Mutational Analysis of the Transmembrane Helix 2-HAMP Domain Connection in the Escherichia coli Aspartate Chemoreceptor Tar

Transmembrane helix 2 (TM2) of the Tar chemoreceptor undergoes an inward piston-like displacement of 1 to 3 Å upon binding aspartate. This signal is transmitted to the kinase-control module via the HAMP domain. Within Tar, the HAMP domain forms a parallel four-helix bundle consisting of a dimer of two amphipathic helices connected by a flexible linker. In the nuclear magnetic resonance structure of an archaeal HAMP domain, residues corresponding to the MLLT sequence between Arg-214 at the end of TM2 and Pro-219 of Tar are an N-terminal helical extension of AS1. We modified this region to test whether it behaves as a continuous helical connection between TM2 and HAMP. First, one to four Gly residues were inserted between Thr-218 and Pro-219. Second, the MLLT sequence was replaced with one to nine Gly residues. Third, the sequence was shortened or extended with residues compatible with helix formation. Cells expressing receptors in which the MLLT sequence was shortened to MLL or in which the MLLT sequence was replaced by four Gly residues performed good aspartate chemotaxis. Other mutant receptors supported diminished aspartate taxis. Most mutant receptors had biased signal outputs and/or abnormal patterns of adaptive methylation. We interpret these results to indicate that a strong, permanent helical connection between TM2 and the HAMP domain is not necessary for normal transmembrane signaling.The HAMP domain is a structural motif commonly found in histidine kinases (HKs), adenylate cyclases, methyl-accepting chemotaxis proteins (MCPs), and phosphatases (2). In Escherichia coli and Salmonella enterica MCPs, the HAMP domain is located between a transmembrane-sensing module composed of ligand-binding and transmembrane regions and a kinase-control module composed of adaptation and kinase-activating regions (Fig. ​(Fig.1A)1A) (19). Therefore, HAMP domains are responsible for bidirectional signal transduction between these modules.Open in a separate windowFIG. 1.Domain architecture of the aspartate chemoreceptor. (A) The cartoon, based on a figure from Hazelbauer et al. (2008) (19), illustrates the architecture of the aspartate chemoreceptor. Protein structural domains are labeled on the left, and functional modules are labeled on the right. (B) Schematic of TM2 and the control cable region attached to a ribbon diagram of the solution NMR structure of the Af1503 HAMP domain four-helix bundle (22). TM2 is shown in purple within the membrane. The control cable of Tar_Ec consists of 5 amino acyl residues (Gly-Ile-Arg-Arg-Met) that connect TM2 and AS1 of HAMP. AS1 is shown in blue, AS2 is shown in red, and the 14-residue AS1-AS2 connector (CTR) is shown in black. The residue equivalent to Arg-214 in Tar_Ec is also highlighted in blue, the conserved Pro residue (Pro-219 in Tar_Ec) is highlighted in yellow, and residues equivalent to the MLLT sequence between TM2 and Pro-219 in Tar_Ec are highlighted in cyan.The determination of a high-resolution three-dimensional structure of a HAMP domain from an MCP remains elusive. However, a solution nuclear magnetic resonance (NMR) structure of a HAMP domain has been determined for the Af1503 protein of unknown function from the archaeal thermophile Archeoglobus fulgidus (22). The domain forms a parallel four-helix bundle, with two amphipathic helices, AS1 and AS2, being contributed by each subunit (Fig. ​(Fig.1B).1B). In this structure, the helices pack in an unusual x-da configuration, commonly referred to as knobs-to-knobs packing, in which the large hydrophobic x residues stabilize both intrasubunit and intersubunit interactions.Evidence for the existence of a four-helix HAMP bundle within intact receptors comes from disulfide cross-linking experiments with Salmonella enterica Tar (Tar_Se) (41) and the E. coli Aer redox receptor (Aer_Ec) (44). In vivo genetic studies (1, 48) are also consistent with the existence of a four-helix bundle in the E. coli Tsr receptor (Tsr_Ec).Tar_Ec functions as the aspartate chemoreceptor in E. coli (39). Each monomer within the homodimeric (15, 29) receptor possess a periplasmic ligand-binding domain composed of four antiparallel α helices that form four-helix bundles (8). The transmembrane regions that flank this periplasmic domain (transmembrane helix 1 [TM1] and TM2) are extensions of the periplasmic helices PD1 and PD4 (27, 33, 37, 40). Aspartate binds at either of two rotationally symmetrical sites at the dimer interface. Each binding site contains residues from PD1 of one subunit and PD1′ and PD4′ of the other. Aspartate binding generates a small (∼1- to 3-Å) vertical displacement into the cytoplasm of one contiguous PD4-TM2 helix relative to the other (14).E. coli Tar (Tar_Ec) and other chemoreceptors normally activate the histidine protein kinase CheA (5), which is coupled to the receptors via the adapter protein CheW. CheA autophosphorylates, and the phosphoryl group is subsequently transferred to the response regulator CheY (21). CheY-P interacts with FliM within the flagellar motor to promote clockwise (CW) rotation of the flagella (36, 46). Counterclockwise (CCW) motor rotation allows the flagellar filaments to coalesce into a bundle that propels the cell in a run (38). CW rotation of one or more flagella disrupts the bundle and generates a tumble (43). Therefore, the relative activities of CheA and the CheY-P phosphatase, CheZ, establish the ratio of CheY to CheY-P within the cell and hence the frequency of tumbling (11, 21).The conformational changes induced by aspartate convert Tar_Ec from a stimulator of CheA activity into an inhibitor (6). The resulting drop in CheY-P activity, which is accelerated by CheZ, suppresses tumbling and lengthens the average run. Inhibition of CheA activity is reversed by covalent methylation of the cognate receptor (17). Methylation is facilitated by a transient decrease in the level of the active, phosphorylated form of the CheB methylesterase (26), which is another substrate for phosphotransfer from CheA (21). The well-studied properties of this system, and the possibility of monitoring several different in vivo parameters, make it amenable for examining signal transduction between TM2 and the adjoining HAMP domain.The apical region of AS1 in Tar_Ec is composed of the tetrapeptide Met-Leu-Leu-Thr, which connects residue Arg-214 at the end of TM2 with the conserved Pro-219 residue within AS1 of the HAMP domain (Fig. ​(Fig.1B).1B). The corresponding sequence is Thr-Ile-Thr-Arg in the Af1503 HAMP domain. In the NMR structure of the isolated Af1503 HAMP, the corresponding four residues (Thr-Ile-Thr-Arg) comprise an unpaired helical extension of the N terminus of AS1 before Pro-283 (9, 22), but it is unclear how these residues may pack in an intact membrane-spanning protein. In the Af1503 HAMP, Pro-283 packs against residues Glu-311 and Ile-312, which form the N terminus of AS2. The residues at the equivalent positions in Tar_Ec are Glu-246 and Met-247 (Fig. ​(Fig.1B1B).We modified the length and residue composition of the region between Arg-214 and Pro-219 in Tar_Ec in different ways and monitored the ability of the mutant proteins to support chemotactic migration, to generate the clockwise flagellar rotation that reflects CheA activation, and to regulate adaptive methylation. The results support a model in which the structural tension exerted by TM2 on AS1 controls its signaling state, as proposed by Zhou et al. (48). In the context of that model, the results suggest that when the receptor is in the kinase-inhibiting state, the HAMP domain is in a stable four-helix bundle.     

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.     

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.     

Interactions of DNA with Biofilm-Derived Membrane Vesicles

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

Pseudotyping Incompatibility between HIV-1 and Gibbon Ape Leukemia Virus Env Is Modulated by Vpu

The Env protein from gibbon ape leukemia virus (GaLV) has been shown to be incompatible with human immunodeficiency virus type 1 (HIV-1) in the production of infectious pseudotyped particles. This incompatibility has been mapped to the C-terminal cytoplasmic tail of GaLV Env. Surprisingly, we found that the HIV-1 accessory protein Vpu modulates this incompatibility. The infectivity of HIV-1 pseudotyped with murine leukemia virus (MLV) Env was not affected by Vpu. However, the infectivity of HIV-1 pseudotyped with an MLV Env with the cytoplasmic tail from GaLV Env (MLV/GaLV Env) was restricted 50- to 100-fold by Vpu. A Vpu mutant containing a scrambled membrane-spanning domain, Vpu_RD, was still able to restrict MLV/GaLV Env, but mutation of the serine residues at positions 52 and 56 completely alleviated the restriction. Loss of infectivity appeared to be caused by reduced MLV/GaLV Env incorporation into viral particles. The mechanism of this downmodulation appears to be distinct from Vpu-mediated CD4 downmodulation because Vpu-expressing cells that failed to produce infectious HIV-1 particles nonetheless continued to display robust surface MLV/GaLV Env expression. In addition, if MLV and HIV-1 were simultaneously introduced into the same cells, only the HIV-1 particle infectivity was restricted by Vpu. Collectively, these data suggest that Vpu modulates the cellular distribution of MLV/GaLV Env, preventing its recruitment to HIV-1 budding sites.The gammaretrovirus gibbon ape leukemia virus (GaLV) has been widely used for gene therapy because of its wide host cell tropism and nonpathogenicity (1, 6, 10, 12, 13, 20). The host cell receptor for GaLV Env has been cloned and identified as a sodium-dependent phosphate transporter protein (25, 26). Like other retroviruses, GaLV encodes a single transmembrane surface glycoprotein (GaLV Env), which is cleaved into surface (SU) and transmembrane (TM) subunits (Fig. ​(Fig.1).1). The TM domain of GaLV Env contains a short 30-amino-acid C-terminal cytoplasmic tail. Although GaLV Env functions well when coupled (pseudotyped) with murine leukemia virus (MLV)-based retroviral vectors, it has been shown to be completely incompatible with HIV-1 (4, 35). When GaLV Env is expressed with HIV-1, essentially no infectious HIV-1 particles are produced (4, 35). The mechanism for this infectivity downmodulation is unknown, but the component of GaLV Env responsible for the restriction has been mapped to the cytoplasmic tail. Replacing the cytoplasmic tail of GaLV Env with the equivalent sequence from MLV Env ameliorates the restriction. Likewise, replacing the cytoplasmic tail of MLV Env with that from GaLV Env confers the restriction (4).Open in a separate windowFIG. 1.Schematic of MLV Env protein. Sequences are the C-terminal cytoplasmic tails of MLV Env, GaLV Env, and human CD4. GaLV sequences in boldface are residues that have been shown to modulate the HIV-1 incompatibility (4). Underlined sequences in CD4 are amino acids required for Vpu-mediated downmodulation (2, 15). Arrows denote the location of MLV/GaLV tail substitution. SU, surface domain; TM, transmembrane domain.Vpu is an 81-amino-acid HIV-1 accessory protein produced from the same mRNA as the HIV-1 Env gene. The N terminus of Vpu contains a membrane-spanning domain, followed by a 50-amino-acid cytoplasmic domain. Vpu is unique to HIV-1 and a few closely related SIV strains. The best-characterized roles for Vpu in the HIV-1 life cycle are modulation of host proteins CD4 and tetherin (also known as BST-2, CD317, and HM1.24) (24, 38, 39). Vpu promotes the degradation of CD4 in the endoplasmic reticulum through a proteasome-dependent mechanism (29). The cytoplasmic tail of Vpu physically interacts with the cytoplasmic tail of CD4 and recruits the human β-transducing repeat-containing protein (β-TrCP) and E3 ubiquitin ligase components to polyubiquitinate and ultimately trigger the degradation of CD4 (18). Two serine residues at positions 52 and 56 of Vpu are phosphorylated by casein kinase-2 and are required for CD4 degradation (31, 32). The membrane-spanning domain of Vpu is not specifically required for CD4 degradation. A mutant protein containing a scrambled membrane-spanning sequence, Vpu_RD, is still able to trigger the degradation of CD4 (32). The region of CD4 that is targeted by Vpu is approximately 17 to 13 amino acids from the C terminus in the cytoplasmic tail (Fig. ​(Fig.1)1) (2, 15).In addition to degrading CD4, Vpu has also long been known to result in enhanced viral release (EVR) in certain cell lines (14, 36). Recently, the type I interferon-induced host protein tetherin was identified as being responsible for this Vpu-modulated restriction (24, 38). In the absence of Vpu, tetherin causes particles to remain tethered (hence the name) to the host cell postfission. Although Vpu counteracts the function of tetherin, the exact mechanism has not been fully elucidated. However, the mechanism for tetherin antagonism appears to be distinct from that for modulating CD4. Mutation of the serines 52 and 56 of Vpu abolish CD4 degradation, but only reduce EVR activity (5, 17, 21, 32). Some EVR activity remains even when much of the Vpu cytoplasmic tail is deleted (30). In addition, many mutations in the membrane-spanning domain, such as Vpu_RD, do not affect CD4 degradation and yet completely abolish EVR activity (27, 30, 37). The critical residues in tetherin for recognition by Vpu appear to be in the membrane-spanning domain and not the cytoplasmic tail (9, 19, 28). Although β-TrCP is required for complete EVR activity, there is no consensus whether the degradation of tetherin is proteasome or lysosome mediated (5, 7, 21) or whether degradation is required at all. In some cases there can be some EVR activity in the absence of tetherin degradation (17, 22).We demonstrate here that Vpu is responsible for the incompatibility between HIV-1 and GaLV Env. Glycoproteins containing the cytoplasmic tail from GaLV Env are prevented from being incorporated into HIV-1 particles by Vpu, effectively reducing infectious particle production by 50- to 100-fold. The serines at positions 52 and 56 are required for this restriction, but the membrane-spanning domain is not. Although the mechanism for this restriction appears similar to CD4 degradation, there are apparent differences. Vpu does not prevent surface expression, and it does not prevent its incorporation into MLV particles. Therefore, the mechanism of restriction appears to involve a system that does not rely directly on global protein degradation.     

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

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

Reengineering Escherichia coli for Succinate Production in Mineral Salts Medium

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