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.     

RNA Structures Required for Production of Subgenomic Flavivirus RNA

Flaviviruses are a group of single-stranded, positive-sense RNA viruses causing ∼100 million infections per year. We have recently shown that flaviviruses produce a unique, small, noncoding RNA (∼0.5 kb) derived from the 3′ untranslated region (UTR) of the genomic RNA (gRNA), which is required for flavivirus-induced cytopathicity and pathogenicity (G. P. Pijlman et al., Cell Host Microbe, 4: 579-591, 2008). This RNA (subgenomic flavivirus RNA [sfRNA]) is a product of incomplete degradation of gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, which stalls on the rigid secondary structure stem-loop II (SL-II) located at the beginning of the 3′ UTR. Mutations or deletions of various secondary structures in the 3′ UTR resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3). Here, we investigated in detail the importance of West Nile virus Kunjin (WNV_KUN) 3′ UTR secondary structures as well as tertiary interactions for sfRNA formation. We show that secondary structures SL-IV and dumbbell 1 (DB1) downstream of SL-II are able to prevent further degradation of gRNA when the SL-II structure is deleted, leading to production of sfRNA2 and sfRNA3, respectively. We also show that a number of pseudoknot (PK) interactions, in particular PK1 stabilizing SL-II and PK3 stabilizing DB1, are required for protection of gRNA from nuclease degradation and production of sfRNA. Our results show that PK interactions play a vital role in the production of nuclease-resistant sfRNA, which is essential for viral cytopathicity in cells and pathogenicity in mice.Arthropod-borne flaviviruses such as West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) cause major outbreaks of potentially fatal disease and affect over 50 million people every year. The highly pathogenic North American strain of WNV (WNV_NY99) has already claimed more than 1,000 lives with over 27,000 cases reported since its emergence in New York in 1999 and has raised global public health concerns (9). In contrast, the closely related Australian strain of WNV, WNV_KUN, is highly attenuated and does not cause overt disease in humans and animals (11). WNV_KUN has been used extensively as a model virus to study flavivirus replication and flavivirus-host interactions (13, 14, 16-19, 26, 38, 39).The ∼11-kb positive-stranded, capped WNV genomic RNA (gRNA) lacks a poly(A) tail and consists of 5′ and 3′ untranslated regions (UTRs) flanking one open reading frame, which encodes the viral proteins required for the viral life cycle (6, 15, 38, 39). Flavivirus UTRs are involved in translation and initiation of RNA replication and likely determine genome packaging (13, 14, 16, 21, 30, 39-41). Both the 5′ UTR (∼100 nucleotides [nt] in size) and the 3′ UTR (from ∼400 to 700 nucleotides) can form secondary and tertiary structures which are highly conserved among mosquito-borne flaviviruses (1, 8, 10, 14, 29, 32, 34). More specifically, the WNV_KUN 3′ UTR consists of several conserved regions and secondary structures (Fig. ​(Fig.1A)1A) which were previously predicted or shown to exist in various flaviviruses by computational and chemical analyses, respectively (4, 10, 25, 26, 29-32). The 5′ end of the 3′ UTR starts with an AU-rich region which can form stem-loop structure I (SL-I) followed by SL-II, which we previously showed to be vitally important for subgenomic flavivirus RNA (sfRNA) production (26; see also below). SL-II is followed by a short, repeated conserved hairpin (RCS3) and SL-III (26). Further downstream of SL-III are the SL-IV and CS3 structures, which are remarkably similar to the preceding SL-II-RCS3 structure (26, 29). Further downstream of the SL-IV-CS3 structure are dumbbells 1 and 2 (DB1 and DB2, respectively) followed by a short SL and the 3′ SL (25, 26).Open in a separate windowFIG. 1.(A) Model of the WNV_KUN 3′ UTR RNA structure. Highlighted in bold are the secondary structures investigated here. Dashed lines indicate putative PKs. The two sites of the putative PK interactions are shown in open boxes. sfRNA1, -2, -3, and -4 start sites are indicated by arrows. (R)CS, (repeated) conserved sequence; DB, dumbbell structure; PK, pseudoknot; SL, stem-loop. (B) Structural model of PK1 in SL-II with disruptive mutations. Nucleotide numbering is from the end of the 3′ UTR. The sfRNA1 start is indicated by an arrow. Nucleotides forming PK1 are on a gray background, and mutated nucleotides are white on a black background. (C) Sequences mutated in the different constructs. Nucleotides in the wt PK sequences used for mutations are bold and underlined. Introduced mutations are shown under the corresponding nucleotides in the wt sequence.The described structures have been investigated in some detail for their requirement in RNA replication and translation. Generally, a progressive negative effect on viral growth was shown with progressive deletions into the 3′-proximal region of the JEV 3′ UTR (41). However, only a relatively short region of the JEV 3′ UTR, consisting of the 3′-terminal 193 nt, was shown to be absolutely essential for gRNA replication (41). The minimal region for DENV replication was reported to be even shorter (23). Extensive analysis has shown that the most 3′-terminal, essential regions of the 3′ UTR include the cyclization sequence and 3′ SL, which are required for efficient RNA replication (2, 14, 16, 23, 35). As we showed, deletion of SL-II or SL-I did not overtly affect WNV_KUN replication (26). However, deletion of CS2, RCS2, CS3, or RCS3 in WNV replicon RNA significantly reduced RNA replication but not translation (20), indicating that these elements facilitate but are not essential for RNA replication. In addition, it was shown that deletion of DB1 or DB2 resulted in a viable mutant virus that was reduced in growth efficiency, while deletion of both DB structures resulted in a nonviable mutant (23).In addition to the above-mentioned secondary stem-loop structures, computational and chemical analysis of the flavivirus 3′ UTR suggested the presence of 5 pseudoknot (PK) interactions (Fig. ​(Fig.1A)1A) (25, 26, 32). A PK is a structure formed upon base pairing of a single-stranded region of RNA in the loop of a hairpin to a stretch of complementary nucleotides elsewhere in the RNA chain (Fig. ​(Fig.1B).1B). These structures are referred to as hairpin type (H-type) PKs (3), and they usually stabilize secondary RNA structures. Typically, the final tertiary structure does not significantly alter the preformed secondary structure (5). In general, PK interactions have been shown to be important in biological processes such as initiation and/or elongation of translation, initiation of gRNA replication, and ribosomal frameshifting for a number of different viruses, including flaviviruses (reviewed in references 3 and 22). The first PK in the WNV 3′ UTR was predicted to form in SL-II, followed by a similar PK in SL-IV (26) (PK1 and PK2 in Fig. ​Fig.1A).1A). For the DENV, yellow fever virus (YFV), and JEV subgroup of flaviviruses, two PKs further downstream were predicted to form between DB1 and DB2 and corresponding single-stranded RNA regions located further downstream (25) (PK3 and PK4 in Fig. ​Fig.1A).1A). The formation of these structures is supported by covariations in the WNV RNAs. In addition, a PK was proposed to form between a short SL and the 3′ SL at the 3′ terminus of the viral genome (32) (PK5 in Fig. ​Fig.1A1A).Importantly, in addition to its role in viral replication and translation, we have shown that the WNV_KUN 3′ UTR is important for the production of a small noncoding RNA fragment designated sfRNA (26). This short RNA fragment of ∼0.5 kb is derived from the 3′ UTR of the gRNA and exclusively produced by the members of the Flavivirus genus of the Flaviviridae family, where it is required for efficient viral replication, cytopathicity, and pathogenicity (26). Our studies suggested that sfRNA is a product of incomplete degradation of the gRNA presumably by the cellular 5′-3′ exoribonuclease XRN1, resulting from XRN1 stalling on the rigid secondary/tertiary structures located at the beginning of the 3′ UTR (26). XRN1 is an exoribonuclease which usually degrades mRNA from the 5′ to the 3′ end as part of cellular mRNA decay and turnover (33), and it was shown previously that XRN1 can be stalled by SL structures (28). Mutations or deletions of WNV 3′ UTR secondary structures resulted in the loss of full-length sfRNA (sfRNA1) and production of smaller and less abundant sfRNAs (sfRNA2 and sfRNA3) (26). In particular, SL-II (Fig. ​(Fig.1A)1A) was shown to be important for sfRNA1 production; deletion of this structure either alone or in conjunction with other structures located downstream of SL-II abolished sfRNA1 production, leading to the production of the smaller RNA fragments sfRNA2 and sfRNA3.Here, we extended our investigation and studied the importance of several predicted 3′ UTR secondary structures and PK interactions for the production of sfRNA. To further understand the generation mechanism of sfRNA and its requirements, we deleted or mutated a number of RNA structures in the WNV_KUN 3′ UTR and investigated the size and amount of sfRNA generated from these mutant RNAs. The results show that not only SLs but also PK interactions play a vital role in stabilizing the 3′ UTR RNA and preventing complete degradation of viral gRNA to produce nuclease-resistant sfRNA, which is required for efficient virus replication and cytopathicity in cells and virulence in mice.     

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.     

Identification of the Biosynthetic Gene Cluster for the Pseudomonas aeruginosa Antimetabolite l-2-Amino-4-Methoxy-trans-3-Butenoic Acid

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.     

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).     

Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E

The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.Flaviviruses form a genus in the family Flaviviridae (52) and comprise a number of important human pathogens such as yellow fever, dengue, Japanese encephalitis, West Nile, and tick-borne encephalitis (TBE) viruses (30). They are small, enveloped viruses with only three structural proteins, designated C (capsid), M (membrane), and E (envelope). The E protein is oriented parallel to the viral membrane and forms a head-to-tail homodimeric complex (Fig. 1A and B). The structure of the E ectodomain (soluble E [sE])—consisting of about 400 amino acids and lacking the 100 C-terminal amino acids (including the so-called stem and two transmembrane helices)—has been determined by X-ray crystallography for several flaviviruses (Fig. ​(Fig.1A)1A) (25, 34, 36, 38, 44, 55). Both of the essential entry functions—receptor-binding and membrane fusion after uptake by receptor-mediated endocytosis—are mediated by E, which is therefore the primary target for virus-neutralizing antibodies (11, 42, 43, 45).Open in a separate windowFIG. 1.Structures and schematic representations of the TBE virus E protein, virions, and RSPs. In all panels, DI, DII, and DIII of the E protein are shown in red, yellow, and blue, respectively, and the fusion peptide (FP) is in orange. (A) Ribbon diagram of the sE dimer (top view). (B) Schematic of the full-length E dimer in a top view (upper panel) and side view (lower panel). The position of the two transmembrane helices of the membrane anchor and the two helices of the stem are based on Zhang et al. (54) and are shown in green and purple, respectively. (C) Pseudo-atomic structure of the virion based on cryo-EM reconstructions of dengue and West Nile viruses (27, 37, 54). One of the 30 rafts, each consisting of three parallel dimers, is highlighted. DIIIs of three monomers belonging to one icosahedral asymmetric unit are labeled by white stars. (D) Pseudo-atomic structure of RSP based on cryo-EM reconstructions (12).As revealed by cryo-electron microscopy (cryo-EM), mature infectious virions have smooth surfaces, comparable to a golf ball (27, 37). Their envelopes are icosahedrally symmetric and consist of a closed shell of 180 E monomers that are arranged in a herringbone-like pattern of 30 rafts of three dimers each (Fig. ​(Fig.1C)1C) (27). On the other hand, capsid-lacking subviral particles, which can be produced in recombinant form by the coexpression of prM and E, have a different symmetry, with 30 E dimers in a T=1 icosahedral structure (Fig. ​(Fig.1D)1D) (12, 49).The peculiar organization of E in virions is reminiscent of the tight packing of capsid proteins in nonenveloped viruses, for which it was shown that the native antigenic structure is strongly dependent on the intact capsid structure and not completely represented by isolated forms of capsid proteins (1, 41, 53). Such modulations of antigenic structure may be due to conformational changes in the course of packaging the capsid proteins into virions and/or to the fact that antibody binding sites at the virion surface are composed of residues that come together only through the juxtaposition of capsid proteins or neighboring protein subunits. Even in the case of spiky viral envelope proteins, the dependence of certain epitopes on the quaternary organization of the envelope glycoproteins has been described (8, 47).For flaviviruses, structural studies provide evidence for the considerable flexibility of E, especially at the junctions between the individual domains I, II, and III (DI, DII, and DIII) (7, 35, 55), suggesting that soluble forms may display differences in antigenic structure compared to those fixed in the closed envelope shell of whole virions. Furthermore, because of the tight packing of E at the virion surface, certain epitopes may be cryptic in the context of whole virus particles but accessible in soluble forms of E (40, 51).Studies on the antigenic structure of flaviviruses have used different antigen preparations including virions, recombinant subviral particles (RSPs), and soluble forms and subunits of E (10, 15-17, 32, 39, 40, 46, 49, 51), but so far no systematic comparative analysis of E in different physical forms and quaternary arrangements has been conducted. It was therefore the objective of our study, using TBE virus as a model, to investigate possible structural and/or antigenic differences between (i) soluble dimeric forms of E, including C-terminally truncated sE and detergent-solubilized full-length E (Fig. 1A and B); (ii) E in the context of whole virions (Fig. ​(Fig.1C);1C); and (iii) E in the context of RSPs (Fig. ​(Fig.1D).1D). For this purpose we used, and further characterized, a set of monoclonal antibodies (MAbs) directed to each of the three domains of E. All of these MAbs have neutralizing activity (17, 24) and therefore, by definition, react with infectious virions.Through these analyses, we demonstrate that the reactivity of several MAbs is significantly dependent on the quaternary arrangement of E and differs between virions, RSPs, and/or sE dimers. We thus provide evidence for previously unrecognized structural factors that have an impact on the antigenicity of the flavivirus E protein.     

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.     

Role of Complex Carbohydrates in Human Immunodeficiency Virus Type 1 Infection and Resistance to Antibody Neutralization

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.     

Crystal Structure of a Novel Conformational State of the Flavivirus NS3 Protein: Implications for Polyprotein Processing and Viral Replication

The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One of these, nonstructural protein 3 (NS3), is an enzyme with both serine protease and NTPase/helicase activities. NS3 plays a central role in the flavivirus life cycle: the NS3 N-terminal serine protease together with its essential cofactor NS2B is involved in the processing of the polyprotein, whereas the NS3 C-terminal NTPase/helicase is responsible for ATP-dependent RNA strand separation during replication. An unresolved question remains regarding why NS3 appears to encode two apparently disconnected functionalities within one protein. Here we report the 2.75-Å-resolution crystal structure of full-length Murray Valley encephalitis virus NS3 fused with the protease activation peptide of NS2B. The biochemical characterization of this construct suggests that the protease has little influence on the helicase activity and vice versa. This finding is in agreement with the structural data, revealing a single protein with two essentially segregated globular domains. Comparison of the structure with that of dengue virus type 4 NS2B-NS3 reveals a relative orientation of the two domains that is radically different between the two structures. Our analysis suggests that the relative domain-domain orientation in NS3 is highly variable and dictated by a flexible interdomain linker. The possible implications of this conformational flexibility for the function of NS3 are discussed.Flaviviruses such as dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) belong to the family Flaviviridae and are the causative agents of a range of serious human diseases including hemorrhagic fever, meningitis, and encephalitis (37). They remain a global health priority, as many viruses are endemic in large parts of the Americas, Africa, Australia, and Asia, and vaccines remain unavailable for most members (31, 46, 57).Flaviviruses have a positive-sense single-stranded RNA (ssRNA) genome (approximately 11 kb) that encodes one large open reading frame containing a 5′ type 1 cap and conserved RNA structures at both the 5′ and 3′ untranslated regions that are important for viral genome translation and replication. The genomic RNA is translated into a single polyprotein precursor (11) consisting of three structural (C [capsid], prM [membrane], and E [envelope]) and seven nonstructural (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5) proteins arranged in the order C-prM-E-NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 (reviewed in reference 33) (Fig. ​(Fig.1).1). Only the structural proteins become part of the mature, infectious virion, whereas the nonstructural proteins are involved in polyprotein processing, viral RNA synthesis, and virus morphogenesis (33, 43). The precursor protein is directed by signal sequences into the host endoplasmic reticulum (ER), where NS1 and the exogenous domains of prM and E face the lumen, while C, NS3, and NS5 are cytoplasmic. NS2A, NS2B, NS4A, and NS4B are largely hydrophobic transmembrane proteins with small hydrophilic segments (Fig. ​(Fig.1).1). The post- and cotranslational cleavage of the polyprotein is performed by NS3 in the cytoplasm and by host proteases in the ER lumen to yield the mature proteins (Fig. ​(Fig.1)1) (33, 43). Of the nonstructural proteins, NS3 and NS5 are the best characterized, and both are essential for viral replication (23, 27, 41). Both proteins are multifunctional. The N-terminal one-third of NS3 contains the viral protease (NS3pro), which requires a portion of NS2B for its activity, while the remaining portion codes for the RNA helicase/NTPase/RTPase domain (NS3hel) (21, 22, 32, 55). NS5, however, contains both an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (16, 51). The functions of NS1, NS2A, NS4A, and NS4B are not well understood, but they appear to play important roles in replication and virus assembly/maturation and have been found to bind to NS3 and NS5, possibly modulating their activity (33, 36).Open in a separate windowFIG. 1.Schematic diagram of flavivirus polyprotein organization and processing. (Top) Linear organization of the structural and nonstructural proteins within the polyprotein. (Middle) Putative membrane topology of the polyprotein predicted from biochemical and cellular analyses, which is then processed by cellular and viral proteases (indicated by arrows). (Bottom) Different complexes that are thought to arise in different cellular compartments during and following polyprotein processing.Because of its enzymatic activities and its critical role in viral replication and polyprotein processing, NS3 constitutes a promising drug target for antiviral therapy (31). NS3pro (residues 1 to 169) is a trypsin-like serine protease with the characteristic catalytic triad (Asp-His-Ser) and a highly specific substrate recognition sequence, conserved in all flaviviruses, consisting of two basic residues in P2 and P1 followed by a small unbranched amino acid in P1′ (11). NS3pro has an aberrant fold compared to the canonical trypsin structure, and its folding and protease activity are dependent on a noncovalent association with a central 47-amino-acid hydrophilic domain of NS2B (19, 21). The remainder of NS2B contains three transmembrane helices involved in membrane associations. NS3 mediates cleavages at the C-terminal side of the highly conserved dibasic residue located at the coding junctions NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 and also between the C terminus of C and NS4A (11, 33) (Fig. ​(Fig.11).The C-terminal portion of NS3 (NS3hel, residues 170 to 619) performs several catalytically related activities, namely, RNA strand separation and (poly)nucleotide hydrolysis (5, 22, 32, 55) at a common, RecA-like NTPase catalytic center that couples the energy released from the hydrolysis of the triphosphate moieties of nucleotides to RNA unwinding. Although the precise role of NS3 in replication has not been established, its helicase activity is thought to separate nascent RNA strands from the template strands and to assist replication initiation by unwinding RNA secondary structure in the 3′ untranslated region (11, 13, 15, 33). NS3 is a member of the DEAH/D box family within helicase superfamily 2 (SF2) and is characterized by seven conserved sequence motifs involved in nucleic acid binding and hydrolysis (45). In addition, its RNA triphosphatase activity is thought to be involved in the capping of the viral RNA. In the process of replication, NS3 interacts, most likely via its C-terminal domain, with NS5 (13, 15, 24, 26, 58, 62). The NS3 5′ triphosphatase and NS5 methyltransferase activities probably cooperate in cap formation by removing the terminal γ-phosphate and performing sequential N7 and 2′ O methylations, respectively (16, 28, 46, 56). The guanylyltransferase activity required for cap formation remains elusive at present, although recent evidence suggests that it may be present in NS5 (8, 17). In addition, the interaction between NS3 and NS5 can stimulate NS3 helicase/NTPase activity (15, 62).The atomic structures of NS3pro in the presence and absence of ligands and/or the NS2B activating domain (2, 19, 47) and NS3hel (35, 38, 39, 49, 58-60) are known, and recently, the structure of full-length DENV4 (one of four dengue virus serotypes) NS3 fused to an 18-residue NS2B cofactor (NS2B₁₈NS3) was reported (34). This structure revealed an elongated conformation, with the protease domain interfacing with the NTP binding pocket and being separated from NS3hel by a relatively flexible linker, which suggested that the protease domain may have a positive effect on the activity of the NTPase/helicase domain. However, other reports suggested that NS3pro has no or a very limited effect on the activity of NS3hel (32, 62). In addition, since current evidence suggests that NS2B is not part of the replication complex (Fig. ​(Fig.1)1) (36), and it is known that in the absence of the NS2B cofactor, NS3pro is unfolded and inactive, it becomes hard to envisage what effect the NS3 protease domain may have on the helicase domain in a biologically relevant context. Equally, it is still not clear what role the helicase domain plays during polyprotein processing by NS3pro and, in general, why these two apparently distinct and unrelated catalytic activities are harbored within a single polypeptide.In order to gain further insights into these questions, we report the biochemical analysis and crystallographic structure at a 2.75-Å resolution of full-length NS3 from Murray Valley encephalitis virus (MVEV), a member of the JEV group of flaviviruses, fused to the entire protease activation peptide of the NS2B cofactor (NS2B₄₅NS3). The structure reveals the protease and helicase domains to be structurally independent and differs dramatically from the structure observed for DENV4 NS2B₁₈NS3. We discuss the implications of this unexpectedly different configuration of the NS3 protein and argue that the structural flexibility observed is likely to be crucial for its multifunctional nature.     

Critical Evaluation of the Volumetric “Bottle Effect” on Microbial Batch Growth

We have analyzed the impact of surface-to-volume ratio on final bacterial concentrations after batch growth. We examined six bottle sizes (20 to 1,000 ml) using three independent enumeration methods to quantify growth. We found no evidence of a so-called volumetric bottle effect, thus contradicting numerous previous reports.Microbial batch growth during confined incubation in bottles of various sizes is used daily in a broad variety of microbiological studies and methods, including bioassays such as the assimilable organic carbon (AOC) assay (6, 10, 18) and the analysis of pure culture or microbial community growth in freshwater (3, 11, 19, 20). In this context, “bottle effect” or “volume effect” is a term that has cropped up frequently in aquatic microbiology papers (e.g., references 12, 13, and 21) during the last 100 years to explain inexplicable phenomena and variations in results obtained from such batch growth studies. The uncertainty surrounding this apparent effect was clearly summarized in a recent paper by Pernthaler and Amann (16): “Such investigations are often plagued by the mysterious ‘bottle effect’, a hard-to-define concept that reflects the worry of whether phenomena observed in confined assemblages are nonspecific consequences of the confinement rather than a result of the planned manipulation.” The “bottle effect” alludes to an apparent reaction of bacteria to batchwise incubation in a confined environment, and this concept has intermittently been linked to influences on final cell concentrations (3) and grazing/bacterivory (13), a change in viability/activity parameters (9), a change in cultivability (5), and a change in population composition (1).The fact that microbiological processes during confined incubation differ from those in the environment is indisputable. However, a particular section of “bottle effect” literature focuses specifically on a volumetric “bottle effect”, where the above-mentioned effects are linked specifically to the size (or surface-to-volume ratio) of the incubation vessel (3, 8, 11-13, 15, 21). One of the oldest and best-known studies summarized clearly: “It will be observed that the densest bacterial populations appear in the bottles of water which offer the largest area of glass surface per unit volume of water” (21). This idea has established itself as dogma during the last century, with only a few differing opinions (4). However, precious little empirical data that actually quantify and explain the volumetric “bottle effect” are ever presented. In one example, Bischofberger et al. (3) observed that incubation of groundwater led to significantly more growth (about 2 log units) in small bottles (100 ml) than in big ones (10 liters). More often, however, the “bottle effect” is merely mentioned, as if it is self-explanatory and indisputable (2, 11, 12). In the present study, we took a simple but detailed look at the effect of bottle size on the outcome of short-term (<5-day) batch growth assays and compared the data critically to information in the literature and current opinion on this topic.Three batch growth experiments were conducted to assess the volumetric bottle effect on final cell concentrations after growth into stationary phase. Six different bottle sizes were used, covering the ranges most often reported in “bottle effect” literature. All glassware and Teflon-coated caps were cleaned comprehensively as described elsewhere (6) to remove any traces of organic carbon that might have been present on surfaces. The bottle sizes were as follows (water volumes and surface area-to-volume ratios [square centimeters to milliliters] are respectively included in parentheses): 1,000 ml (900 ml, 0.3:1), 500 ml (400 ml, 0.4:1), 250 ml (200 ml, 0.6:1), 100 ml (90 ml, 0.8:1), 40 ml (35 ml, 1.5:1), and 20 ml (15 ml, 2.4:1). In the first experiment, a sample of natural river water (dissolved organic carbon [DOC], 3.8 mg/liter; AOC, 0.3 mg/liter) from a small oligotrophic stream was obtained, filter sterilized with a 50-kDa dialysis filter (Fresenius Medical Care), and inoculated (at 10³ cells/ml) with a microbial community used for AOC assays (19). In the second experiment, a sample of the effluent (DOC, 1.2 mg/liter; AOC, 0.03 mg/liter; total cell concentration [TCC], 3 × 10⁵ cells/ml) from a granulated active carbon filter situated in a drinking water pilot plant (7) was collected and used directly for the experiment without additional treatment or inoculation. For the third experiment, sterile Luria-Bertani (LB) medium (diluted 1:10,000; DOC, 0.7 mg/liter; AOC, 0.46 mg/liter) was inoculated with Vibrio cholerae O1 (10³ cells/ml) as described previously (19). The water from each experiment was distributed into triplicate flasks of each size and incubated (at 30°C) until stationary phase was reached. Stationary phase was indicated by no significant increase in the TCC (measured after 3, 4, and 5 days) on consecutive days. Samples from all experiments were analyzed (i) for TCCs after being stained with SYBR green I and subjected to flow cytometry (7, 19), (ii) for ATP by using a commercial luciferin-luciferase assay (Promega Corporation) (7), and (iii) for heterotrophic plate counts (HPC) on R2A agar by a pour plate method with incubation at 30°C for 10 days. Possible biofilm growth was checked by applying sonication to selected samples. However, no wall growth in bottles of any size was observed.Growth was observed in all three experiments. The results show the net growth after subtraction of the initial cell/ATP/HPC concentrations from the final concentrations (Fig. ​(Fig.1).1). The proposed concept of the volumetric bottle effect implies that more growth should occur in smaller bottles. All data sets were subjected to regression analysis, and we observed no significant correlation (P < 0.01) between bottle size and final growth in any of the experiments by any of the three independent methods used for quantification. Figure ​Figure1A1A shows the batch growth results for a natural microbial community in prefiltered river water. This experimental setup is reflective of a typical AOC assay (6) or batch cultivation of natural microbial communities (20). Figure ​Figure1B1B shows the results for direct incubation of a treated drinking water sample. This sample and experimental setup were chosen specifically to assess any potential volumetric “bottle effect” on an indigenous microbial community in a biologically stable water sample, where only limited growth is expected. Indeed, the final cell concentration in the sample was only about 25% higher than the original cell concentration. The cultivability (HPC/TCC × 100) at day 0 was 0.4%, and at the end of the experimental period it had increased to 2.5%. This points to increased cultivability as a result of growth during confinement (5), yet it does not relate at all to the size of the incubation vessel. Figure ​Figure1C1C shows the data for V. cholerae grown in sterile LB medium (diluted 1:10,000) to stationary phase. Again, this particular setup is of specific relevance since a recently published paper on the growth of V. cholerae referred directly to the volumetric “bottle effect” to explain rather large differences between growth results from two separate studies (11, 19). The data from Fig. ​Fig.1C1C suggest at least that a “bottle effect” should be ruled out as an interfering factor in this case.Open in a separate windowFIG. 1.Effects of bottle size on bacterial batch growth of a natural microbial community in filter-sterilized surface water (A), growth of bacteria during direct incubation of water from a drinking water treatment plant (B), and batch growth of a V. cholerae pure culture in diluted LB medium (C). Growth (expressed as the net growth) was quantified by flow cytometric total cell counting (circles), total ATP analysis (diamonds), and conventional plating (squares). All data points represent averages of triplicate measurements.The results presented in this study clearly dispute the concept of a volumetric “bottle effect” on the outcome of short-term batch growth assays, be it for pure cultures or natural microbial communities. These findings contradict evidence reported by many other researchers (3, 8, 11-13, 15, 21). Although the volumetric “bottle effect” is often cited as a somewhat mysterious occurrence, it is imperative that clear experimental data are required for the critical appraisal thereof. The main experimental theory behind the phenomenon is that organic carbon adsorbs to clean glass surfaces, thus locally concentrating the carbon and creating more favorable growth conditions (2, 14). This adsorption and the fact that bacteria can utilize such adsorbed carbon have been demonstrated experimentally (14). What has, in our opinion, not been shown conclusively is that these effects can be so dramatic that they would alter the growth of samples to the extent that different sizes of bottles would render different final cell numbers after growth. Since we have not observed any volumetric “bottle effect” in our work, we can only speculate on the possible reasons why this has been observed previously. One explanation may be that glassware contaminated with organic carbon can contribute to the perception of a volumetric “bottle effect,” as large surface-to-volume ratios (found in small bottles) would account for increased contamination compared to that in bottles with smaller ratios. Hence, more additional available carbon would be introduced into smaller bottles, giving rise to higher final cell numbers after growth. In this context, it is essential that a comprehensive glassware-cleaning protocol be followed, including heating to a high temperature (>500°C) and storage away from volatile organics (6). In addition, it is important that such experiments at low carbon concentrations are complemented with the inclusion of correct and sensitive controls to assess potential organic carbon contamination. For example, the use of deionized water as a negative control should be avoided, since the absence of inorganic nutrients is bound to lead to no growth and thus false-negative results (10). A good negative control would be water that is only carbon limited, e.g., bottled drinking water (17). Moreover, the use of multiple tools for analyzing growth, including cultivation-independent methods, is encouraged.In conclusion, we did not observe evidence of a volumetric bottle effect on short-term (<5-day) batch incubations. The findings of this study suggest that reference to the so-called volumetric bottle effect should be considered carefully unless supported by clear experimental data. This study does not dispute the fact that many authors have observed results implying apparent bottle effects during growth studies, but it questions the interpretation and understanding of this concept and the random use of the term “bottle effect” to explain uncertainty in results, specifically in relation to bottle size. Hopefully, these data will assist with experimental setups and comparison of data among different groups and stimulate discussion of and future research on this interesting, but slightly controversial, topic.