMB26 and pBMB28, were observed to recover the SCA phenotype in an acrystalliferous mutant, B. thuringiensis strain BMB171. Furthermore, deletion analysis indicated that the crystal protein gene cry26Aa from pBMB26, along with five genes from pBMB28, were indispensable to the SCA phenotype. Gene disruption and frame-shift mutation analyses revealed that two of the five genes from pBMB28, which showed low similarity to crystal proteins, determined the location of crystals inside the exosporium. Gene disruption revealed that the three remaining genes, similar to spore germination genes, contributed to the stability of the SCA phenotype in strain YBT-020. Our results thus identified the genes determining the SCA phenotype in B. thuringiensis subsp. finitimus

Early events of Bacillus anthracis germination identified by time-course quantitative proteomics

Germination of Bacillus anthracis spores involves rehydration of the spore interior and rapid degradation of several of the protective layers, including the spore coat. Here, we examine the temporal changes that occur during B. anthracis spore germination using an isobaric tagging system. Over the course of 17 min from the onset of germination, the levels of at least 19 spore proteins significantly decrease. Included are acid-soluble proteins, several known and predicted coat proteins, and proteins of unknown function. Over half of these proteins are small (less than 100 amino acids) and would have been undetectable by conventional gel-based analysis. We also identified 20 proteins, whose levels modestly increased at the later time points when metabolism has likely resumed. Taken together, our data show that isobaric labeling of complex mixtures is particularly effective for temporal studies. Furthermore, we describe a rigorous statistical approach to define relevant changes that takes into account the nature of data obtained from multidimensional protein identification technology coupled with the use of isobaric tags. This study provides an expanded list of the proteins that may be involved in germination of the B. anthracis spore and their relative levels during germination.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55849/1/5199_ftp.pd

Fabrication of Atomically Precise Nanopores in Hexagonal Boron Nitride

We demonstrate the fabrication of individual nanopores in hexagonal boron nitride (hBN) with atomically precise control of the pore size. Previous methods of pore production in other 2D materials create pores of irregular geometry with imprecise diameters. By taking advantage of the preferential growth of boron vacancies in hBN under electron beam irradiation, we are able to observe the pore growth via transmission electron microscopy, and terminate the process when the pore has reached its desired size. Careful control of beam conditions allows us to nucleate and grow individual triangular and hexagonal pores with diameters ranging from subnanometer to 6nm over a large area of suspended hBN using a conventional TEM. These nanopores could find application in molecular sensing, DNA sequencing, water desalination, and molecular separation. Furthermore, the chemical edge-groups along the hBN pores can be made entirely nitrogen terminated or faceted with boron-terminated edges, opening avenues for tailored functionalization and extending the applications of these hBN nanopores.Comment: 5 pages, 6 figure