Robust Markers Reflecting Phylogeny and Taxonomy of Rhizobia

Genomic ANI (Average Nucleotide Identity) has been found to be able to replace DNA-DNA hybridization in prokaryote taxonomy. The ANI of each of the core genes that has a phylogeny congruent with the reference species tree of rhizobia was compared to the genomic ANI. This allowed us to identify three housekeeping genes ({"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA) whose ANI reflected the intraspecies and interspecies genomic ANI among rhizobial strains, revealing an ANI gap (≥2%) between the inter- and intra-species comparisons. The intraspecies (96%) and interspecies (94%) ANI boundaries calculated from three genes ({"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA) provided a criterion for bacterial species definition and confirmed 621/629 of known interspecies relationships within Bradyrhizobium, Mesorhizobium, Sinorhizobium and Rhizobium. Some widely studied strains should be renamed. The {"type":"entrez-protein","attrs":{"text":"SMc00019","term_id":"1174089704","term_text":"SMC00019"}}SMc00019-truA-thrA ANI also correlates well with the genomic ANI of strains in Agrobacterium, Methylobacterium, Ralstonia, Rhodopseudomonas, Cupriavidus and Burkholderia, suggesting their wide applicability in other bacteria.     

The Sinorhizobium meliloti Essential Porin RopA1 Is a Target for Numerous Bacteriophages

The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti harbors a gene, {"type":"entrez-protein","attrs":{"text":"SMc02396","term_id":"1174172413","term_text":"SMC02396"}}SMc02396, which encodes a predicted outer membrane porin that is conserved in many symbiotic and pathogenic bacteria in the order Rhizobiales. Here, this gene (renamed ropA1) is shown to be required for infection by two commonly utilized transducing bacteriophages (ΦM12 and N3). Mapping of S. meliloti mutations conferring resistance to ΦM12, N3, or both phages simultaneously revealed diverse mutations mapping within the ropA1 open reading frame. Subsequent tests determined that RopA1, lipopolysaccharide, or both are required for infection by all of a larger collection of Sinorhizobium-specific phages. Failed attempts to disrupt or delete ropA1 suggest that this gene is essential for viability. Phylogenetic analysis reveals that ropA1 homologs in many Rhizobiales species are often found as two genetically linked copies and that the intraspecies duplicates are always more closely related to each other than to homologs in other species, suggesting multiple independent duplication events.     

Dinucleotide compositional analysis of Sinorhizobium meliloti using the genome signature: distinguishing chromosomes and plasmids

The symbiotic N₂-fixing α-proteobacterium Sinorhizobium meliloti has three replicons: a circular chromosome (3.7 Mb) and two smaller replicons, pSymA (1.4 Mb) and pSymB (1.7 Mb). Sequence analysis has revealed that an essential gene is carried on pSymB, which brings into question whether pSymB should be considered a chromosome or a plasmid. Based on the criterion that essential genes define a chromosome, several species have been shown to have multiple chromosomes. Many of these species are part of the α subdivision of the Proteobacteria family. Here, additional justification is presented for designating the pSymB replicon as a chromosome. It is shown that chromosomes within a species share a more similar dinucleotide composition, or genome signature, than plasmids do with the host chromosome(s). Dinucleotide signatures were determined for each of the S. meliloti replicons, and, consistent with the suggestion that pSymB is a chromosome, it is shown that the pSymB signature more closely resembles that of the S. meliloti chromosome, while the pSymA signature is typical of other α-proteobacterial plasmids. Electronic Publication     

Examination of Prokaryotic Multipartite Genome Evolution through Experimental Genome Reduction

Many bacteria carry two or more chromosome-like replicons. This occurs in pathogens such as Vibrio cholerea and Brucella abortis as well as in many N₂-fixing plant symbionts including all isolates of the alfalfa root-nodule bacteria Sinorhizobium meliloti. Understanding the evolution and role of this multipartite genome organization will provide significant insight into these important organisms; yet this knowledge remains incomplete, in part, because technical challenges of large-scale genome manipulations have limited experimental analyses. The distinct evolutionary histories and characteristics of the three replicons that constitute the S. meliloti genome (the chromosome (3.65 Mb), pSymA megaplasmid (1.35 Mb), and pSymB chromid (1.68 Mb)) makes this a good model to examine this topic. We transferred essential genes from pSymB into the chromosome, and constructed strains that lack pSymB as well as both pSymA and pSymB. This is the largest reduction (45.4%, 3.04 megabases, 2866 genes) of a prokaryotic genome to date and the first removal of an essential chromid. Strikingly, strains lacking pSymA and pSymB (ΔpSymAB) lost the ability to utilize 55 of 74 carbon sources and various sources of nitrogen, phosphorous and sulfur, yet the ΔpSymAB strain grew well in minimal salts media and in sterile soil. This suggests that the core chromosome is sufficient for growth in a bulk soil environment and that the pSymA and pSymB replicons carry genes with more specialized functions such as growth in the rhizosphere and interaction with the plant. These experimental data support a generalized evolutionary model, in which non-chromosomal replicons primarily carry genes with more specialized functions. These large secondary replicons increase the organism''s niche range, which offsets their metabolic burden on the cell (e.g. pSymA). Subsequent co-evolution with the chromosome then leads to the formation of a chromid through the acquisition of functions core to all niches (e.g. pSymB).     

Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia

A multilocus sequence typing (MLST) analysis was used to examine the genetic structure and diversity within the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and Sinorhizobium medicae). The allelic diversity within these replicons was high compared to the reported diversity within the corresponding chromosomes of the same strains (P. van Berkum et al., J. Bacteriol. 188:5570-5577, 2006). Also, there was strong localized linkage disequilibrium (LD) between certain pSymA loci: e.g., nodC and nifD. Although both of these observations could be explained by positive (or diversifying) selection by plant hosts, results of tests for positive selection did not provide consistent support for this hypothesis. The strong LD observed between the nodC and nifD genes could also be explained by their close proximity on the pSymA replicon. Evidence was obtained that some nodC alleles had a history of intragenic recombination, while other alleles of this locus had a history of intergenic recombination. Both types of recombination were associated with a decline in symbiotic competence with Medicago sativa as the host plant. The combined observations of LD between the nodC and nifD genes and intragenic recombination within one of these loci indicate that the symbiotic gene region on the pSymA plasmid has evolved as a clonal segment, which has been laterally transferred within the natural populations.Plants of the genus Medicago are legumes that often benefit from a mutualistic symbiosis with rhizobia. The most agriculturally significant species of rhizobia that nodulate these plants are Sinorhizobium meliloti (9) and Sinorhizobium medicae (22). Previously reported population genetic analyses of these bacteria have focused on the study of how allelic variants at multiple loci are distributed within and among natural populations (2, 3, 10, 26, 31, 32). This was also the focus of the present study, but it was extended by examining more loci in many more strains of both species of Sinorhizobium coupled with an analysis having a range of symbiotic genotypes. One goal was to determine if there were any obvious correlations between the megaplasmid genotypes observed and their symbiotic competence. A second goal was to determine if selection by their host plants may have influenced the evolution of their symbiotic relationships.The genes for symbiosis reside on the extrachromosomal replicons pSymA (1,354,226 nucleotides [nt]) and pSMED02 (1,245,408 nt) in the genomes of S. meliloti Rm1021 and S. medicae WSM419, respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE006469","term_id":"25168258","term_text":"AE006469"}}AE006469 and {"type":"entrez-nucleotide","attrs":{"text":"CP000740","term_id":"150031715","term_text":"CP000740"}}CP000740, respectively). Besides these two plasmids, these two strains each harbor one other large extrachromosomal replicon, pSymB (1,683,333 nt) and pSMED01 (1,570,951 nt), respectively (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AL591985","term_id":"30407151","term_text":"AL591985"}}AL591985 and {"type":"entrez-nucleotide","attrs":{"text":"CP000739","term_id":"150030273","term_text":"CP000739"}}CP000739, respectively).Multilocus sequence typing (MLST) (16) is a form of genomic indexing that is commonly used to study the population genetic structure and phylogenetic relatedness within diverse groups of bacteria. In this method, nucleotide sequences of a fixed set of common loci are obtained from a collection of strains, and polymorphic sites among these sequences are used to derive an allelic profile or sequence type (ST) for each genome. Comparisons of the resulting data can be used to infer phylogenetic relationships among the organisms in the sample population, and they also can be used to infer how evolutionary processes, such as recombination and selection, have shaped the genetic structure of the population. For example, levels of intergenic recombination among chromosomal genes in natural populations of Neisseria meningitidis reportedly are relatively high, while corresponding levels within populations of Staphylococcus aureus were low (28). Depending on the specific pairs of loci examined, the levels of linkage disequilibrium (LD) (a lack of intergenic recombination) among several chromosomally carried core genes of S. meliloti were reported to be generally moderate to high (26).The MLST approach has been used to confirm that the chromosomes of S. meliloti and S. medicae are sexually isolated (2, 3, 31) and to provide evidence that horizontal gene transfer (HGT) does occur between the symbiotic megaplasmids of these species (3, 32). It has also been used to demonstrate that levels of intergenic recombination, as indicated by linkage disequilibrium, differ between the three replicons of S. meliloti (26). Levels of intergenic recombination within the pSymB replicons of these strains are generally high, unlike the chromosomes and pSymA replicons within the same strains (26). Bailly et al. (3) hypothesized that the region of the pSymA plasmid that contains the nodulation (nod) genes is frequently transferred in natural populations. They also suggested that selective pressures from the host plant may have influenced both nod gene diversity and patterns of polymorphism across the entire nod gene region.In the present study, multilocus allelic variation of the two megaplasmids was examined among 231 Medicago-nodulating rhizobia that originated primarily from southwest Asia (10). Previously, 91 different chromosomal sequence types (STs) were identified among the same strains from sequence variation in 10 loci (31). This collection of strains had earlier been divided into two closely related groups based on results of multilocus enzyme electrophoresis (10), and this result was subsequently cited in support of separating the Medicago-nodulating rhizobia into the two species S. meliloti and S. medicae (22).The objectives of this study were (i) to use MLST to examine the genetic relationships within and among the large extrachromosomal replicons in S. meliloti and S. medicae, (ii) to estimate levels of intergenic and intragenic recombination in these replicons, (iii) to evaluate the nitrogen-fixing competence of representative symbiotic genotypes with Medicago sativa, and (iv) to determine whether positive (or diversifying) selection may have influenced the genetic structure of the megaplasmids.     

A Putative Bifunctional Histidine Kinase/Phosphatase of the HWE Family Exerts Positive and Negative Control on the Sinorhizobium meliloti General Stress Response

The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene ({"type":"entrez-protein","attrs":{"text":"SMc01507","term_id":"1174171461","term_text":"SMC01507"}}SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam:HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria.     

Allosteric Modulation of a Cannabinoid G Protein-coupled Receptor: BINDING SITE ELUCIDATION AND RELATIONSHIP TO G PROTEIN SIGNALING*

The cannabinoid 1 (CB₁) allosteric modulator, 5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide) ({"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569), has the paradoxical effect of increasing the equilibrium binding of [³H](−)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl]cyclohexan-1-ol (CP55,940, an orthosteric agonist) while at the same time decreasing its efficacy (in G protein-mediated signaling). {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 also decreases basal signaling, acting as an inverse agonist for the G protein-mediated signaling pathway. In ligand displacement assays, {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 can displace the CB₁ antagonist/inverse agonist, N-(piperidiny-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide(SR141716A). The goal of this work was to identify the binding site of {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 at CB₁. To this end, we used computation, synthesis, mutation, and functional studies to identify the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-binding site in the CB₁ TMH3-6-7 region. This site is consistent with the results of K3.28¹⁹²A, F3.36²⁰⁰A, W5.43²⁷⁹A, W6.48³⁵⁶A, and F3.25¹⁸⁹A mutation studies, which revealed the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-binding site overlaps with our previously determined binding site of SR141716A but extends extracellularly. Additionally, we identified a key electrostatic interaction between the {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 piperidine ring nitrogen and K3.28¹⁹² that is important for {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 to act as an inverse agonist. At this allosteric site, {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 promotes an intermediate conformation of the CB₁ receptor, explaining {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569''s ability to increase equilibrium binding of CP55,940. This site also explains {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569''s ability to antagonize the efficacy of CP55,940 in three complementary ways. 1) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 sterically blocks movements of the second extracellular loop that have been linked to receptor activation. 2) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 sterically blocks a key electrostatic interaction between the third extracellular loop residue Lys-373 and D2.63¹⁷⁶. 3) {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 packs against TMH6, sterically hindering movements of this helix that have been shown to be important for receptor activation.     

Arylbenzazepines Are Potent Modulators for the Delayed Rectifier K+ Channel: A Potential Mechanism for Their Neuroprotective Effects

(±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, like many other arylbenzazepines, elicits powerful neuroprotection in vitro and in vivo. The neuroprotective action of the compound was found to partially depend on its D₁-like dopamine receptor agonistic activity. The precise mechanism for the (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959-mediated neuroprotection remains elusive. We report here that (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is a potent blocker for delayed rectifier K⁺ channel. (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 inhibited the delayed rectifier K⁺ current (I _K) dose-dependently in rat hippocampal neurons. The IC ₅₀ value for inhibition of I _K was 41.9±2.3 µM (Hill coefficient = 1.81±0.13, n = 6), whereas that for inhibition of I _A was 307.9±38.5 µM (Hill coefficient = 1.37±0.08, n = 6). Thus, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 is 7.3-fold more potent in suppressing I _K than I _A. Moreover, the inhibition of I _K by (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 was voltage-dependent and not related to dopamine receptors. The rapidly onset of inhibition and recovery suggests that the inhibition resulted from a direct interaction of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 with the K⁺ channel. The intracellular application of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 had no effects of on I _K, indicating that the compound most likely acts at the outer mouth of the pore of K⁺ channel. We also tested the enantiomers of (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, R-(+) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-201), and S-(−) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (MCL-202), as well as {"type":"entrez-protein","attrs":{"text":"SKF38393","term_id":"1157151916","term_text":"SKF38393"}}SKF38393; all these compounds inhibited I _K. However, (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959, at either 0.1 or 1 mM, exhibited the strongest inhibition on the currents among all tested drug. The present findings not only revealed a new potent blocker of I _K , but also provided a novel mechanism for the neuroprotective action of arylbenzazepines such as (±) {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959.     

Role for the A Domain of Unprocessed Accumulation-Associated Protein (Aap) in the Attachment Phase of the Staphylococcus epidermidis Biofilm Phenotype

The polysaccharide intercellular adhesin or the cell wall-anchored accumulation-associated protein (Aap) mediates cellular accumulation during Staphylococcus epidermidis biofilm maturation. Mutation of sortase, which anchors up to 11 proteins (including Aap) to the cell wall, blocked biofilm development by the cerebrospinal fluid isolate {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498. Aap was implicated in this phenotype when Western blots and two-dimensional (2D) electrophoresis revealed increased levels of the protein in culture supernatants. Unexpectedly, reduced levels of primary attachment were associated with impaired biofilm formation by {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 srtA and aap mutants. In contrast to previous studies, which implicated Aap proteolytic cleavage and, specifically, the Aap B domains in biofilm accumulation, the {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 Aap protein was unprocessed. Furthermore, aap appeared to play a less important role in the biofilm phenotype of S. epidermidis 1457, in which the Aap protein is processed. Anti-Aap A-domain IgG inhibited primary attachment and biofilm formation in strain {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 but not in strain 1457. The nucleotide sequences of the aap gene A-domain region and cleavage site in strains {"type":"entrez-protein","attrs":{"text":"CSF41498","term_id":"904025233","term_text":"CSF41498"}}CSF41498 and 1457 were identical, implicating altered protease activity in the differential Aap processing results in the two strains. These data reveal a new role for the A domain of unprocessed Aap in the attachment phase of biofilm formation and suggest that extracellular protease activity can influence whether Aap contributes to the attachment or accumulation phases of the S. epidermidis biofilm phenotype.     

Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the {"type":"entrez-protein","attrs":{"text":"STK17810","term_id":"1438759421"}}STK17810/{"type":"entrez-protein","attrs":{"text":"STK17830","term_id":"1438888158"}}STK17830/{"type":"entrez-protein","attrs":{"text":"STK17820","term_id":"1438888149"}}STK17820 hetero-trimer, GAOR2 is a dimer of the {"type":"entrez-protein","attrs":{"text":"STK23390","term_id":"1439642066"}}STK23390/{"type":"entrez-protein","attrs":{"text":"STK05620","term_id":"1438918159"}}STK05620/{"type":"entrez-protein","attrs":{"text":"STK05610","term_id":"1439044704"}}STK05610 hetero-trimer, and GAOR3 is the {"type":"entrez-protein","attrs":{"text":"STK24840","term_id":"1439962493"}}STK24840/{"type":"entrez-protein","attrs":{"text":"STK05620","term_id":"1438918159"}}STK05620/{"type":"entrez-protein","attrs":{"text":"STK05610","term_id":"1439044704"}}STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system.     

Analysis of the Arabinose-5-Phosphate Isomerase of Bacteroides fragilis Provides Insight into Regulation of Single-Domain Arabinose Phosphate Isomerases

Arabinose-5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate, the first step in the biosynthesis of 3-deoxy-d-manno-octulosonic acid (Kdo), an essential component of the lipopolysaccharide in Gram-negative bacteria. Classical APIs, such as Escherichia coli KdsD, contain a sugar isomerase domain and a tandem cystathionine beta-synthase domain. Despite substantial effort, little is known about structure-function relationships in these APIs. We recently reported an API containing only a sugar isomerase domain. This protein, c3406 from E. coli CFT073, has no known physiological function. In this study, we investigated a putative single-domain API from the anaerobic Gram-negative bacterium Bacteroides fragilis. This putative API (UniProt ID {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1) is the only protein encoded by the B. fragilis genome with significant identity to any known API, suggesting that it is responsible for lipopolysaccharide biosynthesis in B. fragilis. We tested this hypothesis by preparing recombinant {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 protein (here referred to by the UniProt ID {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1), characterizing its API activity in vitro, and demonstrating that the gene encoding {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 (GenBank ID {"type":"entrez-protein","attrs":{"text":"YP_209877.1","term_id":"60679733","term_text":"YP_209877.1"}}YP_209877.1) was able to complement an API-deficient E. coli strain. We demonstrated that {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 is inhibited by cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid, the final product of the Kdo biosynthesis pathway, with a K_i of 1.91 μM. These results support the assertion that {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 is the API that supports lipopolysaccharide biosynthesis in B. fragilis and is subject to feedback regulation by CMP-Kdo. The sugar isomerase domain of E. coli KdsD, lacking the two cystathionine beta-synthase domains, demonstrated API activity and was further characterized. These results suggest that {"type":"entrez-protein","attrs":{"text":"Q5LIW1","term_id":"81317194","term_text":"Q5LIW1"}}Q5LIW1 may be a suitable system to study API structure-function relationships.     

Anti-inflammatory effects of N-cyclooctyl-5-methylthiazol-2-amine hydrobromide on lipopolysaccharide-induced inflammatory response through attenuation of NLRP3 activation in microglial cells

Microglial activation is closely associated with neuroinflammatory pathologies. The nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasomes are highly organized intracellular sensors of neuronal alarm signaling. NLRP3 inflammasomes activate nuclear factor kappa-B (NF-κB) and reactive oxygen species (ROS), which induce inflammatory responses. Moreover, NLRP3 dysfunction is a common feature of chronic inflammatory diseases. The present study investigated the effect of a novel thiazol derivative, N-cyclooctyl-5-methylthiazol-2-amine hydrobromide ({"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700), on inflammatory responses in lipopolysaccharide (LPS)-treated BV-2 microglial cells. {"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700 significantly attenuated the expression of several pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-1β, and interleukin-6, in these cells, as well as the LPS-induced increases in NLRP3, NF-κB, and phospho-IkBα levels. {"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700 also suppressed the LPS-induced increases in protein levels of autophagy protein 5 (ATG5), microtubule-associated protein 1 light chain 3 (LC3), and beclin-1, as well as downregulating the LPS-enhanced levels of ROS, lipid peroxidation, and nitric oxide. These results suggest that the anti-inflammatory effects of {"type":"entrez-protein","attrs":{"text":"KHG26700","term_id":"728847257"}}KHG26700 may be due, at least in part, to the regulation of the NLRP3-mediated signaling pathway during microglial activation.     

Electrophysiological Effects of SKF83959 on Hippocampal CA1 Pyramidal Neurons: Potential Mechanisms for the Drug's Neuroprotective Effects

Although the potent anti-parkinsonian action of the atypical D₁-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D₁ receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D₁, D₂, 5-HT_2A/2C receptors and α₁-adrenoceptor, nor by intracellular dialysis of GDP-β-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959. In voltage-clamp experiments, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D₁ receptor activation. Moreover, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 increased Ih current through a D₁ receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug''s neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson''s disease.     

USP7 inhibitor P22077 inhibits neuroblastoma growth via inducing p53-mediated apoptosis

Neuroblastoma (NB) is a common pediatric cancer and contributes to more than 15% of all pediatric cancer-related deaths. Unlike adult tumors, recurrent somatic mutations in NB, such as tumor protein 53 (p53) mutations, occur with relative paucity. In addition, p53 downstream function is intact in NB cells with wild-type p53, suggesting that reactivation of p53 may be a viable therapeutic strategy for NB treatment. Herein, we report that the ubiquitin-specific protease 7 (USP7) inhibitor, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077, potently induces apoptosis in NB cells with an intact USP7-HDM2-p53 axis but not in NB cells with mutant p53 or without human homolog of MDM2 (HDM2) expression. In this study, we found that {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 stabilized p53 by inducing HDM2 protein degradation in NB cells. {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 also significantly augmented the cytotoxic effects of doxorubicin (Dox) and etoposide (VP-16) in NB cells with an intact USP7-HDM2-p53 axis. Moreover, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 was found to be able to sensitize chemoresistant LA-N-6 NB cells to chemotherapy. In an in vivo orthotopic NB mouse model, {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 significantly inhibited the xenograft growth of three NB cell lines. Database analysis of NB patients shows that high expression of USP7 significantly predicts poor outcomes. Together, our data strongly suggest that targeting USP7 is a novel concept in the treatment of NB. USP7-specific inhibitors like {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077 may serve not only as a stand-alone therapy but also as an effective adjunct to current chemotherapeutic regimens for treating NB with an intact USP7-HDM2-p53 axis.     

Distinct Roles of β-Arrestin 1 and β-Arrestin 2 in ORG27569-induced Biased Signaling and Internalization of the Cannabinoid Receptor 1 (CB1)

The cannabinoid receptor 1 (CB1) is a G protein-coupled receptor primarily expressed in brain tissue that has been implicated in several disease states. CB1 allosteric compounds, such as {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569, offer enormous potential as drugs over orthosteric ligands, but their mechanistic, structural, and downstream effects upon receptor binding have not been established. Previously, we showed that {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 enhances agonist binding affinity to CB1 but inhibits G protein-dependent agonist signaling efficacy in HEK293 cells and rat brain expressing the CB1 receptor (Ahn, K. H., Mahmoud, M. M., and Kendall, D. A. (2012) J. Biol. Chem. 287, 12070–12082). Here, we identify the mediators of CB1 receptor internalization and {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced G protein-independent signaling. Using siRNA technology, we elucidate an {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569-induced signaling mechanism for CB1 wherein β-arrestin 1 mediates short term signaling to ERK1/2 with a peak at 5 min and other upstream kinase components including MEK1/2 and c-Src. Consistent with these findings, we demonstrate co-localization of CB1-GFP with red fluorescent protein-β-arrestin 1 upon {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569 treatment using confocal microscopy. In contrast, we show the critical role of β-arrestin 2 in CB1 receptor internalization upon treatment with CP55940 (agonist) or treatment with {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593","term_text":"ORG27569"}}ORG27569. These results demonstrate for the first time the involvement of β-arrestin in CB1-biased signaling by a CB1 allosteric modulator and also define the differential role of the two β-arrestin isoforms in CB1 signaling and internalization.     

Survival in Nuclear Waste,Extreme Resistance,and Potential Applications Gleaned from the Genome Sequence of Kineococcus radiotolerans SRS30216

Kineococcus radiotolerans {"type":"entrez-protein","attrs":{"text":"SRS30216","term_id":"1414251709","term_text":"SRS30216"}}SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits γ-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energy''s Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible.