Solar-forced diurnal regulation of cave drip rates via phreatophyte evapotranspiration

We present results of a detailed study of drip rate variations at 12 drip discharge sites in Glory Hole Cave, New South Wales, Australia. Our novel time series analysis, using the wavelet synchrosqueezed transform, reveals pronounced oscillations at daily and sub-daily frequencies occurring in 8 out of the 12 monitored sites. These oscillations were not spatially or temporally homogenous, with different drip sites exhibiting such behaviour at different times of year in different parts of the cave. We test several hypotheses for the cause of the oscillations, including variations in pressure gradients between karst and cave due to cave breathing effects or atmospheric and earth tides, variations in hydraulic conductivity due to changes in viscosity of water with daily temperature oscillations, and solar-driven daily cycles of vegetative (phreatophytic) transpiration. We conclude that the only hypothesis consistent with the data and hydrologic theory is that daily oscillations are caused by solar-driven pumping by phreatophytic trees which are abundant at the site. The daily oscillations are not continuous and occur sporadically in short bursts (2–14 days) throughout the year due to non-linear modification of the solar signal via complex karst architecture. This is the first indirect observation leading to the hypothesis of tree water use in cave drip water. It has important implications for karst hydrology in regards to developing a new protocol to determine the relative importance of trends in drip rate, such as diurnal oscillations, and how these trends change over timescales of weeks to years. This information can also be used to infer karst architecture. This study demonstrates the importance of vegetation on recharge dynamics, information that will inform both process-based karst models and empirical estimation approaches. Our findings support a growing body of research exploring the impact of trees on speleothem paleoclimate proxies

Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in \u3cem\u3ePseudomonas aeruginosa\u3c/em\u3e

Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro4-green fluorescent protein (Pro4GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival

Mycobacterium tuberculosis nucleoid-associated DNA-binding protein H-NS binds with high-affinity to the Holliday junction and inhibits strand exchange promoted by RecA protein

A number of studies have shown that the structure and composition of bacterial nucleoid influences many a processes related to DNA metabolism. The nucleoid-associated proteins modulate not only the DNA conformation but also regulate the DNA metabolic processes such as replication, recombination, repair and transcription. Understanding of how these processes occur in the context of Mycobacterium tuberculosis nucleoid is of considerable medical importance because the nucleoid structure may be constantly remodeled in response to environmental signals and/or growth conditions. Many studies have concluded that Escherichia coli H-NS binds to DNA in a sequence-independent manner, with a preference for A-/T-rich tracts in curved DNA; however, recent studies have identified the existence of medium- and low-affinity binding sites in the vicinity of the curved DNA. Here, we show that the M. tuberculosis H-NS protein binds in a more structure-specific manner to DNA replication and repair intermediates, but displays lower affinity for double-stranded DNA with relatively higher GC content. Notably, M. tuberculosis H-NS was able to bind Holliday junction (HJ), the central recombination intermediate, with substantially higher affinity and inhibited the three-strand exchange promoted by its cognate RecA. Likewise, E. coli H-NS was able to bind the HJ and suppress DNA strand exchange promoted by E. coli RecA, although much less efficiently compared to M. tuberculosis H-NS. Our results provide new insights into a previously unrecognized function of H-NS protein, with implications for blocking the genome integration of horizontally transferred genes by homologous and/or homeologous recombination

Differential Regulation of Horizontally Acquired and Core Genome Genes by the Bacterial Modulator H-NS

Horizontal acquisition of DNA by bacteria dramatically increases genetic diversity and hence successful bacterial colonization of several niches, including the human host. A relevant issue is how this newly acquired DNA interacts and integrates in the regulatory networks of the bacterial cell. The global modulator H-NS targets both core genome and HGT genes and silences gene expression in response to external stimuli such as osmolarity and temperature. Here we provide evidence that H-NS discriminates and differentially modulates core and HGT DNA. As an example of this, plasmid R27-encoded H-NS protein has evolved to selectively silence HGT genes and does not interfere with core genome regulation. In turn, differential regulation of both gene lineages by resident chromosomal H-NS requires a helper protein: the Hha protein. Tight silencing of HGT DNA is accomplished by H-NS-Hha complexes. In contrast, core genes are modulated by H-NS homoligomers. Remarkably, the presence of Hha-like proteins is restricted to the Enterobacteriaceae. In addition, conjugative plasmids encoding H-NS variants have hitherto been isolated only from members of the family. Thus, the H-NS system in enteric bacteria presents unique evolutionary features. The capacity to selectively discriminate between core and HGT DNA may help to maintain horizontally transmitted DNA in silent form and may give these bacteria a competitive advantage in adapting to new environments, including host colonization

Lsr2 of Mycobacterium tuberculosis is a DNA-bridging protein

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2–DNA interactions and structure–function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo. These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2

The potential science and engineering value of samples delivered to Earth by Mars sample return

© The Meteoritical Society, 2019. Executive Summary: Return of samples from the surface of Mars has been a goal of the international Mars science community for many years. Affirmation by NASA and ESA of the importance of Mars exploration led the agencies to establish the international MSR Objectives and Samples Team (iMOST). The purpose of the team is to re-evaluate and update the sample-related science and engineering objectives of a Mars Sample Return (MSR) campaign. The iMOST team has also undertaken to define the measurements and the types of samples that can best address the objectives. Seven objectives have been defined for MSR, traceable through two decades of previously published international priorities. The first two objectives are further divided into sub-objectives. Within the main part of the report, the importance to science and/or engineering of each objective is described, critical measurements that would address the objectives are specified, and the kinds of samples that would be most likely to carry key information are identified. These seven objectives provide a framework for demonstrating how the first set of returned Martian samples would impact future Martian science and exploration. They also have implications for how analogous investigations might be conducted for samples returned by future missions from other solar system bodies, especially those that may harbor biologically relevant or sensitive material, such as Ocean Worlds (Europa, Enceladus, Titan) and others. Summary of Objectives and Sub-Objectives for MSR Identified by iMOST: Objective 1 Interpret the primary geologic processes and history that formed the Martian geologic record, with an emphasis on the role of water. Intent To investigate the geologic environment(s) represented at the Mars 2020 landing site, provide definitive geologic context for collected samples, and detail any characteristics that might relate to past biologic processesThis objective is divided into five sub-objectives that would apply at different landing sites. 1.1 Characterize the essential stratigraphic, sedimentologic, and facies variations of a sequence of Martian sedimentary rocks. Intent To understand the preserved Martian sedimentary record. Samples A suite of sedimentary rocks that span the range of variation. Importance Basic inputs into the history of water, climate change, and the possibility of life 1.2 Understand an ancient Martian hydrothermal system through study of its mineralization products and morphological expression. Intent To evaluate at least one potentially life-bearing “habitable” environment Samples A suite of rocks formed and/or altered by hydrothermal fluids. Importance Identification of a potentially habitable geochemical environment with high preservation potential. 1.3 Understand the rocks and minerals representative of a deep subsurface groundwater environment. Intent To evaluate definitively the role of water in the subsurface. Samples Suites of rocks/veins representing water/rock interaction in the subsurface. Importance May constitute the longest-lived habitable environments and a key to the hydrologic cycle. 1.4 Understand water/rock/atmosphere interactions at the Martian surface and how they have changed with time. Intent To constrain time-variable factors necessary to preserve records of microbial life. Samples Regolith, paleosols, and evaporites. Importance Subaerial near-surface processes could support and preserve microbial life. 1.5 Determine the petrogenesis of Martian igneous rocks in time and space. Intent To provide definitive characterization of igneous rocks on Mars. Samples Diverse suites of ancient igneous rocks. Importance Thermochemical record of the planet and nature of the interior. Objective 2 Assess and interpret the potential biological history of Mars, including assaying returned samples for the evidence of life. Intent To investigate the nature and extent of Martian habitability, the conditions and processes that supported or challenged life, how different environments might have influenced the preservation of biosignatures and created nonbiological “mimics,” and to look for biosignatures of past or present life.This objective has three sub-objectives: 2.1 Assess and characterize carbon, including possible organic and pre-biotic chemistry. Samples All samples collected as part of Objective 1. Importance Any biologic molecular scaffolding on Mars would likely be carbon-based. 2.2 Assay for the presence of biosignatures of past life at sites that hosted habitable environments and could have preserved any biosignatures. Samples All samples collected as part of Objective 1. Importance Provides the means of discovering ancient life. 2.3 Assess the possibility that any life forms detected are alive, or were recently alive. Samples All samples collected as part of Objective 1. Importance Planetary protection, and arguably the most important scientific discovery possible. Objective 3 Quantitatively determine the evolutionary timeline of Mars. Intent To provide a radioisotope-based time scale for major events, including magmatic, tectonic, fluvial, and impact events, and the formation of major sedimentary deposits and geomorphological features. Samples Ancient igneous rocks that bound critical stratigraphic intervals or correlate with crater-dated surfaces. Importance Quantification of Martian geologic history. Objective 4 Constrain the inventory of Martian volatiles as a function of geologic time and determine the ways in which these volatiles have interacted with Mars as a geologic system. Intent To recognize and quantify the major roles that volatiles (in the atmosphere and in the hydrosphere) play in Martian geologic and possibly biologic evolution. Samples Current atmospheric gas, ancient atmospheric gas trapped in older rocks, and minerals that equilibrated with the ancient atmosphere. Importance Key to understanding climate and environmental evolution. Objective 5 Reconstruct the processes that have affected the origin and modification of the interior, including the crust, mantle, core and the evolution of the Martian dynamo. Intent To quantify processes that have shaped the planet's crust and underlying structure, including planetary differentiation, core segregation and state of the magnetic dynamo, and cratering. Samples Igneous, potentially magnetized rocks (both igneous and sedimentary) and impact-generated samples. Importance Elucidate fundamental processes for comparative planetology. Objective 6 Understand and quantify the potential Martian environmental hazards to future human exploration and the terrestrial biosphere. Intent To define and mitigate an array of health risks related to the Martian environment associated with the potential future human exploration of Mars. Samples Fine-grained dust and regolith samples. Importance Key input to planetary protection planning and astronaut health. Objective 7 Evaluate the type and distribution of in-situ resources to support potential future Mars exploration. Intent To quantify the potential for obtaining Martian resources, including use of Martian materials as a source of water for human consumption, fuel production, building fabrication, and agriculture. Samples Regolith. Importance Production of simulants that will facilitate long-term human presence on Mars. Summary of iMOST Findings: Several specific findings were identified during the iMOST study. While they are not explicit recommendations, we suggest that they should serve as guidelines for future decision making regarding planning of potential future MSR missions. The samples to be collected by the Mars 2020 (M-2020) rover will be of sufficient size and quality to address and solve a wide variety of scientific questions. Samples, by definition, are a statistical representation of a larger entity. Our ability to interpret the source geologic units and processes by studying sample sub sets is highly dependent on the quality of the sample context. In the case of the M-2020 samples, the context is expected to be excellent, and at multiple scales. (A) Regional and planetary context will be established by the on-going work of the multi-agency fleet of Mars orbiters. (B) Local context will be established at field area- to outcrop- to hand sample- to hand lens scale using the instruments carried by M-2020. A significant fraction of the value of the MSR sample collection would come from its organization into sample suites, which are small groupings of samples designed to represent key aspects of geologic or geochemical variation. If the Mars 2020 rover acquires a scientifically well-chosen set of samples, with sufficient geological diversity, and if those samples were returned to Earth, then major progress can be expected on all seven of the objectives proposed in this study, regardless of the final choice of landing site. The specifics of which parts of Objective 1 could be achieved would be different at each of the final three candidate landing sites, but some combination of critically important progress could be made at any of them. An aspect of the search for evidence of life is that we do not know in advance how evidence for Martian life would be preserved in the geologic record. In order for the returned samples to be most useful for both understanding geologic processes (Objective 1) and the search for life (Objective 2), the sample collection should contain BOTH typical and unusual samples from the rock units explored. This consideration should be incorporated into sample selection and the design of the suites. The retrieval missions of a MSR campaign should (1) minimize stray magnetic fields to which the samples would be exposed and carry a magnetic witness plate to record exposure, (2) collect and return atmospheric gas sample(s), and (3) collect additional dust and/or regolith sample mass if possible

Listeria pathogenesis and molecular virulence determinants

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research

Cyclic Rhamnosylated Elongation Factor P Establishes Antibiotic Resistance in Pseudomonas aeruginosa

Elongation factor P (EF-P) is a ubiquitous bacterial protein that is required for the synthesis of poly-proline motifs during translation. In Escherichia coli and Salmonella enterica, the posttranslational β-lysylation of Lys34 by the PoxA protein is critical for EF-P activity. PoxA is absent from many bacterial species such as Pseudomonas aeruginosa, prompting a search for alternative EF-P posttranslation modification pathways. Structural analyses of P. aeruginosa EF-P revealed the attachment of a single cyclic rhamnose moiety to an Arg residue at a position equivalent to that at which β-Lys is attached to E. coli EF-P. Analysis of the genomes of organisms that both lack poxA and encode an Arg32-containing EF-P revealed a highly conserved glycosyltransferase (EarP) encoded at a position adjacent to efp. EF-P proteins isolated from P. aeruginosa ΔearP, or from a ΔrmlC::acc1 strain deficient in dTDP-l-rhamnose biosynthesis, were unmodified. In vitro assays confirmed the ability of EarP to use dTDP-l-rhamnose as a substrate for the posttranslational glycosylation of EF-P. The role of rhamnosylated EF-P in translational control was investigated in P. aeruginosa using a Pro(4)-green fluorescent protein (Pro(4)GFP) in vivo reporter assay, and the fluorescence was significantly reduced in Δefp, ΔearP, and ΔrmlC::acc1 strains. ΔrmlC::acc1, ΔearP, and Δefp strains also displayed significant increases in their sensitivities to a range of antibiotics, including ertapenem, polymyxin B, cefotaxim, and piperacillin. Taken together, our findings indicate that posttranslational rhamnosylation of EF-P plays a key role in P. aeruginosa gene expression and survival