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.     

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.     

Malonic Semialdehyde Reductase,Succinic Semialdehyde Reductase,and Succinyl-Coenzyme A Reductase from Metallosphaera sedula: Enzymes of the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Sulfolobales

A 3-hydroxypropionate/4-hydroxybutyrate cycle operates during autotrophic CO₂ fixation in various members of the Crenarchaea. In this cycle, as determined using Metallosphaera sedula, malonyl-coenzyme A (malonyl-CoA) and succinyl-CoA are reductively converted via their semialdehydes to the corresponding alcohols 3-hydroxypropionate and 4-hydroxybutyrate. Here three missing oxidoreductases of this cycle were purified from M. sedula and studied. Malonic semialdehyde reductase, a member of the 3-hydroxyacyl-CoA dehydrogenase family, reduces malonic semialdehyde with NADPH to 3-hydroxypropionate. The latter compound is converted via propionyl-CoA to succinyl-CoA. Succinyl-CoA reduction to succinic semialdehyde is catalyzed by malonyl-CoA/succinyl-CoA reductase, a promiscuous NADPH-dependent enzyme that is a paralogue of aspartate semialdehyde dehydrogenase. Succinic semialdehyde is then reduced with NADPH to 4-hydroxybutyrate by succinic semialdehyde reductase, an enzyme belonging to the Zn-dependent alcohol dehydrogenase family. Genes highly similar to the Metallosphaera genes were found in other members of the Sulfolobales. Only distantly related genes were found in the genomes of autotrophic marine Crenarchaeota that may use a similar cycle in autotrophic carbon fixation.The thermoacidophilic autotrophic crenarchaeum Metallosphaera sedula uses a 3-hydroxypropionate/4-hydroxybutyrate cycle for CO₂ fixation (9, 28, 29, 35) (Fig. ​(Fig.1).1). A similar cycle may operate in other autotrophic members of the Sulfolobales (31) and in mesophilic marine group I Crenarchaea (Cenarchaeum sp., Nitrosopumilus sp.). This cycle uses elements of the 3-hydroxypropionate cycle that was originally discovered in the phototrophic bacterium Chloroflexus aurantiacus (15, 22-25, 41, 42). It involves the carboxylation of acetyl coenzyme A (acetyl-CoA) to malonyl-CoA by a biotin-dependent acetyl-CoA carboxylase (12, 29). The carboxylation product is reduced to malonic semialdehyde by malonyl-CoA reductase (1). Malonic semialdehyde is further reduced to 3-hydroxypropionate, the characteristic intermediate of the pathway (9, 31, 35). 3-Hydroxypropionate is further reductively converted to propionyl-CoA (3), which is carboxylated to (S)-methylmalonyl-CoA by propionyl-CoA carboxylase. Only one copy of the genes encoding the acetyl-CoA/propionyl-CoA carboxylase subunits is present in most Archaea, indicating that this enzyme is a promiscuous enzyme that acts on both acetyl-CoA and propionyl-CoA (12, 29). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, which is followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle in M. sedula and other autotrophic Sulfolobales. Enzymes: 1, acetyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonate semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, propionyl-CoA carboxylase, identical to acetyl-CoA carboxylase; 8, (S)-methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH), identical to malonyl-CoA reductase; 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14, crotonyl-CoA hydratase; 15, (S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase. The highlighted steps are catalyzed by the enzymes studied here.Succinyl-CoA is converted via succinic semialdehyde and 4-hydroxybutyrate to two molecules of acetyl-CoA (9), thus regenerating the starting CO₂ acceptor molecule and releasing another acetyl-CoA molecule for biosynthesis. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (Fig. ​(Fig.1)1) can be divided into two parts. The first part transforms one acetyl-CoA molecule and two bicarbonate molecules into succinyl-CoA (Fig. ​(Fig.1,1, steps 1 to 9), and the second part converts succinyl-CoA to two acetyl-CoA molecules (Fig. ​(Fig.1,1, steps 10 to 16).The second part of the autotrophic cycle also occurs in the dicarboxylate/4-hydroxybutyrate cycle, which operates in autotrophic CO₂ fixation in Desulfurococcales and Thermoproteales (Crenarchaea) (27, 37), raising the question of whether the enzymes in these two lineages have common roots (37). The first part of the cycle also occurs in the 3-hydroxypropionate cycle for autotrophic CO₂ fixation in Chloroflexus aurantiacus and a few related green nonsulfur phototrophic bacteria (19, 22, 23, 32, 49).The two-step reduction of malonyl-CoA to 3-hydroxpropionate in Chloroflexus is catalyzed by a single bifunctional 300-kDa enzyme (30). The M. sedula malonyl-CoA reductase is completely unrelated and forms only malonic semialdehyde (1), and the enzyme catalyzing the second malonic semialdehyde reduction step that forms 3-hydroxypropionate is unknown. In the second part of the 3-hydroxypropionate/4-hydroxybutyrate cycle a similar reduction of succinyl-CoA via succinic semialdehyde to 4-hydroxybutyrate takes place. The enzymes responsible for these reactions also have not been characterized.In this work we purified the enzymes malonic semialdehyde reductase, succinyl-CoA reductase, and succinic semialdehyde reductase from M. sedula. The genes coding for these enzymes were identified in the genome, and recombinant proteins were studied in some detail. Interestingly, succinyl-CoA reductase turned out to be identical to malonyl-CoA reductase. We also show here that enzymes that are highly similar to succinyl-CoA reductase in Thermoproteus neutrophilus do not function as succinyl-CoA reductases in M. sedula.     

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.     

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.     

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).     

The Carboxy-Terminal Segment of the Human Cytomegalovirus DNA Polymerase Accessory Subunit UL44 Is Crucial for Viral Replication

The amino-terminal 290 residues of UL44, the presumed processivity factor of human cytomegalovirus DNA polymerase, possess all of the established biochemical activities of the full-length protein, while the carboxy-terminal 143 residues contain a nuclear localization signal (NLS). We found that although the amino-terminal domain was sufficient for origin-dependent synthesis in a transient-transfection assay, the carboxy-terminal segment was crucial for virus replication and for the formation of DNA replication compartments in infected cells, even when this segment was replaced with a simian virus 40 NLS that ensured nuclear localization. Our results suggest a role for this segment in viral DNA synthesis.Human cytomegalovirus (HCMV) encodes a DNA polymerase which is composed of two subunits, UL54, the catalytic subunit, and UL44, an accessory protein (8, 12, 21). UL44 can be divided into two regions, a 290-residue amino (N)-terminal domain and a 143-residue carboxy (C)-terminal segment. The overall fold of the N-terminal domain is markedly similar to that of processivity factors such as herpes simplex virus type 1 (HSV-1) UL42 and eukaryotic proliferating cell nuclear antigen (6, 22, 41), which function to tether catalytic subunits to DNA to ensure long-chain DNA synthesis. In vitro, the N-terminal domain of UL44 is sufficient for all of the established biochemical activities of full-length UL44, including dimerization, binding to double-stranded DNA, interaction with UL54, and stimulation of long-chain DNA synthesis, consistent with a role as a processivity factor (4, 5, 8, 11, 23, 24, 39). In contrast, little is known about the functions of the C-terminal segment of UL44 other than its having been reported from transfection experiments to be important for downregulation of transactivation of a non-HCMV promoter (7) and to contain a nuclear localization signal (NLS) (3). Neither the importance of this NLS nor the role of the entire C-terminal segment has been investigated in HCMV-infected cells.We first examined whether the N-terminal domain is sufficient to support DNA synthesis from HCMV oriLyt in cells using a previously described cotransfection-replication assay (27, 28). A DpnI-resistant fragment, indicative of oriLyt-dependent DNA synthesis, was detected in the presence of wild-type (WT) UL44 (pSI-UL44) (34) and in the presence of the UL44 N-terminal domain (pSI-UL44ΔC290), but not in the presence of UL44-F121A (6, 34), a mutant form previously shown not to support oriLyt-dependent DNA synthesis (34) (Fig. ​(Fig.1A).1A). Thus, the N-terminal domain alone is sufficient to support oriLyt-dependent DNA synthesis in a transient-transfection assay.Open in a separate windowFIG. 1.Effects of UL44 C-terminal truncations in various assays. (A) HFF cells were cotransfected with the pSP50 plasmid (containing the oriLyt DNA replication origin), a plasmid expressing WT or mutant UL44 (as indicated at the top of the panel), and plasmids expressing all of the other essential HCMV DNA replication proteins. At 5 days posttransfection, total DNA was extracted and cleaved with DpnI to digest unreplicated DNA and a Southern blot assay was performed to detect replicated pSP50. An arrow indicates DpnI-resistant, newly synthesized pSP50 fragments. (B) FLAG-tagged constructs analyzed in panel C are cartooned as horizontal bars. The names of the constructs are above the bars. The lengths of the constructs in amino acids are indicated by the scale at the bottom of the panel. The positions of residues required but not necessarily sufficient for features of the constructs are designated by shading, as indicated at the bottom of the panel. (C) Vero cells were transfected with plasmids expressing WT UL44 (parts a to c), FLAG-UL44 (parts d to f), FLAG-UL44-290stop (parts g to i), or FLAG-UL44-290NLSstop (parts j to l). At 48 h posttransfection, cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nucleus (blue) (parts a, d, g, and j) and by IF with anti-UL44 (part b) or anti-FLAG (parts e, h, and k) and a secondary antibody conjugated with Alexa 488 (green). Parts c, f, i, and l are merged from images in the left and middle columns. Magnification: ×1,000. (D) Replication kinetics of rescued viruses. Rescued derivatives of UL44 mutant viruses (UL44-290stop-R and UL44-290NLSstop-R) or WT AD169 viruses were used to infect HFF cells at an MOI of 1 PFU/cell. The supernatants from infected cells were collected every 24 h, and viral titers were determined by plaque assays on HFF cells.These results were somewhat unexpected, as the C-terminal segment contains a functional NLS identified in transfection assays (3). We therefore assayed the intracellular localization of WT and mutant UL44 following transient transfection using pcDNA3-derived expression plasmids. Since the anti-UL44 antibodies that we have tested do not recognize the N-terminal domain of UL44, we constructed UL44 genes to encode N-terminally FLAG-tagged full-length UL44 (FLAG-UL44) or a FLAG-tagged N-terminal domain, the latter by inserting three in-frame tandem stop codons after codon 290 (FLAG-UL44-290stop, Fig. ​Fig.1B).1B). We also constructed a mutant form encoding a FLAG-tagged N-terminal domain, followed by the simian virus 40 (SV40) T-antigen NLS (15-17), followed by three tandem stop codons (FLAG-UL44-290NLSstop, Fig. ​Fig.1B).1B). Vero cells were transfected with each construct using Lipofectamine 2000, fixed with 4% formaldehyde at 48 h posttransfection, and assayed by indirect immunofluorescence (IF) using anti-UL44 (Virusys) or anti-FLAG antibody (Sigma). We observed mostly nuclear localization of WT UL44 or FLAG-UL44 with either diffuse or more localized intranuclear distribution (Fig. ​(Fig.1C,1C, parts a to c and d to f, respectively) and some occasional perinuclear staining, which may be due to protein overexpression. In cells expressing FLAG-UL44-290NLSstop, we observed mostly diffuse nuclear localization with little to no perinuclear staining (Fig. ​(Fig.1C,1C, parts j to l). In cells expressing FLAG-UL44-290stop, we observed mostly cytoplasmic staining, but with some cells exhibiting some nuclear staining (Fig. ​(Fig.1C,1C, parts g to i), which may explain the ability of truncated UL44 to support oriLyt-dependent DNA replication in a transient-transfection assay (Fig. ​(Fig.1A1A).We next investigated whether the C-terminal segment of UL44 is necessary for viral replication. We reasoned that we could investigate whether any requirement for this segment could be due to a requirement for an NLS by testing whether the SV40 NLS could substitute for the loss of the UL44 C terminus. We therefore constructed HCMV UL44 mutant viruses by introducing the UL44-290stop and UL44-290NLSstop mutations into a WT AD169 bacterial artificial chromosome (BAC) using two-step red-mediated recombination as previously described (35, 38). We also constructed the same mutants with a FLAG epitope at the N terminus of UL44 (BAC-FLAG-UL44-290stop and BAC-FLAG-UL44-290NLSstop) to monitor UL44 expression, and we constructed rescued derivatives of the mutant BACs by replacing the mutated sequences with WT UL44 sequences, as described previously (35). We introduced BACs into human foreskin fibroblast (HFF) cells using electroporation (35, 38). In several experiments using at least two independent clones for each mutant, cells electroporated with any of the mutant BACs did not exhibit any cytopathic effect (CPE) within 21 days. In contrast, within 7 to 10 days, cells electroporated with the WT AD169 BAC, a BAC expressing WT UL44 with an N-terminal FLAG tag [AD169-BACF44 (35)], or any of the rescued derivatives began displaying a CPE and yielded infectious virus. The rescued derivatives of the nontagged mutants displayed replication kinetics similar to those of the WT virus following infection at a multiplicity of infection (MOI) of 1 PFU/cell (Fig. ​(Fig.1D).1D). The rescued derivatives of the FLAG-tagged mutants also replicated to WT levels (data not shown). Thus, the replication defects of the mutants were due to the introduced mutations that result in truncated UL44 either with or without the SV40 NLS. We therefore conclude that the C-terminal segment of UL44 is required for viral replication.To investigate the stage of viral replication at which the UL44 C-terminal segment is important, we first assayed the subcellular localization of immediate-early proteins IE1 and IE2 and FLAG-UL44 in cells electroporated with BAC DNA expressing the FLAG-tagged WT or the two mutant UL44s using IF at 2 days postelectroporation. IE1/IE2 could be detected diffusely distributed in nuclei of cells electroporated with all three BACs (Fig. 2b, f, and j). In cells electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, FLAG-UL44 was localized largely within the nucleus (Fig. 2c and k, respectively). In contrast, in cells electroporated with BAC-FLAG-UL44-290stop, the FLAG epitope was mainly localized diffusely in the cytoplasm, with only a small amount diffusely distributed in the nucleus (Fig. ​(Fig.2g).2g). These data indicate that IE proteins expressed from mutant BACs are properly localized and suggest that without its C-terminal segment, which includes the NLS identified in transfection assays (3), UL44 cannot efficiently localize to the nucleus in HCMV-infected cells. However, addition of the SV40 NLS was sufficient to efficiently localize the N-terminal domain of UL44 to the nucleus. Thus, the requirement for the C-terminal segment of UL44 for viral replication is not due solely to its NLS.Open in a separate windowFIG. 2.Localization of IE1/IE2 and FLAG-UL44 proteins in electroporated cells. HFF cells were electroporated with AD169-BACF44 (panels a to d), BAC-UL44-290stop (panels e to h), or BAC-FLAG-UL44-290NLSstop (panels i to l). At 48 h posttransfection, cells were fixed and probed with anti-IE1/2 (Virusys) or anti-FLAG (Sigma). Secondary antibodies coupled to fluorophores were used for visualization of IE1/2 (anti-mouse Alexa 594; panels b, f, and j) and FLAG (anti-rabbit Alexa 488; panels c, g, and k) antibodies. DAPI was used to counterstain the nucleus (panels a, e, and i). Panels d, h, and l are merged images of the panels in the other columns. Magnification: ×1,000.We next investigated if the block in viral replication due to the loss of the C-terminal segment could be attributed to a defect in viral DNA synthesis. Cells were electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, and viral DNA accumulation was assayed by quantitative real-time PCR at various times postelectroporation (Fig. ​(Fig.3)3) as previously described (32, 35). In HFFs electroporated with AD169-BACF44, viral DNA began to accumulate above the input levels by 8 days postelectroporation and increased over time, with as much as a 350-fold increase over the input DNA level by 18 days postelectroporation. In contrast, levels of viral DNA in cells electroporated with BAC-UL44-290NLSstop did not increase above input levels, even by 18 days postelectroporation. These data are consistent with the notion that the UL44 C-terminal segment is required for viral DNA synthesis, although we caution that the assay did not detect DNA synthesis from AD169-BACF44 until day 8, when viral spread had likely occurred (see below).Open in a separate windowFIG. 3.Quantification of viral DNA accumulation in electroporated cells. HFF cells were electroporated with AD169-BACF44 or BAC-FLAG-UL44-290NLSstop, and total DNA was harvested on the days postelectroporation indicated. Viral DNA accumulation was assessed by real-time PCR by assessing levels of the UL83 gene and normalizing to levels of the cellular β-actin gene (32). The data are presented as the fold increase in normalized viral DNA levels over the amount of input DNA (day 1).We also analyzed the localization patterns of UL44 and UL57, the viral single-stranded DNA binding protein, which is a marker for viral DNA replication compartments (1, 2, 18, 26, 29). At 8 days postelectroporation with AD169-BACF44, UL57 and FLAG-UL44 largely colocalized within a single large intranuclear structure that likely represents a fully formed replication compartment, with some cells containing multiple smaller globular structures within the nucleus that likely represent earlier stages of replication compartments (1, 2, 29) (Fig. 4a to d). Neighboring cells also stained for UL57 and FLAG-UL44, indicative of viral spread. In contrast, in cells electroporated with BAC-FLAG-UL44-290NLSstop, UL57 (Fig. ​(Fig.4f)4f) was found in either punctate or small globular structures. This pattern of UL57 staining resembled that observed at very early stages of viral DNA synthesis in HCMV-infected cells, but the structures were larger and less numerous than those observed in HCMV-infected cells in the presence of a viral DNA polymerase inhibitor (2, 29). Staining for FLAG-UL44 was nuclear and largely diffuse, with some areas of more concentrated staining (Fig. ​(Fig.4g),4g), which could also be observed in some cells at day 2 postelectroporation (Fig. ​(Fig.3k).3k). This pattern of UL44 localization was generally similar to that observed in HCMV-infected cells at very early stages of infection or when HCMV DNA synthesis is blocked and also similar to the pattern in cells transfected with a UL84 null mutant BAC (2, 29, 33, 40). Importantly, little colocalization of UL57 and UL44 was observed, with areas of concentration of UL57 or UL44 occupying separate regions in the nuclei of these cells (Fig. ​(Fig.4h).4h). We are unaware of any other examples of this pattern of localization of these proteins in HCMV-infected cells and suggest that it may be a result of the loss of the UL44 C-terminal segment. These results indicate that this segment is important for efficient formation of viral DNA replication compartments, again consistent with a requirement for this portion of UL44 for viral DNA synthesis.Open in a separate windowFIG. 4.Localization of UL57 and FLAG-UL44 proteins in electroporated cells. HFF cells were electroporated with AD169-BACF44 (panels a to d) or BAC-FLAG-UL44-290NLSstop (panels e to h). At 8 days posttransfection, cells were fixed and then stained with antibodies specific for UL57 (Virusys) or FLAG (Sigma), followed by a secondary antibody coupled to fluorophores to detect UL57 (anti-mouse Alexa 594; panels b and f) and FLAG (anti-rabbit Alexa 488; panels c and g) antibodies. DAPI stain was used to counterstain the nucleus (panels a and e). Panels d and h are merged images of the panels in the other columns. White arrows identify punctate UL57 staining. Yellow arrows identify areas of concentration of FLAG-UL44 staining. Magnification: ×1,000.Our results, taken together, argue for a role for the C-terminal segment of UL44 in HCMV-infected cells in efficient nuclear localization of UL44 and a role in viral DNA synthesis beyond its role in nuclear localization. It is possible that this segment interacts with host or viral proteins involved in DNA replication. Of the various proteins reported to interact with UL44 (10, 19, 30, 31, 35-37), interesting candidates include the host protein nucleolin, which has been shown to associate with UL44 and be important for viral DNA synthesis (35), and the viral UL112-113 proteins, which in transfection assays were shown to recruit UL44 to early sites of DNA replication (2, 29, 33). After this paper was submitted, Kim and Ahn reported that the C-terminal segment of UL44 is necessary for interaction with a UL112-113 protein and, similar to our findings, crucial for viral replication (19). However, contrary to our findings, they reported that this segment was not necessary for efficient nuclear localization of UL44 (19). It may well be that the C-terminal segment of UL44 also has some other role later in viral replication, perhaps in gene expression, as has been suggested (7, 13, 14).A virus with a deletion of the C-terminal 150 amino acids of the HSV-1 polymerase accessory subunit UL42 displays no obvious defect in replication (9). Thus, it appears that HSV-1 and HCMV exhibit different requirements for the C-terminal segments of their respective accessory proteins. This and many other differences between these functionally and structurally orthologous proteins (5, 6, 20, 24, 25) suggest considerable selection for different features during evolution.     

Latency of the Lipid A Deacylase PagL Is Involved in Producing a Robust Permeation Barrier in the Outer Membrane of Salmonella enterica

Lipid A deacylase PagL, which detoxifies endotoxin, is latent in Salmonella enterica. This study determined the biological significance of this latency. PagL latency was beneficial for bacteria in producing a robust permeation barrier through lipid A modifications under host-mimetic conditions that induced the modification enzymes, including PagL.The outer layer of the outer membrane in enteric Gram-negative bacteria is exclusively occupied by lipopolysaccharide (LPS), which contains lipid A as the membrane anchor, while the inner layer contains phospholipids. This asymmetric lipid bilayer serves as a permeation barrier to a large number of noxious compounds. The strength of this barrier is due to the strong lateral interactions between LPS molecules and the low fluidity of the saturated fatty acid portion of lipid A in the outer membrane (reviewed in reference 20). Large hydrophilic compounds are excluded by narrow porin channels, and lipophilic compounds cross the asymmetric bilayer very slowly.The prototype lipid A structure synthesized in Salmonella enterica serovar Typhimurium (S. Typhimurium) is shown in Fig. ​Fig.11 A. In S. Typhimurium, lipid A is further modified by enzymes that are induced upon activation of the two-component regulatory system PhoP-PhoQ (Fig. ​(Fig.1B)1B) (9). PhoP-PhoQ is essential for Salmonella virulence (3, 6, 18), and PhoP-PhoQ-regulated lipid A modifications are involved in many aspects of virulence. PhoQ is a sensor histidine kinase that responds to environmental conditions, including those within mammalian tissues. The host environment is experimentally mimicked by magnesium limitation and/or mild acid pH in the culture medium (3, 4, 6, 18, 21). In response to specific environmental signals, PhoQ phosphorylates PhoP, leading to the activation of pagL and pagP, which encode outer membrane lipid A 3-O-deacylase and outer membrane lipid A palmitoyltransferase, respectively (2, 22). Lipid A 3-O-deacylation by PagL and palmitoylation by PagP reduce the ability of lipid A to activate host Toll-like receptor 4, indicating that PhoP-PhoQ-dependent lipid A modifications help pathogens evade innate immune recognition (12). The regulation of lpxO, which encodes lipid A hydroxylase, is also mediated, at least in part, by PhoP-PhoQ (5, 9). Activation of PhoP-PhoQ leads to the activation of a second two-component regulatory system, PmrA-PmrB (8, 10). PmrA-PmrB promotes the attachment of aminoarabinose and phosphoethanolamine to phosphate groups on lipid A, which are involved in bacterial resistance to cationic antimicrobial peptides (7, 15). Furthermore, PhoP-PhoQ activation produces a more robust permeation barrier in the outer membrane, and lipid A modifications are involved in the generation of this enhanced barrier (19). Mg²⁺ ions decreased membrane permeability strongly in a phoP-null strain but only modestly in a PhoP-constitutive strain (19), implying a biological relevance of lipid A modifications by magnesium limitation.Open in a separate windowFIG. 1.Structures of the prototype lipid A (A) and modified lipid A (B) of S. Typhimurium.Previous studies did not detect PagL-dependent lipid A deacylation when S. Typhimurium was grown under PhoP-PhoQ-activating conditions that induce PagL expression (11, 13, 22). In contrast, PagL-dependent lipid A deacylation was observed in pmrA-null and pmrE-null strains, both of which lacked aminoarabinose modification of lipid A (11, 13). These findings cannot be simply ascribed to the substrate specificity of PagL, since many lipid A species that are not modified with aminoarabinose exist in S. Typhimurium grown under PhoP-PhoQ-activating conditions (13). Therefore, it is thought that PagL is latent under these conditions and that aminoarabinose modification of lipid A is involved in the regulation of latency (13). PagL latency is consistent with an emerging paradigm of outer membrane enzyme regulation (1). It should be noted that PagL-dependent lipid A deacylation, which is beneficial for invading bacteria by allowing them to avoid Toll-like receptor 4 responses, would occur under some specific conditions such as those which activate PhoP-PhoQ without induction of lipid A aminoarabinose modification. Furthermore, we have identified several amino acid residues in the extracellular loops of PagL that are essential for latency but not for deacylase activity (17). The amino acid residues essential for latency were also necessary for PagL to associate with LPS (16). However, the biological significance of latency remains unknown.The influx rate of a lipophilic agent, ethidium bromide, is increased by a pmrA-null mutation in an S. Typhimurium strain with a PhoP-constitutive phenotype (19). The rate-limiting step of this influx is crossing of the asymmetric bilayer in the outer membrane. Therefore, these observations suggest that pmrA-dependent lipid A modifications, such as aminoarabinose and phosphoethanolamine attachment, help generate a more robust permeation barrier through PhoP-PhoQ activation. On the other hand, lipid A is deacylated by PagL in a pmrA strain under PhoP-PhoQ-activating conditions (13). These observations led us to examine whether PagL-dependent lipid A deacylation increases the membrane permeability of the pmrA mutant strain.     

Antibody Recognition Force Microscopy Shows that Outer Membrane Cytochromes OmcA and MtrC Are Expressed on the Exterior Surface of Shewanella oneidensis MR-1

Antibody recognition force microscopy showed that OmcA and MtrC are expressed on the exterior surface of living Shewanella oneidensis MR-1 cells when Fe(III), including solid-phase hematite (Fe₂O₃), was the terminal electron acceptor. OmcA was localized to the interface between the cell and mineral. MtrC displayed a more uniform distribution across the cell surface. Both cytochromes were associated with an extracellular polymeric substance.Shewanella oneidensis MR-1 is a dissimilatory metal-reducing bacterium that is well known for its ability to use a variety of anaerobic terminal electron acceptors (TEAs), including solid-phase iron oxide minerals, such as goethite and hematite (8, 10). Previous studies suggest that S. oneidensis MR-1 uses outer membrane cytochromes OmcA and MtrC to catalyze the terminal reduction of Fe(III) through direct contact with the extracellular iron oxide mineral (2, 8, 10, 15, 16, 20, 21, 23). However, it has yet to be shown whether OmcA or MtrC is actually targeted to the external surface of live S. oneidensis MR-1 cells when Fe(III) serves as the TEA.In the present study, we used atomic force microscopy (AFM) to probe the surface of live S. oneidensis MR-1 cells, using AFM tips that were functionalized with cytochrome-specific polyclonal antibodies (i.e., anti-OmcA or anti-MtrC). This technique, termed antibody recognition force microscopy (Ig-RFM), detects binding events that occur between antibodies (e.g., anti-OmcA) on an AFM tip and antigens (e.g., OmcA) that are exposed on a cell surface. While this is a relatively new technique, Ig-RFM has been used to map the nanoscale spatial location of single molecules in complex biological structures under physiological conditions (5, 9, 11, 13).Anti-MtrC or anti-OmcA molecules were covalently coupled to silicon nitride (Si₃N₄) cantilevers (Veeco or Olympus) via a flexible, heterofunctional polyethylene glycol (PEG) linker molecule. The PEG linker consists of an NHS (N-hydroxysuccinimide) group at one end and an aldehyde group at the other end (i.e., NHS-PEG-aldehyde). AFM tips were functionalized with amine groups, using ethanolamine (6, 7). The active NHS ester of the NHS-PEG-aldehyde linker molecule was then used to form a covalent linkage between PEG-aldehyde and the amine groups on the AFM tips (6, 7). Next, anti-MtrC or anti-OmcA molecules were covalently tethered to these tips via the linker molecule''s aldehyde group. This was accomplished by incubating the tips with antibody (0.2 mg/ml) and NaCNBH₃ as described previously (7). The cantilevers were purchased from Veeco and had spring constant values between 0.06 and 0.07 N/m, as determined by the thermal method of Hutter and Bechhoefer (12).Prior to conducting the Ig-RFM experiments, the specificity of each polyclonal antibody (i.e., anti-OmcA and anti-MtrC) for OmcA or MtrC was verified by Western blot analysis as described previously (24, 28). Proteins were resolved by both denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Briefly, 2.5 μg of purified OmcA or MtrC (23) was resolved by sodium dodecyl sulfate-PAGE or native PAGE, transferred to a polyvinylidene difluoride membrane, incubated with either anti-OmcA or anti-MtrC, and then visualized using the Amersham ECL Plus Western blotting detection kit. Anti-OmcA bound exclusively to OmcA, anti-MtrC bound exclusively to MtrC, and neither antibody showed cross-reactivity with the other cytochrome. Antibody specificities of anti-OmcA and anti-MtrC were also validated by immunoblot analysis of S. oneidensis whole-cell lysate (28).To determine if MtrC or OmcA was expressed on the external surface of live bacteria when Fe(III) served as the TEA, Ig-RFM was conducted on wild-type versus ΔomcA ΔmtrC double mutant cells. For these experiments, bacteria were cultivated anaerobically with Fe(III), in the form of Fe(III) chelated to nitrilotriacetic acid (NTA), serving as the TEA (19, 23). Growth conditions have been described elsewhere (3, 15) and were based on previous studies (3, 15, 16, 18) that suggest that S. oneidensis MR-1 targets OmcA and MtrC to the cell surface when Fe(III) serves as the TEA.An Asylum Research MFP-3D-BIO AFM or a Digital Instruments Bioscope AFM (16, 17) was used for these experiments. The z-piezoelectric scanners were calibrated as described previously (17). Cells were deposited on a hydrophobic glass coverslip and immersed in imaging buffer (i.e., phosphate-buffered saline [pH 7.4]). The hydrophobic glass coverslips were made as described previously (17) using a self-assembling silane compound called octadecyltrichlorosilane (OTS; Sigma-Aldrich). S. oneidensis MR-1 cells readily adsorbed onto OTS glass coverslips and remained attached to the coverslips during the entire experiment. No lateral cell movement was observed during the experiment, consistent with previous studies that used OTS glass to immobilize bacteria (15, 17, 18, 27).The AFM tip was brought into contact with the surface of a bacterium, and the antibody-functionalized tip was repeatedly brought into and out of contact with the sample, “fishing” for a binding reaction with cytochrome molecules that were exposed on the external cell surface. Binding events were observed upon separating anti-OmcA- or anti-MtrC-functionalized tips from wild-type S. oneidensis MR-1 cells (Fig. ​(Fig.1).1). For the wild-type cells, we observed both nonspecific and specific interactions (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Retraction force curves for anti-MtrC-functionalized tips (A) and anti-OmcA-functionalized tips (B) that are being pulled away from the surface of living ΔomcA ΔmtrC double mutant (gray dotted line) or wild-type (solid black line) S. oneidensis MR-1. These bacteria were adsorbed onto OTS glass coverslips. (C) Retraction curves exhibiting nonspecific binding, specific binding, or no binding between the AFM tip and the cell surface.The distinction between “specific” and “nonspecific” adhesion is made by observing the change in slope of the force curve during the retraction process (26). During specific binding (Fig. ​(Fig.1C),1C), the cantilever is initially relaxed as it is pulled away from the sample. Upon further retraction, the ligand-receptor complex becomes stretched and unravels, resulting in a nonlinear force profile as noted in references 26 and 16. On the other hand, nonspecific adhesion (Fig. ​(Fig.1C)1C) maintains the same slope during the retraction process because only the cantilever flexes (26).Figure ​Figure22 summarizes the frequency or probability of observing a binding event for both anti-OmcA and anti-MtrC tips. Each bar in Fig. ​Fig.22 represents one experiment in which 500 to 1,000 force curves were collected between one AFM tip and two to four live bacterial cells. This figure does not make a distinction between specific and nonspecific binding. It simply shows the frequency of observing an attractive interaction as the antibody-functionalized tip was pulled away from the surface of S. oneidensis MR-1. Binding events occurred with roughly the same frequency when wild-type S. oneidensis MR-1 cells were probed with anti-MtrC-functionalized tips as when they were probed with anti-OmcA-functionalized tips (Fig. ​(Fig.22).Open in a separate windowFIG. 2.Histograms showing the frequency of observing a binding event for anti-MtrC-functionalized (blue) or anti-OmcA-functionalized (red) AFM tips on live wild-type S. oneidensis MR-1 (solid bars) or ΔomcA ΔmtrC double mutant (diagonally hatched bars) cells. The downward arrows designate injection of free antibody into the imaging buffer. The solid gray bars correspond to results obtained with unbaited AFM tips.A number of control experiments were performed to verify the detection of OmcA and MtrC on the surface of wild-type S. oneidensis MR-1. First, 0.1 μM of free anti-OmcA (or anti-MtrC) was added to the imaging fluid to block binding between the antibody-functionalized AFM tip and surface-exposed cytochromes (11, 16). This decreased the adhesion that was observed between the antibody-functionalized tip and the cell surface (Fig. ​(Fig.22).Second, we performed force measurements on ΔomcA ΔmtrC double mutant S. oneidensis MR-1 cells. This mutant is deficient in both OmcA and MtrC (19, 23, 24) but produces other proteins native to the outer surface of S. oneidensis MR-1. The resulting force spectra showed a noticeable reduction in binding events for the ΔomcA ΔmtrC double mutant cells (Fig. ​(Fig.2).2). The binding events that were observed for the double mutant were only nonspecific in nature (Fig. ​(Fig.1).1). This indicates that the antibodies on the tip do not participate in specific interactions with other proteins on the surface of S. oneidensis MR-1 cells.As a final control experiment, force measurements were conducted on wild-type S. oneidensis MR-1 cells, using Si₃N₄ tips conjugated with the PEG linker but not functionalized with polyclonal antibody (unbaited tips). Like the results with the double mutant, the unbaited tips were largely unreactive with the surface of the bacteria (Fig. ​(Fig.2).2). Those binding events that were observed were nonspecific in nature. Taken together, these results demonstrate that the antibody-coated tips have a specific reactivity with OmcA and MtrC molecules. Furthermore, these force measurements show that MtrC and OmcA are present on the external cell surface when Fe(III) serves as the TEA.To map the distribution of cytochromes on living cells, Ig-RFM was conducted on living S. oneidensis MR-1 cells that were growing on a hematite (α-Fe₂O₃) thin film. The conditions for these experiments were as follows. A hematite film was grown on a 10-mm by 10-mm by 1-mm oxide substrate via oxygen plasma-assisted molecular beam epitaxy (14, 16). The cells were grown anaerobically to mid-log phase with Fe(III)-NTA serving as the TEA. Cells were deposited onto the hematite thin film along with anaerobic growth medium that lacked Fe(III)-NTA. The cells were allowed to attach to the hematite surface (without drying) overnight in an anaerobic chamber. The following day, the liquid was carefully removed and immediately replaced with fresh anaerobic solution (pH 7.4). Ig-RFM was performed on the cells by raster scanning an antibody-functionalized AFM tip across the sample surface, thereby creating an affinity map (1). Force curves were collected for a 32-by-32 array. The raw pixilated force-volume data were deconvoluted using a regularized filter algorithm. The total time to acquire a complete image was approximately 20 min.As noted above, attractive interactions between an antibody tip and cell resulted in relatively short-range, nonspecific and longer-range, specific adhesive forces (Fig. ​(Fig.1C).1C). To distinguish between these two interactions, we integrated each force curve beginning at >20 nm and ending at the full retraction of the piezoelectric motor (∼1,800 nm). This integration procedure quantifies the work of binding, measured in joules, between the antibody tip and a particular position on the sample. While this integration procedure does not totally exclude nonspecific binding, it does select for those events associated primarily with specific antibody-antigen binding. Figure ​Figure33 is the antibody-cytochrome recognition images for MtrC and OmcA. The corresponding height (or topography) images of the bacterial cells are also shown in Fig. ​Fig.33.Open in a separate windowFIG. 3.Ig-RFM of live S. oneidensis MR-1 cells deposited on a hematite (α-Fe₂O₃) thin film. Height image (A) and corresponding Ig-RFM image (B) for a bare unfunctionalized Si₃N₄ tip. Height and corresponding Ig-RFM image for a tip functionalized with anti-MtrC (C and D) or anti-OmcA (E and F). Each panel contains a thin white oval showing the approximate location of the bacterium on the hematite surface. A color-coded scale bar is shown on the right (height in micrometers [μm], and the work required to separate the tip from the surface in attojoules [aJ]).OmcA molecules were concentrated at the boundary between the bacterial cell and hematite surface (Fig. 3E and F). MtrC molecules were also detected at the edge of a cell (Fig. 3C and D). Some MtrC, unlike OmcA, was observed on the cell surface distal from the point of contact with the mineral (Fig. 3C and D). Both OmcA and MtrC were also present in an extracellular polymeric substance (EPS) on the hematite surface (Fig. 3D and F), which is consistent with previous results showing MtrC and OmcA in an EPS produced by cells under anaerobic conditions (19, 24). This discovery is interesting in light of the research by Rosso et al. (22) and Bose et al. (4), who found that Shewanella can implement a nonlocal electron transfer strategy to reduce the surface of hematite at locations distant from the point of cell attachment. Rosso et al. (22) proposed that the bacteria utilize unknown extracellular factors to access the most energetically favorable regions of the Fe(III) oxide surface. The Ig-AFM results (Fig. ​(Fig.3)3) suggest the possibility that MtrC and/or OmcA are the “unknown extracellular factors” that are synthesized by Shewanella to reduce crystalline Fe(III) oxides at points distal from the cell. Additional experiments showing reductive dissolution features coinciding with the extracellular location of MtrC and/or OmcA would need to be performed to test this hypothesis.It is important to note that these affinity maps were collected on only a few cells because it so challenging to produce large numbers of quality images. Future work should be conducted on a population of cells. Until this time, these affinity maps can be used to provide a crude, lowest-order estimate of the number of cytochromes on the outer surface of living S. oneidensis MR-1. For example, there were 236 force curves collected on the bacterium shown in Fig. ​Fig.3D.3D. Thirty-eight of these curves exhibited a distinct, sawtooth-shaped, antibody-antigen binding event. In other words, MtrC molecules were detected in one out of every six force curves (16%) that were collected on the cell surface.This probability can be compared to other independent studies that estimated the density and size of MtrC and OmcA molecules from S. oneidensis MR-1. Lower et al. (16) estimated that S. oneidensis has 4 × 10¹⁵ to 7 × 10¹⁵ cytochromes per square meter by comparing AFM measurements for whole cells to force curves on purified MtrC and OmcA molecules. Wigginton et al. (25) used scanning tunneling microscopy to determine that the diameter of an individual cytochrome is 5 to 8 nm. These values can be used to create a simple, geometric, close-packing arrangement of MtrC or OmcA molecules on a surface. Using this approach, cytochromes could occupy 8 to 34% of the cell surface.This estimate is consistent with the observed number of putative MtrC molecules shown in Fig. ​Fig.3D.3D. Therefore, it appears that these affinity maps can be used as a lowest-order estimate for the number of cytochromes on S. oneidensis MR-1 even though we do not know a priori the exact configuration of the antibody tip (e.g., the concentration of antibody on the tip, the exact shape of the tip, the binding epitopes within the antibody).In summary, the data presented here show that S. oneidensis MR-1 localizes OmcA and MtrC molecules to the exterior cell surface, including an EPS, when Fe(III) is the TEA. Here, the cytochromes presumably serve as terminal reductases that catalyze the reduction of Fe(III) through direct contact with the extracellular iron-oxide mineral.     

Isoprenoids Are Essential for Fruiting Body Formation in Myxococcus xanthus

It was recently shown that Myxococcus xanthus harbors an alternative and reversible biosynthetic pathway to isovaleryl coenzyme A (CoA) branching from 3-hydroxy-3-methylglutaryl-CoA. Analyses of various mutants in these pathways for fatty acid profiles and fruiting body formation revealed for the first time the importance of isoprenoids for myxobacterial development.Myxobacteria are unique among the prokaryotes as (i) they can form highly complex fruiting bodies under starvation conditions, even up to microscopic tree-like structures (28); (ii) they can move on solid surfaces using different motility mechanisms (16); (iii) they produce some of the most cytotoxic secondary metabolites, with epothilone already in clinical use against cancer (2, 3); and (iv) they harbor the largest prokaryotic genomes found so far (15, 27). The large genome might be directly related to their complex life-style and the diverse secondary (3) and primary (9) metabolisms. Already in 2002 we found that myxobacteria are able to produce isovaleryl coenzyme A (IV-CoA) and compounds derived thereof via a new pathway that branches from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), which is the central intermediate of the well-known mevalonate-dependent isoprenoid biosynthesis (Fig. ​(Fig.1)1) (22, 23). Usually IV-CoA is derived from leucine degradation via the branched-chain keto acid dehydrogenase (BKD) complex (24), which is also the preferred pathway to IV-CoA in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca (Fig. ​(Fig.2A).2A). However, in bkd mutants, where no or only residual leucine degradation is possible (30), the alternative pathway is induced (Fig. ​(Fig.2B),2B), presumably to ensure the production of iso-fatty acids (iso-FAs) (5). A possible reason for this alternative pathway is the importance of IV-CoA-derived compounds in the complex myxobacterial life cycle, which is the starvation-induced formation of fruiting bodies in which the cells differentiate into myxospores. We showed that this pathway is induced during fruiting body formation in M. xanthus when leucine is limited. Under these conditions, this pathway might be more important for protein synthesis than for lipid remodeling, as lipids are present in excess during development due to the surface reduction from vegetative rods to round myxospores as described previously (29). Examples of IV-CoA-derived compounds are the unusual iso-branched ether lipids, which are almost exclusively produced in the developing myxospores. They might serve as structural lipids and signaling compounds during fruiting body formation (26).Open in a separate windowFIG. 1.Biosynthesis of IV-CoA and compounds derived thereof and biosynthesis of isoprenoids in M. xanthus. Broken arrows indicate multistep reactions; supplementation (double-lined arrows) with MVL and IVA can be used to complement selected mutants.Open in a separate windowFIG. 2.Short representations of proposed metabolic fluxes through the IV-CoA/isoprenoid network. Broken arrows indicate no metabolic flux. (A) DK1622 (wild type); (B) DK5643 (Δbkd); (C) DK5624 (Δbkd mvaS::kan); (D) HB002 (Δbkd liuC::kan); (E) HB002 with 1 mM IVA; (F) HB002 with 1 mM MVL. Ac-CoA, acetyl-CoA; MVA, mevalonic acid.In M. xanthus, we could recently identify candidate genes involved in the alternative pathway from HMG-CoA to IV-CoA. We also described the genes required for the degradation pathway of leucine and subsequently also those involved in the transformation of IV-CoA to HMG-CoA (4). In myxobacteria leucine is an important precursor for isoprenoid biosynthesis, as was already shown elsewhere for the biosynthesis of steroids (7) and prenylated secondary metabolites like aurachin (22) or leupyrrins (6), as well as volatiles like geosmin or germacradienol in M. xanthus and S. aurantiaca (11, 13). The interconnection of iso-FAs and isoprenoid biosynthesis made it difficult to assign functions to these compound classes during fruiting body formation in M. xanthus because it cannot be excluded that reduced leucine degradation also impairs isoprenoid biosynthesis. A mutant strain of M. xanthus that was blocked in the degradation of leucine and the alternative pathway had a deletion in the bkd locus as well as a plasmid insertion in the mvaS gene encoding the HMG-CoA synthase (strain DK5624). This double mutation severely affected isoprenoid biosynthesis (5), and cultures of DK5624 must be supplemented with mevalonolactone (MVL; the cyclized form of mevalonic acid) in order to enable growth (Fig. ​(Fig.2C).2C). Since we have identified the genes involved in IV-CoA biosynthesis and the mevalonate pathway (4), we can now start to identify differences between strains that show deficiencies in iso-FAs and strains that show deficiencies in isoprenoids via simple analysis of the FA profile and analysis of the myxobacterial development of selected mutants.All mutants used in this study (HB002 [Δbkd liuC::kan], HB015 [Δbkd MXAN_4265::kan], DK5624 [Δbkd mvaS::kan], HB019 [Δbkd mvaS::kan mvaS⁺], and HB020 [Δbkd MXAN_4265::kan mvaS⁺]) have been published previously (4), and FA analysis as well as myxobacterial fruiting body formation has also been described previously (26).M. xanthus HB002 (Δbkd liuC) shows only residual amounts of iso-FAs, as both leucine degradation and the alternative pathway to IV-CoA are blocked (Fig. ​(Fig.2D)2D) and its capability to form fruiting bodies is strongly reduced (Fig. ​(Fig.3).3). The residual amount of iso-FAs results from a second BKD activity in M. xanthus that has been identified by residual leucine incorporation as well as by residual enzymatic activity in bkd mutants (23, 30). This second BKD activity might be a side activity of the pyruvate dehydrogenase or a related chemical oxidative decarboxylation, as no second bkd locus could be identified in the genome (unpublished results). Moreover, growth of HB002 is not MVL dependent because the block in the alternative pathway does not affect isoprenoid biosynthesis, as liuC encodes a dehydratase/hydratase that is involved in the conversion of HMG-CoA to 3-methylglutaconyl-CoA and vice versa (4). As expected, the FA profile (4) as well as the developmental phenotype (data not shown) can be complemented (Fig. ​(Fig.2E)2E) by the addition of isovaleric acid (IVA), the free acid of IV-CoA, indicating the importance of iso-branched compounds for development in M. xanthus. Unexpectedly, addition of MVL (Fig. ​(Fig.2F)2F) also partially restored fruiting body formation without restoring the FA profile (Fig. ​(Fig.3).3). Similarly, M. xanthus HB015 (Δbkd MXAN_4265::kan) can produce only traces of iso-FAs, as both pathways to IV-CoA are blocked. MXAN_4265 encodes a protein with similarity to a glutaconyl-CoA transferase subunit, but from our previous results, we postulated it to be involved in the alternative pathway to IV-CoA (Fig. ​(Fig.1)1) (4). The respective mutant shows a severely impaired developmental phenotype, which can be complemented not only by the addition of IVA (not shown) but also by the addition of MVL (Fig. ​(Fig.3).3). Again, no change in the FA profile was observed after the addition of MVL. However, a plasmid insertion into MXAN_4265 has a polar effect on mvaS, which is the last gene in this five-gene operon and which is crucial for HMG-CoA formation from acetoacetyl-CoA and acetyl-CoA. Therefore, we assume that both pathways to HMG-CoA are blocked in HB015: no HMG-CoA can be made from acetyl-CoA and hardly any can be made via leucine degradation. In order to prove this hypothesis, we complemented HB015 with an additional copy of mvaS under the constitutive T7A1 promoter as described previously, using the plasmid pCK4267exp (4). The resulting strain, HB020 (Δbkd MXAN_4265::kan mvaS⁺), showed a restored developmental phenotype but still produced only trace amounts of iso-FAs.Open in a separate windowFIG. 3.Fruiting body formation on TPM agar in selected mutants at 24, 48, and 72 h after starvation. Numbers refer to the relative amounts (in percentages) of the most abundant iso-FA, iso-15:0, which is indicative of iso-FAs in general. Strains were DK1622 (wild type), HB002 (Δbkd liuC::kan), HB015 (Δbkd MXAN_4265::kan), DK5624 (Δbkd mvaS::kan), HB019 (Δbkd mvaS::kan mvaS⁺), and HB020 (Δbkd MXAN_4265::kan mvaS⁺). DK5624 was grown with 0.3 mM MVL prior to starvation, and the cells were washed and plated on TPM with or without 1 mM of MVL.The data from HB002, HB015, and HB020 indicate an important function of the mevalonate-dependent isoprenoid pathway for fruiting body formation in M. xanthus. Therefore, MVL addition can at least partially complement the developmental phenotype of DK5624, which cannot form fruiting bodies without MVL (Fig. ​(Fig.3).3). However, genetic complementation with mvaS in HB019 resulted in the expected complementation of the fruiting body formation and the FA profile (Fig. ​(Fig.3,3, bottom row).Leucine is one of the most abundant proteinogenic amino acids. It is also an essential amino acid for M. xanthus (8), which has a predatory life-style (1), as it lives on other bacteria and fungi that contain a lot of leucine. Moreover, leucine is very efficiently incorporated into isoprenoids like geosmin and aurachin (10, 22). Thus, one can conclude that in fact leucine degradation is the major pathway for HMG-CoA biosynthesis instead of the usual formation via acetoacetyl-CoA and acetyl-CoA by the HMG-CoA synthase MvaS as indicated in Fig. ​Fig.2A.2A. No difference in growth was observed between culture with and culture without MVL for HB002 (Δbkd liuC::kan) and HB015 (Δbkd MXAN_4265::kan) in rich medium (data not shown), probably due to the complete MvaS activity (in HB002) or residual BKD activity (in HB002 and HB015), resulting in all precursors for the mevalonate-dependent isoprenoid biosynthesis still being present in excess under these conditions. However, under starvation conditions a small reduction in HMG-CoA biosynthesis caused by completely blocked leucine degradation (as in HB002 due to the mutation in liuC [Fig. ​[Fig.2D])2D]) or reduced leucine degradation and a mutation in mvaS (as in HB015) might each result in a reduced isoprenoid level, which can be complemented at least partially by the addition of MVL. This would also explain the difference in the developmental phenotypes of HB002 and HB015, with the phenotype being more severe in HB002 (Fig. ​(Fig.3).3). The fact that complementation with IVA is in all cases more efficient than that with MVL can be explained by the role of the already-mentioned isolipids. They can be produced only after IVA addition, which also complements the (developmental) phenotype of some of these mutants (26).As isoprenoids represent probably the most diverse class of natural products (14), it is very hard to predict which particular isoprenoids might be responsible for the observed effects. Several isoprenoids (7, 11-13), prenylated secondary metabolites (6, 22), and carotenoids (18-21) are known from myxobacteria in general, and a major volatile compound from M. xanthus is the terpenoid geosmin (13). In order to test whether geosmin might be required for fruiting body formation, we constructed a plasmid insertion mutant in MXAN_6247, which is involved in the cyclization of farnesyl diphosphate to geosmin, following published procedures (4, 5). The resulting strain, HB022, showed the expected loss in geosmin production but no developmental phenotype (data not shown).Additionally, it cannot be excluded that prenylated proteins, sugars, or quinones from the respiratory chain are important for fruiting body formation. Moreover, stigmolone has been described as a pheromone involved in fruiting body formation in S. aurantiaca (25). Although its biosynthesis has not been elucidated yet, stigmolone could be an isoprenoid as well, which is deducible from the two iso-branched residues within its chemical structure (17). Nevertheless, the importance of isoprenoids for M. xanthus is evident from the data presented, and clearly more work is needed to identify the compound(s) involved.     

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.