Mechanism and Consequences of Anaerobic Respiration of Cobalt by Shewanella oneidensis Strain MR-1

Bacteria from the genus Shewanella are the most diverse respiratory organisms studied to date and can utilize a variety of metals and metal(loid)s as terminal electron acceptors. These bacteria can potentially be used in bioremediation applications since the redox state of metals often influences both solubility and toxicity. Understanding molecular mechanisms by which metal transformations occur and the consequences of by-products that may be toxic to the organism and thus inhibitory to the overall process is significant to future applications for bioremediation. Here, we examine the ability of Shewanella oneidensis to catalyze the reduction of chelated cobalt. We describe an unexpected ramification of [Co(III)-EDTA]⁻ reduction by S. oneidensis: the formation of a toxic by-product. We found that [Co(II)-EDTA]²⁻, the product of [Co(III)-EDTA]⁻ respiration, inhibited the growth of S. oneidensis strain MR-1 and that this toxicity was partially abolished by the addition of MgSO₄. We demonstrate that [Co(III)-EDTA]⁻ reduction by S. oneidensis requires the Mtr extracellular respiratory pathway and associated pathways required to develop functional Mtr enzymes (the c-type cytochrome maturation pathway) and ensure proper localization (type II secretion). The Mtr pathway is known to be required for a variety of substrates, including some chelated and insoluble metals and organic compounds. Understanding the full substrate range for the Mtr pathway is crucial for developing S. oneidensis strains as a tool for bioremediation.     

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.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Substrate-Level Phosphorylation Is the Primary Source of Energy Conservation during Anaerobic Respiration of Shewanella oneidensis Strain MR-1

It is well established that respiratory organisms use proton motive force to produce ATP via F-type ATP synthase aerobically and that this process may reverse during anaerobiosis to produce proton motive force. Here, we show that Shewanella oneidensis strain MR-1, a nonfermentative, facultative anaerobe known to respire exogenous electron acceptors, generates ATP primarily from substrate-level phosphorylation under anaerobic conditions. Mutant strains lacking ackA (SO2915) and pta (SO2916), genes required for acetate production and a significant portion of substrate-level ATP produced anaerobically, were tested for growth. These mutant strains were unable to grow anaerobically with lactate and fumarate as the electron acceptor, consistent with substrate-level phosphorylation yielding a significant amount of ATP. Mutant strains lacking ackA and pta were also shown to grow slowly using N-acetylglucosamine as the carbon source and fumarate as the electron acceptor, consistent with some ATP generation deriving from the Entner-Doudoroff pathway with this substrate. A deletion strain lacking the sole F-type ATP synthase (SO4746 to SO4754) demonstrated enhanced growth on N-acetylglucosamine and a minor defect with lactate under anaerobic conditions. ATP synthase mutants grown anaerobically on lactate while expressing proteorhodopsin, a light-dependent proton pump, exhibited restored growth when exposed to light, consistent with a proton-pumping role for ATP synthase under anaerobic conditions. Although S. oneidensis requires external electron acceptors to balance redox reactions and is not fermentative, we find that substrate-level phosphorylation is its primary anaerobic energy conservation strategy. Phenotypic characterization of an ackA deletion in Shewanella sp. strain MR-4 and genomic analysis of other sequenced strains suggest that this strategy is a common feature of Shewanella.Shewanella oneidensis strain MR-1 is a nonfermentative, facultative anaerobe which respires various substrates, including oxygen, soluble metals, insoluble iron and manganese oxide minerals, electrodes, and organic compounds (8, 12, 18, 22). Other bacteria with the ability to respire electrodes and oxide minerals, such as Geobacter and Geothrix, oxidize acetate to carbon dioxide (4, 7, 9), consistent with these organisms generating ATP primarily from oxidative phosphorylation rather than substrate-level phosphorylation. Yet, an examination of metabolic end products and a variety of central metabolism and flux analyses of MR-1 show that acetate is the major product under anaerobic conditions (18, 27, 29, 31). The general anaerobic metabolism model for MR-1, as depicted in Fig. ​Fig.1,1, has key features of glycolysis via the Entner-Doudoroff pathway as well as acetyl coenzyme A (acetyl-CoA) flux toward acetate anaerobically via phosphate acetyltransferase (Pta) and acetate kinase (AckA) (27, 29, 31). High-performance liquid chromatography (HPLC) studies in our lab and others have shown that pyruvate may be excreted during lactate utilization both aerobically and anaerobically (30, 31), and MR-1 has not been shown to maintain significant flux through the tricarboxylic acid (TCA) cycle under anaerobic conditions (31).Open in a separate windowFIG. 1.Simplified model of S. oneidensis central metabolism. Entner-Doudoroff glycolysis yields two molecules of pyruvate. Under aerobic conditions, pyruvate facilitates the reduction of NAD⁺ to NADH before being completely oxidized to carbon dioxide in the TCA cycle. Anaerobically, pyruvate oxidation to acetyl-CoA yields formate before the pyruvate is converted to acetate. Formate is subsequently oxidized to carbon dioxide. Reactions catalyzed by acetate kinase and phosphate acetyltransferase are denoted AckA and Pta, respectively. QH₂ is reduced quinone. The model is based on several references (5, 24, 29, 31, 36).Characterization studies of proton motive force (PMF) in MR-1 have not definitively determined whether the source of anaerobic proton pumping or translocation is electron transport, ATP synthase, or metabolite transport (13, 19). Myers et al. demonstrated that anaerobic MR-1 cells starved of electron acceptor generate PMF in response to fumarate addition (19). However, the directionality of the ATP synthase (i.e., generation of ATP or ATPase to pump protons) was not characterized. Previous work has confirmed that proteorhodopsin (PR), a light-dependent, proton-pumping integral membrane protein, can be used in MR-1 to supplement PMF (13). However, the observed increase in PMF in wild-type cells expressing PR did not result in higher optical densities (ODs) or in a higher growth rate. Though all known bacteria depend on PMF, whether MR-1 uses that PMF for ATP production or uses ATP to help generate PMF under anaerobic conditions has yet to be determined.To examine ATP production in MR-1, growth on carbon sources that offer various amounts of substrate-level-derived ATP and reducing equivalents (NADH, formate, or quinones) in their oxidation was characterized. Two carbon sources entering central metabolism at different locations are N-acetylglucosamine (NAG) and lactate, which enter before and after glycolysis, respectively (Fig. ​(Fig.1)1) (24, 36). Both are oxidized to acetate and carbon dioxide anaerobically, though lactate yields one ATP and two reducing equivalents per molecule, while NAG yields three ATPs and four reducing equivalents per molecule. The differences in ATP yields derived from utilization of NAG versus lactate, combined with modification of those yields through gene deletions, allowed for characterization of ATP production in MR-1.The goal of this work was to elucidate the primary source of ATP generation under anaerobic conditions in MR-1. Data presented here support a model of anaerobic metabolism where substrate-level phosphorylation is the primary mechanism for ATP generation and where some amount of the ATP pool is used to generate PMF. Paradoxically, the most diverse respiratory organism characterized to date (8, 12, 22) does not generate ATP from electron transport reactions and PMF. Our finding highlights a critical difference in metabolic strategies between Shewanella and other organisms that are able to reduce insoluble substrates, such as Geobacter and Geothrix.     

Respiration and growth of Shewanella oneidensis MR-1 using vanadate as the sole electron acceptor

Shewanella oneidensis MR-1 is a free-living gram-negative gamma-proteobacterium that is able to use a large number of oxidizing molecules, including fumarate, nitrate, dimethyl sulfoxide, trimethylamine N-oxide, nitrite, and insoluble iron and manganese oxides, to drive anaerobic respiration. Here we show that S. oneidensis MR-1 is able to grow on vanadate as the sole electron acceptor. Oxidant pulse experiments demonstrated that proton translocation across the cytoplasmic membrane occurs during vanadate reduction. Proton translocation is abolished in the presence of protonophores and the inhibitors 2-heptyl-4-hydroxyquinoline N-oxide and antimycin A. Redox difference spectra indicated the involvement of membrane-bound menaquinone and cytochromes c, which was confirmed by transposon mutagenesis and screening for a vanadate reduction-deficient phenotype. Two mutants which are deficient in menaquinone synthesis were isolated. Another mutant with disruption in the cytochrome c maturation gene ccmA was unable to produce any cytochrome c and to grow on vanadate. This phenotype could be restored by complementation with the pEC86 plasmid expressing ccm genes from Escherichia coli. To our knowledge, this is the first report of E. coli ccm genes being functional in another organism. Analysis of an mtrB-deficient mutant confirmed the results of a previous paper indicating that OmcB may function as a vanadate reductase or may be part of a vanadate reductase complex.     

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 10⁴ CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 10³ CFU/g.     

Siderophores Are Not Involved in Fe(III) Solubilization during Anaerobic Fe(III) Respiration by Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 respires a wide range of anaerobic electron acceptors, including sparingly soluble Fe(III) oxides. In the present study, S. oneidensis was found to produce Fe(III)-solubilizing organic ligands during anaerobic Fe(III) oxide respiration, a respiratory strategy postulated to destabilize Fe(III) and produce more readily reducible soluble organic Fe(III). In-frame gene deletion mutagenesis, siderophore detection assays, and voltammetric techniques were combined to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration were synthesized via siderophore biosynthesis systems and (ii) if the Fe(III)-siderophore reductase was required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. Genes predicted to encode the siderophore (hydroxamate) biosynthesis system (SO3030 to SO3032), the Fe(III)-hydroxamate receptor (SO3033), and the Fe(III)-hydroxamate reductase (SO3034) were identified in the S. oneidensis genome, and corresponding in-frame gene deletion mutants were constructed. ΔSO3031 was unable to synthesize siderophores or produce soluble organic Fe(III) during aerobic respiration yet retained the ability to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. ΔSO3034 retained the ability to synthesize siderophores during aerobic respiration and to solubilize and respire Fe(III) at wild-type rates during anaerobic Fe(III) oxide respiration. These findings indicate that the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are not synthesized via the hydroxamate biosynthesis system and that the Fe(III)-hydroxamate reductase is not essential for respiration of Fe(III)-citrate or Fe(III)-nitrilotriacetic acid (NTA) as an anaerobic electron acceptor.Bacterial electron transfer to sparingly soluble electron acceptors is a critical component of a wide variety of environmental and energy-generating processes, including biogeochemical cycling of metals, degradation of natural and contaminant organic matter, weathering of clays and minerals, biomineralization of Fe-bearing minerals, reductive precipitation of toxic metals and radionuclides, and electricity generation in microbial fuel cells (17, 33, 34). Anaerobic and facultatively anaerobic bacteria capable of respiring sparingly soluble (<10⁻²⁵ M at pH 7) Fe(III) oxides are ubiquitous in nature and may be found in marine, freshwater, and terrestrial environments, including metal- and radionuclide-contaminated subsurface aquifers (25, 34). Fe(III)-respiring prokaryotes are also deeply rooted and scattered throughout the domains Bacteria and Archaea (possibly indicating an ancient metabolic process) and include hyperthermophiles, psychrophiles, acidophiles, and extreme barophiles (34). Despite their potential environmental, energy-generating, and evolutionary significance, the molecular details of microbial Fe(III) respiration remain unclear.Fe(III)-respiring, neutrophilic bacteria are presented with a unique physiological challenge: they are required to respire anaerobically on electron acceptors found largely as sparingly soluble Fe(III) oxides presumably unable to contact periplasm- or inner membrane (IM)-localized electron transport systems. To overcome this problem, Fe(III)-respiring bacteria are postulated to employ novel respiratory strategies not found in other bacteria (e.g., aerobes, denitrifiers, sulfate-reducing bacteria, and methanogens) that respire soluble electron acceptors (17, 38). The novel respiratory strategies include (i) a direct-contact pathway in which terminal Fe(III) reductases are secreted to the cell outer membrane (OM), where they contact and deliver electrons directly to external Fe(III) oxides (18, 23, 40, 42, 48, 57, 64, 67), (ii) a two-step electron shuttling pathway in which bacterially reduced endogenous or exogenous electron shuttles deliver electrons to external Fe(III) oxides in a second (abiotic) electron transfer reaction (11, 26, 39, 45), and (iii) a two-step Fe(III) chelation (solubilization) pathway in which Fe(III) oxides are first nonreductively dissolved by endogenously synthesized organic ligands prior to reduction of the resulting soluble organic Fe(III) [Fe(III) bound to an organic molecule] complexes (36, 59).Candidate organic ligands for production of soluble organic Fe(III) during anaerobic Fe(III) oxide respiration include siderophores, the Fe(III)-chelating compounds synthesized and secreted by a wide variety of bacteria and fungi for solubilization and subsequent assimilation of otherwise inaccessible Fe(III) substrates (12, 44, 49, 63). Hydroxamate-type siderophores are produced via N⁶ hydroxylation and N⁶ acylation of l-ornithine and, in some cases, cyclization to macrocyclic ring structures (13). The macrocyclic siderophores bisucaberin and putrebactin, for example, are two structural analogs of the cyclic bis(hydroxamate) siderophore alcaligin, synthesized by Aliivibrio salmonicida and Shewanella putrefaciens strain 200, respectively (27, 32, 65). After transport across the cell envelope via a TonB-dependent pathway, Fe(III) is subsequently released from the Fe(III)-siderophore complex by ligand exchange reactions promoted by siderophore ligand hydrolysis and/or protonation or by Fe(III)-siderophore reduction and release of Fe(II) to acceptor ligands (9, 66).The main objectives of the present study were to determine (i) if the Fe(III)-solubilizing organic ligands produced by S. oneidensis during anaerobic Fe(III) oxide respiration are synthesized by Fe(III)-siderophore biosynthesis systems and (ii) if Fe(III)-siderophore reductases are required for respiration of soluble organic Fe(III) as an anaerobic electron acceptor. The experimental strategy for this study included (i) identification of genes encoding the siderophore biosynthesis and Fe(III)-siderophore reductase systems in the S. oneidensis genome, (ii) generation of in-frame deletions in the corresponding siderophore biosynthesis and Fe(III)-siderophore reductase genes, (iii) tests of the resulting siderophore biosynthesis mutants for production of siderophores and soluble organic Fe(III) during aerobic and anaerobic Fe(III) oxide respiration, and (iv) tests of the resulting Fe(III)-siderophore reductase mutants for respiration of soluble organic Fe(III) as an anaerobic electron acceptor.     

一株奥奈达希瓦氏菌烈性噬菌体的分离、纯化及鉴定

【目的】从环境中分离获得希瓦氏菌烈性噬菌体,并对其性质进行研究。【方法】以4株希瓦氏菌为宿主菌,采用双层平板法从污水样品中分离得到奥奈达希瓦氏菌MR-1烈性噬菌体M1;观察噬菌斑特征;利用超速离心法浓缩M1颗粒,进一步用氯化铯密度梯度离心纯化;采用透射电子显微镜观察纯化的M1颗粒;提取M1核酸,通过核酸酶处理分析其核酸类型及结构;绘制一步生长曲线。【结果】噬菌体M1在双层平板上形成圆形的噬菌斑,清晰透明,边缘光滑,直径为2.3 mm-2.5 mm;经电镜观察,噬菌体M1头部呈二十面体,直径约为55 nm,尾长约为170 nm,尾部可收缩,属于肌尾噬菌体科(Myoviridae);通过酶切分析表明噬菌体M1核酸为线形双链DNA;一步生长曲线显示该噬菌体感染后完成一个复制循环所需要的时间约为15-20 min。【结论】噬菌体M1属肌尾噬菌体科,研究结果为后续研究病毒在地球微生物成岩过程中所起的作用提供了实验材料。     

Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide

Nitric oxide (NO) signaling in animals controls processes such as smooth muscle relaxation and neurotransmission by activation of soluble guanylate cyclase (sGC). Prokaryotic homologues of the sGC heme domain, called H-NOX domains, have been identified and are generally found in a predicted operon in conjunction with a histidine kinase. Here, we show that an H-NOX protein (SO2144) from Shewanella oneidensis directly interacts with the sensor histidine kinase (SO2145), binds NO in a 5-coordinate complex similar to mammalian sGC, and in that form inhibits the activity of a histidine kinase (SO2145). We also describe the first account of NO formation by S. oneidensis under anaerobic growth conditions derived from nitrate and nitrite. These observations suggest that the S. oneidensis H-NOX and histidine kinase pair function as part of a novel two-component signaling pathway that is responsive to NO formation from higher nitrogen oxides used as electron acceptors when oxygen is low and thereby functioning as an environmental sensor.     

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.     

Vanadium(V) Reduction by Shewanella oneidensis MR-1 Requires Menaquinone and Cytochromes from the Cytoplasmic and Outer Membranes

The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.     

Expression of cspH, Encoding the Cold Shock Protein in Salmonella enterica Serovar Typhimurium UK-1

Both Salmonella enterica serovar Typhimurium and Escherichia coli contain the cspH gene encoding CspH, one of the cold shock proteins (CSPs). In this study, we investigated the expression of cspH in S. enterica serovar Typhimurium and found that it was induced in response to a temperature downshift during exponential phase. The cspH promoter was activated at 37 degrees C, and its mRNA was more stable than the other csp mRNAs at 37 degrees C. Moreover, lacZ expression of the translational cspH-lacZ fusion was induced at that temperature. Interestingly, the cspH mRNA had a much shorter 5'-untranslated region than those in the other cold-shock-inducible genes, and the promoter sequence, which was only 55 bp, was sufficient for cspH expression. The 14-base downstream box located 12 bases downstream of the initiation codon of cspH mRNA was essential for its cold shock activation.     

A Biochemical Approach to Study the Role of the Terminal Oxidases in Aerobic Respiration in Shewanella oneidensis MR-1

The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb ₃-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb ₃-type oxidase is the major terminal oxidase under aerobic conditions while both cbb ₃-type and bd-type oxidases are involved in respiration at low-O₂ tensions. On the contrary, the low O₂-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa ₃-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa ₃-type oxidase in S. oneidensis MR-1 are discussed.     

Indirect modulation of the intracellular c-Di-GMP level in Shewanella oneidensis MR-1 by MxdA

The GGDEF domain protein MxdA, which is important for biofilm formation in Shewanella oneidensis MR-1, was hypothesized to possess diguanylate cyclase activity. Here, we demonstrate that while MxdA controls the cellular level of c-di-GMP in S. oneidensis, it modulates the c-di-GMP pool indirectly.     

Construction and elementary mode analysis of a metabolic model for Shewanella oneidensis MR-1

A stoichiometric model describing the central metabolism of Shewanella oneidensis MR-1 wild-type and derivative strains was developed and used in elementary mode analysis (EMA). Shewanella oneidensis MR-1 can anaerobically respire a diverse pool of electron acceptors, and may be applied in several biotechnology settings, including bioremediation of toxic metals, electricity generation in microbial fuel cells, and whole-cell biocatalysis. The metabolic model presented here was adapted and verified by comparing the growth phenotypes of 13 single- and 1 double-knockout strains, while considering respiration via aerobic, anaerobic fumarate, and anaerobic metal reduction (Mtr) pathways, and utilizing acetate, n-acetylglucosamine (NAG), or lactate as carbon sources. The gene ppc, which encodes phosphoenolpyruvate carboxylase (Ppc), was determined to be necessary for aerobic growth on NAG and lactate, while not essential for growth on acetate. This suggests that Ppc is the only active anaplerotic enzyme when cultivated on lactate and NAG. The application of regulatory and substrate limitations to EMA has enabled creation of metabolic models that better reflect biological conditions, and significantly reduce the solution space for each condition, facilitating rapid strain optimization. This wild-type model can be easily adapted to include utilization of different carbon sources or secretion of different metabolic products, and allows the prediction of single- and multiple-knockout strains that are expected to operate under defined conditions with increased efficiency when compared to wild type cells.     

Nutritional and Metabolic Requirements for the Infection of HeLa Cells by Salmonella enterica Serovar Typhimurium

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food - and water - borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.     

Modulation of Biofilm-Formation in Salmonella enterica Serovar Typhimurium by the Periplasmic DsbA/DsbB Oxidoreductase System Requires the GGDEF-EAL Domain Protein STM3615

In Salmonella enterica serovar Typhimurium (S. Typhimurium), biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3′, 5′-di- guanosine monophosphate (c-di-GMP) through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either dsbA or dsbB, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a red, dry and rough (rdar) colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting csgD in either dsb mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the dsb mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene STM3615 (yhjK), previously not connected to rdar morphotype development, also abrogated the escalated rdar morphotype formation in dsb mutant backgrounds. Enhanced biofilm-formation in dsb mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both dsb mutants initially showed an escalated rdar morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in S. Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s) as signaling mediators.