Active site rearrangement and structural divergence in prokaryotic respiratory oxidases.

Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.

Assessing the fitness-for-purpose of satellite multi-mission ocean color climate data records: A protocol applied to OC-CCI chlorophyll-a data.

In this work, trend estimates are used as indicators to compare the multi-annual variability of different satellite chlorophyll-a (Chla) data and to assess the fitness-for-purpose of multi-mission Chla products as climate data records (CDR). Under the assumption that single-mission products are free from spurious temporal artifacts and can be used as benchmark time series, multi-mission CDRs should reproduce the main trend patterns observed by single-mission series when computed over their respective periods. This study introduces and applies quantitative metrics to compare trend distributions from different data records. First, contingency matrices compare the trend diagnostics associated with two satellite products when expressed in binary categories such as existence, significance and signs of trends. Contingency matrices can be further summarized by metrics such as Cohen's κ index that rates the overall agreement between the two distributions of diagnostics. A more quantitative measure of the discrepancies between trends is provided by the distributions of differences between trend slopes. Thirdly, maps of the level of significance P of a t-test quantifying the degree to which two trend estimates differ provide a statistical, spatially-resolved, evaluation. The proposed methodology is applied to the multi-mission Ocean Colour-Climate Change Initiative (OC-CCI) Chla data. The agreement between trend distributions associated with OC-CCI data and single-mission products usually appears as good as when single-mission products are compared. As the period of analysis is extended beyond 2012 to 2015, the level of agreement tends to be degraded, which might be at least partly due to the aging of the MODIS sensor on-board Aqua. On the other hand, the trends displayed by the OC-CCI series over the short period 2012-2015 are very consistent with those observed with VIIRS. These results overall suggest that the OC-CCI Chla data can be used for multi-annual time series analysis (including trend detection), but with some caution required if recent years are included, particularly in the central tropical Pacific. The study also recalls the challenges associated with creating a multi-mission ocean color data record suitable for climate research.

Comparative studies in series of cytochrome c oxidase models.

This study compares the behavior as cytochrome c oxidase (CcO) functional and structural models of a series of reported and unreported ligands that provide either a binding site for copper without a built-in proximal base, or both a flexible binding site for copper and a built-in proximal base, or a fixed binding site for copper with a built-in proximal base. The comparisons of the models show that the relative position of the two metal sites is not only a crucial parameter in the control of the catalytic behavior but also essential in mimicking other features of the enzyme such as CO exchange between the ferrous heme a(3) and the cuprous Cu(B) center.

Transcription enhancer factor-1 (TEF-1) DNA binding sites can specifically enhance gene expression at the beginning of mouse development.

In an effort to identify transcriptional elements that are recognized at different stages of early mouse development, polyomavirus (PyV) enhancer mutations were selected for their ability to support PyV transcription and replication in various mouse undifferentiated embryonal carcinoma (EC) and embryonic stem (ES) cell lines. Several of these enhancer mutations were then isolated, sequenced and tested for their ability to stimulate the PyV early gene promoter in plasmid DNA that was either transfected into EC, ES and fibroblast cell lines, or injected into the nuclei of mouse 1-cell and 2-cell embryos. EC, ES and fibroblast cell lines showed clear preferences for different enhancer configurations, and cleavage-stage embryos (2- to 8-cell stage) strongly preferred the same enhancer configuration favored by ES cells. This 'embryo responsive' (ER) enhancer configuration was characterized by a tandem duplication of the region containing a single point mutation that created a DNA binding site for Transcription Enhancer Factor-1 (TEF-1). ER enhancers stimulated the PyV promoter up to 350-fold in embryos, and were up to 74-fold more active than the wild-type PyV enhancer. Most of the activity from PyER enhancers could be duplicated in 2-cell embryos by synthesizing only the tandemly repeated sequence. Comparison of these synthetic enhancers with ER enhancers confirmed that TEF-1 DNA binding sites were highly preferred in ES cells and cleavage-stage embryos, and suggested that ER enhancer activity resulted primarily from cooperative interaction between either two closely spaced TEF-1 DNA binding sites or two TEF-1 DNA binding sites separated by a third, as yet unidentified, transcription factor binding site. These results provide a prototype of a mammalian embryo responsive enhancer, and suggest that TEF-1 plays an important role in activation of gene expression at the beginning of mammalian development.

Analysis of transcription factors binding to the duplicated PEA1 and PEA3 sites that are required for polyomavirus mutant expression in PCC4 embryonic carcinoma cells.

Embryonic carcinoma (EC) cell lines, representative of early embryonic undifferentiated cells, are nonpermissive for polyomavirus (PyV) infection as a result of a blockade of viral DNA early transcription and replication. All enhancers of PyV mutants (Py EC-PCC4), selected for the ability to grow on PCC4 EC cells, display a duplication of PEA1 and PEA3 binding sites (sites 1 and 3). However, the Py EC-PCC4 rearrangement is complex and results in variable mutant enhancer activities. We demonstrate here that duplication of sites 1 and 3 is absolutely required for a cooperative cis activation of early Py EC-PCC4 mutant transcription in PCC4 EC cells. In addition, we detect in PCC4 EC cells significant amounts of site 1- and 3-binding proteins, which we characterize as related to the Fos/Jun and Ets protein families, respectively. Wild-type PyV restriction in PCC4 EC cells may be relieved by a cooperation between site 2- and 3-binding proteins that would thereby be activated. Since site 1- or 3-binding factors could be derepressed, we improved the analysis of UV cross-linked DNA-protein complexes and were able to detect a novel factor, called PEA1/2 (for PyV enhancer A site 1- and 2-binding factor). Its DNA binding sequence overlaps sites 1 and 2 (PEA2 binding site) and is not duplicated in the M1 mutant, which exhibits the highest Py EC-PCC4 enhancer activity. he suggest that PEA1/2 is also involved in the regulation of PyV enhancer activity by repressing the site 1-binding activity.

Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos.

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.

Common features of polyomavirus mutants selected on PCC4 embryonal carcinoma cells.

The genomic rearrangements of six polyomavirus mutants selected on PCC4 embryonal carcinoma cells have been compared and their common characteristics pointed out. All mutants show a duplication which includes at least the adenovirus type 5 (Ad5) E1A-like enhancer core sequence plus a deletion of variable size and location. The presence of the second enhancer core sequence, the SV40-like enhancer, is not required for expression of the PyEC PCC4 phenotype. Two of these mutants are also able to express polyomavirus T antigen on F9 and LT1 cells. Multiadaptation seems to require the duplication of the Ad5 E1A-like core sequence, the maintenance of the SV40-like core sequence and a local change in DNA stability.

Isolation of polyomavirus mutants multiadapted to murine embryonal carcinoma cells.

Polyomavirus mutants were isolated from PCC4 embryonal carcinoma cells infected with a variant strain of polyomavirus (ev 1001h) and were found to contain a tandem duplication overlapping the enhancers and the origin of replication. These mutants were able to infect several lines of embryonal carcinoma cells, including PCC4, F9, and LT1. The sequence and structure of one of these mutants are presented and compared with those of other PyEC PCC4 mutants previously described.