The authors wish to note the following: ''We wish to add direct references to a stochastic model of DNA replication previously applied to the Xenopus laevis early embryonic divisions. That model was applied to molecular combing experiments on cellfree extracts from Xenopus laevis embryos.'' The additional references appear below. www.pnas.org/cgi

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.

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.

The solubility of orthophosphate (PO 4 3؊ ) 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 Ca 2؉ -independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca 2؉ 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 Ca 2؉ -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.Phosphorus (P) is a key element that often limits bacterial growth in various freshwater and marine habitats (13,40,44,51,53). Inorganic phosphate (P i ), or orthophosphate (PO 4 3Ϫ ), can serve as a direct source of P for essentially all physiological groups of microorganisms in both natural environments and laboratory media. Measurements of soluble phosphate in different aquatic environments, however, suggest that concentrations of bioavailable P i are very low. Recent studies have revealed that P i represents only a small fraction of soluble reactive P in natural waters and that even in eutrophic systems, its concentration may be as low as 27 pM (2,20,25). This is not surprising, since in a variety of aquatic systems, soils, and sediments, P i bioavailability can be controlled by adsorption to metal oxides (3, 16, 59) and through chemical reactions with hydrous oxides, amorphous and crystalline complexes of Fe, Al, and Ca, and organic matter (2, 41, 49). In particular, the reaction of P i with Fe(III) oxides such as goethite can result in the precipitation of tinticite [Fe 6 (PO 4 ) 4 (OH) 6 · 7H 2 O] (22) or griphite [Fe 3 Mn 2 (PO 4 ) 2 · 5(OH) 2 ] (32) depending on the solution and surface conditions. The latter findings entail important physiological implications for dissimilatory metal-reducing bacteria residing in zones with high concentration...

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 O 2 , 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 O 2 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.Shewanella oneidensis MR-1 is a facultatively anaerobic, Gram-negative bacterium that generates energy by coupling the oxidation of organic compounds to the reduction of a wide range of electron acceptors, including O 2 , fumarate, and Fe(III) (15,17). The diverse metabolic capabilities of Shewanella species provide a competitive advantage in a range of environments that are subject to spatial and temporal variations in the type and concentration of electron acceptors (for a review, see reference 7). Considered strictly respiratory organisms, oxidative phosphorylation is thought to be the primary pathway for ATP synthesis in shewanellae (18,29). A recent report, however, demonstrated that S. oneidensis MR-1 could gain energy for survival by fermenting pyruvate (15). Although the physiological significance of this process in S. oneidensis MR-1 is yet to be understood, fermentative metabolism in obligatory respiratory bacteria may represent an important mechanism of survival in the absence of available electron acceptors. Long-term survival via pyruvate fermentation was also reported previously for Pseudomonas aeruginosa (6).A common metabolic trait displayed by shewanellae ...