Developing a genetic manipulation system for the Antarctic archaeon, Halorubrum lacusprofundi: investigating acetamidase gene function.

No systems have been reported for genetic manipulation of cold-adapted Archaea. Halorubrum lacusprofundi is an important member of Deep Lake, Antarctica (~10% of the population), and is amendable to laboratory cultivation. Here we report the development of a shuttle-vector and targeted gene-knockout system for this species. To investigate the function of acetamidase/formamidase genes, a class of genes not experimentally studied in Archaea, the acetamidase gene, amd3, was disrupted. The wild-type grew on acetamide as a sole source of carbon and nitrogen, but the mutant did not. Acetamidase/formamidase genes were found to form three distinct clades within a broad distribution of Archaea and Bacteria. Genes were present within lineages characterized by aerobic growth in low nutrient environments (e.g. haloarchaea, Starkeya) but absent from lineages containing anaerobes or facultative anaerobes (e.g. methanogens, Epsilonproteobacteria) or parasites of animals and plants (e.g. Chlamydiae). While acetamide is not a well characterized natural substrate, the build-up of plastic pollutants in the environment provides a potential source of introduced acetamide. In view of the extent and pattern of distribution of acetamidase/formamidase sequences within Archaea and Bacteria, we speculate that acetamide from plastics may promote the selection of amd/fmd genes in an increasing number of environmental microorganisms.

Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii.

Cold environments dominate the Earth's biosphere and the resident microorganisms play critical roles in fulfilling global biogeochemical cycles. However, only few studies have examined the molecular basis of thermosensing; an ability that microorganisms must possess in order to respond to environmental temperature and regulate cellular processes. Two component regulatory systems have been inferred to function in thermal regulation of gene expression, but biochemical studies assessing these systems in Bacteria are rare, and none have been performed in Archaea or psychrophiles. Here we examined the LtrK/LtrR two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii, assessing kinase and phosphatase activities of wild-type and mutant proteins. LtrK was thermally unstable and had optimal phosphorylation activity at 10 °C (the lowest optimum activity for any psychrophilic enzyme), high activity at 0 °C and was rapidly thermally inactivated at 30 °C. These biochemical properties match well with normal environmental temperatures of M. burtonii (0-4 °C) and the temperature this psychrophile is capable of growing at in the laboratory (-2 to 28 °C). Our findings are consistent with a role for LtrK in performing phosphotransfer reactions with LtrR that could lead to temperature-dependent gene regulation.

Single TRAM domain RNA-binding proteins in Archaea: functional insight from Ctr3 from the Antarctic methanogen Methanococcoides burtonii.

TRAM domain proteins present in Archaea and Bacteria have a ß-barrel shape with anti-parallel ß-sheets that form a nucleic acid binding surface; a structure also present in cold shock proteins (Csps). Aside from protein structures, experimental data defining the function of TRAM domains is lacking. Here, we explore the possible functional properties of a single TRAM domain protein, Ctr3 (cold-responsive TRAM domain protein 3) from the Antarctic archaeon Methanococcoides burtonii that has increased abundance during low temperature growth. Ribonucleic acid (RNA) bound by Ctr3 in vitro was determined using RNA-seq. Ctr3-bound M. burtonii RNA with a preference for transfer (t)RNA and 5S ribosomal RNA, and a potential binding motif was identified. In tRNA, the motif represented the C loop; a region that is conserved in tRNA from all domains of life and appears to be solvent exposed, potentially providing access for Ctr3 to bind. Ctr3 and Csps are structurally similar and are both inferred to function in low temperature translation. The broad representation of single TRAM domain proteins within Archaea compared with their apparent absence in Bacteria, and scarcity of Csps in Archaea but prevalence in Bacteria, suggests they represent distinct evolutionary lineages of functionally equivalent RNA-binding proteins.

Disentangling Electronic and Vibrational Coherence in the Phycocyanin-645 Light-Harvesting Complex.

Energy transfer between chromophores in photosynthesis proceeds with near-unity quantum efficiency. Understanding the precise mechanisms of these processes is made difficult by the complexity of the electronic structure and interactions with different vibrational modes. Two-dimensional spectroscopy has helped resolve some of the ambiguities and identified quantum effects that may be important for highly efficient energy transfer. Many questions remain, however, including whether the coherences observed are electronic and/or vibrational in nature and what role they play. We utilize a two-color, four-wave mixing experiment with control of the wavelength and polarization to selectively excite specific coherence pathways. For the light-harvesting complex PC645, from cryptophyte algae, we reveal and identify specific contributions from both electronic and vibrational coherences and determine an excited-state structure based on two strongly coupled electronic states and two vibrational modes. Separation of the coherence pathways also uncovers the complex evolution of these coherences and the states involved.

Coherent Vibronic Coupling in Light-Harvesting Complexes from Photosynthetic Marine Algae.

Observations of long-lived coherences in photosynthetic light-harvesting complexes utilize short pulses with broad spectral bandwidths to coherently excite multiple transitions and coherent superpositions. In order to identify the role that such quantum effects might play in efficient energy transfer, however, an alternative approach is required. We have developed a technique for two-color photon echo spectroscopy to selectively excite the pathway of interest and measure its evolution in the absence of any other excitation. We use this technique to excite a coherence pathway in phycocyanin-645 from cryptophyte algae and measure the dynamics of this coherence. A decoherence time of 500 fs was measured, and clear signatures for strong coupling between the electronic states and phonon modes were observed, allowing coherent coupling between otherwise nonresonant transitions. This provides detailed experimental evidence of the long-lived coherences and the nature of the quantum mechanical interactions between electronic states and phonon modes in phycocyanin-645 from cryptophyte marine algae.