Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Analysis of the genome of Anabaena sp. PCC7120 reveals that it lacks the gene, ccaA, which encodes the bonafide carboxysomal, β-class carbonic anhydrase (CA) CcaA. However, the carboxysome enriched fraction of Anabaena PCC7120 exhibits CA activity. Bioinformatic analysis reveals that the N-terminal region of the carboxysome protein CcmM has high sequence and structural similarity to the γ-class CA of Methanosarcina thermophila. Recombinantly expressed CcmM is found to be inactive in in-vitro CA assays. E. coli cell extracts containing an overexpressed form of CcmM comprised of the N-terminal 209 amino acids (CcmM209) are also inactive. However, CcmM209 displays CA activity after incubation with the thiol oxidizing agent diamide or when bound to an affinity matrix. It appears that CcmM is indeed a functional γ-CA which is active under oxidizing condition. It is hypothesized that the C-terminal RbcS like domain in CcmM may regulate activity by allowing CcmM activation only when sequestered within the carboxysome.

Abstract The gammaproteobacterium Thiomicrospira crunogena XCL–2 is a hydrothermal vent chemolithoautotroph that has a carbon concentrating mechanism (CCM), which is functionally similar to that of cyanobacteria. At hydrothermal vents, dissolved inorganic carbon (DIC) concentrations and pH values fluctuate over time, with CO2 concentrations ranging from 20 μM to greater than 1 mM, therefore having a CCM would provide an advantage when CO2 availability is very low as CCMs generate intracellular DIC concentrations much higher than extracellular, thereby providing sufficient substrate for carbon fixation. The CCM in T. crunogena includes α–carboxysomes (intracellular inclusions containing form IA RubisCO and carbonic anhydrase), and also presumably requires at least one active HCO3 µ transporter to generate the elevated intracellular concentrations of DIC. To determine whether RubisCO itself might be adapted to low CO2 concentrations, the KCO2 for purified carboxysomal RubisCO was measured (250 μM SD ±; 40) and was much greater than that of whole cells (1.03 μM). This finding suggests that the primary adaptation by T. crunogena to low–DIC conditions has been to enhance DIC uptake, presumably by energy–dependent membrane transport systems that are either ATP–dependent and/or dependent on membrane potential (δ ψ). To determine the mechanism for active DIC uptake, cells were incubated in the presence of inhibitors targeting ATP synthesis andδ ψ. After separate incubations with the ATP synthase inhibitor DCCD and the protonophore CCCP, intracellular ATP was diminished, as was the concentration of intracellular DIC and fixed carbon, despite the absence of an inhibitory effect on δ ψ in the DCCD–incubated cells. In some organisms, DCCD inhibits the NDH–1 and bc1 complexes so it was necessary to verify that ATP synthase was the primary target of DCCD in T. crunogena. Both electron transport complex activities were assayed in the presence and absence of DCCD and there was no significant difference between inhibited (309.0 μmol/s for NDH–1 and 3.4 μmol/s for bc1) and uninhibited treatments (271.7 μmol/s for NDH–1 and 3.6 μmol/s for bc1). These data support the hypothesis that an ATP–dependent transporter is primarily responsible for HCO3 µ transport in T. crunogena. The ATP–dependent transporter solute–binding protein gene (cmpA) from Synechococcus elongatus PCC 7942, was used to perform a BLAST query. Tcr_1153 was the closest match in the T. crunogena genome. However, the gene neighborhood and the result of a maximum likelihood tree suggest that Tcr_1153 is a nitrate transporter protein. Work is underway to find the genes responsible for this ATP–dependent transporter.

While much is known about the structure and interactions of the β-carboxysomal proteins, interactions of the proposed vertex protein CcmL with the other components have not yet been directly demonstrated. A fluorescence resonance energy transfer based method combined with other complementary spectrophotometry techniques as well as x-ray crystallography and transmission electron microscopy was used in a Thermosynechococcus elongatus BP-1 model to study these interactions. CcmL was found to interact with the various CcmK shell proteins with a clear binding preference for CcmK2; the previously proposed interaction of CcmL with CcmM was shown to not occur in vitro, and a possible CcmL-CcmL interaction was observed tentatively. In addition, analysis of a novel x-ray crystal structure of Nostoc sp. PCC7120 CcmL in a decameric form suggests a possibility of a CcmL-CcmL back-to-back interaction. This study gives the first direct experimental evidence for the biological role of CcmL as the carboxysomal vertex protein.

The newly emerging technology of X-ray free-electron lasers (XFELs) has the potential to revolutionise molecular imaging. XFELs generate very intense X-ray pulses and predictions suggest that they may be used for structure determination to atomic resolution even for single molecules. XFELs produce femtosecond pulses that outrun processes of radiation damage and permit the study of structures at room temperature and of structural dynamics. While the first demonstrations of flash X-ray diffractive imaging (FXI) on biological particles were encouraging, they also revealed technical challenges. In this work we demonstrated how some of these challenges can be overcome. We exemplified, with heterogeneous cell organelles, how tens of thousands of FXI diffraction patterns can be collected, sorted, and analysed in an automatic data processing pipeline. We improved  image resolution and reduced problems with missing data. We validated, described, and deposited the experimental data in the Coherent X-ray Imaging Data Bank. We demonstrated that aerosol injection can be used to collect FXI data at high hit ratios and with low background. We reduced problems with non-volatile sample contaminants by decreasing aerosol droplet sizes from ~1000 nm to ~150 nm. We achieved this by adapting an electrospray aerosoliser to the Uppsala sample injector. Mie scattering imaging was used as a diagnostic tool to measure positions, sizes, and velocities of individual injected particles. XFEL experiments generate large amounts of data at high rates. Preparation, execution, and data analysis of these experiments benefits from specialised software. In this work we present new open-source software tools that facilitates prediction, online-monitoring, display, and pre-processing of XFEL diffraction data. We hope that this work is a valuable contribution in the quest of transitioning FXI from its first experimental demonstration into a technique that fulfills its potentials.