Grigoriy E. Pinchuk

Towards Environmental Systems Biology of Shewanella

Bacteria of the genus Shewanella are known for their versatile electron-accepting capacities, which allow them to couple the decomposition of organic matter to the reduction of the various terminal electron acceptors that they encounter in their stratified environments. Owing to their diverse metabolic capabilities, shewanellae are important for carbon cycling and have considerable potential for the remediation of contaminated environments and use in microbial fuel cells. Systems-level analysis of the model species Shewanella oneidensis MR-1 and other members of this genus has provided new insights into the signal-transduction proteins, regulators, and metabolic and respiratory subsystems that govern the remarkable versatility of the shewanellae.

Constraint-based model of Shewanella oneidensis MR-1 metabolism: a tool for data analysis and hypothesis generation.

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level.

Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1

ABSTRACT Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe2O3), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.

Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction

ABSTRACT The solubility of orthophosphate (PO43−) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus Shewanella. Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca2+-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca2+ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca2+-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5′ nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.

Genome-Scale Modeling of Light-Driven Reductant Partitioning and Carbon Fluxes in Diazotrophic Unicellular Cyanobacterium Cyanothece sp. ATCC 51142

Genome-scale metabolic models have proven useful for answering fundamental questions about metabolic capabilities of a variety of microorganisms, as well as informing their metabolic engineering. However, only a few models are available for oxygenic photosynthetic microorganisms, particularly in cyanobacteria in which photosynthetic and respiratory electron transport chains (ETC) share components. We addressed the complexity of cyanobacterial ETC by developing a genome-scale model for the diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142. The resulting metabolic reconstruction, iCce806, consists of 806 genes associated with 667 metabolic reactions and includes a detailed representation of the ETC and a biomass equation based on experimental measurements. Both computational and experimental approaches were used to investigate light-driven metabolism in Cyanothece sp. ATCC 51142, with a particular focus on reductant production and partitioning within the ETC. The simulation results suggest that growth and metabolic flux distributions are substantially impacted by the relative amounts of light going into the individual photosystems. When growth is limited by the flux through photosystem I, terminal respiratory oxidases are predicted to be an important mechanism for removing excess reductant. Similarly, under photosystem II flux limitation, excess electron carriers must be removed via cyclic electron transport. Furthermore, in silico calculations were in good quantitative agreement with the measured growth rates whereas predictions of reaction usage were qualitatively consistent with protein and mRNA expression data, which we used to further improve the resolution of intracellular flux values.

Pyruvate and Lactate Metabolism by Shewanella oneidensis MR-1 under Fermentation, Oxygen Limitation, and Fumarate Respiration Conditions

ABSTRACT Shewanella oneidensis MR-1 is a facultative anaerobe that derives energy by coupling organic matter oxidation to the reduction of a wide range of electron acceptors. Here, we quantitatively assessed the lactate and pyruvate metabolism of MR-1 under three distinct conditions: electron acceptor-limited growth on lactate with O2, lactate with fumarate, and pyruvate fermentation. The latter does not support growth but provides energy for cell survival. Using physiological and genetic approaches combined with flux balance analysis, we showed that the proportion of ATP produced by substrate-level phosphorylation varied from 33% to 72.5% of that needed for growth depending on the electron acceptor nature and availability. While being indispensable for growth, the respiration of fumarate does not contribute significantly to ATP generation and likely serves to remove formate, a product of pyruvate formate-lyase-catalyzed pyruvate disproportionation. Under both tested respiratory conditions, S. oneidensis MR-1 carried out incomplete substrate oxidation, whereby the tricarboxylic acid (TCA) cycle did not contribute significantly. Pyruvate dehydrogenase was not involved in lactate metabolism under conditions of O2 limitation but was required for anaerobic growth, likely by supplying reducing equivalents for biosynthesis. The results suggest that pyruvate fermentation by S. oneidensis MR-1 cells represents a combination of substrate-level phosphorylation and respiration, where pyruvate serves as an electron donor and an electron acceptor. Pyruvate reduction to lactate at the expense of formate oxidation is catalyzed by a recently described new type of oxidative NAD(P)H-independent d-lactate dehydrogenase (Dld-II). The results further indicate that pyruvate reduction coupled to formate oxidation may be accompanied by the generation of proton motive force.

Oxygen-dependent autoaggregation in Shewanella oneidensis MR-1.

In aerobic chemostat cultures maintained at 50% dissolved O(2) tension (3.5 mg l(-1) dissolved O(2)), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl(2) and retained this multicellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein and glycoconjugates. Upon onset of O(2)-limited growth (dissolved O(2) below detection) however, the Ca(2+)-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O(2)-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O(2) concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologues of metal reduction genes, including mtrD (SO1782), mtrE (SO1781) and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H(2)O(2). Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

Inference of interactions in cyanobacterial- heterotrophic co-cultures via transcriptome sequencing

We used deep sequencing technology to identify transcriptional adaptation of the euryhaline unicellular cyanobacterium Synechococcus sp. PCC 7002 and the marine facultative aerobe Shewanella putrefaciens W3-18-1 to growth in a co-culture and infer the effect of carbon flux distributions on photoautotroph–heterotroph interactions. The overall transcriptome response of both organisms to co-cultivation was shaped by their respective physiologies and growth constraints. Carbon limitation resulted in the expansion of metabolic capacities, which was manifested through the transcriptional upregulation of transport and catabolic pathways. Although growth coupling occurred via lactate oxidation or secretion of photosynthetically fixed carbon, there was evidence of specific metabolic interactions between the two organisms. These hypothesized interactions were inferred from the excretion of specific amino acids (for example, alanine and methionine) by the cyanobacterium, which correlated with the downregulation of the corresponding biosynthetic machinery in Shewanella W3-18-1. In addition, the broad and consistent decrease of mRNA levels for many Fe-regulated Synechococcus 7002 genes during co-cultivation may indicate increased Fe availability as well as more facile and energy-efficient mechanisms for Fe acquisition by the cyanobacterium. Furthermore, evidence pointed at potentially novel interactions between oxygenic photoautotrophs and heterotrophs related to the oxidative stress response as transcriptional patterns suggested that Synechococcus 7002 rather than Shewanella W3-18-1 provided scavenging functions for reactive oxygen species under co-culture conditions. This study provides an initial insight into the complexity of photoautotrophic–heterotrophic interactions and brings new perspectives of their role in the robustness and stability of the association.

Sustained H2 Production Driven by Photosynthetic Water Splitting in a Unicellular Cyanobacterium

ABSTRACT The relationship between dinitrogenase-driven H2 production and oxygenic photosynthesis was investigated in a unicellular cyanobacterium, Cyanothece sp. ATCC 51142, using a novel custom-built photobioreactor equipped with advanced process control. Continuously illuminated nitrogen-deprived cells evolved H2 at rates up to 400 µmol ⋅ mg Chl−1 ⋅ h−1 in parallel with uninterrupted photosynthetic O2 production. Notably, sustained coproduction of H2 and O2 occurred over 100 h in the presence of CO2, with both gases displaying inverse oscillations which eventually dampened toward stable rates of 125 and 90 µmol ⋅ mg Chl−1 ⋅ h−1, respectively. Oscillations were not observed when CO2 was omitted, and instead H2 and O2 evolution rates were positively correlated. The sustainability of the process was further supported by stable chlorophyll content, maintenance of baseline protein and carbohydrate levels, and an enhanced capacity for linear electron transport as measured by chlorophyll fluorescence throughout the experiment. In situ light saturation analyses of H2 production displayed a strong dose dependence and lack of O2 inhibition. Inactivation of photosystem II had substantial long-term effects but did not affect short-term H2 production, indicating that the process is also supported by photosystem I activity and oxidation of endogenous glycogen. However, mass balance calculations suggest that carbohydrate consumption in the light may, at best, account for no more than 50% of the reductant required for the corresponding H2 production over that period. Collectively, our results demonstrate that uninterrupted H2 production in unicellular cyanobacteria can be fueled by water photolysis without the detrimental effects of O2 and have important implications for sustainable production of biofuels. IMPORTANCE The study provides an important insight into the photophysiology of light-driven H2 production by the nitrogen-fixing cyanobacterium Cyanothece sp. strain ATCC 51142. This work is also of significance for biotechnology, supporting the feasibility of “direct biophotolysis.” The sustainability of the process, highlighted by prolonged gas evolution with no clear sign of significant decay or apparent photodamage, provides a foundation for the future development of an effective, renewable, and economically efficient bio-H2 production process. The study provides an important insight into the photophysiology of light-driven H2 production by the nitrogen-fixing cyanobacterium Cyanothece sp. strain ATCC 51142. This work is also of significance for biotechnology, supporting the feasibility of “direct biophotolysis.” The sustainability of the process, highlighted by prolonged gas evolution with no clear sign of significant decay or apparent photodamage, provides a foundation for the future development of an effective, renewable, and economically efficient bio-H2 production process.