Phylogenetically Driven Sequencing of Extremely Halophilic Archaea Reveals Strategies for Static and Dynamic Osmo-response

© 2014. Organisms across the tree of life use a variety of mechanisms to respond to stress-inducing fluctuations in osmotic conditions. Cellular response mechanisms and phenotypes associated with osmoadaptation also play important roles in bacterial virulence, human health, agricultural production and many other biological systems. To improve understanding of osmoadaptive strategies, we have generated 59 high-quality draft genomes for the haloarchaea (a euryarchaeal clade whose members thrive in hypersaline environments and routinely experience drastic changes in environmental salinity) and analyzed these new genomes in combination with those from 21 previously sequenced haloarchaeal isolates. We propose a generalized model for haloarchaeal management of cytoplasmic osmolarity in response to osmotic shifts, where potassium accumulation and sodium expulsion during osmotic upshock are accomplished via secondary transport using the proton gradient as an energy source, and potassium loss during downshock is via a combination of secondary transport and non-specific ion loss through mechanosensitive channels. We also propose new mechanisms for magnesium and chloride accumulation. We describe the expansion and differentiation of haloarchaeal general transcription factor families, including two novel expansions of the TATA-binding protein family, and discuss their potential for enabling rapid adaptation to environmental fluxes. We challenge a recent high-profile proposal regarding the evolutionary origins of the haloarchaea by showing that inclusion of additional genomes significantly reduces support for a proposed large-scale horizontal gene transfer into the ancestral haloarchaeon from the bacterial domain. The combination of broad (17 genera) and deep (≥5 species in four genera) sampling of a phenotypically unified clade has enabled us to uncover both highly conserved and specialized features of osmoadaptation. Finally, we demonstrate the broad utility of such datasets, for metagenomics, improvements to automated gene annotation and investigations of evolutionary processes

A prospective comparative evaluation of persistent respiratory morbidity in esophageal atresia and congenital diaphragmatic hernia survivors

__Purpose:__ The aim of the study was to compare long-term respiratory morbidity in children after repair of esophageal atresia (EA) or congenital diaphragmatic hernia (CDH). __Patients and Methods:__ Children were seen at 6, 12, and 24 months and 5 years within a prospective longitudinal follow-up program in a tertiary children's hospital. Respiratory morbidity and physical condition were evaluated at all moments. At age 5 years, pulmonary function and maximal exercise performance were tested. __Results:__ In 3 of 23 atresia patients and 10 of 20 hernia patients, bronchopulmonary dysplasia was developed. Seventeen atresia and 11 hernia patients had recurrent respiratory tract infections mainly in the first years of life. At age 5, 25% of EA and CDH patients measured showed reduced forced expiratory volume in 1 second (z-score < -2). Both atresia and hernia patients showed impaired growth, with catch-up growth at 5 years in patients with EA but not in those with hernia. Maximal exercise performance was significantly below normal for both groups

Structural Basis for the Aminoacid Composition of Proteins from Halophilic Archea

In order to survive in highly saline environments, proteins from halophilic archea have evolved with biased amino acid compositions that have the capacity to reduce contacts with the solvent

Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

Background: Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings: A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance: The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity

Functional Insight into the C-Terminal Extension of Halolysin SptA from Haloarchaeon Natrinema sp. J7

Halolysin SptA from haloarchaeon Natrinema sp. J7 consists of a subtilisin-like catalytic domain and a C-terminal extension (CTE) containing two cysteine residues. In this report, we have investigated the function of the CTE using recombinant enzymes expressed in Haloferax volcanii WFD11. Deletion of the CTE greatly reduced but did not abolish protease activity, which suggests that the CTE is not essential for enzyme folding. Mutational analysis suggests that residues Cys303 and Cys338 within the CTE form a disulfide bond that make this domain resistant to autocleavage and proteolysis under hypotonic conditions. Characterization of full-length and CTE-truncation enzymes indicates the CTE not only confers extra stability to the enzyme but also assists enzyme activity on protein substrates by facilitating binding at high salinities. Interestingly, homology modeling of the CTE yields a β-jelly roll-like structure similar to those seen in Claudin-binding domain of Clostridium perfringens enterotoxin (clostridial C-CPE) and collagen binding domain (CBD), and the CTE also possesses collagen-binding activity, making it a potential candidate as an anchoring unit in drug delivery systems