一株海洋产电菌Shewanella sp. S2的筛选和产电分析

以厦门白城海域的潮间带表面沉积物为菌种来源筛选得到一株具有电催化活性的菌株S2,该菌株的16S rRNA和gyrB基因发育树与Shewanella oneidensis MR-1同支,相似性分别为98.5%和87%,葡萄糖、木糖、半乳糖等碳源利用及最佳生长的NaCl浓度与S.oneidensis MR-1有显著差别,因此初步鉴定为Shewanella属菌株,命名为Shewanella sp.S2。初步研究了菌株S2产电活性,在以乳酸作为碳源产电时,电压最高为150mV,相应的电流密度为66.1mA/m2。     

High frequency of glucose-utilizing mutants in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 has conventionally been considered unable to use glucose as a carbon substrate for growth. The genome sequence of S. oneidensis MR-1 however suggests the ability to use glucose. Here, we demonstrate that during initial glucose exposure, S. oneidensis MR-1 quickly and frequently gains the ability to utilize glucose as a sole carbon source, in contrast to wild-type S. oneidensis, which cannot immediately use glucose as a sole carbon substrate. High-performance liquid chromatography and (14)C glucose tracer studies confirm the disappearance in cultures and assimilation and respiration, respectively, of glucose. The relatively short time frame with which S. oneidensis MR-1 gained the ability to use glucose raises interesting ecological implications.     

Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions.     

Aggrandizing power output from Shewanella oneidensis MR-1 microbial fuel cells using calcium chloride

There are several interconnected metabolic pathways in bacteria essential for the conversion of carbon electron sources directly into electrical currents using microbial fuel cells (MFCs). This study establishes a direct exogenous method to increase power output from a Shewanella oneidensis MR-1 containing MFC by adding calcium chloride to the culture medium. The current output from each CaCl(2) concentration tested revealed that the addition of CaCl(2) to 1400 μM increased the current density by >80% (0.95-1.76 μA/cm(2)) using sodium lactate as the sole carbon source. Furthermore, polarization curves showed that the maximum power output could be increased from 157 to 330 μW with the addition of 2080 μM CaCl(2). Since the conductivity of the culture medium did not change after the addition of CaCl(2) (confirmed by EIS and bulk conductivity measurements), this increase in power was primarily biological and not based on ionic effects. Thus, controlling the concentration of CaCl(2) is a pathway to increase the efficiency and performance of S. oneidensis MR-1 MFCs.     

Hydrogen metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

Functional specificity of extracellular nucleases of Shewanella oneidensis MR-1

Bacterial species such as Shewanella oneidensis MR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases of S. oneidensis MR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease of Shewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg(2+) or Mn(2+)) were required for function. endA is cotranscribed with phoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion of endA abolished both extracellular degradation of DNA by S. oneidensis MR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM, endA deletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases of S. oneidensis exert specific functions required under different conditions.     

Current production and metal oxide reduction by Shewanella oneidensis MR-1 wild type and mutants

Shewanella oneidensis MR-1 is a gram-negative facultative anaerobe capable of utilizing a broad range of electron acceptors, including several solid substrates. S. oneidensis MR-1 can reduce Mn(IV) and Fe(III) oxides and can produce current in microbial fuel cells. The mechanisms that are employed by S. oneidensis MR-1 to execute these processes have not yet been fully elucidated. Several different S. oneidensis MR-1 deletion mutants were generated and tested for current production and metal oxide reduction. The results showed that a few key cytochromes play a role in all of the processes but that their degrees of participation in each process are very different. Overall, these data suggest a very complex picture of electron transfer to solid and soluble substrates by S. oneidensis MR-1.     

The role of 4-hydroxyphenylpyruvate dioxygenase in enhancement of solid-phase electron transfer by Shewanella oneidensis MR-1

We hypothesized that Shewanella oneidensis MR-1, a model dissimilatory metal-reducing bacterium, could utilize environmentally relevant concentrations of tyrosine to produce pyomelanin for enhanced Fe(III) oxide reduction. Because homogentisate is an intermediate of the tyrosine degradation pathway, and a precursor of a redox-cycling metabolite, pyomelanin, we evaluated the process of homogentisate production by S. oneidensis MR-1, in order to identify the key steps involved in pyomelanin production. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. We used genetic analysis and physiological characterization of MR-1 strains either deficient in or displaying substantially increased pyomelanin production. The relative significance imparted by pyomelanin on solid-phase electron transfer was also addressed using electrochemical techniques, which allowed us to extend the genetic and physiological findings to biogeochemical cycling of metals. Based on our findings, environmental production of pyomelanin from available organic precursors could contribute to the survival of S. oneidensis MR-1 when dissolved oxygen concentrations become low, by providing an increased capacity for solid-phase metal reduction. This study demonstrates the role of organic precursors and their concentrations in pyomelanin production, solid phase metal reduction and biogeochemical cycling of iron.     

Shewanella oneidensis MR-1 fluxome under various oxygen conditions

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.     

Anaerobic biodecolorization mechanism of methyl orange by Shewanella oneidensis MR-1

In this work, we investigated the anaerobic decolorization of methyl orange (MO), a typical azo dye, by Shewanella oneidensis MR-1, which can use various organic and inorganic substances as its electron acceptor in natural and engineered environments. S. oneidensis MR-1 was found to be able to obtain energy for growth through anaerobic respiration accompanied with dissimilatory azo-reduction of MO. Chemical analysis shows that MO reduction occurred via the cleavage of azo bond. Block of Mtr respiratory pathway, a transmembrane electron transport chain, resulted in a reduction of decolorization rate by 80%, compared to the wild type. Knockout of cymA resulted in a substantial loss of its azo-reduction ability, indicating that CymA is a key c-type cytochrome in the electron transfer chain to MO. Thus, the MtrA-MtrB-MtrC respiratory pathway is proposed to be mainly responsible for the anaerobic decolorization of azo dyes such as MO by S. oneidensis.     

产电微生物基因调控网络的构建和特异性通路分析

研究产电微生物胞外电子传递过程和机制,发现与产电效率相关的关键基因、通路和代谢物,是微生物燃料电池研究中的关键技术。为了发现在胞外电子传递过程中起到关键作用的基因以及通路,首先利用比较基因组学的方法,以模式微生物大肠杆菌和同属希瓦氏菌的其他菌株为参考,构建了Shewanella.onedensis MR-1的全基因组基因转录调控网络,大大扩展了目前已知的基因调控关系。然后以此网络为基础,结合基于蛋白质相互作用分析得到的胞外电子传递通路,构建了与胞外电子传递直接传递密切相关的细胞色素C编码基因及其相关调控基因构成的子网络,结合全基因组基因表达数据,研究了特异性条件下胞外电子传递的可能通路和基因调控过程。     

Using genome-context data to identify specific types of functional associations in pathway/genome databases

BACKGROUND: Hundreds of genes lacking homology to any protein of known function are sequenced every day. Genome-context methods have proved useful in providing clues about functional annotations for many proteins. However, genome-context methods detect many biological types of functional associations, and do not identify which type of functional association they have found. RESULTS: We have developed two new genome-context-based algorithms. Algorithm 1 extends our previous algorithm for identifying missing enzymes in predicted metabolic pathways (pathway holes) to use genome-context features. The new algorithm has significantly improved scope because it can now be applied to pathway reactions to which sequence similarity methods cannot be applied due to an absence of known sequences for enzymes catalyzing the reaction in other organisms. The new method identifies at least one known enzyme in the top ten hits for 58% of EcoCyc reactions that lack enzyme sequences in other organisms. Surprisingly, the addition of genome-context features does not improve the accuracy of the algorithm when sequences for the enzyme do exist in other organisms. Algorithm 2 uses genome-context methods to predict three distinct types of functional relationships between pairs of proteins: pairs that occur in the same protein complex, the same pathway, or the same operon. This algorithm performs with varying degrees of accuracy on each type of relationship, and performs best in predicting pathway and protein complex relationships.     

Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions

Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple pathways within a single organism leading to Cr(VI) reduction has not been reported previously.     

Enzymes related to fructose utilization in Pseudomonas cepacia

Growth of Pseudomonas cepacia on fructose, mannitol, or sorbitol depended on formation of an inducible fructokinase (forming fructose-6-phosphate) and the presence of enzymes of the Entner-Doudoroff pathway. Mutants deficient in any of these enzymes failed to utilize the aforementioned carbohydrates. Fructokinase deficiency did not affect growth of the bacteria on glucose. Fructose was accumulated intracellularly by active transport. Mutants blocked in transport of fructose grew normally on mannitol or sorbitol despite their inability to utilize fructose. Growth on either of these hexitols or on galactitol was accompanied by induction of two hexitol dehydrogenases, one active primarily with mannitol and the other active with sorbitol and galactitol. As expected, a mutant deficient in mannitol dehydrogenase failed to utilize mannitol as a carbon and energy source but grew normally on sorbitol and galactitol. Extracts of bacteria grown on fructose, mannitol, or sorbitol and higher levels of phosphoglucose isomerase than extracts of bacteria grown on alternate carbon sources such as citrate or phthalate. The higher levels were due to appearance of a second phosphoglucose isomerase species not present in cells with the lower activity. The results indicate that the initial steps in fructose utilization by P. cepacia differ from those of most other pseudomonads, which transport fructose by phosphoenolpyruvate-dependent translocation, forming fructose-1-phosphate, and suggest that degradation of fructose, mannitol, and sorbitol occurs primarily via the Entner-Doudoroff pathway.     

厌氧条件下Shewanella oneidensis MR-1对2,4-二硝基甲苯的还原转化

【目的】研究Shewanella oneidensis MR-1厌氧生物转化2,4-二硝基甲苯(2,4-DNT)的能力、转化过程和影响因素。【方法】以乳酸钠为电子供体, 2,4-DNT为电子受体, S. oneidensis MR-1为降解菌, 黄素为胞外电子载体, 设立四个不同的对照体系并监测各体系在转化过程中2,4-DNT及其产物的动态变化。同时研究不同2,4-DNT浓度下细胞的生长情况, 以及不同黄素浓度下2,4-DNT的降解情况。【结果】S. oneidensis MR-1菌能够高效还原转化2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT), 并将其进一步还原为2,4-二氨基甲苯(2,4-DAT), 黄素能加速转化过程。【结论】S. oneidensis MR-1菌具备高效还原转化2,4-DNT的能力, 为实际环境中硝基苯污染的原位修复提供科学依据。     

Pyruvate and lactate metabolism by Shewanella oneidensis MR-1 under fermentation, oxygen limitation, and fumarate respiration conditions

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.     

Identification of mobile elements and pseudogenes in the Shewanella oneidensis MR-1 genome

Shewanella oneidensis MR-1 is the first of 22 different Shewanella spp. whose genomes have been or are being sequenced and thus serves as the model organism for studying the functional repertoire of the Shewanella genus. The original MR-1 genome annotation revealed a large number of transposase genes and pseudogenes, indicating that many of the genome's functions may be decaying. Comparative analyses of the sequenced Shewanella strains suggest that 209 genes in MR-1 have in-frame stop codons, frameshifts, or interruptions and/or are truncated and that 65 of the original pseudogene predictions were erroneous. Among the decaying functions are that of one of three chemotaxis clusters, type I pilus production, starch utilization, and nitrite respiration. Many of the mutations could be attributed to members of 41 different types of insertion sequence (IS) elements and three types of miniature inverted-repeat transposable elements identified here for the first time. The high copy numbers of individual mobile elements (up to 71) are expected to promote large-scale genome recombination events, as evidenced by the displacement of the algA promoter. The ability of MR-1 to acquire foreign genes via reactions catalyzed by both the integron integrase and the ISSod25-encoded integrases is suggested by the presence of attC sites and genes whose sequences are characteristic of other species downstream of each site. This large number of mobile elements and multiple potential sites for integrase-mediated acquisition of foreign DNA indicate that the MR-1 genome is exceptionally dynamic, with many functions and regulatory control points in the process of decay or reinvention.