Inoculation of hybrid poplar with the endophytic bacterium Enterobacter sp. 638 increases biomass but does not impact leaf level physiology

Endophytic bacteria have been shown to provide several advantages to their host, including enhanced growth. Inoculating biofuel species with endophytic bacteria is therefore an attractive option to increase the productivity of biofuel feedstocks. Here, we investigated the effect of inoculating hard wood cuttings of Populus deltoides Bartr. × Populus. nigra L. clone OP367 with Enterobacter sp. 638. After 17 weeks, plants inoculated with Enterobacter sp. 638 had 55% greater total biomass than un‐inoculated control plants. Study of gas exchange and fluorescence in developing and mature leaves over a diurnal cycle and over a 5 week measurement campaign revealed no effects of inoculation on photosynthesis, stomatal conductance, photosynthetic water use efficiency or the maximum and operating efficiency of photosystem II. However, plants inoculated with Enterobacter sp. 638 had a canopy that was 39% larger than control plants indicating that the enhanced growth was fueled by increased leaf area, not by improved physiology. Leaf nitrogen content was determined at two stages over the 5 week measurement period. No effect of Enterobacter sp. 638 on leaf nitrogen content was found indicating that the larger plants were acquiring sufficient nitrogen. Enterobacter sp. 638 lacks the genes for N₂ fixation, therefore the increased availability of nitrogen likely resulted from enhanced nitrogen acquisition by the 84% larger root system. These data show that Enterobacter sp. 638 has the potential to dramatically increase productivity in poplar. If fully realized in the production environment, these results indicate that an increase in the environmental and economic viability of poplar as a biofuel feedstock is possible when inoculated with endophytic bacteria like Enterobacter sp. 638.     

The violacein biosynthetic enzyme VioE shares a fold with lipoprotein transporter proteins

VioE, an unusual enzyme with no characterized homologues, plays a key role in the biosynthesis of violacein, a purple pigment with antibacterial and cytotoxic properties. Without bound cofactors or metals, VioE, from the bacterium Chromobacterium violaceum, mediates a 1,2 shift of an indole ring and oxidative chemistry to generate prodeoxyviolacein, a precursor to violacein. Our 1.21 A resolution structure of VioE shows that the enzyme shares a core fold previously described for lipoprotein transporter proteins LolA and LolB. For both LolB and VioE, a bound polyethylene glycol molecule suggests the location of the binding and/or active site of the protein. Mutations of residues near the bound polyethylene glycol molecule in VioE have identified the active site and five residues important for binding or catalysis. This structural and mutagenesis study suggests that VioE acts as a catalytic chaperone, using a fold previously associated with lipoprotein transporters to catalyze the production of its prodeoxyviolacein product.     

B cell overexpression of FCRL5 and PD-1 is associated with low antibody titers in HCV infection

Antibodies targeting the hepatitis C virus (HCV) envelope glycoprotein E2 are associated with delayed disease progression, and these antibodies can also facilitate spontaneous clearance of infection in some individuals. However, many infected people demonstrate low titer and delayed anti-E2 antibody responses. Since a goal of HCV vaccine development is induction of high titers of anti-E2 antibodies, it is important to define the mechanisms underlying these suboptimal antibody responses. By staining lymphocytes with a cocktail of soluble E2 (sE2) glycoproteins, we detected HCV E2-specific (sE2+) B cells directly ex vivo at multiple acute infection timepoints in 29 HCV-infected subjects with a wide range of anti-E2 IgG titers, including 17 persistently infected subjects and 12 subjects with spontaneous clearance of infection. We performed multi-dimensional flow cytometric analysis of sE2+ and E2-nonspecific (sE2-) class-switched B cells (csBC). In sE2+ csBC from both persistence and clearance subjects, frequencies of resting memory B cells (rMBC) were reduced, frequencies of activated MBC (actMBC) and tissue-like MBC (tlMBC) were increased, and expression of FCRL5, an IgG receptor, was significantly upregulated. Across all subjects, plasma anti-E2 IgG levels were positively correlated with frequencies of sE2+ rMBC and sE2+ actMBC, while anti-E2 IgG levels were negatively correlated with levels of FCRL5 expression on sE2+ rMBC and PD-1 expression on sE2+ actMBC. Upregulation of FCRL5 on sE2+ rMBC and upregulation of PD-1 on sE2+ actMBC may limit anti-E2 antibody production in vivo. Strategies that limit upregulation of these molecules could potentially generate higher titers of protective antibodies against HCV or other pathogens.     

Epitope-focused immunogen design based on the ebolavirus glycoprotein HR2-MPER region

The three human pathogenic ebolaviruses: Zaire (EBOV), Bundibugyo (BDBV), and Sudan (SUDV) virus, cause severe disease with high fatality rates. Epitopes of ebolavirus glycoprotein (GP) recognized by antibodies with binding breadth for all three ebolaviruses are of major interest for rational vaccine design. In particular, the heptad repeat 2 –membrane-proximal external region (HR2-MPER) epitope is relatively conserved between EBOV, BDBV, and SUDV GP and targeted by human broadly-neutralizing antibodies. To study whether this epitope can serve as an immunogen for the elicitation of broadly-reactive antibody responses, protein design in Rosetta was employed to transplant the HR2-MPER epitope identified from a co-crystal structure with the known broadly-reactive monoclonal antibody (mAb) BDBV223 onto smaller scaffold proteins. From computational analysis, selected immunogen designs were produced as recombinant proteins and functionally validated, leading to the identification of a sterile alpha motif (SAM) domain displaying the BDBV-HR2-MPER epitope near its C terminus as a promising candidate. The immunogen was fused to one component of a self-assembling, two-component nanoparticle and tested for immunogenicity in rabbits. Robust titers of cross-reactive serum antibodies to BDBV and EBOV GPs and moderate titers to SUDV GP were induced following immunization. To confirm the structural composition of the immunogens, solution NMR studies were conducted and revealed structural flexibility in the C-terminal residues of the epitope. Overall, our study represents the first report on an epitope-focused immunogen design based on the structurally challenging BDBV-HR2-MPER epitope.     

Expression of Ca2+-ATPase and Na+/Ca2+-exchanger is upregulated during epithelial Ca2+ transport in hypodermal cells of the isopod Porcellio scaber

It is thought that a plasma membrane Ca(2+)-transport ATPase (PMCA) and a Na(+)/Ca(2+)-exchange (NCE) mechanism are involved in epithelial Ca(2+) transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca(2+)-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca(2+)-transporting condition to the stages of CaCO(3) deposit formation and degradation. During Ca(2+)-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.     

The Genetic Basis of a Rare Flower Color Polymorphism in Mimulus lewisii Provides Insight into the Repeatability of Evolution

A long-standing question in evolutionary biology asks whether the genetic changes contributing to phenotypic evolution are predictable. Here, we identify a genetic change associated with segregating variation in flower color within a population of Mimulus lewisii. To determine whether these types of changes are predictable, we combined this information with data from other species to investigate whether the spectrum of mutations affecting flower color transitions differs based on the evolutionary time-scale since divergence. We used classic genetic techniques, along with gene expression and population genetic approaches, to identify the putative, loss-of-function mutation that generates rare, white flowers instead of the common, pink color in M. lewisii. We found that a frameshift mutation in an anthocyanin pathway gene is responsible for the white-flowered polymorphism found in this population of M. lewisii. Comparison of our results with data from other species reveals a broader spectrum of flower color mutations segregating within populations relative to those that fix between populations. These results suggest that the genetic basis of fixed differences in flower color may be predictable, but that for segregating variation is not.     

Diverse aerobic anoxygenic phototrophs synthesize bacteriochlorophyll in oligotrophic rather than copiotrophic conditions,suggesting ecological niche

While investigating aerobic anoxygenic phototrophs (AAP) from Lake Winnipeg's bacterial community, over 500 isolates were obtained. Relatives of 20 different species were examined simultaneously, identifying conditions for optimal growth or pigment production to determine features that may unify this group of phototrophs. All were distributed among assorted α-Proteobacterial families including Erythrobacteraceae, Sphingomonadaceae, Sphingosinicellaceae, Acetobacteraceae, Methylobacteriaceae, and Rhodobacteraceae. Major phenotypic characteristics matched phylogenetic association, including pigmentation, morphology, metal transformations, tolerances, lipid configurations, and enzyme activities, which distinctly separated each taxonomic family. While varying pH and temperature had a limited independent impact on pigment production, bacteriochlorophyll synthesis was distinctly promoted under low nutrient conditions, whereas copiotrophy repressed its production but enhanced carotenoid yield. New AAP diversity was also reported by revealing strains related to non-phototrophic Rubellimicrobium and Sphingorhabdus, as well as spread throughout Roseomonas, Sphingomonas, and Methylobacterium/Methylorubrum, which previously only had a few known photosynthetic members. This study exemplified the overwhelming diversity of AAP in a single aquatic environment, confirming cultivation continues to be of importance in microbial ecology to discover functionality in both new and previously reported cohorts of bacteria as specific laboratory conditions were required to promote aerobic bacteriochlorophyll production.     

Definition of the carbohydrate response element of the rat S14 gene. Evidence for a common factor required for carbohydrate regulation of hepatic genes.

The 5'-flanking region of the S14 gene from -4316 to +18 contains regulatory sequences responsible for activation of promoter activity in response to elevated carbohydrate metabolism in primary hepatocytes. To map these sequences, a series of constructs containing various internal deletions of the S14 5'-flanking sequence were assayed in primary hepatocytes. The region from -1601 to -1395 was found to be essential for this response. Comparison of the sequence of this S14 region to a region of the L-type pyruvate kinase gene that has been shown to mediate carbohydrate regulation (Thompson, K. S., and Towle, H. C. (1991) J. Biol. Chem. 266, 8679-8682) revealed a segment with 9 out of 10 identity. In both cases, this conserved sequence aligned with a DNase I footprint formed with hepatic nuclear extract. Oligonucleotides (approximately 30 base pairs) from either S14 or pyruvate kinase genes containing the conserved element bound to a hepatic nuclear factor(s) that gave identical complexes by mobility shift assay. Furthermore, these two oligonucleotides cross-competed for binding to the nuclear factor(s), suggesting that a common factor(s) binds to this conserved element. Reinsertion of the S14 oligonucleotide into an unresponsive S14 promoter construct restored the carbohydrate control. Moreover, this oligonucleotide could confer a glucose response when fused to a heterologous promoter. Thus, the S14 segment from -1457 to -1428 is a carbohydrate response element essential for the binding of nuclear factor(s) regulated by increased carbohydrate metabolism. This factor(s) may be common to the carbohydrate regulation of the S14 and pyruvate kinase genes.