Folded Alpha Helical Putative New Proteins from Apilactobacillus kunkeei.

The emergence of new proteins is a central question in biology. Most tertiary protein folds known to date appear to have an ancient origin, but it is clear from bioinformatic analyses that new proteins continuously emerge in all organismal groups. However, there is a paucity of experimental data on new proteins regarding their structure and biophysical properties. We performed a detailed phylogenetic analysis and identified 48 putative open reading frames in the honeybee-associated bacterium Apilactobacillus kunkeei for which no or few homologs could be identified in closely-related species, suggesting that they could be relatively new on an evolutionary time scale and represent recently evolved proteins. Using circular dichroism-, fluorescence- and nuclear magnetic resonance (NMR) spectroscopy we investigated six of these proteins and show that they are not intrinsically disordered, but populate alpha-helical dominated folded states with relatively low thermodynamic stability (0-3 kcal/mol). The NMR and biophysical data demonstrate that small new proteins readily adopt simple folded conformations suggesting that more complex tertiary structures can be continuously re-invented during evolution by fusion of such simple secondary structure elements. These findings have implications for the general view on protein evolution, where de novo emergence of folded proteins may be a common event.

Apilactobacillus kunkeei releases RNA-associated membrane vesicles and proteinaceous nanoparticles.

Extracellularly released particles, including membrane vesicles, have increasingly been recognized as important for bacterial community functions and host-interaction processes, but their compositions and functional roles differ between species and also between strains of the same species. In this study, we have determined the composition of membrane vesicles and protein particles identified in the cell-free pellets of two strains of Apilactobacillus kunkeei, a defensive symbiont of honeybees. The membrane vesicles were separated from the extracellular particles using density gradient ultracentrifugation. The peaks of the RNA and protein distributions were separated from each other and the highest concentration of RNA was observed in the fractions that contained the membrane vesicles while the highest protein concentration coincided with the fractions that contained extracellular particles. A comparative proteomics analysis by LC-MS/MS showed that 37 proteins with type-I signal peptides were consistently identified across the fractionated samples obtained from the cell-free pellets, of which 29 were orthologs detected in both strains. Functional predictions of the extracellular proteins revealed the presence of glycoside hydrolases, glycosyltransferases, giant proteins and peptidases. The extracellular transcriptomes mapped to a broad set of genes with a similar functional profile as the whole cell transcriptome. This study provides insights into the composition of membrane vesicles and extracellular proteins of a bee-associated symbiont.

Genome Evolution of a Symbiont Population for Pathogen Defense in Honeybees.

The honeybee gut microbiome is thought to be important for bee health, but the role of the individual members is poorly understood. Here, we present closed genomes and associated mobilomes of 102 Apilactobacillus kunkeei isolates obtained from the honey crop (foregut) of honeybees sampled from beehives in Helsingborg in the south of Sweden and from the islands Gotland and Åland in the Baltic Sea. Each beehive contained a unique composition of isolates and repeated sampling of similar isolates from two beehives in Helsingborg suggests that the bacterial community is stably maintained across bee generations during the summer months. The sampled bacterial population contained an open pan-genome structure with a high genomic density of transposons. A subset of strains affiliated with phylogroup A inhibited growth of the bee pathogen Melissococcus plutonius, all of which contained a 19.5 kb plasmid for the synthesis of the antimicrobial compound kunkecin A, while a subset of phylogroups B and C strains contained a 32.9 kb plasmid for the synthesis of a putative polyketide antibiotic. This study suggests that the mobile gene pool of A. kunkeei plays a key role in pathogen defense in honeybees, providing new insights into the evolutionary dynamics of defensive symbiont populations.

The Subcellular Proteome of a Planctomycetes Bacterium Shows That Newly Evolved Proteins Have Distinct Fractionation Patterns.

The Planctomycetes bacteria have unique cell architectures with heavily invaginated membranes as confirmed by three-dimensional models reconstructed from FIB-SEM images of Tuwongella immobilis and Gemmata obscuriglobus. The subcellular proteome of T. immobilis was examined by differential solubilization followed by LC-MS/MS analysis, which identified 1569 proteins in total. The Tris-soluble fraction contained mostly cytoplasmic proteins, while inner and outer membrane proteins were found in the Triton X-100 and SDS-soluble fractions, respectively. For comparisons, the subcellular proteome of Escherichia coli was also examined using the same methodology. A notable difference in the overall fractionation pattern of the two species was a fivefold higher number of predicted cytoplasmic proteins in the SDS-soluble fraction in T. immobilis. One category of such proteins is represented by innovations in the Planctomycetes lineage, including unique sets of serine/threonine kinases and extracytoplasmic sigma factors with WD40 repeat domains for which no homologs are present in E. coli. Other such proteins are members of recently expanded protein families in which the newly evolved paralog with a new domain structure is recovered from the SDS-soluble fraction, while other paralogs may have similar domain structures and fractionation patterns as the single homolog in E. coli. The expanded protein families in T. immobilis include enzymes involved in replication-repair processes as well as in rRNA and tRNA modification and degradation. These results show that paralogization and domain shuffling have yielded new proteins with distinct fractionation characteristics. Understanding the molecular intricacies of these adaptive changes might aid in the development of a model for the evolution of cellular complexity.

Evolutionary Remodeling of the Cell Envelope in Bacteria of the Planctomycetes Phylum.

Bacteria of the Planctomycetes phylum have many unique cellular features, such as extensive membrane invaginations and the ability to import macromolecules. These features raise intriguing questions about the composition of their cell envelopes. In this study, we have used microscopy, phylogenomics, and proteomics to examine the composition and evolution of cell envelope proteins in Tuwongella immobilis and other members of the Planctomycetes. Cryo-electron tomography data indicated a distance of 45 nm between the inner and outer membranes in T. immobilis. Consistent with the wide periplasmic space, our bioinformatics studies showed that the periplasmic segments of outer-membrane proteins in type II secretion systems are extended in bacteria of the order Planctomycetales. Homologs of two highly abundant cysteine-rich cell wall proteins in T. immobilis were identified in all members of the Planctomycetales, whereas genes for peptidoglycan biosynthesis and cell elongation have been lost in many members of this bacterial group. The cell wall proteins contain multiple copies of the YTV motif, which is the only domain that is conserved and unique to the Planctomycetales. Earlier diverging taxa in the Planctomycetes phylum contain genes for peptidoglycan biosynthesis but no homologs to the YTV cell wall proteins. The major remodeling of the cell envelope in the ancestor of the Planctomycetales coincided with the emergence of budding and other unique cellular phenotypes. The results have implications for hypotheses about the process whereby complex cellular features evolve in bacteria.

Noelin1 Affects Lateral Mobility of Synaptic AMPA Receptors.

Lateral diffusion on the neuronal plasma membrane of the AMPA-type glutamate receptor (AMPAR) serves an important role in synaptic plasticity. We investigated the role of the secreted glycoprotein Noelin1 (Olfactomedin-1 or Pancortin) in AMPAR lateral mobility and its dependence on the extracellular matrix (ECM). We found that Noelin1 interacts with the AMPAR with high affinity, however, without affecting rise- and decay time and desensitization properties. Noelin1 co-localizes with synaptic and extra-synaptic AMPARs and is expressed at synapses in an activity-dependent manner. Single-particle tracking shows that Noelin1 reduces lateral mobility of both synaptic and extra-synaptic GluA1-containing receptors and affects short-term plasticity. While the ECM does not constrain the synaptic pool of AMPARs and acts only extrasynaptically, Noelin1 contributes to synaptic potentiation by limiting AMPAR mobility at synaptic sites. This is the first evidence for the role of a secreted AMPAR-interacting protein on mobility of GluA1-containing receptors and synaptic plasticity.

Tuwongella immobilis gen. nov., sp. nov., a novel non-motile bacterium within the phylum Planctomycetes.

A gram-negative, budding, catalase negative, oxidase positive and non-motile bacterium (MBLW1T) with a complex endomembrane system has been isolated from a freshwater lake in southeast Queensland, Australia. Phylogeny based on 16S rRNA gene sequence analysis places the strain within the family Planctomycetaceae, related to Zavarzinella formosa (93.3 %), Telmatocola sphagniphila (93.3 %) and Gemmata obscuriglobus (91.9 %). Phenotypic and chemotaxonomic analysis demonstrates considerable differences to the type strains of the related genera. MBLW1T displays modest salt tolerance and grows optimally at pH values of 7.5-8.0 and at temperatures of 32-36 °C. Transmission electron microscopy analysis demonstrates the presence of a complex endomembrane system, however, without the typically condensed nucleoid structure found in related genera. The major fatty acids are 16 : 1 ω5c, 16 : 0 and 18 : 0. Based on discriminatory results from 16S rRNA gene sequence analysis, phenotypic, biochemical and chemotaxonomic analysis, MBLW1T should be considered as a new genus and species, for which the name Tuwongella immobilis gen. nov., sp. nov. is proposed. The type strain is MBLW1T (=CCUG 69661T=DSM 105045T).

Biophysical analysis of the dynamics of calmodulin interactions with neurogranin and Ca2+ /calmodulin-dependent kinase II.

Calmodulin (CaM) functions depend on interactions with CaM-binding proteins, regulated by Ca2+. Induced structural changes influence the affinity, kinetics, and specificities of the interactions. The dynamics of CaM interactions with neurogranin (Ng) and the CaM-binding region of Ca2+/calmodulin-dependent kinase II (CaMKII290-309 ) have been studied using biophysical methods. These proteins have opposite Ca2+ dependencies for CaM binding. Surface plasmon resonance biosensor analysis confirmed that Ca2+ and CaM interact very rapidly, and with moderate affinity ( KDSPR=3µM). Calmodulin-CaMKII290-309 interactions were only detected in the presence of Ca2+, exhibiting fast kinetics and nanomolar affinity ( KDSPR=7.1nM). The CaM-Ng interaction had higher affinity under Ca2+-depleted ( KDSPR=480nM,k1=3.4×105M-1s-1 and k-1 = 1.6 × 10-1 s-1 ) than Ca2+-saturated conditions ( KDSPR=19µM). The IQ motif of Ng (Ng27-50 ) had similar affinity for CaM as Ng under Ca2+-saturated conditions ( KDSPR=14µM), but no interaction was seen under Ca2+-depleted conditions. Microscale thermophoresis using fluorescently labeled CaM confirmed the surface plasmon resonance results qualitatively, but estimated lower affinities for the Ng ( KDMST=890nM) and CaMKII290-309 ( KDMST=190nM) interactions. Although CaMKII290-309 showed expected interaction characteristics, they may be different for full-length CaMKII. The data for full-length Ng, but not Ng27-50 , agree with the current model on Ng regulation of Ca2+/CaM signaling.

Analysis of the leakage of gene repression by an artificial TetR-regulated promoter in cyanobacteria.

BACKGROUND: There is a need for strong and tightly regulated promoters to construct more reliable and predictable genetic modules for synthetic biology and metabolic engineering. For this reason we have previously constructed a TetR regulated L promoter library for the cyanobacterium Synechocystis PCC 6803. In addition to the L03 promoter showing wide dynamic range of transcriptional regulation, we observed the L09 promoter as unique in high leaky gene expression under repressed conditions. In the present study, we attempted to identify the cause of L09 promoter leakage. TetR binding to the promoter was studied by theoretical simulations of DNA breathing dynamics and by surface plasmon resonance (SPR) biosensor technology to analyze the kinetics of the DNA-protein interactions. RESULTS: DNA breathing dynamics of a promoter was computed with the extended nonlinear Peyrard-Bishop-Dauxois mesoscopic model to yield a DNA opening probability profile at a single nucleotide resolution. The L09 promoter was compared to the L10, L11, and L12 promoters that were point-mutated and different in repressed promoter strength. The difference between DNA opening probability profiles is trivial on the TetR binding site. Furthermore, the kinetic rate constants of TetR binding, as measured by SPR biosensor technology, to the respective promoters are practically identical. This suggests that a trivial difference in probability as low as 1 × 10(-4) cannot lead to detectable variations in the DNA-protein interactions. Higher probability at the downstream region of transcription start site of the L09 promoter compared to the L10, L11, and L12 promoters was observed. Having practically the same kinetics of binding to TetR, the leakage problem of the L09 promoter might be due to enhanced RNA Polymerase (RNAP)-promoter interactions in the downstream region. CONCLUSIONS: Both theoretical and experimental analyses of the L09 promoter's leakage problem exclude a mechanism of reduced TetR binding but instead suggest enhanced RNAP binding. These results assist in creating more tightly regulated promoters for realizing synthetic biology and metabolic engineering in biotechnological applications.

MyHealthAssistant: an event-driven middleware for multiple medical applications on a smartphone-mediated body sensor network.

An ever-growing range of wireless sensors for medical monitoring has shown that there is significant interest in monitoring patients in their everyday surroundings. It however remains a challenge to merge information from several wireless sensors and applications are commonly built from scratch. This paper presents a middleware targeted for medical applications on smartphone-like platforms that relies on an event-based design to enable flexible coupling with changing sets of wireless sensor units, while posing only a minor overhead on the resources and battery capacity of the interconnected devices. We illustrate the requirements for such middleware with three different healthcare applications that were deployed with our middleware solution, and characterize the performance with energy consumption, overhead caused for the smartphone, and processing time under real-world circumstances. Results show that with sensing-intensive applications, our solution only minimally impacts the phone's resources, with an added CPU utilization of 3% and a memory usage under 7 MB. Furthermore, for a minimum message delivery ratio of 99.9%, up to 12 sensor readings per second are guaranteed to be handled, regardless of the number of applications using our middleware.

Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function.

The kinetic and mechanistic details of the interaction between caldendrin, calmodulin and the B-domain of AKAP79 were determined using a biosensor-based approach. Caldendrin was found to compete with calmodulin for binding at AKAP79, indicating overlapping binding sites. Although the AKAP79 affinities were similar for caldendrin (K(D) = 20 nM) and calmodulin (K(D) = 30 nM), their interaction characteristics were different. The calmodulin interaction was well described by a reversible one-step model, but was only detected in the presence of Ca(2+). Caldendrin interacted with a higher level of complexity, deduced to be an induced fit mechanism with a slow relaxation back to the initial encounter complex. It interacted with AKAP79 also in the absence of Ca(2+), but with different kinetic rate constants. The data are consistent with a similar initial Ca(2+)-dependent binding step for the two proteins. For caldendrin, a second Ca(2+)-independent rearrangement step follows, resulting in a stable complex. The study shows the importance of establishing the mechanism and kinetics of protein-protein interactions and that minor differences in the interaction of two homologous proteins can have major implications in their functional characteristics. These results are important for the further elucidation of the roles of caldendrin and calmodulin in synaptic function.

AKAP79/150 interacts with the neuronal calcium-binding protein caldendrin.

The A kinase-anchoring protein AKAP79/150 is a postsynaptic scaffold molecule and a key regulator of signaling events. At the postsynapse it coordinates phosphorylation and dephosphorylation of receptors via anchoring kinases and phosphatases near their substrates. Interactions between AKAP79 and two Ca(2+) -binding proteins caldendrin and calmodulin have been investigated here. Calmodulin is a known interaction partner of AKAP79/150 that has been shown to regulate activity of the kinase PKC in a Ca(2+) -dependent manner. Pull-down experiments and surface plasmon resonance biosensor analyses have been used here to demonstrate that AKAP79 can also interact with caldendrin, a neuronal calcium-binding protein implicated in regulation of Ca(2+) -influx and release. We demonstrate that calmodulin and caldendrin compete for a partially overlapping binding site on AKAP79 and that their binding is differentially dependent on calcium. Therefore, this competition is regulated by calcium levels. Moreover, both proteins have different binding characteristics suggesting that the two proteins might play complementary roles. The postsynaptic enrichment, the complex binding mechanism, and the competition with calmodulin, makes caldendrin an interesting novel player in the signaling toolkit of the AKAP interactome.

Histaminergic pharmacology of homo-oligomeric ß3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology.

A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric ß3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to ß3 GABA(A) receptors with micromolar affinity (K(D)<300 µM), thus extending previous evidence that ß3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with ß3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric ß3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for ß3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether ß3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.

Identification of a novel scaffold for allosteric inhibition of wild type and drug resistant HIV-1 reverse transcriptase by fragment library screening.

A novel scaffold inhibiting wild type and drug resistant variants of human immunodeficiency virus type 1 reverse transcriptase (HIV-1RT) has been identified in a library consisting of 1040 fragments. The fragments were significantly different from already known non-nucleoside reverse transcriptase inhibitors (NNRTIs), as indicated by a Tversky similarity analysis. A screening strategy involving SPR biosensor-based interaction analysis and enzyme inhibition was used. Primary biosensor-based screening, using short concentration series, was followed by analysis of nevirapine competition and enzyme inhibition, thus identifying inhibitory fragments binding to the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding site. Ten hits were discovered, and their affinities and resistance profiles were evaluated with wild type and three drug resistant enzyme variants (K103N, Y181C, and L100I). One fragment exhibited submillimolar K(D) and IC(50) values against all four tested enzyme variants. A substructure comparison between the fragment and 826 structurally diverse published NNRTIs confirmed that the scaffold was novel. The fragment is a bromoindanone with a ligand efficiency of 0.42 kcal/mol(-1).