Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO2 through geologic time should be reevaluated.

The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

Modeling and mutagenesis of amino acid residues critical for CO2 hydration by specialized NDH-1 complexes in cyanobacteria.

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO2-concentrating mechanism (CCM). This includes specialized Type-1 NDH complexes that function to couple photosynthetic redox energy to CO2 hydration forming the bicarbonate that accumulates to high cytoplasmic concentrations during the operation of the CCM, required for effective carbon fixation. Here we used a Synechococcus PCC7942 expression system to investigate the role of conserved histidine and cysteine residues in the CupB (also designated, ChpX) protein, which has been hypothesized to participate in a vectoral CO2 hydration reaction near the interface between CupB protein and the proton-pumping subunits of the NDH-1 complex. A homology model has been constructed and most of the targeted conserved residues are in the vicinity of a Zn ion modeled to form the catalytic site of deprotonation and CO2 hydration. Growth and CO2 uptake assays show that the most severe defects in activity among the targeted residues are due to a substitution of the predicted Zn ligand, CupB-His86. Mutations at other sites produced intermediate effects. Proteomic analysis revealed that some amino acid substitution mutations of CupB caused the induction of bicarbonate uptake proteins to a greater extent than complete deletion of CupB, despite growth under CO2-enriched conditions. The results are discussed in terms of hypotheses on the catalytic function of this unusual enzyme.

Engineering living building materials for enhanced bacterial viability and mechanical properties.

Living building materials (LBMs) utilize microorganisms to produce construction materials that exhibit mechanical and biological properties. A hydrogel-based LBM containing bacteria capable of microbially induced calcium carbonate precipitation (MICP) was recently developed. Here, LBM design factors, i.e., gel/sand ratio, inclusion of trehalose, and MICP pathways, are evaluated. The results show that non-saturated LBM (gel/sand = 0.13) and gel-saturated LBM (gel/sand = 0.30) underwent distinct failure modes. The inclusion of trehalose maintains bacterial viability under ambient conditions with low relative humidity, without affecting mechanical properties of the LBM. Comparison of biotic and abiotic LBM shows that MICP efficiency in this material is subject to the pathway selected: the LBM with heterotrophic ureolytic Escherichia coli demonstrated the most mechanical enhancement from the abiotic controls, compared with either ureolytic or CO2-concentrating mechanisms from Synechococcus. The study shows that tailoring of LBM properties can be accomplished in a manner that considers both LBM microstructure and MICP pathways.

Periplasm-enriched fractions from Xanthomonas citri subsp. citri type A and X. fuscans subsp. aurantifolii type B present distinct proteomic profiles under in vitro pathogenicity induction.

The causative agent of Asiatic citrus canker, the Gram-negative bacterium Xanthomonas citri subsp. citri (XAC), produces more severe symptoms and attacks a larger number of citric hosts than Xanthomonas fuscans subsp. aurantifolii XauB and XauC, the causative agents of cancrosis, a milder form of the disease. Here we report a comparative proteomic analysis of periplasmic-enriched fractions of XAC and XauB in XAM-M, a pathogenicity- inducing culture medium, for identification of differential proteins. Proteins were resolved by two-dimensional electrophoresis combined with liquid chromatography-mass spectrometry. Among the 12 proteins identified from the 4 unique spots from XAC in XAM-M (p<0.05) were phosphoglucomutase (PGM), enolase, xylose isomerase (XI), transglycosylase, NAD(P)H-dependent glycerol 3-phosphate dehydrogenase, succinyl-CoA synthetase ß subunit, 6-phosphogluconate dehydrogenase, and conserved hypothetical proteins XAC0901 and XAC0223; most of them were not detected as differential for XAC when both bacteria were grown in NB medium, a pathogenicity non-inducing medium. XauB showed a very different profile from XAC in XAM-M, presenting 29 unique spots containing proteins related to a great diversity of metabolic pathways. Preponderant expression of PGM and XI in XAC was validated by Western Blot analysis in the periplasmic-enriched fractions of both bacteria. This work shows remarkable differences between the periplasmic-enriched proteomes of XAC and XauB, bacteria that cause symptoms with distinct degrees of severity during citrus infection. The results suggest that some proteins identified in XAC can have an important role in XAC pathogenicity.

Synthetic DNA system for structure-function studies of the high affinity CO2 uptake NDH-13 protein complex in cyanobacteria.

The CO2-concentrating mechanism (CCM) in cyanobacteria supports high rates of photosynthesis by greatly increasing the concentration of CO2 around the major carbon fixing enzyme, Rubisco. However, the CCM remains poorly understood, especially in regards to the enigmatic CO2-hydration enzymes which couple photosynthetically generated redox energy to the hydration of CO2 to bicarbonate. This CO2-hydration reaction is catalysed by specialized forms of NDH-1 thylakoid membrane complexes that contain phylogenetically unique extrinsic proteins that appear to couple CO2 hydration to NDH-1 proton pumping. The development of the first molecular genetic system to probe structure-function relationships of this important enzyme system is described. A CO2-hydration deficient strain was constructed as a recipient for DNA constructs containing different forms of the CO2-hydration system. This was tested by introducing a construct to an ectopic location that gives constitutive expression, rather than native inducible expression, of the ndhF3-ndhD3-cupA-cupS, (cupA operon) encoding high affinity CO2-hydration complex, NDH-13. Uptake assays show the restoration of high affinity for CO2 uptake, but demonstrate that the CupA complex can drive only modest uptake fluxes, underlining the importance of its tandem operation with the CupB-containing complex NDH-14, the complementary high flux, low affinity CO2 hydration system. Experiments with the carbonic anhydrase inhibitor, ethoxyzolamide, indicate that the NDH-13 complex is strongly inhibited, yet the remaining NDH-14 activity in the wild-type is less so, suggesting structural differences between the low affinity and high affinity CO2-hydration systems. This new construct will be an important tool to study and better understand cyanobacterial CO2 uptake systems.

Glycogen Synthesis and Metabolite Overflow Contribute to Energy Balancing in Cyanobacteria.

Understanding how living cells manage high-energy metabolites such as ATP and NADPH is essential for understanding energy transformations in the biosphere. Using light as the energy input, we find that energy charge (ratio of ATP over ADP+ATP) in the cyanobacterium Synechocystis sp. PCC 6803 varies in different growth stages, with a peak upon entry into the rapid growth phase, as well as a positive correlation with light intensity. In contrast, a mutant that can no longer synthesize the main carbon storage compound glycogen showed higher energy charge. The overflow of organic acids in this mutant under nitrogen depletion could also be triggered under high light in nitrogen-replete conditions, with an energy input level dependency. These findings suggest that energy charge in cyanobacteria is tightly linked to growth and carbon partition and that energy management is of key significance for their application as photosynthetic carbon dioxide-assimilating cell factories.

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction.

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.

Xanthomonas citri subsp. citri surface proteome by 2D-DIGE: Ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction.

Xanthomonas citri subsp. citri (XAC) is the causative agent of citrus canker, a disease of great economic impact around the world. Understanding the role of proteins on XAC cellular surface can provide new insights on pathogen-plant interaction. Surface proteome was performed in XAC grown in vivo (infectious) and in vitro (non-infectious) conditions, by labeling intact cells followed by cellular lysis and direct 2D-DIGE analysis. Seventy-nine differential spots were analyzed by mass spectrometry. Highest relative abundance for in vivo condition was observed for spots containing DnaK protein, 60kDa chaperonin, conserved hypothetical proteins, malate dehydrogenase, phosphomannose isomerase, and ferric enterobactin receptors. Elongation factor Tu, OmpA-related proteins, Oar proteins and some Ton-B dependent receptors were found in spots decreased in vivo. Some proteins identified on XAC's surface in infectious condition and predicted to be cytoplasmic, such as DnaK and 60KDa chaperonin, have also been previously found at cellular surface in other microorganisms. This is the first study on XAC surface proteome and results point to mediation of molecular chaperones in XAC-citrus interaction. The approach utilized here can be applied to other pathogen-host interaction systems and help to achieve new insights in bacterial pathogenicity toward promising targets of biotechnological interest. BIOLOGICAL SIGNIFICANCE: This research provides new insights for current knowledge of the Xanthomonas sp. pathogenicity. For the first time the 2D-DIGE approach was applied on intact cells to find surface proteins involved in the pathogen-plant interaction. Results point to the involvement of new surface/outer membrane proteins in the interaction between XAC and its citrus host and can provide potential targets of biotechnological interest for citrus canker control.

Interaction network and mass spectrometry data of Xanthomonas citri subsp. citri surface proteins from differential proteomic analysis of infectious and non-infectious cells.

Here we provide the mass-spectrometry and in silico interaction network dataset of proteins identified on our research article on surface proteomic analysis from Xanthomonas citri subsp. citri (XAC) cells grown in vivo (infectious) and in vitro (non-infectious, control) by 2D-DIGE approach. Fluorescence labeling of proteins were performed on intact cells followed by cellular lysis and labeled spots from 2D gel differing in abundance between the two conditions (ANOVA, p-value<0.05) were analyzed by a nano-electrospray tandem mass spectrometry Q-Tof Ultima API mass spectrometer (MicroMass/Waters) (LC-ESI-MS/MS). This article contains raw data of proteins detected in the 79 spots analyzed by LC-ESI-MS/MS approach and also an enrichment analysis on the resulting protein-protein interaction network performed with the Integrated Interactome System (IIS) platform and Cytoscape software. The data are supplementary to our original research article, "Xanthomonas citri subsp. citri surface proteome by 2D-DIGE: ferric enterobactin receptor and other outer membrane proteins potentially involved in citric host interaction" (Carnielli et al., 2016) [1], and raw data are available via Peptide Atlas (ftp://PASS00850:ZJ7425v@ftp.peptideatlas.org/).