Mechanisms for Electron Uptake by \u3cem\u3eMethanosarcina acetivorans\u3c/em\u3e during Direct Interspecies Electron Transfer

Direct interspecies electron transfer (DIET) between bacteria and methanogenic archaea appears to be an important syntrophy in both natural and engineered methanogenic environments. However, the electrical connections on the outer surface of methanogens and the subsequent processing of electrons for carbon dioxide reduction to methane are poorly understood. Here, we report that the genetically tractable methanogen Methanosarcina acetivorans can grow via DIET in coculture with Geobacter metallireducens serving as the electron-donating partner. Comparison of gene expression patterns in M. acetivorans grown in coculture versus pure-culture growth on acetate revealed that transcripts for the outer-surface multiheme c-type cytochrome MmcA were higher during DIET-based growth. Deletion of mmcA inhibited DIET. The high aromatic amino acid content of M. acetivorans archaellins suggests that they might assemble into electrically conductive archaella. A mutant that could not express archaella was deficient in DIET. However, this mutant grew in DIET-based coculture as well as the archaellum-expressing parental strain in the presence of granular activated carbon, which was previously shown to serve as a substitute for electrically conductive pili as a conduit for long-range interspecies electron transfer in other DIET-based cocultures. Transcriptomic data suggesting that the membrane-bound Rnf, Fpo, and HdrED complexes also play a role in DIET were incorporated into a charge-balanced model illustrating how electrons entering the cell through MmcA can yield energy to support growth from carbon dioxide reduction. The results are the first genetics-based functional demonstration of likely outersurface electrical contacts for DIET in a methanogen

Geobacter Strains Expressing Poorly Conductive Pili Reveal Constraints on Direct Interspecies Electron Transfer Mechanisms

Cytochrome-to-cytochrome electron transfer and electron transfer along conduits of multiple extracellular magnetite grains are often proposed as strategies for direct interspecies electron transfer (DIET) that do not require electrically conductive pili (e-pili). However, physical evidence for these proposed DIET mechanisms has been lacking. To investigate these possibilities further, we constructed Geobacter metallireducens strain Aro-5, in which the wild-type pilin gene was replaced with the aro-5 pilin gene that was previously shown to yield poorly conductive pili in Geobacter sulfurreducens strain Aro-5. G. metallireducens strain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. Like G. sulfurreducens strain Aro-5, G. metallireducensstrain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-type G. metallireducens as the electron-donating strain and G. sulfurreducens strain Aro-5 as the electron-accepting strain grew via DIET. However, G. metallireducens Aro-5/G. sulfurreducenswild-type cocultures did not. Cocultures initiated with the Aro-5 strains of both species grew only when amended with granular activated carbon (GAC), a conductive material known to be a conduit for DIET. Magnetite could not substitute for GAC. The inability of the two Aro-5 strains to adapt for DIET in the absence of GAC suggests that there are physical constraints on establishing DIET solely through cytochrome-to-cytochrome electron transfer or along chains of magnetite. The finding that DIET is possible with electron-accepting partners that lack highly conductive pili greatly expands the range of potential electron-accepting partners that might participate in DIET. IMPORTANCE DIET is thought to be an important mechanism for interspecies electron exchange in natural anaerobic soils and sediments in which methane is either produced or consumed, as well as in some photosynthetic mats and anaerobic digesters converting organic wastes to methane. Understanding the potential mechanisms for DIET will not only aid in modeling carbon and electron flow in these geochemically significant environments but will also be helpful for interpreting meta-omic data from as-yet-uncultured microbes in DIET-based communities and for designing strategies to promote DIET in anaerobic digesters. The results demonstrate the need to develop a better understanding of the diversity of types of e-pili in the microbial world to identify potential electron-donating partners for DIET. Novel methods for recovering as-yet-uncultivated microorganisms capable of DIET in culture will be needed to further evaluate whether DIET is possible without e-pili in the absence of conductive materials such as GAC

Potential Role of Protein Kinase B in Insulin-induced Glucose Transport, Glycogen Synthesis, and Protein Synthesis

Various biological responses stimulated by insulin have been thought to be regulated by phosphatidylinosi-tol 3-kinase, including glucose transport, glycogen syn-thesis, and protein synthesis. However, the molecular link between phosphatidylinositol 3-kinase and these biological responses has been poorly understood. Re-cently, it has been shown that protein kinase B (PKB/c-Akt/ Rac) lies immediately downstream from phosphati-dylinositol 3-kinase. Here, we show that expression of a constitutively active form of PKB induced glucose up-take, glycogen synthesis, and protein synthesis in L6 myotubes downstream of phosphatidylinositol 3-kinase and independent of Ras and mitogen-activated protein kinase activation. Introduction of constitutively active PKB induced glucose uptake and protein synthesis but not glycogen synthesis in 3T3L-1 adipocytes, which lack expression of glycogen synthase kinase 3 different from L6 myotubes. Furthermore, we show that deactivation of glycogen synthase kinase 3 and activation of rapamy-cin- sensitive serine/threonine kinase by PKB in L6 myo-tubes might be involved in the enhancement of glycogen synthesis and protein synthesis, respectively. These re-sults suggest that PKB acts as a key enzyme linking phosphatidylinositol 3-kinase activation to multiple bi-ological functions of insulin through regulation of downstream kinases in skeletal muscle, a major target tissue of insulin

Control of pre-replicative complex during the division cycle in Chlamydomonas reinhardtii

DNA replication is fundamental to all living organisms. In yeast and animals, it is triggered by an assembly of pre-replicative complex including ORC, CDC6 and MCMs. Cyclin Dependent Kinase (CDK) regulates both assembly and firing of the pre-replicative complex. We tested temperature-sensitive mutants blocking Chlamydomonas DNA replication. The mutants were partially or completely defective in DNA replication and did not produce mitotic spindles. After a long G1, wild type Chlamydomonas cells enter a division phase when it undergoes multiple rapid synchronous divisions (‘multiple fission’). Using tagged transgenic strains, we found that MCM4 and MCM6 were localized to the nucleus throughout the entire multiple fission division cycle, except for transient cytoplasmic localization during each mitosis. Chlamydomonas CDC6 was transiently localized in nucleus in early division cycles. CDC6 protein levels were very low, probably due to proteasomal degradation. CDC6 levels were severely reduced by inactivation of CDKA1 (CDK1 ortholog) but not the plant-specific CDKB1. Proteasome inhibition did not detectably increase CDC6 levels in the cdka1 mutant, suggesting that CDKA1 might upregulate CDC6 at the transcriptional level. All of the DNA replication proteins tested were essentially undetectable until late G1. They accumulated specifically during multiple fission and then were degraded as cells completed their terminal divisions. We speculate that loading of origins with the MCM helicase may not occur until the end of the long G1, unlike in the budding yeast system. We also developed a simple assay for salt-resistant chromatin binding of MCM4, and found that tight MCM4 loading was dependent on ORC1, CDC6 and MCM6, but not on RNR1 or CDKB1. These results provide a microbial framework for approaching replication control in the plant kingdom

Systematic Study of Short Range Antiferromagnetic Order and The Spin-Glass State in Lightly Doped La2-xSrxCuO4

Systematic measurements of the magnetic susceptibility were performed on single crystals of lightly doped La2-xSrxCuO4 (x=0.03, 0.04 and 0.05). For all samples the temperature dependence of the in-plane magnetic susceptibility shows typical spin-glass features with spin-glass transition temperatures Tg of 6.3K, 5.5K and 5.0K for x=0.03, 0.04 and 0.05, respectively. The canonical spin-glass order parameter extracted from the in-plane susceptibility of all the samples follows a universal scaling curve. On the other hand, the out-of-plane magnetic susceptibility deviates from Curie law below a temperature Tdv, higher than Tg. Comparing with previous neutron scattering results with an instrumental energy resolution of 0.25 meV from Wakimoto et al., the x-dependence of Tdv is qualitatively the same as that of Tel, the temperature below which the elastic magnetic scattering develops around (pi, pi). Thus, a revised magnetic phase diagram in the lightly doped region of La2-xSrxCuO4 is proposed. The Curie constants calculated from the in-plane susceptibility are independent of the Sr concentration. On the basis of the cluster spin-glass model, this fact might reflect an inhomogeneous distribution of doped holes in the CuO2 plane, such as in a stripe structure.Comment: 7 pages, 6 figure

Observation of New Incommensurate Magnetic Correlations at the Lower Critical Concentration for Superconductivity (x=0.05) in La(2-x)Sr(x)CuO4

Neutron-scattering experiments have been performed on lightly-doped La(2-x)Sr(x)CuO4 single crystals in both the insulating (x=0.03,0.04,0.05) and superconducting (x=0.06) regions. Elastic magnetic peaks are observed at low temperatures in all samples with the maximum peak linewidth occuring at the critical concentration x_c=0.05. New incommensurate peaks are observed only at x=0.05, the positions of which are rotated by 45 degrees in reciprocal space about (pi,pi) from those observed for x>=0.06 in the superconducting phase.Comment: 5 pages, LaTeX, 4 figures include

The GATA1s isoform is normally down-regulated during terminal haematopoietic differentiation and over-expression leads to failure to repress MYB, CCND2 and SKI during erythroid differentiation of K562 cells

Background: Although GATA1 is one of the most extensively studied haematopoietic transcription factors little is currently known about the physiological functions of its naturally occurring isoforms GATA1s and GATA1FL in humans—particularly whether the isoforms have distinct roles in different lineages and whether they have non-redundant roles in haematopoietic differentiation. As well as being of general interest to understanding of haematopoiesis, GATA1 isoform biology is important for children with Down syndrome associated acute megakaryoblastic leukaemia (DS-AMKL) where GATA1FL mutations are an essential driver for disease pathogenesis. <p/>Methods: Human primary cells and cell lines were analyzed using GATA1 isoform specific PCR. K562 cells expressing GATA1s or GATA1FL transgenes were used to model the effects of the two isoforms on in vitro haematopoietic differentiation. <p/>Results: We found no evidence for lineage specific use of GATA1 isoforms; however GATA1s transcripts, but not GATA1FL transcripts, are down-regulated during in vitro induction of terminal megakaryocytic and erythroid differentiation in the cell line K562. In addition, transgenic K562-GATA1s and K562-GATA1FL cells have distinct gene expression profiles both in steady state and during terminal erythroid differentiation, with GATA1s expression characterised by lack of repression of MYB, CCND2 and SKI. <p/>Conclusions: These findings support the theory that the GATA1s isoform plays a role in the maintenance of proliferative multipotent megakaryocyte-erythroid precursor cells and must be down-regulated prior to terminal differentiation. In addition our data suggest that SKI may be a potential therapeutic target for the treatment of children with DS-AMKL

Gene Silencing of Phogrin Unveils Its Essential Role in Glucose-Responsive Pancreatic β-Cell Growth

OBJECTIVE—Phogrin and IA-2, autoantigens in insulin-dependent diabetes, have been shown to be involved in insulin secretion in pancreatic β-cells; however, implications at a molecular level are confusing from experiment to experiment. We analyzed biological functions of phogrin in β-cells by an RNA interference technique