Generation of synthetic shuttle vectors enabling modular genetic engineering of cyanobacteria

Cyanobacteria have raised great interest in biotechnology due to their potential for a sustainable, photosynthesis-driven production of fuels and value-added chemicals. This has led to a concomitant development of molecular tools to engineer the metabolism of those organisms. In this regard, however, even cyanobacterial model strains lag behind compared to their heterotrophic counterparts. For instance, replicative shuttle vectors that allow gene transfer independent of recombination into host DNA are still scarce. Here, we introduce the pSOMA shuttle vector series comprising 10 synthetic plasmids for comprehensive genetic engineering of Synechocystis sp. PCC 6803. The series is based on the small endogenous plasmids pCA2.4 and pCB2.4, each combined with a replicon from Escherichia coli, different selection markers as well as features facilitating molecular cloning and the insulated introduction of gene expression cassettes. We made use of genes encoding green fluorescent protein (GFP) and a Baeyer–Villiger monooxygenase (BVMO) to demonstrate functional gene expression from the pSOMA plasmids in vivo. Moreover, we demonstrate the expression of distinct heterologous genes from individual plasmids maintained in the same strain and thereby confirmed compatibility between the two pSOMA subseries as well as with derivatives of the broad-host-range plasmid RSF1010. We also show that gene transfer into the filamentous model strain Anabaena sp. PCC 7120 is generally possible, which is encouraging to further explore the range of cyanobacterial host species that could be engineered via pSOMA plasmids. Altogether, the pSOMA shuttle vector series displays an attractive alternative to existing plasmid series and thus meets the current demand for the introduction of complex genetic setups and to perform extensive metabolic engineering of cyanobacteria

Long-Term Follow-Up of the Telemonitoring Weight-Reduction Program “Active Body Control”

The Active Body Control (ABC) weight-reduction program is based on telemonitoring of physical activity and nutrition together with telecoaching by weekly counseling letters sent by post or by e-mail. The study presented here reports the results of a 1-year follow-up of 49 patients with the metabolic syndrome who had lost weight with the aid of the ABC program in the preceding year. The weight regain after the second year in patients not receiving any further care (“ABC discontinued” group; n=24) and the potential benefit of continuing with the ABC program with monthly counseling letters (“ABC continued” group; n=25) were investigated. The relative weight changes after the first year had been, respectively, −13.4% and −11.4% in the “ABC discontinued” and “ABC continued” groups, and after the second year they decreased by, respectively, 4.4 and 2.8%. However, this difference in weight regains between the two groups was not statistically significant. It is concluded that three-quarters of the weight loss after 1 year is maintained after the second year. The decision whether to continue with the ABC program after 1 year should be made individually

Rapid evolution of the primate larynx?

Tissue vibrations in the larynx produce most sounds that comprise vocal communication in mammals. Larynx morphology is thus predicted to be a key target for selection, particularly in species with highly developed vocal communication systems. Here, we present a novel database of digitally modeled scanned larynges from 55 different mammalian species, representing a wide range of body sizes in the primate and carnivoran orders. Using phylogenetic comparative methods, we demonstrate that the primate larynx has evolved more rapidly than the carnivoran larynx, resulting in a pattern of larger size and increased deviation from expected allometry with body size. These results imply fundamental differences between primates and carnivorans in the balance of selective forces that constrain larynx size and highlight an evolutionary flexibility in primates that may help explain why we have developed complex and diverse uses of the vocal organ for communication

Identification of full length bovine TLR1 and functional characterization of lipopeptide recognition by bovine TLR2/1 heterodimer

Toll-like receptors (TLR) are highly conserved pattern recognition receptors of the innate immune system. Toll-like receptor 2 (TLR2) recognizes bacterial lipopeptides in a heterodimeric complex with TLR6 or TLR1, thereby discriminating between di- or triacylated lipopeptides, respectively. Previously, we found that HEK293 cells transfected with bovine TLR2 (boTLR2) were able to respond to diacylated lipopeptides but did not recognize triacylated lipopeptides, even after cotransfection with the so far published sequence of boTLR1. In this study we now could show that primary bovine cells were in general able to detect triacylated lipopetides. A closer investigation of the boTLR1 gene locus revealed an additional ATG 195 base pairs upstream from the published start codon. Its transcription would result in an N-terminus with high identity to human and murine TLR1 (huTLR1, muTLR1). Cloning and cotransfection of this longer boTLR1 with boTLR2 now resulted in the recognition of triacylated lipopeptides by HEK293 cells, thereby resembling the ex vivo observation. Analysis of the structure-activity relationship showed that the ester-bound acid chains of these lipopeptides need to consist of at least 12 carbon atoms to activate the bovine heterodimer showing similarity to the recognition by huTLR2/huTLR1. In contrast, HEK293 cell cotransfected with muTLR2 and muTLR1 could already be activated by lipopeptides with shorter fatty acids of only 6 carbon atoms. Thus, our data indicate that the additional N-terminal nucleotides belong to the full length and functionally active boTLR1 (boTLR1-fl) which participates in a species-specific recognition of bacterial lipopeptides

Duplicated fie Genes in Maize: Expression Pattern and Imprinting Suggest Distinct Functions

Two maize genes with predicted translational similarity to the Arabidopsis FIE (Fertilization-Independent Endosperm) protein, a repressor of endosperm development in the absence of fertilization, were cloned and analyzed. Genomic sequences of fie1 and fie2 show significant homology within coding regions but none within introns or 5′ upstream. The fie1 gene is expressed exclusively in the endosperm of developing kernels starting at ∼6 days after pollination. fie1 is an imprinted gene showing no detectable expression of the paternally derived fie1 allele during kernel development. Conversely, fie2 is expressed in the embryo sac before pollination. After pollination, its expression persists, predominantly in the embryo and at lower levels in the endosperm. The paternal fie2 allele is not expressed early in kernel development, but its transcription is activated at 5 days after pollination. fie2 is likely to be a functional ortholog of the Arabidopsis FIE gene, whereas fie1 has evolved a distinct function. The maize FIE2 and sorghum FIE proteins form a monophyletic group, sharing a closer relationship to each other than to the FIE1 protein, suggesting that maize fie genes originated from two different ancestral genomes

Cytokinin Oxidase Gene Expression in Maize Is Localized to the Vasculature, and Is Induced by Cytokinins, Abscisic Acid, and Abiotic Stress

Cytokinins are hormones that play an essential role in plant growth and development. The irreversible degradation of cytokinins, catalyzed by cytokinin oxidase, is an important mechanism by which plants modulate their cytokinin levels. Cytokinin oxidase has been well characterized biochemically, but its regulation at the molecular level is not well understood. We isolated a cytokinin oxidase open reading frame from maize (Zea mays), called Ckx1, and we used it as a probe in northern and in situ hybridization experiments. We found that the gene is expressed in a developmental manner in the kernel, which correlates with cytokinin levels and cytokinin oxidase activity. In situ hybridization with Ckx1 and transgenic expression of a transcriptional fusion of the Ckx1 promoter to the Escherichia coli β-glucuronidase reporter gene revealed that the gene is expressed in the vascular bundles of kernels, seedling roots, and coleoptiles. We show that Ckx1 gene expression is inducible in various organs by synthetic and natural cytokinins. Ckx1 is also induced by abscisic acid, which may control cytokinin oxidase expression in the kernel under abiotic stress. We hypothesize that under non-stress conditions, cytokinin oxidase in maize plays a role in controlling growth and development via regulation of cytokinin levels transiting in the xylem. In addition, we suggest that under environmental stress conditions, cytokinin oxidase gene induction by abscisic acid results in aberrant degradation of cytokinins therefore impairing normal development