Exploring the functional soil-microbe interface and exoenzymes through soil metaexoproteomics

Functionally important proteins at the interface of cell and soil are of potentially low abundance when compared with commonly recovered intracellular proteins. A novel approach was developed and used to extract the metaexoproteome, the subset of proteins found outside the cell, in the context of a soil enriched with the nitrogen-containing recalcitrant polymer chitin. The majority of proteins recovered was of bacterial origin and localized to the outer membrane or extracellular milieu. A wide variety of transporter proteins were identified, particularly those associated with amino-acid and phosphate uptake. The metaexoproteome extract retained chitinolytic activity and we were successful in detecting Nocardiopsis-like chitinases that correlated with the glycoside hydrolase family 18 (GH18) chi gene data and metataxonomic analysis. Nocardiopsis-like chitinases appeared to be solely responsible for chitinolytic activity in soil. This is the first study to detect and sequence bacterial exoenzymes with proven activity in the soil enzyme pool.Metaproteomics is an emerging technique for directly assessing cellular function and interactions within an environment. In complex environments such as soil, there is a vast dynamic range of microbial species abundance and protein expression levels. Data acquisition is biased towards high-abundance proteins, for example, chaperonins, ribosomal proteins, elongation factors and ATP synthases (Benndorf et al., 2007; Dill et al., 2010). Removal of these intracellular proteins could allow access to functionally important low-abundance proteins in the soil enzyme pool and at the interface of cell and soil, the soil metaexoproteome.Chitin provides one of the dominant sources of organic nitrogen in soil (Gooday, 1990) and chitinases are implicated in its mineralization in a wide range of contexts (Rhazi et al., 2000; Muzzarelli, 2011), especially in nitrogen-poor soils (Olander and Vitousek, 2000). The molecular diversity of chitinases in soil microbial communities has been studied (Williamson et al., 2000; Metcalfe et al., 2002; Hjort et al., 2010) but very few have focused on the functional contributions of members of the chitinolytic bacterial community. We report here the first attempt to recover and analyse extracellular proteins in soil adopting a novel approach to extract the metaexoproteome. Our data indicate that one actinobacterial group was disproportionately responsible for chitin breakdown.Soil was sampled from an island off the north coast of Cuba known for its high biodiversity and wide range of chitinolytic bacteria (Williamson et al., 2000; Williamson, 2001). Microcosms were constructed and amended with 1% crude crab shell (α-chitin) or squid pen (β-chitin) to enrich the microbial community, an unamended control was included for the 16S rRNA gene metataxonomic analysis (Supplementary Method S1). Community DNA was extracted and sequenced on a 454 GS FLX instrument with titanium reagents (Roche, Basel, Switzerland) using eubacterial primers Gray28F and Gray519R (Dowd et al., 2008) and GH18 Group A chi primers GASQF and GASQR (Williamson et al., 2000); the data were analysed with the bioinformatics package QIIME (Caporaso et al., 2010) (Supplementary Method S2). The metaexoproteome extraction is a modification of Masciandaro et al. (2008). In brief, 100 g soil was gently agitated with a K₂SO₄-based extraction solution (1:3 w/v) then the solid fraction and cells removed by centrifugation and filter sterilization before dilution (3:1 v/v) with 18.2 MΩ cm water and dialysis overnight. The retentate was concentrated to a final volume of ∼1 ml by ultrafiltration and using a centrifugal concentrator for direct loading onto a one-dimensional SDS–polyacrylamide gel electrophoresis gel. Gel-dependent nanoflow liquid chromatography-tandem MS (nanoLC-MS/MS) analysis was performed and the resultant Micromass peak list files interrogated with the NCBInr database using the MASCOT search engine (Matrix Science, London, UK). The full list of proteins was filtered to remove the few eukaryotic proteins and hits with <2 significant unique peptides (Supplementary Methods S3 and S4).To successfully target the metaexoproteome, cell integrity must be maintained. Minimal cell lysis during the extraction was demonstrated experimentally by spiking soil with Escherichia coli overexpressing His-tagged phosphoribosyl isomerase A in the cytoplasm and attempting to detect the His-tag in the extract by western blot (Supplementary Method S5), as no protein was detected we believe the method did not lyse cells. The majority of 52 recovered proteins were Gram-negative in origin and attributed to the extracellular fraction or outer membrane (Supplementary Tables S1 and S2). Across both amendments, 73% of proteins were predicted to have a signal peptide (Nielsen et al., 1997), 13% to have transmembrane helices (Sonnhammer et al., 1998; Krogh et al., 2001), 17% to be TRAP transporters and 52% to be ABC transporters. These features are suggestive of export or being membrane bound and indicate that the metaexoproteome is representing the functional interface between cell and environment. In vitro secretomes commonly feature a similar range of TRAP and ABC transporters in addition to selected extracellular enzymes depending on the enrichment (Adav et al., 2010; Christie-Oleza and Armengaud, 2010; Christie-Oleza et al., 2012). The only extracellular enzymes identified were chitinases.Recovered proteins were affiliated with three phyla, Proteobacteria, Actinobacteria and Bacteroidetes, this correlated well with the 16S rRNA gene data set (Figure 1). Only two genera dominated the metaexoproteome, both in terms of number of proteins recovered and protein abundance measured by emPAI (Ishihama et al., 2005), the actinomycete Nocardiopsis and the rhizobiale Nitratireductor. Approximately 17% of the identified proteins were matched to Nocardiopsis. The family Nocardiopsaceae was undetected in the unamended 16S rRNA gene data set but was one of the few actinobacterial groups to increase in abundance with α-chitin amendment, accounting for 3.7% of the community.Open in a separate windowFigure 1A visual summary of the assigned bacterial community structure, recovered metaexoproteome community and GH18 chi gene taxonomic matches for the combined α- and β-chitin-amended soil. For clarity, low-abundance taxa have been grouped under ‘Other'' and for the GH18 chi gene pie chart Stenotrophomonas, Amycolatopsis and Verrucosispora are not labelled as each account for <0.06% of their respective class segment.The majority of proteins were related to the transport and metabolism of amino acids, carbohydrates and inorganic ions, namely phosphate and phosphonate. Two GH18 chitinases were identified by peptides from within their catalytic domains, ChiA from Nocardiopsis lucentensis and N. dassonvillei (Supplementary Table S1). Corresponding Nocardiopsis chiA-like sequences were identified in the GH18 chi gene pyrosequencing data set (Figure 1). Nocardiopsis chitinases have been shown to have chitinolytic activity against α- and β-chitin (Tsujibo et al., 2003) and to be capable of fast and complete degradation of crystalline chitin in liquid media (Sorokin et al., 2012).A fluorogenic chitinase assay (Sigma-Aldrich, St Louis, MO, USA) was performed on the extracts from α-chitin-amended microcosm soil and metaexoproteome (Supplementary Method S6). Both extracts showed activity against the monomeric substrate but the metaexoproteome extract had proportionally higher activity against the more representative di-NAG and tri-NAG substrates. It is probable that the chitinase activity detected in the metaexoproteome extract is attributable to the Nocardiopsis chiA-like chitinases detected in the sequenced aliquot of the extract and represents the first example of an active exoenzyme extracted, assayed and sequenced from a soil.The efficiency of mass spectrometry via in-gel digestion would preclude recovery of low-abundance peptides. Nocardiopsis-like proteins must therefore contribute disproportionately to the functional activity of the soil and thus the degradation of chitin. This is in marked contrast to the prevalence data for 16S rRNA gene analysis and GH18 chi gene analysis. Despite numerous attempts it was not possible to cultivate Nocardiopsis-like strains directly from the soil.     

New trends in assessing the outcomes of mental health interventions

Assessing the outcomes of interventions in mental health care is both important and challenging. The aim of this paper is to advance the field of outcomes research by proposing a taxonomy of the decisions that clinicians and researchers need to consider when evaluating outcomes. Our taxonomy has eight components, framed as decisions: Whose outcome will be considered? Which scientific stage is being investigated? What outcome domain(s) matter? What level of assessment will be used? Will clinical and/or recovery outcomes be assessed? Whose perspective will be considered? Will deficits and/or strengths be the focus? Will invariant or individualized measures be preferred? We propose a future focus on understanding what matters most to people using mental health services, and on the use of measures rated by service users as the primary approach to evaluating outcome.     

Quantitative analysis of the postcontractile blood-oxygenation-level-dependent (BOLD) effect in skeletal muscle

Previous studies show that transient increases in both blood flow and magnetic resonance image signal intensity (SI) occur in human muscle after brief, single contractions, and that the SI increases are threefold larger in physically active compared with sedentary subjects. This study examined the relationship between these transient changes by measuring anterior tibial artery flow (Doppler ultrasound), anterior muscle SI (3T, one-shot echo-planar images, TR/TE = 1,000/35), and muscle blood volume and hemoglobin saturation [near-infrared spectroscopy (NIRS)] in the same subjects after 1-s-duration maximum isometric ankle dorsiflexion contractions. Arterial flow increased to a peak 5.9 ± 0.7-fold above rest (SE, n = 11, range 2.6-10.2) within 7 s and muscle SI increased to a peak 2.7 ± 0.6% (range 0.0-6.0%) above rest within 12 s after the contractions. The peak postcontractile SI change was significantly correlated with both peak postcontractile flow (r = 0.61, n = 11) and with subject activity level (r = 0.63, n = 10) estimated from 7-day accelerometer recordings. In a subset of 7 subjects in which NIRS data acquisition was successful, the peak magnitude of the postcontractile SI change agreed well with SI calculated from the NIRS blood volume and saturation changes (r = 0.80, slope = 1.02, intercept = 0.16), confirming the blood-oxygenation-level-dependent (BOLD) mechanism underlying the SI change. The magnitudes of postcontractile changes in blood saturation and SI were reproduced by a simple one-compartment muscle vascular model that incorporated the observed pattern of postcontractile flow, and which assumed muscle O(2) consumption peaks within 2 s after a brief contraction. The results show that muscle postcontractile BOLD SI changes depend critically on the balance between O(2) delivery and O(2) consumption, both of which can be altered by chronic physical activity.     

Peripheral microvascular response to muscle contraction is unaltered by early diabetes but decreases with age

Long-term or untreated diabetes leads to micro- and macrovascular complications. However, there are few tests to evaluate microvascular function. A postcontraction blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) technique was exploited to measure peripheral microvascular function in diabetics and healthy controls matched with respect to age, body mass index, and physical activity. Postcontraction BOLD microvascular response was measured following 1-s maximal isometric ankle dorsiflexion in individuals with diabetes mellitus type I [DMI, n = 15, age 33 ± 3 yr (means ± SE), median diabetes duration = 5.5 yr] and type II (DMII, n = 16, age 45 ± 2 yr, median duration = 2.4 yr); responses were compared with controls (CONI and CONII). Peripheral macrovascular function of the popliteal and tibial arteries was assessed during exercise hyperemia with phase contrast magnetic resonance angiography following repetitive exercise. There were no group differences as a result of diabetes in peripheral microvascular function (peak BOLD response: DMI = 2.04 ± 0.38% vs. CONI = 2.08 ± 0.48%; DMII = 0.93 ± 0.24% vs. CONII = 1.13 ± 0.24%; mean ± SE), but the BOLD response was significantly influenced by age (partial r = -0.384, P = 0.003), supporting its sensitivity as a measure of microvascular function. Eleven individuals had no microvascular BOLD response, including three diabetics with neuropathy and four controls with a family history of diabetes. There were no differences in peripheral macrovascular function between groups when assessing exercise hyperemia or the pulsitility and resistive indexes. Although the BOLD microvascular response was not impaired in early diabetes, these results encourage further investigation of muscle BOLD as it relates to peripheral microvascular health.     

Identification of macrodomain proteins as novel O-acetyl-ADP-ribose deacetylases

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD(+) to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD(+)-dependent deacetylation.     

A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.     

A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Thermostable proteins are advantageous in industrial applications, as pharmaceuticals or biosensors, and as templates for directed evolution. As protein-design methodologies improve, bioengineers are able to design proteins to perform a desired function. Although many rationally designed proteins end up being thermostable, how to intentionally design de novo, thermostable proteins is less clear. UVF is a de novo-designed protein based on the backbone structure of the Engrailed homeodomain (EnHD) and is highly thermostable (T_m > 99°C vs. 52°C for EnHD). Although most proteins generally have polar amino acids on their surfaces and hydrophobic amino acids buried in their cores, protein engineers followed this rule exactly when designing UVF. To investigate the contributions of the fully hydrophobic core versus the fully polar surface to UVF’s thermostability, we built two hybrid, chimeric proteins combining the sets of buried and surface residues from UVF and EnHD. Here, we determined a structural, dynamic, and thermodynamic explanation for UVF’s thermostability by performing 4 μs of all-atom, explicit-solvent molecular dynamics simulations at 25 and 100°C, Tanford-Kirkwood solvent accessibility Monte Carlo electrostatic calculations, and a thermodynamic analysis of 40 temperature runs by the weighted-histogram analysis method of heavy-atom, structure-based models of UVF, EnHD, and both chimeric proteins. Our models showed that UVF was highly dynamic because of its fully hydrophobic core, leading to a smaller loss of entropy upon folding. The charged residues on its surface made favorable electrostatic interactions that contributed enthalpically to its thermostability. In the chimeric proteins, both the hydrophobic core and charged surface independently imparted thermostability.