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.     

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.     

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.     

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.     

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.     

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.     

A Putative ABC Transporter,HatABCDE, Is among Molecular Determinants of Pyomelanin Production in Pseudomonas aeruginosa

Pyomelanin overproduction is a common phenotype among Pseudomonas aeruginosa isolates recovered from cystic fibrosis and urinary tract infections. Its prevalence suggests that it contributes to the persistence of the producing microbial community, yet little is known about the mechanisms of its production. Using transposon mutagenesis, we identified factors that contribute to melanogenesis in a clinical isolate of P. aeruginosa. In addition to two enzymes already known to be involved in its biosynthesis (homogentisate dioxygenase and hydroxyphenylpyruvate dioxygenase), we identified 26 genes that encode regulatory, metabolic, transport, and hypothetical proteins that contribute to the production of homogentisic acid (HGA), the monomeric precursor of pyomelanin. One of these, PA14_57880, was independently identified four times and is predicted to encode the ATP-binding cassette of an ABC transporter homologous to proteins in Pseudomonas putida responsible for the extrusion of organic solvents from the cytosol. Quantification of HGA production by P. aeruginosa PA14 strains missing the predicted subcomponents of this transporter confirmed its role in HGA production: mutants unable to produce the ATP-binding cassette (PA14_57880) or the permease (PA14_57870) produced substantially less extracellular HGA after growth for 20 h than the parental strain. In these mutants, concurrent accumulation of intracellular HGA was observed. In addition, quantitative real-time PCR revealed that intracellular accumulation of HGA elicits upregulation of these transport genes. Based on their involvement in homogentisic acid transport, we rename the genes of this operon hatABCDE.Pseudomonas aeruginosa is a metabolically versatile, opportunistic pathogen that is a major cause of life-threatening infections in patients with burn wounds, compromised immunity, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) (23, 41). A major contributor to P. aeruginosa''s pathogenicity is its remarkable genomic plasticity, which often results is a wide range of phenotypic variation among isolates obtained from both acute and chronic infections. These phenotypes include small colony variant formation (24), alginate overproduction (36), hyperpigmentation (22), autoaggregation (13), and autolysis (64). Many of these phenotypes evolve as infections progress, and most have been ascribed to “loss-of-function” genome diversification that promotes long-term survival in the host environment (54). In this regard, recent studies have stimulated interest in another example of a loss-of-function phenotype, the mutation or deletion of hmgA, which encodes the homogentisate 1,2-dioxygenase enzyme. The absence of this protein leads to the accumulation and subsequent export of homogentisic acid (HGA), which ultimately aggregates into the pyomelanin polymer that manifests as a reddish brown pigmentation of P. aeruginosa colonies and their surrounding milieu (Fig. ​(Fig.1A)1A) (5, 49).Open in a separate windowFIG. 1.Pyomelanin production by the PA14 ΔhmgA and DKN343 strains. (A) Homogentisate pathway for the catabolism of chorismate and aromatic amino acids. Enzyme names are shown above the arrows for each step. A mutation or deletion of the hmgA gene (encoding homogentisate 1,2-dioxygenase) leads to the accumulation of pyomelanin. (B) Pyomelanin overproduction by the PA14 ΔhmgA mutant is abolished when complemented with an intact hmgA gene. Complementation of a melanogenic clinical P. aeruginosa isolate, DKN343, with hmgA results in no phenotypic change, indicating that other factors contribute to its pigmentation.Production of pyomelanin (and other forms of melanin) has been described to occur in a wide range of bacterial species, including Aeromonas (4), Azotobacter (51), Azospirillum (50), Bacillus (3), Legionella (8), Marinomonas (33), Micrococcus (40), Mycobacterium (45), Proteus (1), Rhizobium (12), Shewanella (61), Sinorhizobium (38), Streptomyces (67), and Vibrio (63) species. Notably, isolates of other bacterial species associated with chronic infections of the CF lung, Burkholderia cenocepacia and Stenotrophomonas maltophilia, can also be melanogenic (28, 58), suggesting a possible role for this pigment in the establishment and/or persistence of infection. Some genera produce melanin under normal conditions via polyphenol oxidases or laccases, while others synthesize the pigment only in response to specific environmental conditions (17, 35). Many species, however, including P. aeruginosa, overproduce pyomelanin as a result of a point mutation in hmgA or large chromosomal deletions of loci containing the homogentisate operon (2, 19). While these genetic variations have been frequently reported, there is little understanding of the competitive advantage, if any, that this pigment confers to the producing bacterium.Proposed roles for pyomelanin include the enhancement of bacterial surface attachment (20), extracellular electron transfer (61), iron reduction/acquisition (8), induction of virulence factor expression (63), heavy metal binding (21), and protection from environmental stress (11, 28, 32, 44, 53, 65). A protective role has also been proposed to occur in P. aeruginosa PA14, where pyomelanin was shown to contribute to the persistence of an overproducing strain in a chronic CF infection model in mice (49). However, given that melanogenic isolates have been recovered from laboratory-grown communities of P. aeruginosa PAO1 (5, 56), it is probable that pyomelanin plays other roles in addition to protection against host defense mechanisms.As a first step toward gaining a better understanding of pyomelanin function, we sought to identify the molecular determinants of its production in P. aeruginosa. By screening a library of pTnTet/minimariner transposon mutants of a pyomelanin-overproducing clinical isolate for alterations in pigmentation, we identified several genes whose products are involved in tyrosine catabolism, central metabolic pathways, nucleotide biosynthesis, regulation, and membrane transport, in addition to hypothetical proteins of unknown function. We chose to further characterize the gene identified most frequently in our screen, one annotated as encoding a putative ATP-binding cassette of an ABC-type transporter. Here, we demonstrate that this transporter is involved in HGA transport and the subsequent extracellular formation of pyomelanin.