Interactions of DNA with Biofilm-Derived Membrane Vesicles

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.     

Metabolic Engineering of Saccharomyces cerevisiae for Production of Novel Cyanophycins with an Extended Range of Constituent Amino Acids

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.     

Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

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.     

The S Helix Mediates Signal Transmission as a HAMP Domain Coiled-Coil Extension in the NarX Nitrate Sensor from Escherichia coli K-12

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.     

Inhibition of the Activity of Both Domains of Lysostaphin through Peptidoglycan Modification by the Lysostaphin Immunity Protein

Resistance to lysostaphin, a staphylolytic glycylglycine endopeptidase, is due to a FemABX-like immunity protein that inserts serines in place of some glycines in peptidoglycan cross bridges. These modifications inhibit both binding of the recombinant cell wall targeting domain and catalysis by the recombinant catalytic domain of lysostaphin.Lysostaphin is a glycylglycine endopeptidase produced by Staphylococcus simulans biovar staphylolyticus (18) that lyses susceptible staphylococci by hydrolyzing the polyglycine cross bridges in their cell wall peptidoglycans (3). The lysostaphin gene sequence was independently determined in 1987 by two groups (8, 13). BLAST analysis (1) of mature lysostaphin revealed two domains: an N-terminal catalytic domain (CAT), which is a member of the M23 family of zinc metalloendopeptidases, and a C-terminal cell wall targeting domain (CWT), which is a member of the SH3b domain family (Fig. ​(Fig.11 A).Open in a separate windowFIG. 1.(A) Schematic diagram of mature lysostaphin, the recombinant catalytic domain (rCAT) (lysostaphin residues 1 to 148), and the recombinant cell wall targeting domain (rCWT) (lysostaphin residues 149 to 246). The numbers represent the beginning and end of the domains, and the solid boxes indicate the N-terminal His₆ tag of the recombinant proteins. (B) SDS-PAGE analysis of rCAT and rCWT purified by a nickel affinity column. Mobilities of molecular mass standards are given on the left side of the gel.The lysostaphin endopeptidase resistance gene (epr or lif) encodes a FemABX-like immunity protein that is located adjacent to the lysostaphin gene on the plasmid pACK1 in S. simulans bv. staphylolyticus (4, 7, 20). Members of the FemABX family of proteins are nonribosomal peptidyl transferases that are involved in the addition of cross bridge amino acids during peptidoglycan subunit synthesis in the cytoplasm (15). In S. simulans bv. staphylolyticus, the lysostaphin immunity protein inserts serines in place of some glycines during peptidoglycan synthesis, which provides resistance to lysostaphin (4, 20).Originally it was suggested that the incorporation of serines in these peptidoglycan cross bridges gave increased resistance to lysostaphin because of the inability of the enzyme to hydrolyze glycyl-serine or seryl-glycine bonds (4, 14, 16). Others later reported that the CWT specifically binds to the polyglycine cross bridges in staphylococci (6) and the binding of CWT to producer-strain cells was less than that to susceptible cells (2). However, the ability of the enzyme or its targeting domain to bind to purified peptidoglycans from staphylococci containing the lysostaphin resistance gene has not been determined. Therefore, we determined if the modification to staphylococcal peptidoglycan cross bridges made by the lysostaphin immunity protein affected the activity of the binding domain, the catalytic domain, or both.     

The Chemoreceptor Dimer Is the Unit of Conformational Coupling and Transmembrane Signaling

Transmembrane chemoreceptors are central components in bacterial chemotaxis. Receptors couple ligand binding and adaptational modification to receptor conformation in processes that create transmembrane signaling. Homodimers, the fundamental receptor structural units, associate in trimers and localize in patches of thousands. To what degree do conformational coupling and transmembrane signaling require higher-order interactions among dimers? To what degree are they altered by such interactions? To what degree are they inherent features of homodimers? We addressed these questions using nanodiscs to create membrane environments in which receptor dimers had few or no potential interaction partners. Receptors with many, few, or no interaction partners were tested for conformational changes and transmembrane signaling in response to ligand occupancy and adaptational modification. Conformation was assayed by measuring initial rates of receptor methylation, a parameter independent of receptor-receptor interactions. Coupling of ligand occupancy and adaptational modification to receptor conformation and thus to transmembrane signaling occurred with essentially the same sensitivity and magnitude in isolated dimers as for dimers with many neighbors. Thus, we conclude that the chemoreceptor dimer is the fundamental unit of conformational coupling and transmembrane signaling. This implies that in signaling complexes, coupling and transmembrane signaling occur through individual dimers and that changes between dimers in a receptor trimer or among trimer-based signaling complexes are subsequent steps in signaling.In motile bacterial cells, thousands of transmembrane chemoreceptor proteins cluster in polar patches (8, 13, 14, 30, 42). The fundamental structural unit of these receptors is a homodimer (18, 32). Dimers interact at their membrane-distal tips to create trimers (18, 38, 39). Interactions among receptor homodimers in trimers and in higher-order associations (Fig. ​(Fig.1A)1A) are thought to be important for function (36, 37), particularly for the high-performance features of the chemotaxis sensory system (15). Understanding the role of receptor-receptor interactions in chemoreceptor function will require definition of the extent to which each receptor activity is an inherent property of individual receptor dimers and the extent to which activities require or are influenced by interactions with neighboring receptors. These issues had not been addressed experimentally because the receptor-receptor interactions could not be easily controlled in vivo or in vitro. However, we found that nanodiscs (2, 5) could be utilized to manipulate the potential for interactions among membrane-embedded chemoreceptors and thus to investigate the influence of receptor-receptor interactions upon chemoreceptor activities (4).Open in a separate windowFIG. 1.Chemoreceptors. (A) Cartoon of interactions of membrane-embedded chemoreceptors showing a homodimer, a trimer of dimers, and a patch of chemoreceptors. (B) Cartoon of a nanodisc with a single receptor dimer inserted in the plug of the lipid bilayer. (C) Diagram of the chemoreceptor conformational equilibrium.     

Metabolic Engineering of the Tricarboxylic Acid Cycle for Improved Lysine Production by Corynebacterium glutamicum

In the present work, lysine production by Corynebacterium glutamicum was improved by metabolic engineering of the tricarboxylic acid (TCA) cycle. The 70% decreased activity of isocitrate dehydrogenase, achieved by start codon exchange, resulted in a >40% improved lysine production. By flux analysis, this could be correlated to a flux shift from the TCA cycle toward anaplerotic carboxylation.With an annual market volume of more than 1,000,000 tons, lysine is one of the dominating products in biotechnology. In recent years, rational metabolic engineering has emerged as a powerful tool for lysine production (16, 18, 22). Hereby, different target enzymes and pathways in the central metabolism could be identified and successfully modified to create superior production strains (1, 2, 5, 8, 10, 17-20). The tricarboxylic acid (TCA) cycle has not been rationally engineered so far, despite its major role in Corynebacterium glutamicum (6). From metabolic flux studies, however, it seems that the TCA cycle might offer a great potential for optimization (Fig. ​(Fig.1),1), which is also predicted from in silico pathway analysis (13, 22). Experimental evidence comes from studies with Brevibacterium flavum exhibiting increased lysine production due to an induced bottleneck toward the TCA cycle (21). In the present work, we performed TCA cycle engineering by downregulation of isocitrate dehydrogenase (ICD). ICD is the highest expressed TCA cycle enzyme in C. glutamicum (7). Downregulation was achieved by start codon exchange, controlling ICD expression on the level of translation.Open in a separate windowFIG. 1.Stoichiometric correlation of lysine yield (%), biomass yield (g/mol) and TCA cycle flux (%; entry flux through citrate synthase) determined by ¹³C metabolic flux analysis achieved by paraboloid fitting of the data set (parameters were determined with Y0 = 105.1, a = −1.27, b = 0.35, c = −9.35 × 10⁻³, d = −11.16 × 10⁻³). The data displayed represent values from 18 independent experiments with different C. glutamicum strains taken from previous studies (1-3, 11, 12, 15, 23).     

Genome-Scale Phylogenetic Analyses of Chikungunya Virus Reveal Independent Emergences of Recent Epidemics and Various Evolutionary Rates

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has traditionally circulated in Africa and Asia, causing human febrile illness accompanied by severe, chronic joint pain. In Africa, epidemic emergence of CHIKV involves the transition from an enzootic, sylvatic cycle involving arboreal mosquito vectors and nonhuman primates, into an urban cycle where peridomestic mosquitoes transmit among humans. In Asia, however, CHIKV appears to circulate only in the endemic, urban cycle. Recently, CHIKV emerged into the Indian Ocean and the Indian subcontinent to cause major epidemics. To examine patterns of CHIKV evolution and the origins of these outbreaks, as well as to examine whether evolutionary rates that vary between enzootic and epidemic transmission, we sequenced the genomes of 40 CHIKV strains and performed a phylogenetic analysis representing the most comprehensive study of its kind to date. We inferred that extant CHIKV strains evolved from an ancestor that existed within the last 500 years and that some geographic overlap exists between two main enzootic lineages previously thought to be geographically separated within Africa. We estimated that CHIKV was introduced from Africa into Asia 70 to 90 years ago. The recent Indian Ocean and Indian subcontinent epidemics appear to have emerged independently from the mainland of East Africa. This finding underscores the importance of surveillance to rapidly detect and control African outbreaks before exportation can occur. Significantly higher rates of nucleotide substitution appear to occur during urban than during enzootic transmission. These results suggest fundamental differences in transmission modes and/or dynamics in these two transmission cycles.Chikungunya virus (CHIKV; Togaviridae: Alphavirus) is an arbovirus (arthropod-borne virus) vectored by Aedes mosquitoes to humans in tropical and subtropical regions of Africa and Asia (Fig. ​(Fig.1;1; reviewed in references 26 and 46). CHIKV has a single-stranded, positive-sense RNA genome of ∼12 kb and causes chikungunya fever (CHIK), a febrile illness associated with severe arthralgia and rash (2, 15, 31, 35); the name is derived from a Bantu language word describing the severe arthritic signs (32), which can persist for years. Thus, CHIK has enormous economic costs in addition to its public health impact (9). Because the signs and symptoms of CHIK overlap with those of dengue and because CHIKV is transmitted sympatrically in urban areas by the same mosquito vectors, it is grossly underreported in the absence of laboratory diagnostics (10, 37).Open in a separate windowFIG. 1.Distribution of the CHIKV strains used in this study. The map, based on a world map template from http://www.presentationmagazine.com, was edited with permission.CHIKV was first isolated during a 1953 outbreak in present-day Tanzania by Ross (48, 49). Since then, outbreaks have been documented in Africa and Asia, including the Indian subcontinent (Fig. ​(Fig.1)1) (1, 4). In 2005, CHIKV emerged from East Africa to cause an explosive urban epidemic in popular tourist island destinations in the Indian Ocean (Fig. ​(Fig.1;1; reviewed in reference 31). In late 2005, CHIKV spread into the Indian subcontinent, where millions of people have been affected (5). However, the geographic source of spread into India, from the mainland of Africa or from the Indian Ocean Islands, has not been delineated. India had seen large epidemics of CHIK in the past (reviewed in reference 30), but CHIKV apparently disappeared during the 1970s (5). Since 2006, CHIKV has been imported into Europe and the western hemisphere (including the United States) via many viremic travelers, and an epidemic was initiated in Italy by a traveler from India (4, 11, 47). The dramatic spread since 1980 of dengue viruses (DENV) throughout tropical America, via the same vectors, portends the severity of the public health problem if CHIKV becomes established in the western hemisphere.The first phylogenetic analysis of CHIKV (45) identified three geographically associated genotypes: the West African (WAf), East/Central/South African (ECSA), and Asian genotypes. More recent analyses indicate that the recent Indian Ocean and Indian strains form a monophyletic group within the ECSA lineage (5, 12, 14, 27, 40, 51, 52). However, most CHIKV phylogenetic studies (1, 14, 28, 29, 38, 40, 41, 47, 52) have utilized only partial sequences from the envelope glycoprotein E1 gene, preventing a robust assessment of some of the relationships among strains and of their evolutionary dynamics.The CHIKV strains represented in different geographic lineages apparently circulate in different ecological cycles. In Asia, CHIKV appears to circulate primarily in an urban transmission cycle involving the peridomestic mosquitoes Aedes aegypti and A. albopictus, as well as humans (25, 45). Asian epidemics typically infect thousands-to-millions of people over the course of several years (46). In contrast, African CHIKV circulates primarily in a sylvatic/enzootic cycle, transmitted by arboreal primatophilic Aedes mosquitoes (e.g., A. furcifer and A. africanus) and probably relies on nonhuman primates as reservoir hosts (reviewed in reference 16). Epidemics in rural Africa usually occur on a much smaller scale than in Asia, likely a result of the lower human population densities, and possibly more stable herd immunity. Although the assignments of “urban” and “sylvatic/enzootic” are based on the most common mode of transmission, CHIKV strains of African origin are capable of urban transmission by A. aegypti and A. albopictus, as evidenced by outbreaks in the Democratic Republic of the Congo (41), Nigeria (36), Kenya (27), and Gabon (42). The ecological differences between the sylvatic/enzootic (henceforth called enzootic) and urban/endemic/epidemic transmission cycles (henceforth called epidemic) such as seasonality of vector larval habitats, vertebrate host abundance and herd immunity, and vector host preferences, prompted us to hypothesize that the evolutionary dynamics of CHIKV may differ between the two transmission cycles. To test this hypothesis, to provide more robust estimates of the evolutionary relationships among the CHIKV strains including the sources of the recent epidemics, and to elucidate the temporal and spatial history of CHIKV evolution, we performed an extensive, genome-scale phylogenetic analysis, utilizing complete open reading frame (ORF) sequences of a large collection of 80 isolates with broad temporal, spatial, and host coverage.     

Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k_cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K_m) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k_cat = 1.9 × 10² s⁻¹ on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.Aldohexuronate catabolism in bacteria is reported to involve two different pathways, one initiating with an isomerization step and the other with an oxidation step. In the isomerization pathway, aldohexuronate (glucuronate and galacturonate) is isomerized to ketohexuronate by uronate isomerase and ultimately degraded to pyruvate and 3-phosphoglyceraldehyde. The isomerization pathway has been previously reported to occur in bacteria, including Escherichia coli (7), Erwinia carotovora (18), Erwinia chrysanthemi (15), Klebsiella pneumoniae (9, 23), and Serratia marcescens (28). In the oxidation pathway, aldohexuronate is oxidized to aldohexarate by uronate dehydrogenase (Udh) and further catabolized to pyruvate (2, 5, 7, 9, 18, 19, 24). Uronate dehydrogenase, the key enzyme of this pathway, has been investigated in two plant pathogen bacteria, Pseudomonas syringae and Agrobacterium tumefaciens. To date, only limited studies pertaining to the properties of Udh have been reported in the literature (3, 6, 38, 43), and no sequence has yet been identified. Udh is classified as an NAD-linked oxidoreductase (EC 1.1.1.203), with a total molecular weight of about 60,000. It is a homodimer composed of two subunits with molecular weights of about 30,000 each (38). Udh is a thermally unstable, reversible enzyme, with an optimum pH of about 8.0 (3, 6, 38).In E. coli MG1655 that has the isomerization pathway for aldohexuronate catabolism, glucuronate is transported by an aldohexuronate transporter encoded by exuT and converted to fructuronate by uronate isomerase, encoded by uxaC (22, 30) (Fig. ​(Fig.1).1). Fructuronate is transferred to the Entner-Doudoroff pathway to be utilized as an energy source via 2-keto-3-deoxy-6-phospho-gluconate (7, 27, 31, 32). Therefore, E. coli MG1655 with a uxaC deletion cannot use glucuronate as a carbon source. In this strain, glucarate is converted to 5-keto-4-deoxy-d-glucarate by d-glucarate dehydratase, encoded by gudD, and then transferred to glycolysis via pyruvate or 2-phosphoglycerate (27, 33). Recently, a number of bacterial genome sequences have been published, including those of the Udh-containing P. syringae pv. tomato strain DC3000 and A. tumefaciens strain C58 (4, 10). A shotgun library of P. syringae was constructed to identify the gene encoding Udh. Screening for Udh was conducted in E. coli MG1655 ΔuxaC. Since uronate dehydrogenase converts glucuronate to glucarate, uxaC deletion strains of E. coli harboring the shotgun library of P. syringae that can grow in a minimal medium containing glucuronate as a sole carbon source may carry the gene encoding Udh (Fig. ​(Fig.1).1). Once an initial Udh is identified from P. syringae, a BLAST homology search may lead to the identification of Udhs from other bacteria.Open in a separate windowFIG. 1.Catabolism of glucuronate and glucarate in bacteria. Glucuronate consumption is prevented by knockout of the uxaC gene. The presence of uronate dehydrogenase in a uxaC knockout enables growth of E. coli on glucuronate.