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1.
Although members of the genus Shewanella have common features (e.g., the presence of decaheme c-type cytochromes [c-cyts]), they are widely variable in genetic and physiological features. The present study compared the current-generating ability of S. loihica PV-4 in microbial fuel cells (MFCs) with that of well-characterized S. oneidensis MR-1 and examined the roles of c-cyts in extracellular electron transfer. We found that strains PV-4 and MR-1 exhibited notable differences in current-generating mechanisms. While the MR-1 MFCs maintained a constant current density over time, the PV-4 MFCs continued to increase in current density and finally surpassed the MR-1 MFCs. Coulombic efficiencies reached 26% in the PV-4 MFC but 16% in the MR-1 MFCs. Although both organisms produced quinone-like compounds, anode exchange experiments showed that anode-attached cells of PV-4 produced sevenfold more current than planktonic cells in the same chamber, while planktonic cells of MR-1 produced twice the current of the anode-attached cells. Examination of the genome sequence indicated that PV-4 has more c-cyt genes in the metal reductase-containing locus than MR-1. Mutational analysis revealed that PV-4 relied predominantly on a homologue of the decaheme c-cyt MtrC in MR-1 for current generation, even though it also possesses two homologues of the decaheme c-cyt OmcA in MR-1. These results suggest that current generation in a PV-4 MFC is in large part accomplished by anode-attached cells, in which the MtrC homologue constitutes the main path of electrons toward the anode.Some species of dissimilatory metal-reducing bacteria (DMRB) are able to reduce solid metal oxides as terminal electron acceptors and generate currents in microbial fuel cells (MFCs) (2, 11, 14, 30, 46). Although mixed cultures are often used in MFC experiments (13), studies seeking a mechanistic understanding of electron transfer to electrode surfaces typically target pure cultures of such DMRB, due to the complexity in microbial communities. Presently, two model DMRB, Shewanella oneidensis MR-1 and Geobacter sulfurreducens PCA (2, 3, 12, 18, 31), are used in most investigations.S. oneidensis MR-1 is a metabolically diverse DMRB that has been studied extensively for its potential use in bioremediation applications. For this reason, MR-1 was the first Shewanella species to have its genome completely sequenced and annotated (10). In addition, since the first report in 1999 when this microorganism was shown to have the ability to transfer electrons to the electrode without an exogenously added mediator (14), it has also become one of the model organisms for the study of electron transfer mechanisms in MFCs.Although the molecular mechanisms for extracellular electron transfer have not yet been elucidated fully, c-type cytochromes (c-cyts) appear to be the key cellular components involved in this process (38). In S. oneidensis MR-1, OmcA and MtrC are outer membrane (OM), decaheme c-cyts that are considered to be involved in the direct (directly attached) electron transfer to solid metal oxides and anodes of MFCs (9, 20, 22, 23, 47). Several pieces of evidence suggest that OmcA and MtrC form a complex and act in a cooperative manner (33, 37, 42), and these results correlate with the fact that the genes encoding these proteins constitute an operon-like cluster in the chromosome (1). It has also been shown that MtrC and OmcA have overlapping functions as terminal reductases of metal oxides (25, 38). OmcA and MtrC are also present on the surface of nanowires and may be involved in the long-range transfer of electrons (8). In addition to direct electron transfer, MR-1 has the ability to produce water-soluble electron-shuttle compounds (quinones and flavins) that are involved in the mediated electron transfer from cells to distant solid electron acceptors (metal oxides or MFC anodes) (21, 27, 44).Recently, the genome sequences of nearly 20 Shewanella strains have been completed and annotated, opening the door to study the diversity of their extracellular electron transfer mechanisms. A comparison of their genomes has shown that although they have some consensus OM c-cyt genes, variations exist in the number and order of these genes in their metal reductase-containing loci (6). One such species is S. loihica strain PV-4, which was recently isolated from an iron-rich microbial mat near a deep-sea hydrothermal vent located on the Loihi Seamount in Hawaii (7, 32). The phenotypic and phylogenetic characteristics of PV-4 were determined, with a subsequent study focusing on the metal reduction and iron biomineralization capabilities of this bacterium (32). Initial experiments performed in our laboratory revealed that PV-4 developed a c-cyt-dependent deep red color that was much more striking than that of strain MR-1 when grown anaerobically with iron oxide as the terminal electron acceptor (26). This allowed us to assume that PV-4 could have a high extracellular electron transfer ability. Accordingly, the present study evaluated the current-producing ability of strain PV-4 in MFCs and examined the roles of some c-cyts in extracellular electron transfer. Special attention was paid to the comparison of PV-4 with MR-1 to reveal differences in mechanisms for extracellular electron transfer. We report herein differences between these strains in the roles of OM c-cyts for extracellular electron transfer, the behaviors and metabolic patterns of MFC, and the resultant MFC performances.  相似文献   

2.
We have used scaling kinetics and the concept of kinetic competence to elucidate the role of hemeproteins OmcA and MtrC in iron reduction by Shewanella oneidensis MR-1. Second-order rate constants for OmcA and MtrC were determined by single-turnover experiments. For soluble iron species, a stopped-flow apparatus was used, and for the less reactive iron oxide goethite, a conventional spectrophotometer was used to measure rates. Steady-state experiments were performed to obtain molecular rate constants by quantifying the OmcA and MtrC contents of membrane fractions and whole cells by Western blot analysis. For reduction of soluble iron, rates determined from transient-state experiments were able to account for rates obtained from steady-state experiments. However, this was not true with goethite; rate constants determined from transient-state experiments were 100 to 1,000 times slower than those calculated from steady-state experiments with membrane fractions and whole cells. In contrast, addition of flavins to the goethite experiments resulted in rates that were consistent with both transient- and steady-state experiments. Kinetic simulations of steady-state results with kinetic constants obtained from transient-state experiments supported flavin involvement. Therefore, we show for the first time that OmcA and MtrC are kinetically competent to account for catalysis of soluble iron reduction in whole Shewanella cells but are not responsible for electron transfer via direct contact alone with insoluble iron-containing minerals. This work supports the hypothesis that electron shuttles are important participants in the reduction of solid Fe phases by this organism.Microbial processes play a central role in the cycling of organic and inorganic compounds on Earth. A select number of these compounds are assimilated by organisms and used as building material for the cell. In addition, a large number of organic and inorganic compounds are used as electron donors and acceptors for respiratory metabolism. Iron, one of the most abundant elements in the Earth''s crust, is both assimilated by life and utilized as an electron donor (Fe2+) and acceptor (Fe3+) in respiratory metabolism. Due to the importance of iron, microorganisms directly impact the fate and transport of iron, and these Fe impacts indirectly influence the (bio)geochemical cycles of many other elements, including the carbon cycle (38). Furthermore, respiratory metabolism evolved in microorganisms prior to the emergence of oxygenic photosynthesis and was very versatile in using a large number of both organic and inorganic terminal electron acceptors (TEAs) (29). Therefore, the use of iron as a TEA predates the use of molecular oxygen among organisms on Earth. When metals are used as the TEA, this process is referred to as dissimilatory metal reduction (DMR) or, in the case of iron, dissimilatory iron reduction (DIR). Interest in microbial DMR has intensified upon discovery of the ability of DMR bacteria to catalyze reactions of environmentally relevant contaminants, including radionuclides such as U(VI) (1, 19, 20, 24, 30, 45, 47).DMR has been extensively studied in the facultative anaerobe Shewanella oneidensis MR-1 due to its respiratory versatility (for a review, see references 22, 23, 39, and 49). This bacterium also has the ability to utilize soluble and insoluble forms of iron and manganese as TEAs (12, 17, 25, 29, 33, 36). Genetic and biochemical studies show that reduction of the soluble TEAs occurs at the cytoplasmic membrane (CM), the location of the proton motive force-generating electron transport. However, in utilization of insoluble oxides, CM-localized electrons need to traverse the periplasm and the outer membrane (OM) to reach the extracellular matrix. Three mechanisms of transfer of OM-localized electrons to metal oxides have been hypothesized: (i) electron transfer through direct contact with the metal oxide, (ii) cyclic reduction/oxidation of electron shuttles, and (iii) solubilization of the metals with chelators, followed by diffusion to and across the OM to the CM. For a review of the proposed mechanisms, see reference 15.While there are very few reports of bacteria producing chelators for DIR (51), there are more data to support the other two mechanisms. For example, using atomic force microscopy (AFM), Lower et al. (26) measured binding between both OM hemeproteins OmcA and MtrC and the iron oxide hematite and reported that OmcA shows a greater affinity toward hematite than does MtrC. These workers suggested that this tight binding is a prerequisite for direct electron transfer. Xiong et al. (55) also showed tight binding between OmcA and hematite by using dynamic light scattering and fluorescence correlation spectroscopy. Previous work in our laboratory (43, 44) demonstrating the rates of metal oxide reduction by membrane fractions from Shewanella also supported a direct-contact mechanism. Direct contact between cellular components and iron oxides is also consistent with the nanowire mechanism proposed by Gorby et al. (14).In support of the electron shuttle hypothesis, two laboratories independently showed that S. oneidensis produces extracellular flavins, which may act as electron-shuttling agents (31, 52). The involvement of flavins as an electron shuttle would explain previous reports that Shewanella secretes a small, quinone-like compound involved in electron transfer (41) and can utilize sterically sequestered iron located in alginate beads (18).While the previous studies described above provide compelling evidence for their respective mechanisms, none of the mechanisms have been subjected to detailed kinetic analysis. Kinetic studies, while not able to prove a mechanism, can be used to eliminate proposed mechanisms if rates of a reaction measured with purified enzymes, as determined in vitro, cannot account for the rates observed in whole cells. We previously described the importance of kinetic studies and how to apply these kinetic studies to DIR (6, 43, 44). Briefly, rate constants are determined by in vitro studies with purified enzymes. The kinetics of the enzyme of interest is then studied in the whole cell (or in subcellular fractions). The rate constants are then compared between these systems to determine whether the rate of the reactions catalyzed by the enzyme can account for the rates observed in the cell.In our previous work, we performed kinetic studies on membrane fractions of S. oneidensis (43, 44). In the present study, this kinetic characterization is extended to purified enzymes and with whole cells for the purpose of determining kinetic competence. While our major focus is on the reduction of insoluble iron forms, for comparative purposes and as a control to validate our studies with the insoluble oxides, our study also includes a kinetic analysis of soluble chelated iron forms. We have focused our kinetic studies on two OM-localized hemeproteins, OmcA and MtrC. Previous genetic knockout studies show the importance of these enzymes in metal oxide reduction as potential terminal metal reductases (2, 35, 37). We describe both transient- and steady-state kinetic analyses. While both hemeproteins have been studied by stopped-flow analysis (48, 53) and by steady-state methods in whole cells (4), no study has yet attempted to infer mechanisms from scale-up kinetic studies (where kinetic constants from transient-state experiments are compared with those obtained from steady-state experiments). Our studies were thus performed at three different kinetic scales: (i) transient-state iron reduction using purified OmcA and MtrC, (ii) steady-state kinetics with purified total membrane (TM) fractions, and (iii) steady-state whole-cell kinetics.  相似文献   

3.
The Mtr respiratory pathway of Shewanella oneidensis strain MR-1 is required to effectively respire both soluble and insoluble forms of oxidized iron. Flavins (riboflavin and flavin mononucleotide) recently have been shown to be excreted by MR-1 and facilitate the reduction of insoluble substrates. Other Shewanella species tested accumulated flavins in supernatants to an extent similar to that of MR-1, suggesting that flavin secretion is a general trait of the species. External flavins have been proposed to act as both a soluble electron shuttle and a metal chelator; however, at biologically relevant concentrations, our results suggest that external flavins primarily act as electron shuttles for MR-1. Using deletion mutants lacking various Mtr-associated proteins, we demonstrate that the Mtr extracellular respiratory pathway is essential for the reduction of flavins and that decaheme cytochromes found on the outer surface of the cell (MtrC and OmcA) are required for the majority of this activity. Given the involvement of external flavins in the reduction of electrodes, we monitored current production by Mtr respiratory pathway mutants in three-electrode bioreactors under controlled flavin concentrations. While mutants lacking MtrC were able to reduce flavins at 50% of the rate of the wild type in cell suspension assays, these strains were unable to grow into productive electrode-reducing biofilms. The analysis of mutants lacking OmcA suggests a role for this protein in both electron transfer to electrodes and attachment to surfaces. The parallel phenotypes of Mtr mutants in flavin and electrode reduction blur the distinction between direct contact and the redox shuttling strategies of insoluble substrate reduction by MR-1.Shewanella oneidensis strain MR-1 (MR-1) is a facultative anaerobe capable of respiring a variety of substrates, including various metals and metal oxides, a phenotype that is important for bioremediation and metal cycling in natural environments (22, 53). At near-neutral pH, Fe(III) and Mn(IV) often are present as insoluble oxide minerals. Dissimilatory metal-reducing bacteria such as MR-1 have developed pathways to transfer electrons from the interior of the cell to these external terminal electron acceptors. In some bacteria, these pathways also can transfer electrons to electrodes, which can be harnessed for renewable energy and remote biosensor applications (23, 26, 27). Beyond increasing our understanding of this unusual process, applying anaerobic microbial extracellular respiration to new technologies requires a thorough understanding of the molecular dynamics and cellular physiology of electron source utilization (substrate oxidation) and the reduction of insoluble terminal electron acceptor(s). There are four proposed mechanisms to explain how insoluble substrates are reduced by Shewanella: (i) direct contact, (ii) electron shuttling, (iii) chelation, and (iv) electrically conductive appendages (reviewed in reference 18). We will focus on the first three strategies here.Flavins recently have been discovered to accelerate the reduction of both iron oxide minerals (51) and electrodes (30) by MR-1. Riboflavin (vitamin B2) is a precursor for the biosynthesis of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) (13). Riboflavin and FMN both can be observed to build up in the supernatant of anaerobic and aerobically grown cultures of MR-1 (30, 51). However, the mechanism by which flavins enhance the rate of iron oxide mineral or electrode reduction is unknown, although recent work is consistent with a critical role for these compounds in mediating solid Fe(III) reduction by MR-1 (42). Since soluble (chelated) Fe(III) is reduced faster than insoluble Fe(III) by MR-1 (6), one possible explanation for the enhancement of insoluble iron reduction by flavins is increased available soluble iron via chelation (1, 2, 30). Flavins also may be utilized as redox-active compounds to traffic electrons between extracellular reductases on the surface of the cell and insoluble substrates (30, 51), a process termed electron shuttling (18, 39, 41). The chelation of the terminal electron acceptor during electrode reduction is not relevant when the anode is composed of graphite. Therefore, electron shuttling likely is responsible for the flavin enhancement of current production on poised-potential electrodes (30). However, it is unclear if the chelation of metals by flavins influences insoluble metal reduction by S. oneidensis (30).The Mtr pathway is required for the reduction of metals and electrodes (5, 6, 9, 17). Five primary protein components have been identified in this pathway: OmcA, MtrC, MtrA, MtrB, and CymA (47). Current models of electron transfer in MR-1 assume that electrons from carbon source oxidation are passed via the menaquinone pool to the inner membrane-anchored c-type cytochrome CymA (19, 31). These electrons then are transferred to a periplasmic c-type cytochrome, MtrA, and eventually to outer membrane (OM)-anchored c-type cytochromes MtrC and OmcA, which interact with an integral OM scaffolding protein, MtrB (32, 33, 43). These OM cytochromes then can reduce various substrates, including iron oxides and electrodes (8, 9, 12, 36, 47). Since the Mtr system is required by MR-1 to reduce many different substrates (18), it also could be capable of reducing extracellular flavins. Indeed, electron transfer to carbon electrodes is impaired in strains lacking Mtr pathway components (9, 17), which may be explained simply by a decreased ability to reduce extracellular flavins. The observation that Mtr mutants produce less current on electrodes than the wild type could be due to (i) less current generated per cell (either direct reduction or flavin mediated), (ii) decreased attachment to the electrode surface, (iii) differences in external flavin concentrations, or (iv) a combination of these three possibilities. Determining the specific activity (current produced per unit of attached biomass) of Mtr mutants on electrodes under conditions where flavin levels were controlled would allow for differentiation between these possibilities. To date, this kind of analysis has not been reported.The results presented here extend our knowledge of how S. oneidensis catalyzes the reduction of insoluble substrates. Experiments using a model iron chelator and electron shuttle are consistent with electron shuttling being the primary mechanism by which flavins enhance insoluble iron oxide reduction rates. Moreover, we demonstrate that MR-1 reduces extracellular flavins at physiologically relevant rates and that the Mtr pathway accounts for at least 95% of this activity. The specific activities of various mutant strains lacking Mtr pathway components on poised-potential electrodes also are reported. Our data suggest that MtrC is responsible for most of the electron transfer to carbon electrodes, while OmcA is involved in attachment and has a lesser role in electron transfer. These observations could have broader implications regarding the role of OmcA in the reduction of soluble and insoluble substrates (8, 9, 36).  相似文献   

4.
5.
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.  相似文献   

6.
Cyanophycin (multi-l-arginyl-poly-l-aspartic acid; also known as cyanophycin grana peptide [CGP]) is a putative precursor for numerous biodegradable technically used chemicals. Therefore, the biosynthesis and production of the polymer in recombinant organisms is of special interest. The synthesis of cyanophycin derivatives consisting of a wider range of constituents would broaden the applications of this polymer. We applied recombinant Saccharomyces cerevisiae strains defective in arginine metabolism and expressing the cyanophycin synthetase of Synechocystis sp. strain PCC 6308 in order to synthesize CGP with citrulline and ornithine as constituents. Strains defective in arginine degradation (Car1 and Car2) accumulated up to 4% (wt/wt) CGP, whereas strains defective in arginine synthesis (Arg1, Arg3, and Arg4) accumulated up to 15.3% (wt/wt) of CGP, which is more than twofold higher than the previously content reported in yeast and the highest content ever reported in eukaryotes. Characterization of the isolated polymers by different analytical methods indicated that CGP synthesized by strain Arg1 (with argininosuccinate synthetase deleted) consisted of up to 20 mol% of citrulline, whereas CGP from strain Arg3 (with ornithine carbamoyltransferase deleted) consisted of up to 8 mol% of ornithine, and CGP isolated from strain Arg4 (with argininosuccinate lyase deleted) consisted of up to 16 mol% lysine. Cultivation experiments indicated that the incorporation of citrulline or ornithine is enhanced by the addition of low amounts of arginine (2 mM) and also by the addition of ornithine or citrulline (10 to 40 mM), respectively, to the medium.Cyanophycin (multi-l-arginyl-poly-[l-aspartic acid]), also referred to as cyanophycin grana peptide (CGP), represents a polydisperse nonribosomally synthesized polypeptide consisting of poly(aspartic acid) as backbone and arginine residues bound to each aspartate (49) (Fig. (Fig.1).1). One enzyme only, referred to as cyanophycin synthetase (CphA), catalyzes the synthesis of the polymer from amino acids (55). Several CphAs originating from different bacteria exhibit specific features (2, 7, 5, 32, 50, 51). CphAs from the cyanobacteria Synechocystis sp. strain PCC 6308 and Anabaena variabilis ATCC 29413, respectively, exhibit a wide substrate range in vitro (2, 7), whereas CphA from Acinetobacter baylyi or Nostoc ellipsosporum incorporates only aspartate and arginine (23, 24, 32). CphA from Thermosynechococcus elongatus catalyzes the synthesis of CGP primer independently (5); CphA from Synechococcus sp. strain MA19 exhibits high thermostability (22). Furthermore, two types of CGP were observed concerning its solubility behavior: (i) a water-insoluble type that becomes soluble at high or low pH (34, 48) and (ii) a water-soluble type that was only recently observed in recombinant organisms (19, 26, 42, 50, 56). In the past, bacteria were mainly applied for the synthesis of CGP (3, 14, 18, 53), whereas recently there has been greater interest in synthesis in eukaryotes (26, 42, 50). CGP was accumulated to almost 7% (wt/wt) of dry matter in recombinant Nicotiana tabacum and Saccharomyces cerevisiae (26, 50).Open in a separate windowFIG. 1.Chemical structures of dipeptide building blocks of CGP variants detected in vivo. Structure: 1, aspartate-arginine; 2, aspartate-lysine; 3, aspartate-citrulline; 4, aspartate-ornithine. Aspartic acid is presented in black; the second amino acid of the dipeptide building blocks is shown in gray. The nomenclature of the carbon atoms is given.In S. cerevisiae the arginine metabolism is well understood and has been investigated (30) (see Fig. Fig.2).2). Arginine is synthesized from glutamate via ornithine and citrulline in eight successive steps. The enzymes acetylglutamate synthase, acetylglutamate kinase, N-acetyl-γ-glutamylphosphate reductase, and acetylornithine aminotransferase are involved in the formation of N-α-acetylornithine. The latter is converted to ornithine by acetylornithine acetyltransferase. In the next step, ornithine carbamoyltransferase (ARG3) condenses ornithine with carbamoylphosphate, yielding citrulline. Citrulline is then converted to l-argininosuccinate by argininosuccinate synthetase. The latter is in the final step cleaved into fumarate and arginine by argininosuccinate lyase (ARG4). The first five steps occur in the mitochondria, whereas the last three reactions occur in the cytosol (28, 54). Arginine degradation is initiated by arginase (CAR1) and ornithine aminotransferase (CAR2) (10, 11, 38, 39).Open in a separate windowFIG. 2.Schematic overview of the arginine metabolism in S. cerevisiae. Reactions shown in the shaded area occur in the mitochondria, while the other reactions are catalyzed in the cytosol. Abbreviations: ARG2, acetylglutamate synthase; ARG6, acetylglutamate kinase; ARG5, N-acetyl-γ-glutamyl-phosphate reductase; ARG8, acetylornithine aminotransferase; ECM40, acetylornithine acetyltransferase; ARG1, argininosuccinate synthetase; ARG3, ornithine carbamoyltransferase; ARG4, argininosuccinate lyase; CAR1, arginase; CAR2, ornithine aminotransferase.A multitude of putative applications for CGP derivatives are available (29, 41, 45, 47), thus indicating a need for efficient biotechnological production and for further investigations concerning the synthesis of CGP with alternative properties and different constituents. It is not only the putative application of the polymer as a precursor for poly(aspartic acid), which is used as biodegradable alternative for poly(acrylic acid) or for bulk chemicals, that makes CGP interesting (29, 45-47). In addition, a recently developed process for the production of dipeptides from CGP as a precursor makes the synthesis of CGP variants worthwhile (43). Dipeptides play an important role in medicine and pharmacy, e.g., as additives for malnourished patients, as treatments against liver diseases, or as aids for muscle proliferation (43). Because dipeptides are synthesized chemically (40) or enzymatically (6), novel biotechnological production processes are welcome.  相似文献   

7.
The biofilm matrix contributes to the chemistry, structure, and function of biofilms. Biofilm-derived membrane vesicles (MVs) and DNA, both matrix components, demonstrated concentration-, pH-, and cation-dependent interactions. Furthermore, MV-DNA association influenced MV surface properties. This bears consequences for the reactivity and availability for interaction of matrix polymers and other constituents.The biofilm matrix contributes to the chemistry, structure, and function of biofilms and is crucial for the development of fundamental biofilm properties (46, 47). Early studies defined polysaccharides as the matrix component, but proteins, lipids, and nucleic acids are all now acknowledged as important contributors (7, 15). Indeed, DNA has emerged as a vital participant, fulfilling structural and functional roles (1, 5, 6, 19, 31, 34, 36, 41, 43, 44). The phosphodiester bond of DNA renders this polyanionic at a physiological pH, undoubtedly contributing to interactions with cations, humic substances, fine-dispersed minerals, and matrix entities (25, 41, 49).In addition to particulates such as flagella and pili, membrane vesicles (MVs) are also found within the matrices of gram-negative and mixed biofilms (3, 16, 40). MVs are multifunctional bilayered structures that bleb from the outer membranes of gram-negative bacteria (reviewed in references 4, 24, 27, 28, and 30) and are chemically heterogeneous, combining the known chemistries of the biofilm matrix. Examination of biofilm samples by transmission electron microscopy (TEM) has suggested that matrix material interacts with MVs (Fig. (Fig.1).1). Since MVs produced in planktonic culture have associated DNA (11, 12, 13, 20, 21, 30, 39, 48), could biofilm-derived MVs incorporate DNA (1, 39, 40, 44)?Open in a separate windowFIG. 1.Possible interactions between matrix polymers and particulate structures. Shown is an electron micrograph of a thin section through a P. aeruginosa PAO1 biofilm. During processing, some dehydration occurred, resulting in collapse of matrix material into fibrillate arrangements (black filled arrows). There is a suggestion of interactions occurring with particulate structures such as MVs (hollow white arrow) and flagella (filled white arrows) (identified by the appearance and cross-dimension of these highly ordered structures when viewed at high magnification), which was consistently observed with other embedded samples and also with whole-mount preparations of gently disrupted biofilms (data not shown). The scale bar represents 200 nm.  相似文献   

8.
9.
10.
In the nitrate-responsive, homodimeric NarX sensor, two cytoplasmic membrane α-helices delimit the periplasmic ligand-binding domain. The HAMP domain, a four-helix parallel coiled-coil built from two α-helices (HD1 and HD2), immediately follows the second transmembrane helix. Previous computational studies identified a likely coiled-coil-forming α-helix, the signaling helix (S helix), in a range of signaling proteins, including eucaryal receptor guanylyl cyclases, but its function remains obscure. In NarX, the HAMP HD2 and S-helix regions overlap and apparently form a continuous coiled-coil marked by a heptad repeat stutter discontinuity at the distal boundary of HD2. Similar composite HD2-S-helix elements are present in other sensors, such as Sln1p from Saccharomyces cerevisiae. We constructed deletions and missense substitutions in the NarX S helix. Most caused constitutive signaling phenotypes. However, strongly impaired induction phenotypes were conferred by heptad deletions within the S-helix conserved core and also by deletions that remove the heptad stutter. The latter observation illuminates a key element of the dynamic bundle hypothesis for signaling across the heptad stutter adjacent to the HAMP domain in methyl-accepting chemotaxis proteins (Q. Zhou, P. Ames, and J. S. Parkinson, Mol. Microbiol. 73:801-814, 2009). Sequence comparisons identified other examples of heptad stutters between a HAMP domain and a contiguous coiled-coil-like heptad repeat sequence in conventional sensors, such as CpxA, EnvZ, PhoQ, and QseC; other S-helix-containing sensors, such as BarA and TorS; and the Neurospora crassa Nik-1 (Os-1) sensor that contains a tandem array of alternating HAMP and HAMP-like elements. Therefore, stutter elements may be broadly important for HAMP function.Transmembrane signaling in homodimeric bacterial sensors initiates upon signal ligand binding to the extracytoplasmic domain. In methyl-accepting chemotaxis proteins (MCPs), the resulting conformational change causes a displacement of one transmembrane α-helix (TM α-helix) relative to the other. This motion is conducted by the HAMP domain to control output domain activity (reviewed in references 33 and 39).Certain sensors of two-component regulatory systems share topological organization with MCPs. For example, the paralogous nitrate sensors NarX and NarQ contain an amino-terminal transmembrane signaling module similar to those in MCPs, in which a pair of TM α-helices delimit the periplasmic ligand-binding domain (Fig. (Fig.1)1) (24) (reviewed in references 32 and 62). The second TM α-helix connects to the HAMP domain. Hybrid proteins in which the NarX transmembrane signaling module regulates the kinase control modules of the MCPs Tar, DifA, and FrzCD demonstrate that NarX and MCPs share a mechanism for transmembrane signaling (73, 74, 81, 82).Open in a separate windowFIG. 1.NarX modular structure. Linear representation of the NarX protein sequence, from the amino (N) to carboxyl (C) termini, drawn to scale. The four modules are indicated at the top of the figure and shown in bold typeface, whereas domains within each module are labeled with standard (lightface) typeface. The nomenclature for modules follows that devised by Swain and Falke (67) for MCPs. Overlap between the HAMP domain HD2 and S-helix elements is indicated in gray. The three conserved Cys residues within the central module (62) are indicated. TM1 and TM2 denote the two transmembrane helices. Helices H1 to H4 of the periplasmic domain (24), and the transmitter domain H, N, D, G (79), and X (41) boxes, are labeled. The HPK 7 family of transmitter sequences, including NarX, have no F box and an unconventional G box (79). The scale bar at the bottom of the figure shows the number of aminoacyl residues.The HAMP domain functions as a signal conversion module in a variety of homodimeric proteins, including histidine protein kinases, adenylyl cyclases, MCPs, and certain phosphatases (12, 20, 77). This roughly 50-residue domain consists of a pair of amphiphilic α-helices, termed HD1 and HD2 (formerly AS1 and AS2) (67), joined by a connector (Fig. (Fig.2A).2A). Results from nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy, Cys and disulfide scanning, and mutational analysis converge on a model in which the HD1 and HD2 α-helices form a four-helix parallel coiled-coil (7, 20, 30, 42, 67, 75, 84). The mechanisms through which HAMP domains mediate signal conduction remain to be established (30, 42, 67, 84) (for commentary, see references 43, 49, and 50).Open in a separate windowFIG. 2.HAMP domain extensions. (A) Sequences from representative MCPs (E. coli Tsr and Salmonella enterica serovar Typhimurium Tar) and S-helix-containing sensors (E. coli NarX, NarQ, and BarA, and S. cerevisiae Sln1p). The HAMP domain, S-helix element, and the initial sequence of the MCP adaptation region are indicated. Flanking numbers denote positions of the terminal residues within the overall sequence. Sequential heptad repeats are indicated in alternating bold and standard (lightface) typeface. Numbering for heptad repeats in the methylation region and S-helix sequences has been described previously (4, 8). Numbers within the HD1 and HD2 helices indicate interactions within the HAMP domain (42). Residues at heptad positions a and d are enclosed within boxes, residues at the stutter position a/d are enclosed within a thickly outlined box, and residues in the S-helix ERT signature are in bold typeface. (B) NarX mutational alterations. Deletions are depicted as boxes, and missense substitutions are shown above the sequence. Many of these deletions were reported previously (10) and are presented here for comparison. The phenotypes conferred by the alterations are indicated as follows: impaired induction, black box; constitutive and elevated basal, light gray box; reversed response, dark gray box; wild-type, white box; null, striped box.Coiled-coils result from packing of two or more α-helices (27). The primary sequence of coiled-coils exhibits a characteristic heptad repeat pattern, denoted as a-b-c-d-e-f-g (52, 61), in which positions a and d are usually occupied by nonpolar residues (reviewed in references 1, 47, and 80). For example, the coiled-coil nature of the HAMP domain can be seen in the heptad repeat patterns within the HD1 and HD2 sequences (Fig. (Fig.2A2A).Coiled-coil elements adjacent to the HAMP domain have been identified in several sensors, including Saccharomyces cerevisiae Sln1p (69) and Escherichia coli NarX (60). Recently, this element was defined as a specific type of dimeric parallel coiled-coil, termed the signaling helix (S helix), present in a wide range of signaling proteins (8). Sequence comparisons delimit a roughly 40-residue element with a conserved heptad repeat pattern (Fig. (Fig.2A).2A). Based on mutational analyses of Sln1p and other proteins, the S helix is suggested to function as a switch that prevents constitutive activation of adjacent output domains (8).The term “signaling helix” previously was used to define the α4-TM2 extended helix in MCPs (23, 33). Here, we use the term S helix to denote the element described by Anantharaman et al. (8).The NarX and NarQ sensors encompass four distinct modules (Fig. (Fig.1):1): the amino-terminal transmembrane signaling module, the signal conversion module (including the HAMP domain and S-helix element), the central module of unknown function, and the carboxyl-terminal transmitter module (62). The S-helix element presumably functions together with the HAMP domain in conducting ligand-responsive motions from the transmembrane signaling module to the central module, ultimately regulating transmitter module activity.Regulatory output by two-component sensors reflects opposing transmitter activities (reviewed in reference 55). Positive function results from transmitter autokinase activity; the resulting phosphosensor serves as a substrate for response regulator autophosphorylation. Negative function results from transmitter phosphatase activity, which accelerates phosphoresponse regulator autodephosphorylation (reviewed in references 64 and 65). We envision a homogeneous two-state model for NarX (17), in which the equilibrium between these mutually exclusive conformations is modulated by ligand-responsive signaling.Previous work from our laboratory concerned the NarX and other HAMP domains (9, 10, 26, 77) and separately identified a conserved sequence in NarX and NarQ sensors, the Y box, that roughly corresponds to the S helix (62). Therefore, we were interested to explore the NarX S helix and to test some of the predictions made for its function. Results show that the S helix is critical for signal conduction and suggest that it functions as an extension of the HAMP HD2 α-helix in a subset of sensors exemplified by Sln1p and NarX. Moreover, a stutter discontinuity in the heptad repeat pattern was found to be essential for the NarX response to signal and to be conserved in several distinct classes of HAMP-containing sensors.  相似文献   

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l-2-Amino-4-methoxy-trans-3-butenoic acid (AMB) is a potent antibiotic and toxin produced by Pseudomonas aeruginosa. Using a novel biochemical assay combined with site-directed mutagenesis in strain PAO1, we have identified a five-gene cluster specifying AMB biosynthesis, probably involving a thiotemplate mechanism. Overexpression of this cluster in strain PA7, a natural AMB-negative isolate, led to AMB overproduction.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of human infections and is considered the main pathogen responsible for chronic pneumonia in cystic fibrosis patients (7, 23). P. aeruginosa also infects other organisms, such as insects (4), nematodes (6), plants (18), and amoebae (20). Its ability to thrive as a pathogen and to compete in aquatic and soil environments can be partly attributed to the production and interplay of secreted virulence factors and secondary metabolites. While the importance of many of these exoproducts has been studied, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid (AMB; methoxyvinylglycine) (Fig. (Fig.1)1) has received only limited attention. Identified during a search for new antibiotics, AMB was found to reversibly inhibit the growth of Bacillus spp. (26) and Escherichia coli (25) and was later shown to inhibit the growth and metabolism of cultured Walker carcinosarcoma cells (28). AMB is a γ-substituted vinylglycine, a naturally occurring amino acid with a β,γ-C=C double bond. Other members of this family are aminoethoxyvinylglycine from Streptomyces spp. (19) and rhizobitoxine, made by Bradyrhizobium japonicum (16) and Pseudomonas andropogonis (15) (Fig. (Fig.1).1). As inhibitors of pyridoxal phosphate-dependent enzymes (13, 17, 21, 22), γ-substituted vinylglycines have multiple targets in bacteria, animals, and plants (3, 5, 10, 21, 22, 29). However, the importance of AMB as a toxin in biological interactions with P. aeruginosa has not been addressed, as AMB biosynthesis and the genes involved have not been elucidated.Open in a separate windowFIG. 1.Chemical structures of the γ-substituted vinylglycines AMB, aminoethoxyvinylglycine, and rhizobitoxine.  相似文献   

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The fermentative metabolism of glucose was redirected to succinate as the primary product without mutating any genes encoding the native mixed-acid fermentation pathway or redox reactions. Two changes in peripheral pathways were together found to increase succinate yield fivefold: (i) increased expression of phosphoenolpyruvate carboxykinase and (ii) inactivation of the glucose phosphoenolpyruvate-dependent phosphotransferase system. These two changes increased net ATP production, increased the pool of phosphoenolpyruvate available for carboxylation, and increased succinate production. Modest further improvements in succinate yield were made by inactivating the pflB gene, encoding pyruvate formate lyase, resulting in an Escherichia coli pathway that is functionally similar to the native pathway in Actinobacillus succinogenes and other succinate-producing rumen bacteria.Succinic acid is used as a specialty chemical in the agricultural, food, and pharmaceutical industries (17, 32). It has also been identified by the U.S. Department of Energy as one of the top 12 building block chemicals (30), because it can be converted into a variety of products, including green solvents, pharmaceutical products, and biodegradable plastics (17, 32). Although succinic acid is currently produced from petroleum-derived maleic anhydride, considerable interest in the fermentative production of succinate from sugars has emerged during the past decade (9, 10, 17).Several natural succinate-producing rumen bacteria that have high rates of succinate production and high succinate yields, such as Anaerobiospirillum succiniciproducens (22), Actinobacillus succinogenes (13, 28), and “Mannheimia succiniciproducens” (15, 16), have been isolated. However, these strains require complex organic nutrients that increase the costs associated with production, purification, and waste disposal (15, 22, 28). Low levels of succinate are produced by native strains of Escherichia coli in complex and mineral salts media (1, 4). Most mutant strains of E. coli that have been described previously as succinate producers also require complex organic nutrients (18, 23-26, 29, 31). Many involve two-step aerobic and anaerobic processes (3, 23-25, 29) and the addition of foreign genes (5, 6, 23-26, 29, 31).Novel E. coli biocatalysts (KJ060, KJ071, and KJ073) for the anaerobic production of succinate in mineral salts medium have been developed recently without the use of foreign genes or resident plasmids (9, 10). These biocatalysts were developed by combining constructed mutations to eliminate alternative routes of NADH oxidation in the mixed-acid pathway with growth-based selection (metabolic evolution). In subsequent studies (33), these strains were found to have recruited the glucose-repressed (7), gluconeogenic pck gene (11, 12, 19, 21, 27), encoding phosphoenolpyruvate carboxykinase (PCK) (derepressed via a point mutation in the promoter region), to replace the native phosphoenolpyruvate carboxylase (ppc) and serve as the primary route for CO2 fixation (Fig. (Fig.1).1). A second acquired mutation was also identified as a frameshift mutation in the carboxy terminus of ptsI, inactivating the phosphoenolpyruvate-dependent phosphotransferase system (33). Glucose uptake by the phosphotransferase system was functionally replaced by galactose permease (galP) and glucokinase (glk).Open in a separate windowFIG. 1.Anaerobic metabolism of E. coli using the mixed-acid fermentation pathway (data from reference 1). The native phosphotransferase system pathway for glucose uptake and the mixed-acid pathway for fermentation are shown with black arrows. Peripheral reactions for glucose uptake, carboxylation, and acetyl-CoA synthesis are shown as dotted green arrows and represent new metabolic functions that have been recruited for succinate production from glucose. Reactions that have been blocked by gene deletions or point mutations are marked with an X. pck* indicates a novel mutation that derepressed pck, allowing the enzyme to serve as the primary route for oxaloacetate production. Pyruvate (boxed) appears at two sites but is presumed to exist as a single intracellular pool.Based on these previous studies, we have now determined the core mutations needed to direct carbon flow from glucose to succinate in E. coli and have constructed new succinate-producing strains with a minimum of genetic change.  相似文献   

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Complex N-glycans flank the receptor binding sites of the outer domain of HIV-1 gp120, ostensibly forming a protective “fence” against antibodies. Here, we investigated the effects of rebuilding this fence with smaller glycoforms by expressing HIV-1 pseudovirions from a primary isolate in a human cell line lacking N-acetylglucosamine transferase I (GnTI), the enzyme that initiates the conversion of oligomannose N-glycans into complex N-glycans. Thus, complex glycans, including those that surround the receptor binding sites, are replaced by fully trimmed oligomannose stumps. Conversely, the untrimmed oligomannoses of the silent domain of gp120 are likely to remain unchanged. For comparison, we produced a mutant virus lacking a complex N-glycan of the V3 loop (N301Q). Both variants exhibited increased sensitivities to V3 loop-specific monoclonal antibodies (MAbs) and soluble CD4. The N301Q virus was also sensitive to “nonneutralizing” MAbs targeting the primary and secondary receptor binding sites. Endoglycosidase H treatment resulted in the removal of outer domain glycans from the GnTI- but not the parent Env trimers, and this was associated with a rapid and complete loss in infectivity. Nevertheless, the glycan-depleted trimers could still bind to soluble receptor and coreceptor analogs, suggesting a block in post-receptor binding conformational changes necessary for fusion. Collectively, our data show that the antennae of complex N-glycans serve to protect the V3 loop and CD4 binding site, while N-glycan stems regulate native trimer conformation, such that their removal can lead to global changes in neutralization sensitivity and, in extreme cases, an inability to complete the conformational rearrangements necessary for infection.The intriguing results of a recent clinical trial suggest that an effective HIV-1 vaccine may be possible (97). Optimal efficacy may require a component that induces broadly neutralizing antibodies (BNAbs) that can block virus infection by their exclusive ability to recognize the trimeric envelope glycoprotein (Env) spikes on particle surfaces (43, 50, 87, 90). Env is therefore at the center of vaccine design programs aiming to elicit effective humoral immune responses.The amino acid sequence variability of Env presents a significant challenge for researchers seeking to elicit broadly effective NAbs. Early sequence comparisons revealed, however, that the surface gp120 subunit can be divided into discrete variable and conserved domains (Fig. (Fig.1A)1A) (110), the latter providing some hope for broadly effective NAb-based vaccines. Indeed, the constraints on variability in the conserved domains of gp120 responsible for binding the host cell receptor CD4, and coreceptor, generally CCR5, provide potential sites of vulnerability. However, viral defense strategies, such as the conformational masking of conserved epitopes (57), have made the task of eliciting bNAbs extremely difficult.Open in a separate windowFIG. 1.Glycan biosynthesis and distribution on gp120 and gp41. (A) Putative carbohydrate modifications are shown on gp120 and gp41 secondary structures, based on various published works (26, 42, 63, 74, 119, 128). The gp120 outer domain is indicated, as are residues that form the SOS gp120-gp41 disulfide bridge. The outer domain is divided into neutralizing and silent faces. Symbols distinguish complex, oligomannose, and unknown glycans. Generally, the complex glycans of the outer domain line the receptor binding sites of the neutralizing face, while the oligomannose glycans of the outer domain protect the silent domain (105). Asterisks denote sequons that are unlikely to be utilized, including position 139 (42), position 189 (26, 42), position 406 (42, 74), and position 637 (42). Glycans shown in gray indicate when sequon clustering may lead to some remaining unused, e.g., positions 156 and 160 (42, 119), positions 386, 392, and 397 (42), and positions 611 and 616 (42). There is also uncertainty regarding some glycan identities: glycans at positions 188, 355, 397, and 448 are not classified as predominantly complex or oligomannose (26, 42, 63, 128). The number of mannose moieties on oligomannose glycans can vary, as can the number of antennae and sialic acids on complex glycans (77). The glycan at position 301 appears to be predominantly a tetra-antennary complex glycan, as is the glycan at position 88, while most other complex glycans are biantennary (26, 128). (B) Schematic of essential steps of glycan biosynthesis from the Man9GlcNAc2 precursor to a mature multiantennary complex glycan. Mannosidase I progressively removes mannose moieties from the precursor, in a process that can be inhibited by the drug kifunensine. GnTI then transfers a GlcNAc moiety to the D1 arm of the resulting Man5GlcNAc2 intermediate, creating a hybrid glycan. Mannose trimming of the D2 and D3 arms then allows additional GlcNAc moieties to be added by a series of GnT family enzymes to form multiantennary complexes. This process can be inhibited by swainsonine. The antennae are ultimately capped and decorated by galactose and sialic acid. Hybrid and complex glycans are usually fucosylated at the basal GlcNAc, rendering them resistant to endo H digestion. However, NgF is able to remove all types of glycan.Carbohydrates provide a layer of protection against NAb attack (Fig. (Fig.1A).1A). As glycans are considered self, antibody responses against them are thought to be regulated by tolerance mechanisms. Thus, a glycan network forms a nonimmunogenic “cloak,” protecting the underlying protein from antibodies (3, 13, 20, 29, 39, 54, 65, 67, 74, 85, 96, 98, 117, 119, 120). The extent of this protection can be illustrated by considering the ways in which glycans differ from typical amino acid side chains. First, N-linked glycans are much larger, with an average mass more than 20 times that of a typical amino acid R-group. They are also usually more flexible and may therefore affect a greater volume of surrounding space. In the more densely populated parts of gp120, the carbohydrate field may even be stabilized by sugar-sugar hydrogen bonds, providing even greater coverage (18, 75, 125).The process of N-linked glycosylation can result in diverse structures that may be divided into three categories: oligomannose, hybrid, and complex (56). Each category shares a common Man3GlcNAc2 pentasaccharide stem (where Man is mannose and GlcNAc is N-acetylglucosamine), to which up to six mannose residues are attached in oligomannose N-glycans, while complex N-glycans are usually larger and may bear various sizes and numbers of antennae (Fig. (Fig.1B).1B). Glycan synthesis begins in the endoplasmic reticulum, where N-linked oligomannose precursors (Glc3Man9GlcNAc2; Glc is glucose) are transferred cotranslationally to the free amide of the asparagine in a sequon Asn-X-Thr/Ser, where X is not Pro (40). Terminal glucose and mannose moieties are then trimmed to yield Man5GlcNAc2 (Fig. (Fig.1B).1B). Conversion to a hybrid glycan is then initiated by N-acetylglucosamine transferase I (GnTI), which transfers a GlcNAc moiety to the D1 arm of the Man5GlcNAc2 substrate (19) (Fig. (Fig.1B).1B). This hybrid glycoform is then a substrate for modification into complex glycans, in which the D2 and D3 arm mannose residues are replaced by complex antennae (19, 40, 56). Further enzymatic action catalyzes the addition of α-1-6-linked fucose moiety to the lower GlcNAc of complex glycan stems, but usually not to oligomannose glycan stems (Fig. (Fig.1B)1B) (21, 113).Most glycoproteins exhibit only fully mature complex glycans. However, the steric limitations imposed by the high density of glycans on some parts of gp120 lead to incomplete trimming, leaving “immature” oligomannose glycans (22, 26, 128). Spatial competition between neighboring sequons can sometimes lead to one or the other remaining unutilized, further distancing the final Env product from what might be expected based on its primary sequence (42, 48, 74, 119). An attempt to assign JR-FL gp120 and gp41 sequon use and types, based on various studies, is shown in Fig. Fig.1A1A (6, 26, 34, 35, 42, 63, 71, 74, 119, 128). At some positions, the glycan type is conserved. For example, the glycan at residue N301 has consistently been found to be complex (26, 63, 128). At other positions, considerable heterogeneity exists in the glycan populations, in some cases to the point where it is difficult to unequivocally assign them as predominantly complex or oligomannose. The reasons for these uncertainties might include incomplete trimming (42), interstrain sequence variability, the form of Env (e.g., gp120 or gp140), and the producer cell. The glycans of native Env trimers and monomeric gp120 may differ due to the constraints imposed by oligomerization (32, 41, 77). Thus, although all the potential sequons of HXB2 gp120 were found to be occupied in one study (63), some are unutilized or variably utilized on functional trimers, presumably due to steric limitations (42, 48, 75, 96, 119).The distribution of complex and oligomannose glycans on gp120 largely conforms with an antigenic map derived from structural models (59, 60, 102, 120), in which the outer domain is divided into a neutralizing face and an immunologically silent face. Oligomannose glycans cluster tightly on the silent face of gp120 (18, 128), while complex glycans flank the gp120 receptor binding sites of the neutralizing face, ostensibly forming a protective “fence” against NAbs (105). The relatively sparse clustering of complex glycans that form this fence may reflect a trade-off between protecting the underlying functional domains from NAbs by virtue of large antennae while at the same time permitting sufficient flexibility for the refolding events associated with receptor binding and fusion (29, 39, 67, 75, 98, 117). Conversely, the dense clustering of oligomannose glycans on the silent domain may be important for ensuring immune protection and/or in creating binding sites for lectins such as DC-SIGN (9, 44).The few available broadly neutralizing monoclonal antibodies (MAbs) define sites of vulnerability on Env trimers (reviewed in reference 52). They appear to fall into two general categories: those that access conserved sites by overcoming Env''s various evasion strategies and, intriguingly, those that exploit these very defensive mechanisms. Regarding the first category, MAb b12 recognizes an epitope that overlaps the CD4 binding site of gp120 (14), and MAbs 2F5 and 4E10 (84, 129) recognize adjacent epitopes of the membrane-proximal external region (MPER) at the C-terminal ectodomain of gp41. The variable neutralizing potencies of these MAbs against primary isolates that contain their core epitopes illustrate how conformational masking can dramatically regulate their exposure (11, 118). Conformational masking also limits the activities of MAbs directed to the V3 loop and MAbs whose epitopes overlap the coreceptor binding site (11, 62, 121).A second category of MAbs includes MAb 2G12, which recognizes a tight cluster of glycans in the silent domain of gp120 (16, 101, 103, 112). This epitope has recently sparked considerable interest in exploiting glycan clusters as possible carbohydrate-based vaccines (2, 15, 31, 70, 102, 116). Two recently described MAbs, PG9 and PG16 (L. M. Walker and D. R. Burton, unpublished data), also target epitopes regulated by the presence of glycans that involve conserved elements of the second and third variable loops and depend largely on the quaternary trimer structure and its in situ presentation on membranes. Their impressive breadth and potency may come from the fact that they target the very mechanisms (variable loops and glycans) that are generally thought to protect the virus from neutralization. Like 2G12, these epitopes are likely to be constitutively exposed and thus may not be subject to conformational masking (11, 118).The above findings reveal the importance of N-glycans both as a means of protection against neutralization as well as in directly contributing to unique neutralizing epitopes. Clearly, further studies on the nature and function of glycans in native Env trimers are warranted. Possible approaches may be divided into four categories, namely, (i) targeted mutation, (ii) enzymatic removal, (iii) expression in the presence of glycosylation inhibitors, and (iv) expression in mutant cell lines with engineered blocks in the glycosylation pathway. Much of the available information on the functional roles of glycans in HIV-1 and simian immunodeficiency virus (SIV) infection has come from the study of mutants that eliminate glycans either singly or in combination (20, 54, 66, 71, 74, 91, 95, 96). Most mutants of this type remain at least partially functional (74, 95, 96). In some cases these mutants have little effect on neutralization sensitivity, while in others they can lead to increased sensitivity to MAbs specific for the V3 loop and CD4 binding site (CD4bs) (54, 71, 72, 74, 106). In exceptional cases, increased sensitivity to MAbs targeting the coreceptor binding site and/or the gp41 MPER has been observed (54, 66, 72, 74).Of the remaining approaches for studying the roles of glycans, enzymatic removal is constrained by the extreme resistance of native Env trimers to many common glycosidases, contrasting with the relative sensitivity of soluble gp120 (67, 76, 101). Alternatively, drugs can be used to inhibit various stages of mammalian glycan biosynthesis. Notable examples are imino sugars, such as N-butyldeoxynojirimycin (NB-DNJ), that inhibit the early trimming of the glucose moieties from Glc3Man9GlcNAc2 precursors in the endoplasmic reticulum (28, 38, 51). Viruses produced in the presence of these drugs may fail to undergo proper gp160 processing or fusion (37, 51). Other classes of inhibitor include kifunensine and swainsonine, which, respectively, inhibit the trimming of the Man9GlcNAc2 precursor into Man5GlcNAc2 or inhibit the removal of remaining D2 and D3 arm mannoses from the hybrid glycans, thus preventing the construction of complex glycan antennae (Fig. (Fig.1B)1B) (17, 33, 76, 104, 119). Unlike NB-DNJ, viruses produced in the presence of these drugs remain infectious (36, 76, 79, 100).Yet another approach is to express virus in insect cells that can only modify proteins with paucimannose N-glycans (58). However, the inefficient gp120/gp41 processing by furin-like proteases in these cells prevents their utility in functional studies (123). Another option is provided by ricin-selected GnTI-deficient cell lines that cannot transfer GlcNAc onto the mannosidase-trimmed Man5GlcNAc2 substrate, preventing the formation of hybrid and complex carbohydrates (Fig. (Fig.1B)1B) (17, 32, 36, 94). This arrests glycan processing at a well-defined point, leading to the substitution of complex glycans with Man5GlcNAc2 rather than with the larger Man9GlcNAc2 precursors typically obtained with kifunensine treatment (17, 32, 33, 104). With this in mind, here we produced HIV-1 pseudoviruses in GnTI-deficient cells to investigate the role of complex glycan antennae in viral resistance neutralization. By replacing complex glycans with smaller Man5GlcNAc2 we can determine the effect of “lowering the glycan fence” that surrounds the receptor binding sites, compared to the above-mentioned studies of individual glycan deletion mutants, whose effects are analogous to removing a fence post. Furthermore, since oligomannose glycans are sensitive to certain enzymes, such as endoglycosidase H (endo H), we investigated the effect of dismantling the glycan fence on Env function and stability. Our results suggest that the antennae of complex glycans protect against certain specificities but that glycan stems regulate trimer conformation with often more dramatic consequences for neutralization sensitivity and in extreme cases, infectious function.  相似文献   

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Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.  相似文献   

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