Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic <Emphasis Type="Italic">Halomonas</Emphasis> sp. Y2

Na⁺/H⁺ antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na⁺/H⁺ antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na⁺ and Li⁺ translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0–8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na⁺ and Li⁺, while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.     

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, <Emphasis Type="Italic">Bacillus iranensis (</Emphasis>X5B)

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48–50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50 % of activity at 2.5 M NaCl and about 70 % of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca²⁺. The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K _m and V _max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k _cat was obtained as 3.284 × 10^?2 s^?1. These special and important characteristics make this serine protease as valuable tool for industrial applications.     

Potential applications of stress solutes from extremophiles in protein folding diseases and healthcare

Protein misfolding, aggregation and deposition in the brain, in the form of amyloid, are implicated in the etiology of several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and prion diseases. Drugs available on the market reduce the symptoms, but they are not a cure. Therefore, it is urgent to identify promising targets and develop effective drugs. Preservation of protein native conformation and/or inhibition of protein aggregation seem pertinent targets for drug development. Several studies have shown that organic solutes, produced by extremophilic microorganisms in response to osmotic and/or heat stress, prevent denaturation and aggregation of model proteins. Among these stress solutes, mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, diglycerol phosphate and ectoine are effective in preventing amyloid formation by Alzheimer’s Aβ peptide and/or α-synuclein in vitro. Moreover, mannosylglycerate is a potent inhibitor of Aβ and α-synuclein aggregation in living cells, and mannosylglyceramide and ectoine inhibit aggregation and reduce prion peptide-induced toxicity in human cells. This review focuses on the efficacy of stress solutes from hyper/thermophiles and ectoines to prevent amyloid formation in vitro and in vivo and their potential application in drug development against protein misfolding diseases. Current and envisaged applications of these extremolytes in neurodegenerative diseases and healthcare will also be addressed.     

Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea <Emphasis Type="Italic">Photobacterium phosphoreum</Emphasis> ANT-2200

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.     

Diversity of gene cassettes and the abundance of the class 1 integron-integrase gene in sediment polluted by metals

The integron-gene cassette system has typically been associated with antibiotic-resistant pathogens. However, the diversity of gene cassettes and the abundance of class 1 integrons outside of the clinical context are not fully explored. Primers targeting the conserved segments of attC recombination sites were used to amplify gene cassettes from the sediment of the Mina stream, which exhibited a higher degree of stress to metal pollution in the dry season than the rainy season. Of the 143 total analyzed sequences, 101 had no matches to proteins in the database, where cassette open reading frames could be identified by homology with database entries. There was a predominance of sequences encoding essential cellular functions. Each season that was sampled yielded a specific pool of gene cassettes. Real-time PCR revealed that 8.5 and 41.6 % of bacterial cells potentially harbored a class 1 integron in the rainy and dry seasons, respectively. In summary, our findings demonstrate that most of the gene cassettes have no ascribable function and, apparently, historically metal-contaminated sediment favors the maintenance of bacteria containing the intI1 gene. Thus, the diversity of gene cassettes is far from being fully explored deserving further attention.     

Crystal structure of a hypothetical protein,TTHA0829 from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> HB8, composed of cystathionine-β-synthase (CBS) and aspartate-kinase chorismate-mutase tyrA (ACT) domains

TTHA0829 from Thermus thermophilus HB8 has a molecular mass of 22,754 Da and is composed of 210 amino acid residues. The expression of TTHA0829 is remarkably elevated in the latter half of logarithmic growth phase. TTHA0829 can form either a tetrameric or dimeric structure, and main-chain folding provides an N-terminal cystathionine-β-synthase (CBS) domain and a C-terminal aspartate-kinase chorismate-mutase tyrA (ACT) domain. Both CBS and ACT are regulatory domains to which a small ligand molecule can bind. The CBS domain is found in proteins from organisms belonging to all kingdoms and is observed frequently as two or four tandem copies. This domain is considered as a small intracellular module with a regulatory function and is typically found adjacent to the active (or functional) site of several enzymes and integral membrane proteins. The ACT domain comprises four β-strands and two α-helices in a βαββαβ motif typical of intracellular small molecule binding domains that help control metabolism, solute transport and signal transduction. We discuss the possible role of TTHA0829 based on its structure and expression pattern. The results imply that TTHA0829 acts as a cell-stress sensor or a metabolite acceptor.     

Widespread Distribution of <Emphasis Type="Italic">Dehalococcoides mccartyi</Emphasis> in the Houston Ship Channel and Galveston Bay,Texas, Sediments and the Potential for Reductive Dechlorination of PCDD/F in an Estuarine Environment

Sediments in the Houston Ship Channel and upper Galveston Bay, Texas, USA, are polluted with polychlorinated dibenzo-p-dioxins/furans (PCDD/F; ≤46,000 ng/kg dry weight (wt.)) with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener, contributing >50 % of the total toxic equivalents (TEQ) at most locations. We measured PCDD/F concentrations in sediments and evaluated the potential for enhanced in situ biodegradation by surveying for Dehalococcoides mccartyi, an obligate organohalide respiring bacterium. Dehalococcoides spp. (98 % similar to D. mccartyi) and 22 other members of the class Dehalococcoidia were predominant 16S ribosomal RNA (rRNA) phylotypes. Dehalococcoides spp. were also present in the active fraction of the bacterial community. Presence/absence PCR screening detected D. mccartyi in sediment cores and sediment grab samples having at least 1 ng/kg dry wt. TEQ at salinities ranging from 0.6 to 19.5 PSU, indicating that they are widespread in the estuarine environment. Organic carbon-only and organic carbon + sulfate-amended sediment microcosm experiments resulted in ～60 % reduction of ambient 2,3,7,8-TCDD in just 24 months leading to reductions in total TEQs by 38.4 and 45.0 %, respectively, indicating that 2,3,7,8-TCDD degradation is occurring at appreciable rates.     

Identification and Characterization of the Lysine-Rich Matrix Protein Family in <Emphasis Type="Italic">Pinctada fucata</Emphasis>: Indicative of Roles in Shell Formation

Mantle can secret matrix proteins playing key roles in regulating the process of shell formation. The genes encoding lysine-rich matrix proteins (KRMPs) are one of the most highly expressed matrix genes in pearl oysters. However, the expression pattern of KRMPs is limited and the functions of them still remain unknown. In this study, we isolated and identified six new members of lysine-rich matrix proteins, rich in lysine, glycine and tyrosine, and all of them are basic matrix proteins. Combined with four members of the KRMPs previously reported, all these proteins can be divided into three subclasses according to the results of phylogenetic analyses: KRMP1–3 belong to subclass KPI, KRMP4–5 belong to KPII, and KRMP6–10 belong to KPIII. Three subcategories of lysine-rich matrix proteins are highly expressed in the D-phase, the larvae and adult mantle. Lysine-rich matrix proteins are involved in the shell repairing process and associated with the formation of the shell and pearl. What’s more, they can cause abnormal shell growth after RNA interference. In detail, KPI subgroup was critical for the beginning formation of the prismatic layer; both KPII and KPIII subgroups participated in the formation of prismatic layer and nacreous layer. Compared with different temperatures and salinity stimulation treatments, the influence of changes in pH on KRMPs gene expression was the greatest. Recombinant KRMP7 significantly inhibited CaCO₃ precipitation, changed the morphology of calcite, and inhibited the growth of aragonite in vitro. Our results are beneficial to understand the functions of the KRMP genes during shell formation.     

Drivers of <Emphasis Type="Italic">Bromus tectorum</Emphasis> Abundance in the Western North American Sagebrush Steppe

Bromus tectorum can transform ecosystems causing negative impacts on the ecological and economic values of sagebrush steppe of the western USA. Although our knowledge of the drivers of the regional distribution of B. tectorum has improved, we have yet to determine the relative importance of climate and local factors causing B. tectorum abundance and impact. To address this, we sampled 555 sites distributed geographically and ecologically throughout the sagebrush steppe. We recorded the canopy cover of B. tectorum, as well as local substrate and vegetation characteristics. Boosted regression tree modeling revealed that climate strongly limits the transformative ability of B. tectorum to a portion of the sagebrush steppe with dry summers (that is, July precipitation <10 mm and the driest annual quarter associated with a mean temperature >15°C) and low native grass canopy cover. This portion includes the Bonneville, Columbia, Lahontan, and lower Snake River basins. These areas are likely to require extreme efforts to reverse B. tectorum transformation. Our predictions, using future climate conditions, suggest that the transformative ability of B. tectorum may not expand geographically and could remain within the same climatically suitable basins. We found B. tectorum in locally disturbed areas within or adjacent to all of our sample sites, but not necessarily within sagebrush steppe vegetation. Conversion of the sagebrush steppe by B. tectorum, therefore, is more likely to occur outside the confines of its current climatically optimal region because of site-specific disturbances, including invasive species control efforts and sagebrush steppe mismanagement, rather than climate change.     

Biodegradability of Vegetation-Derived Dissolved Organic Carbon in a Cool Temperate Ombrotrophic Bog

Dissolved organic carbon (DOC) plays a key role in the peatland carbon balance and serves numerous ecological and chemical functions including acting as a microbial substrate. In this study, we quantify the concentration, biodegradability, and intrinsic properties of DOC obtained from peat, fresh material, and litter from nine species of ombrotrophic bog vegetation. Potential biodegradability was assessed by incubating vegetation extracts for 28 days in the dark and measuring percent DOC loss as the fraction of biodegradable DOC (%BDOC) while DOC properties were characterized using UV–Vis absorbance and fluorescence measurements. The mean initial DOC concentration extracted differed significantly among species (P < 0.05) and was significantly higher in fresh material, 217 ± 259 mg DOC l^?1, than either litter or peat extracts with mean concentrations of 82.1 ± 117 mg DOC l^?1 and 12.7 ± 1.0 mg DOC l^?1, respectively (P < 0.05). %BDOC also differed significantly among species (P < 0.05) and ranged from 52 to 73% in fresh cuttings with the greatest fraction observed in S. magellanicum; 22–46% in litter; and 24% in peat. The majority of variability (82.5%) in BDOC was explained by initial absorbance at 254 nm and total dissolved nitrogen concentration which was further resolved into significant non-linear relationships between %BDOC and both humic-like and protein-like DOC fractions (P < 0.05). Our results highlight the extremely heterogeneous nature of the surface vegetation-derived DOC input in peatlands and stress the importance of vegetation species in peatland ecosystem function.