Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880 nm to 915 nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll–protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC–LH1–PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC–LH1–PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC–LH1–PufX arrays, but not with a fixed, stoichiometric cytbc1–RC–LH1–PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1–RC–PufX dimers & 2 RC–LH1–PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities

Sequence of lethal events in HeLa cells exposed to the G2 blocking cytolethal distending toxin

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Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation.

Cytolethal distending toxins (CDT) constitute an emerging heterogeneous family of bacterial toxins whose common biological property is to inhibit the proliferation of cells in culture by blocking their cycle at G2/M phase. In this study, we investigated the molecular mechanisms underlying the block caused by CDT from Escherichia coli on synchronized HeLa cell cultures. To this end, we studied specifically the behavior of the two subunits of the complex that determines entry into mitosis, i.e., cyclin B1, the regulatory unit, and cdc2 protein kinase, the catalytic unit. We thus demonstrate that CDT causes cell accumulation in G2 and not in M, that it does not slow the progression of cells through S phase, and that it does not affect the normal increase of cyclin B1 from late S to G2. On the other hand, we show that CDT inhibits the kinase activity of cdc2 by preventing its dephosphorylation, an event which, in normal cells, triggers mitosis. This inhibitory activity was demonstrated for the three partially related CDTs so far described for E. coli. Moreover, we provide evidence that cells exposed to CDT during G2 and M phases are blocked only at the subsequent G2 phase. This observation means that the toxin triggers a mechanism of cell arrest that is initiated in S phase and therefore possibly related to the DNA damage checkpoint system

The death kiss of pathogenic Escherichia coli strains carrying the Vir plasmid

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Dimerization of core complexes as an efficient strategy for energy trapping in Rhodobacter sphaeroides.

In the purple phototrophic bacterium Rhodobacter sphaeroides, light harvesting LH2 complexes transfer absorbed solar energy to RC-LH1-PufX core complexes, which are mainly found in the dimeric state. Many other purple phototrophs have monomeric core complexes and the basis for requiring dimeric cores is not fully established, so we analysed strains of Rba. sphaeroides that contain either native dimeric core complexes or altered monomeric cores harbouring a deletion of the first 12 residues from the N-terminus of PufX, which retains the PufX polypeptide but removes the major determinant of core complex dimerization. Membranes were purified from strains with dimeric or monomeric cores, and with either high or low levels of the LH2 complex. Samples were interrogated with absorption, steady-state fluorescence, and picosecond time-resolved fluorescence kinetic spectroscopies to reveal their light-harvesting and energy trapping properties. We find that under saturating excitation light intensity the photosynthetic membranes containing LH2 and monomeric core complexes have fluorescence lifetimes nearly twice that of membranes with LH2 plus dimeric core complexes. This trend of increased lifetime is maintained with RCs in the open state as well, and for two different levels of LH2 content. Thus, energy trapping is more efficient when photosynthetic membranes of Rba sphaeroides consist of RC-LH1-PufX dimers and LH2 complexes