Proteorhodopsin-Bearing Bacteria in Antarctic Sea Ice

Proteorhodopsins (PRs) are widespread bacterial integral membrane proteins that function as light-driven proton pumps. Antarctic sea ice supports a complex community of autotrophic algae, heterotrophic bacteria, viruses, and protists that are an important food source for higher trophic levels in ice-covered regions of the Southern Ocean. Here, we present the first report of PR-bearing bacteria, both dormant and active, in Antarctic sea ice from a series of sites in the Ross Sea using gene-specific primers. Positive PR sequences were generated from genomic DNA at all depths in sea ice, and these sequences aligned with the classes Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. The sequences showed some similarity to previously reported PR sequences, although most of the sequences were generally distinct. Positive PR sequences were also observed from cDNA reverse transcribed from RNA isolated from sea ice samples. This finding indicates that these sequences were generated from metabolically active cells and suggests that the PR gene is functional within sea ice. Both blue-absorbing and green-absorbing forms of PRs were detected, and only a limited number of blue-absorbing forms were found and were in the midsection of the sea ice profile in this study. Questions still remain regarding the protein''s ecological functions, and ultimately, field experiments will be needed to establish the ecological and functional role of PRs in the sea ice ecosystem.Proteorhodopsins (PRs) are retinal binding bacterial integral membrane proteins that function as light-driven proton pumps (9, 10) and belong to the microbial rhodopsin superfamily of proteins (54). Since the first reported PR sequence from members of SAR86 clade marine (class Gammaproteobacteria) in 2000 (9), many other PR-bearing bacteria have been identified in a range of marine habitats (5, 18, 20, 24, 25, 46, 62). In the recent Global Ocean Sampling (GOS) expedition, almost 4,000 PR sequences from 41 distinct surface marine environments were acquired, demonstrating that these PR genes are extremely abundant in the genomes of ocean bacterioplankton (46). In fact, PR-containing bacteria account for 13% of the community in the Mediterranean Sea and Red Sea and 70% of the community in the Sargasso Sea (18, 46, 49, 60). These light-harvesting bacteria are present in three major marine classes of bacteria: the Alphaproteobacteria, Gammaproteobacteria, and Flavobacteria. In addition, two distinct PR genes encode pigments with “blue-absorbing” and “green-absorbing” properties, which is achieved by a substitution at a single amino acid position, which thereby functions as a spectral tuning switch (10, 37, 48).Sea ice represents a complex physicochemical environment in polar regions and covers up to 13% of the Earth''s surface (59). Although extreme gradients of temperature, salinity, nutrient availability, and light stratify the ice matrix from the surface to the ice-water interface (41), the sea ice habitat nevertheless supports a diverse microbial community of phytoplankton, Bacteria, Archaea, viruses, and protists that grow in liquid brine channels within the ice (14, 35, 56). This sea ice microbial community (SIMCO) is highly metabolically active despite being unable to avoid the extreme environmental conditions that they experience (39). In fact, very-high-standing stocks of the SIMCO exist in many regions of the Southern Ocean. For example, the concentration of chlorophyll a, a proxy for microalgal biomass, typically reaches 200 mg m⁻² in the Ross Sea, while the concentration of chlorophyll a in the water column below is approximately 2 orders of magnitude less (47), and the percentage of metabolically active bacteria (32% [39]) is significantly higher than the 10% observed for temperate marine systems (36). The SIMCO is thus a major source of biomass in ice-covered regions of the Southern Ocean (59), providing a critical food source for grazing zooplankton (and, consequently, also for higher trophic levels) for much of the year (3, 59). This biomass is of particular importance during the darkness of the polar winter, where the bottom-ice community is the only available food source for juvenile krill. These grazers absolutely rely on the sea ice microbial community to survive, as the water lacks other food sources (6, 28).In the past decade, reports of the widespread occurrence of bacteriochlorophyll and PR pigments in planktonic marine bacteria have challenged the assumption that chlorophyll a is the only principal light-capturing pigment in ocean surface waters. These alternative pigments may in fact play a critical role in light energy harvesting for microbial metabolism in various aquatic ecosystems (5, 10, 25, 40, 49). It has been proposed that energy, rather than nutrient conservation, is important for the regulation of productivity (7). PR-containing phototrophic eubacteria could play a significant role in the energy budget of cells in the photic zone in marine environments (15). PR sequences have been detected in the Southern Ocean (9), but to our knowledge, there have been no reports of PR-bearing bacteria within the sea ice matrix.The majority of the microbial rhodopsin genes found in oceanic samples have been detected by environmental sequencing (30, 46, 48, 60). We have used degenerate PR gene primers (5) in this study to positively identify PR-bearing operational taxonomic units (OTUs) from sea ice. Also, specific bacterial mRNA can now be detected from extracted nucleic acids and used to examine gene expression and, thus, infer metabolic activity (8). With this in mind, we have generated cDNA from RNA extracted from sea ice samples. From these observations, we deduce that PR-bearing bacteria are present in sea ice and may be actively contributing to the ecosystem within this extreme microenvironment.     

Use of amplicon-6 vectors derived from human herpesvirus 6 for efficient expression of membrane-associated and -secreted proteins in T cells

The composite amplicon-6 vectors, which are derived from human herpesvirus 6 (HHV-6), can target hematopoietic cells. In the presence of the respective helper viruses, the amplicons are replicated by the rolling circle mechanism, yielding defective genomes of overall size 135 to 150 kb, composed of multiple repeats of units, containing the viral DNA replication origin, packaging signals, and the selected transgene(s). We report the use of amplicon-6 vectors designed for transgene expression in T cells. The selected transgenes included the green fluorescent protein marker, the herpes simplex virus type 1 glycoprotein D (gD), and the gD gene deleted in the transmembrane region (gDsec). The vectors were tested after electroporation and passage in T cells with or without helper HHV-6A superinfections. The results were as follows. (i)The vectors could be passaged both as cell-associated and as cell-free secreted virions infectious to new cells. (ii)The intact gD accumulated at the cell surface, whereas the gDsec was dispersed at internal locations of the cells or was secreted into the medium. (iii)Analyses of amplicon-6-gD expression by flow cytometry have shown significant expression in cultures with reiterated amplicons and helper viruses. The vector has spread to >60% of the cells, and the efficiency of expression per cell increased 15-fold, most likely due to the presence of concatemeric amplicon repeats. Current studies are designed to test whether amplicon-6 vectors can be used for gene therapy in lymphocytes and whether amplicon-6 vectors expressed in T cells and dendritic cells can induce strong cellular and humoral immune responses.     

Titratable transmembrane residues and a hydrophobic plug are essential for manganese import via the Bacillus anthracis ABC transporter MntBC-A

All extant life forms require trace transition metals (e.g., Fe^2/3+, Cu^1/2+, and Mn²⁺) to survive. However, as these are environmentally scarce, organisms have evolved sophisticated metal uptake machineries. In bacteria, high-affinity import of transition metals is predominantly mediated by ABC transporters. During bacterial infection, sequestration of metal by the host further limits the availability of these ions, and accordingly, bacterial ABC transporters (importers) of metals are key virulence determinants. However, the structure–function relationships of these metal transporters have not been fully elucidated. Here, we used metal-sensitivity assays, advanced structural modeling, and enzymatic assays to study the ABC transporter MntBC-A, a virulence determinant of the bacterial human pathogen Bacillus anthracis. We find that despite its broad metal-recognition profile, MntBC-A imports only manganese, whereas zinc can function as a high-affinity inhibitor of MntBC-A. Computational analysis shows that the transmembrane metal permeation pathway is lined with six titratable residues that can coordinate the positively charged metal, and mutagenesis studies show that they are essential for manganese transport. Modeling suggests that access to these titratable residues is blocked by a ladder of hydrophobic residues, and ATP-driven conformational changes open and close this hydrophobic seal to permit metal binding and release. The conservation of this arrangement of titratable and hydrophobic residues among ABC transporters of transition metals suggests a common mechanism. These findings advance our understanding of transmembrane metal recognition and permeation and may aid the design and development of novel antibacterial agents.     

A novel lipid binding site formed by the MAP kinase insert in p38 alpha

The p38 mitogen-activated protein (MAP) kinases function as signaling molecules essential for many cellular processes, particularly mediating stress response. The activity of p38 MAP kinases is meticulously regulated to reach the desired cellular phenotype. Several alternative activation and attenuation mechanisms have been characterized recently which include new phosphorylation sites. Here we present the crystal structure of p38α MAP kinase in complex with n-octyl-β-glucopyranoside detergent. The complex unveils a novel lipid-binding site formed by a local conformational change of the MAP kinase insert. This binding is the first attribution for a possible role of the MAP kinase insert in p38. The binding site can accommodate a large selection of lipidic molecules. In addition, we also show via biophysical methods that arachidonic acid and its derivatives bind p38α in vitro. Based on our analysis we propose that the binding of lipids could fine-tune p38α catalytic activity towards a preferred phenotype.     

Comparative analyses of actinobacterial genomic fragments from Lake Kinneret

The high genomic G+C group of Actinobacteria possesses a variety of physiological and metabolic properties, and exhibits diverse lifestyles and ecological distribution. In recent years, Actinobacteria have been found to frequently dominate samples obtained from freshwater samples. Furthermore, phylogenetic analyses have shown that 16S rRNA genes from uncultured actinobacterial freshwater samples cluster in four distinct lineages. While these lineages are abundant, little is known about them and currently no pure‐culture representatives or genomic fragments of them are available. In a screen of a genomic library from the moderately eutrophic freshwater Lake Kinneret, five fosmid clones containing actinobacterial genomic fragments were found. Three ～40 kb genomic fragments were chosen for sequencing. Fosmids K003 and K005 showed high similarity and were affiliated with the acIV actinobacterial freshwater lineage. Fosmid K004 was affiliated with the highly abundant acI lineage. A comparative genomic analysis revealed high synteny between the two freshwater clones K003 and K005 but a lower synteny between these two and the K004 fosmid. Fosmids K003 and K005 share an identical arrangement of arginine biosynthesis gene while K004 showed a slightly different arrangement by lacking the argF gene. Fosmid Ant4E12, an Antarctic actinobacterial clone, showed a higher synteny with K003/5 than K004 and a similar arginine operon, but in a different genomic context. The Clusters of Orthologous Groups categories assignment of the three fosmids yielded genes that were mostly involved in amino acid and nucleotide metabolism, as well as transport and ribosomal RNA translation, structure and biogenesis. These genomic fragments represent the first sequences to be published from these lineages, providing a cornerstone for future work on this environmentally dominant group.     

A Contemplative Biofeedback Intervention for Adults with Autism Spectrum Disorder: Feasibility of a Community-Based Treatment

Applied Psychophysiology and Biofeedback - As the number of diagnosed adults living with autism spectrum disorder (ASD) continues to grow, a lack of resources and lack of available interventions...     

Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation

The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk’s activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation.     

Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates

Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria.     

The Butyrylcholinesterase K Variant Confers Structurally Derived Risks for Alzheimer Pathology?

The K variant of butyrylcholinesterase (BChE-K, 20% incidence) is a long debated risk factor for Alzheimer disease (AD). The A539T substitution in BChE-K is located at the C terminus, which is essential both for BChE tetramerization and for its capacity to attenuate β-amyloid (Aβ) fibril formation. Here, we report that BChE-K is inherently unstable as compared with the “usual” BChE (BChE-U), resulting in reduced hydrolytic activity and predicting prolonged acetylcholine maintenance and protection from AD. A synthetic peptide derived from the C terminus of BChE-K (BSP-K), which displayed impaired intermolecular interactions, was less potent in suppressing Aβ oligomerization than its BSP-U counterpart. Correspondingly, highly purified recombinant human rBChE-U monomers suppressed β-amyloid fibril formation less effectively than dimers, which also protected cultured neuroblastoma cells from Aβ neurotoxicity. Dual activity structurally derived changes due to the A539T substitution can thus account for both neuroprotective characteristics caused by sustained acetylcholine levels and elevated AD risk due to inefficient interference with amyloidogenic processes.Butyrylcholinesterase (BChE),³ the secondary acetylcholine (ACh)-hydrolyzing enzyme, is associated with the neurofibrillary tangles and amyloid plaques characteristic of Alzheimer disease (AD) (1), which suggests that it functions as a potential AD modulator. BChE activity increases in the AD brain (2–4), where it co-localizes with β-amyloid (Aβ) fibrils (5, 6). Aβ is a 39–42-amino-acid amphiphilic peptide, derived from the transmembrane domain and extracellular region of the Aβ precursor protein (7). At high concentrations, Aβ acquires a β-sheet structure, becomes insoluble, and accumulates in neurotoxic oligomers and fibrils (8) to become the main constituent of plaques in the brain of AD patients. Recent hypotheses attribute causal roles in AD to presenilin (9), oxidative stress (10), metals (11), double hit origin (12), or mitochondrial damage (13). The alternative theories state that Aβ represents a bystander or even a protector rather than the causative factor of disease and that Aβ amyloidogenesis is secondary to other pathogenic events (14). Nevertheless, a wealth of evidence demonstrates a pivotal role for Aβ in the pathogenesis of AD, yielding the amyloid cascade hypothesis (15). According to this hypothesis, the pathological accumulation of Aβ in the brain leads to oxidative stress, neuronal destruction, and finally, the clinical syndrome of AD. It is within this context that we have studied the interactions of the Kalow variant (BChE-K) with Aβ.The C terminus of BChE functions as a tetramerization domain (16, 17) and is responsible for its quaternary organization. Four BChE monomers are held together by the aromatic interactions of seven highly conserved aromatic residues, termed the tryptophan amphiphilic tetramerization domain (WAT) (16, 17). The WAT domain interacts with proline-rich attachment domains, either via proline-rich membrane anchor in brain neurons (18) or, in neuromuscular junctions, with cholinesterase-associated collagen Q (19). In the serum, BChE tetramerization is supported by an analogous 17-mer proline-rich peptide derived from lamellipodin (20).Analyzing the quaternary organization of cholinesterases is a complicated task. To date, all biologically relevant crystal structures of cholinesterases have been truncated forms that lack the C terminus of the protein (21), apart from a more recent study of full-length BChE that yielded crystal packing, which did not allow C-terminal interactions among subunits and lacked electron densities in the C terminus region, indicating structural disorder Protein Data Bank (PDB) code 1VZJ (22). Of note, the crystal structure of the homologous C terminus of tetrameric synaptic acetylcholinesterase (AChE-S) could only be determined based on synthetic peptides derived from the sequence of the AChE-S tail and stabilized with a proline-rich attachment domain (23).In addition to the “usual” (BChE-U) form, BChE has nearly 40 genomic variants. The most common is BChE-K, with allelic frequencies of 0.13–0.21. BChE-K includes a single nucleotide polymorphism at position 1699 (single nucleotide polymorphism data base (dbSNP) ID: rs1803274; alleles, A/G). This leads to an alanine-to-threonine substitution at position 539, 36 residues upstream to the C terminus of BChE (24), within the tetramerization domain that we previously found to attenuate amyloid fibril formation (25).Ample evidence supports the importance of alanine-to-threonine substitutions and their relevance to amyloidogenic processes, protein stability, and quaternary organization (supplemental Table ST1). Point mutations at the dimer interface of light chain immunoglobulins decrease their stability so that the A34T polymorphism in this protein leads to systemic amyloidosis (26). An A25T mutant of the tetrameric human protein Transthyretin (TTR), associated with central nervous system amyloidosis, is prone to aggregation and exhibits drastically reduced tertiary and quaternary structural stabilities (27). The thermodynamic stability profile of the A25T TTR mutant shows that both monomers and tetramers of this variant are highly destabilized. In addition, A25T TTR tetramers dissociate very rapidly (about 1200-fold faster than the dissociation of wild-type TTR), reflecting a high degree of kinetic destabilization of their quaternary structure. These factors together probably contribute to the high propensity of A25T TTR to aggregate in vitro.The capacity of serum BChE-K to hydrolyze butyrylthiocholine was reported to be reduced by 30% relative to BChE-U, for yet unclear reasons (24). The reduced hydrolytic activity of BChE-K predicts that BChE-K carriers would potentially sustain improved cholinergic transmission as compared with BChE-U carriers and has been shown to correlate with preserved performance of attention and reduced rates of cognitive decline (28). However, BChE-K carriers are refractory to cholinesterase inhibitor therapy, the current leading treatment of AD (29). This raised the question whether BChE-K functions as an AD risk or protection factor. Genotype studies are controversial, with some showing increased risk of AD for homozygote BChE-K carriers (e.g. Ref. 30), whereas others suggest a protective effect (e.g. Ref. 31). A recent meta-analysis concluded that on average, BChE-K is neither a risk factor nor a protection factor for AD (32). Based on our previous findings of the arrest of Aβ fibril formation by BChE and considering the accumulation of monomeric BChE in the most severe AD cases (33), we used a variety of chemical techniques to study the effect of the A539T substitution on BChE stability and tetramerization on the one hand and on its potency in attenuating Aβ oligomerization and fibril formation on the other.