A Rhodobacter capsulatus Member of a Universal Permease Family Imports Molybdate and Other Oxyanions

Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.Molybdenum (Mo) is utilized by many bacteria, archaea, and eukaryotes as a cofactor of redox enzymes catalyzing key reactions in the nitrogen, sulfur, and carbon cycles (62). Nitrogenase, which catalyzes the reduction of dinitrogen to ammonia, carries the unique iron-molybdenum cofactor FeMoco. In contrast to nitrogenase, all other molybdoenzymes harbor the molybdenum cofactor Moco, which transfers either an oxo group or two electrons to or from the substrate in a wide variety of transformations at nitrogen, sulfur, and carbon atoms (47).The phototrophic alphaproteobacterium Rhodobacter capsulatus serves as a model organism to study Mo metabolism because it synthesizes several molybdoenzymes, including dimethyl sulfoxide reductase, xanthine dehydrogenase, and nitrogenase (29, 30, 46). In addition to Mo-dependent nitrogenase (Mo-nitrogenase), R. capsulatus uses an alternative, Mo-free nitrogenase when Mo is limiting (55, 57). Two related Mo-responsive regulators, MopA and MopB, control expression of the alternative nitrogenase and molybdate uptake genes (22, 57, 58).Mo is available for living cells in its oxyanion form, molybdate. The vast majority of Mo-utilizing bacteria is known or predicted to possess ModABC-type high-affinity molybdate uptake systems (62, 63). These importers belong to the family of ATP-binding cassette (ABC) transporters, which couple ATP hydrolysis to substrate translocation across biological membranes (13, 15). ModABC transporters typically consist of a periplasmic molybdate-binding protein (ModA), a membrane-spanning channel protein (ModB), and a cytoplasmic ATP-binding protein (ModC), which specifically interacts with ModB and, upon ATP hydrolysis, energizes the uptake system.ModABC transporters enable bacteria to actively take up molybdate against a concentration gradient and synthesize active molybdoenzymes at nanomolar Mo concentrations in the environment (37, 49). Accordingly, modABC mutants are not able to make use of molybdoenzymes under Mo-limiting conditions, as shown for several bacteria, including Escherichia coli, Anabaena variabilis, Azotobacter vinelandii, and R. capsulatus (16, 33, 55, 61). High Mo concentrations, however, support synthesis of active molybdoenzymes in modABC mutants, indicating the presence of low-affinity molybdate uptake systems in these bacteria. Low-affinity molybdate uptake in E. coli and several other bacteria is mediated (at least in part) by the sulfate-repressed high-affinity sulfate transporter CysTWA (37, 43, 61).In the present study, we describe the identification and characterization of a permease mediating molybdate uptake at micromolar concentrations in R. capsulatus. The permease belongs to the widely distributed family of ArsB/NhaD permeases (27). Several members of this family have been shown to transport various anorganic and organic anions across biological membranes, but molybdate uptake is a previously unrecognized novel function of these permeases. In addition to molybdate, other oxyanions, like sulfate, tungstate, and vanadate, are likely to be imported by the R. capsulatus permease.     

Rhodobacter capsulatus Catalyzes Light-Dependent Fe(II) Oxidation under Anaerobic Conditions as a Potential Detoxification Mechanism

Diverse bacteria are known to oxidize millimolar concentrations of ferrous iron [Fe(II)] under anaerobic conditions, both phototrophically and chemotrophically. Yet whether they can do this under conditions that are relevant to natural systems is understood less well. In this study, we tested how light, Fe(II) speciation, pH, and salinity affected the rate of Fe(II) oxidation by Rhodobacter capsulatus SB1003. Although R. capsulatus cannot grow photoautotrophically on Fe(II), it oxidizes Fe(II) at rates comparable to those of bacteria that do grow photoautotrophically on Fe(II) as soon as it is exposed to light, provided it has a functional photosystem. Chelation of Fe(II) by diverse organic ligands promotes Fe(II) oxidation, and as the pH increases, so does the oxidation rate, except in the presence of nitrilotriacetate; nonchelated forms of Fe(II) are also more rapidly oxidized at higher pH. Salt concentrations typical of marine environments inhibit Fe(II) oxidation. When growing photoheterotrophically on humic substances, R. capsulatus is highly sensitive to low concentrations of Fe(II); it is inhibited in the presence of concentrations as low as 5 μM. The product of Fe(II) oxidation, ferric iron, does not hamper growth under these conditions. When other parameters, such as pH or the presence of chelators, are adjusted to promote Fe(II) oxidation, the growth inhibition effect of Fe(II) is alleviated. Together, these results suggest that Fe(II) is toxic to R. capsulatus growing under strictly anaerobic conditions and that Fe(II) oxidation alleviates this toxicity.Iron is one of the most (photo)redox-active metals involved in biochemical functions, and it can affect the cycling of many other key elements (e.g., C, S, N, and P), trace metals (33), metalloids, and organic compounds (6). It is well appreciated that microorganisms contribute greatly to iron cycling in nature through a diversity of processes, including both oxidation and reduction reactions (16). In the past decade, much attention has been paid to how such reactions can be used to support cellular growth (1, 7, 15, 17, 19, 37, 44-46) and/or iron acquisition (2, 42) under both aerobic and anaerobic conditions, and for some organisms, these processes are understood at the molecular level (10).Our lab has been particularly interested in one branch of the microbial Fe cycle: phototrophic Fe(II) oxidation under anaerobic conditions (9, 11, 12, 23-25). While most of the organisms we and others have studied can grow by coupling Fe(II) oxidation to CO₂ fixation (15, 23, 46), not all strains that oxidize Fe(II) can use it as an electron donor to support growth. An example of this is Rhodobacter capsulatus, which can benefit from Fe(II) oxidation only via an indirect pathway: it grows photoheterotrophically on low-molecular-weight organic compounds that form due to a photochemical reaction between biogenic Fe(III) and organic compounds that it cannot otherwise use (citrate and nitrilotriacetate [NTA]) (4). This observation led us to hypothesize that microbial Fe(II) oxidation might be more broadly useful to microorganisms by making refractory organic compounds, such as humic substances, more bioavailable through photochemical degradation (4).In this work, we set out to test this hypothesis using R. capsulatus. In addition, we sought to increase our understanding of Fe(II) oxidation by this organism by studying the effect of Fe(II) speciation and important environmental variables (e.g., light, pH, and [Cl⁻]) on the rate of Fe(II) oxidation. Along the way, we serendipitously discovered that low levels of Fe(II) are toxic to R. capsulatus when it is growing on humic substances under anaerobic conditions and that Fe(II) oxidation appears to alleviate this toxicity.     

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

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

Interaction of the Colicin K Bactericidal Toxin with Components of Its Import Machinery in the Periplasm of Escherichia coli

Colicins are bacterial antibiotic toxins produced by Escherichia coli cells and are active against E. coli and closely related strains. To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm. Here, we initiated studies to understand how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the periplasm. We first produced KT fused to a signal sequence for periplasm targeting. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent, released periplasmic proteins, and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine. Using a combination of in vivo coimmunoprecipitations and in vitro pulldown experiments, we demonstrated that KT interacts with the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between the TolQ protein and a colicin translocation domain.Colicins are bacterial toxins produced by Escherichia coli strains and are active against E. coli or related strains (17). These bacterial antibiotic toxins play an important role in the E. coli colonization of environmental niches, including the mammal gastrointestinal tract (25, 32, 49, 50). The classification of colicins is based on differences in the mechanisms of action, such as pore formation (colicins A, B, E1, K, Ia, N, 5, etc.), degradation of nucleic acids (including DNases [colicins E2, E7, and E9], 16S RNases [colicins E3, E4, and E6], or tRNases [colicins D and E5]), or degradation of lipid II (colicin M) (17, 34). Colicins are also categorized depending on their import machines: colicins using the Tol proteins are classified as group A (colicins A, E1 to E9, K, N, etc.), whereas colicins using the ExbBD-TonB proteins are classified as group B (colicins B, D, Ia, M, 5, etc.). However, the transport across the periplasm is only one of the three steps of the mechanism of action. Colicins bind to an outer membrane receptor and are translocated through the outer membrane and the periplasm (14, 35, 55, 56). Finally, the C-terminal domain (responsible for the activity) is translocated to its final destination (inner membrane or cytoplasm) depending on its mechanism of action. Colicins are divided into three different structural and functional domains that correspond to the three steps of the mechanism of action: the N-terminal domain is required for translocation, the central domain is involved in receptor binding, and the C-terminal domain carries the activity (4, 5). During the translocation step, the N-terminal domain of the colicin interacts with components of the import machine: colicins A, E1, and N interact with the TolA protein; colicins A, E3, E7, and E9 interact with the TolB protein; and colicins A and E3 interact with TolR (6, 12, 13, 15, 21, 23, 26, 27, 30, 39, 48, 54). In some cases, the domains of the Tol proteins involved in colicin binding have been identified. Reciprocally, the regions of colicins in interaction with the Tol proteins have been delineated. In colicin A, the TolA binding sequence (ABS) is contained within residues 37 to 98 (13, 30), in which a SYNT motif (residues 57 to 60) has been shown to be essential for TolA binding (18, 46). The TolB box and the TolR binding sequences have also been identified in colicin A (27, 30). The TolB box is well conserved within TolB-dependent colicins, including colicins A and E2 to E9, and is composed of residues DG[T,S]GWSSE (12, 13). These residues form a loop penetrating within the TolB beta-propeller (39, 57), mimicking the TolB-Pal interaction (9, 10). Interestingly, the Tol-dependent, pore-forming colicin K does not possess a TolB box (see Fig. ​Fig.1A),1A), raising the hypothesis that its translocation might be TolB independent or that colicin K interacts with TolB differently than do other TolB-dependent colicins. In this study, we tested the Tol requirements for colicin K translocation and showed that colicin K requires the TolA, TolB, TolQ, and TolR proteins. Production of the N-terminal domain of colicin K in the periplasm of wild-type (WT) cells induces specific tol defects and tolerance to Tol-dependent colicins and bacteriophage, suggesting that the colicin K N-terminal domain binds and titrates the Tol proteins. Further in vivo coimmunoprecipitation and in vitro pulldown experiments demonstrated interactions between the colicin K N-terminal domain and the TolA, TolB, and TolR proteins. For the first time, we also identified an interaction between a colicin translocation domain and the fourth component of the Tol complex, the TolQ protein.Open in a separate windowFIG. 1.In the absence of an identifiable TolB-binding sequence, colicin K translocation is TolB dependent. (A) Sequence alignment of colicin K and three TolB-dependent colicins (A, E2, and E9). Conserved residues are indicated by red letters. The characterized TolB binding sequence is indicated by the green box (defined in references 12 and 27). (B) Colicin spot assays using serial dilutions of colicins A (TolB dependent), E1 (TolB independent), and K on a wild-type (WT) strain and its tolB derivative (from left to right, 100, 10, 1, and 0.1 ng of colicins have been spotted, respectively).     

Interaction of an Essential Escherichia coli GTPase,Der, with the 50S Ribosome via the KH-Like Domain

Der, an essential Escherichia coli tandem GTPase, has been implicated in 50S subunit biogenesis. The rrmJ gene encodes a methyltransferase that modifies the U2552 residue of 23S rRNA, and its deletion causes a severe growth defect. Peculiarly, overexpression of Der suppresses growth impairment. In this study, using an rrmJ-deletion strain, we demonstrated that two GTPase domains of Der regulate its association with 50S subunit via the KH-like domain. We also identified a region of Der that is critical for its specific interaction with 50S subunit.Emerging evidence indicates that many Escherichia coli GTPases play critical roles in ribosome biogenesis (6). For example, E. coli Era consists of a conventional GTP-binding domain and a KH domain (an RNA-binding domain) with a consensus VIGXXGXXI sequence (9). The direct interaction between Era and 16S rRNA was demonstrated by structural studies of a Thermus thermophilus 30S ribosomal subunit complexed with Era (24). Peculiarly, Era was shown to suppress the cold-sensitive cell growth of the rbfA-deletion strain whose gene product resembles a KH domain and plays an important role in 30S subunit assembly at low temperature (13, 16). A unique GTPase subfamily of Der (double Era-like GTPase; also known as EngA) is conserved only in eubacteria. We have previously demonstrated that Der is cofractionated with 50S subunits in a GTP-dependent manner and that Der plays a critical role in 50S subunit maturation at a later biogenesis step (15).Interestingly, both GTP-binding domains (G domains) were essential for cell growth; moreover, the two G domains function cooperatively, suggesting that GTP-induced conformational changes and GTPase activity are essential for cell viability as well as function (1, 15). The X-ray crystal structures of two Der orthologs from Thermotoga maritima and Bacillus subtilis revealed that the C-terminal domain has a topology similar to that of a KH domain without a consensus sequence motif and is flanked by two G domains (22, 23). It was suggested that the GTP-bound form of YphC (a Der ortholog in B. subtilis) triggers a dramatic conformational change, which favors an interaction with negatively charged ribonucleic acids by exposing a positively charged KH-like domain with a high pI value (14, 22).Overexpression of E. coli Der functionally suppressed the slow growth defect of a deletion strain of the rrmJ gene, whose gene product is a methyltransferase, modifying the U2552 residue in the A loop of 23S rRNA in an intact 50S subunit (5, 26). Even though ΔrrmJ (strain HB23) is viable, it causes a serious defect of cell growth by accumulating 50S and 30S ribosomal subunits at the expense of 70S ribosomes. Thus, overexpression of Der seems to overcome its weak interaction with 50S subunits that are unmethylated at U2552. In this study, using an ΔrrmJ strain as a genetic background, we tried to elucidate the nature of the functional regulation of two G domains and the KH-like domain. We further characterized the KH-like domain by random mutagenesis and identified crucial residues for its association with 50S subunits. Our data suggest that the unique C-terminal domain indeed plays a role in rRNA-ribosome recognition.     

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.     

Asp1, a Conserved 1/3 Inositol Polyphosphate Kinase,Regulates the Dimorphic Switch in Schizosaccharomyces pombe

The ability to undergo dramatic morphological changes in response to extrinsic cues is conserved in fungi. We have used the model yeast Schizosaccharomyces pombe to determine which intracellular signal regulates the dimorphic switch from the single-cell yeast form to the filamentous invasive growth form. The S. pombe Asp1 protein, a member of the conserved Vip1 1/3 inositol polyphosphate kinase family, is a key regulator of the morphological switch via the cAMP protein kinase A (PKA) pathway. Lack of a functional Asp1 kinase domain abolishes invasive growth which is monopolar, while an increase in Asp1-generated inositol pyrophosphates (PP) increases the cellular response. Remarkably, the Asp1 kinase activity encoded by the N-terminal part of the protein is regulated negatively by the C-terminal domain of Asp1, which has homology to acid histidine phosphatases. Thus, the fine tuning of the cellular response to environmental cues is modulated by the same protein. As the Saccharomyces cerevisiae Asp1 ortholog is also required for the dimorphic switch in this yeast, we propose that Vip1 family members have a general role in regulating fungal dimorphism.Eucaryotic cells are able to define and maintain a particular cellular organization and thus cellular morphology by executing programs modulated by internal and external signals. For example, signals generated within a cell are required for the selection of the growth zone after cytokinesis in the fission yeast Schizosaccharomyces pombe or the emergence of the bud in Saccharomyces cerevisiae (37, 44, 81). Cellular morphogenesis is also subject to regulation by a wide variety of external signals, such as growth factors, temperature, hormones, nutrient limitation, and cell-cell or cell-substrate contact (13, 34, 66, 75, 81). Both types of signals will lead to the selection of growth zones accompanied by the reorganization of the cytoskeleton.The ability to alter the growth form in response to environmental conditions is an important virulence-associated trait of pathogenic fungi which helps the pathogen to spread in and survive the host''s defense system (7, 32). Alteration of the growth form in response to extrinsic signals is not limited to pathogenic fungi but is also found in the model yeasts S. cerevisiae and S. pombe, in which it appears to represent a foraging response (1, 24).The regulation of polarized growth and the definition of growth zones have been studied extensively with the fission yeast S. pombe. In this cylindrically shaped organism, cell wall biosynthesis is restricted to one or both cell ends in a cell cycle-regulated manner and to the septum during cytokinesis (38). This mode of growth requires the actin cytoskeleton to direct growth and the microtubule cytoskeleton to define the growth sites (60). In interphase cells, microtubules are organized in antiparallel bundles that are aligned along the long axis of the cell and grow from their plus ends toward the cell tips. Upon contact with the cell end, microtubule growth will first pause and then undergo a catastrophic event and microtubule shrinkage (21). This dynamic behavior of the microtubule plus end is regulated by a disparate, conserved, microtubule plus end group of proteins, called the +TIPs. The +TIP complex containing the EB1 family member Mal3 is required for the delivery of the Tea1-Tea4 complex to the cell tip (6, 11, 27, 45, 77). The latter complex docks at the cell end and recruits proteins required for actin nucleation (46, 76). Thus, the intricate cross talk between the actin and the microtubule cytoskeleton at specific intracellular locations is necessary for cell cycle-dependent polarized growth of the fission yeast cell.The intense analysis of polarized growth control in single-celled S. pombe makes this yeast an attractive organism for the identification of key regulatory components of the dimorphic switch. S. pombe multicellular invasive growth has been observed for specific strains under specific conditions, such as nitrogen and ammonium limitation and the presence of excess iron (1, 19, 50, 61).Here, we have identified an evolutionarily conserved key regulator of the S. pombe dimorphic switch, the Asp1 protein. Asp1 belongs to the highly conserved family of Vip1 1/3 inositol polyphosphate kinases, which is one of two families that can generate inositol pyrophosphates (PP) (17, 23, 42, 54). The inositol polyphosphate kinase IP6K family, of which the S. cerevisiae Kcs1 protein is a member, is the “classical” family that can phosphorylate inositol hexakisphosphate (IP₆) (70, 71). These enzymes generate a specific PP-IP₅ (IP₇), which has the pyrophosphate at position 5 of the inositol ring (20, 54). The Vip1 family kinase activity was unmasked in an S. cerevisiae strain with KCS1 and DDP1 deleted (54, 83). The latter gene encodes a nudix hydrolase (14, 68). The mammalian and S. cerevisiae Vip1 proteins phosphorylate the 1/3 position of the inositol ring, generating 1/3 diphosphoinositol pentakisphosphate (42). Both enzyme families collaborate to generate IP₈ (17, 23, 42, 54, 57).Two modes of action have been described for the high-energy moiety containing inositol pyrophosphates. First, these molecules can phosphorylate proteins by a nonenzymatic transfer of a phosphate group to specific prephosphorylated serine residues (2, 8, 69). Second, inositol pyrophosphates can regulate protein function by reversible binding to the S. cerevisiae Pho80-Pho85-Pho81 complex (39, 40). This cyclin-cyclin-dependent kinase complex is inactivated by inositol pyrophosphates generated by Vip1 when cells are starved of inorganic phosphate (39, 41, 42).Regulation of phosphate metabolism in S. cerevisiae is one of the few roles specifically attributed to a Vip1 kinase. Further information about the cellular function of this family came from the identification of the S. pombe Vip1 family member Asp1 as a regulator of the actin nucleator Arp2/3 complex (22). The 106-kDa Asp1 cytoplasmic protein, which probably exists as a dimer in vivo, acts as a multicopy suppressor of arp3-c1 mutants (22). Loss of Asp1 results in abnormal cell morphology, defects in polarized growth, and aberrant cortical actin cytoskeleton organization (22).The Vip1 family proteins have a dual domain structure which consists of an N-terminal “rimK”/ATP-grasp superfamily domain found in certain inositol signaling kinases and a C-terminal part with homology to histidine acid phosphatases present in phytase enzymes (28, 53, 54). The N-terminal domain is required and sufficient for Vip1 family kinase activity, and an Asp1 variant with a mutation in a catalytic residue of the kinase domain is unable to suppress mutants of the Arp2/3 complex (17, 23, 54). To date, no function has been described for the C-terminal phosphatase domain, and this domain appears to be catalytically inactive (17, 23, 54).Here we describe a new and conserved role for Vip1 kinases in regulating the dimorphic switch in yeasts. Asp1 kinase activity is essential for cell-cell and cell-substrate adhesion and the ability of S. pombe cells to grow invasively. Interestingly, Asp1 kinase activity is counteracted by the putative phosphatase domain of this protein, a finding that allows us to describe for the first time a function for the C-terminal part of Vip1 proteins.     

Regulation of the Display Ratio of Enzymes on the Saccharomyces cerevisiae Cell Surface by the Immunoglobulin G and Cellulosomal Enzyme Binding Domains

We constructed a novel cell surface display system to control the ratio of target proteins on the Saccharomyces cerevisiae cell surface, using two pairs of protein-protein interactions. One protein pair is the Z domain of protein A derived from Staphylococcus aureus and the Fc domain of human immunoglobulin G. The other is the cohesin (Coh) and dockerin (Dock) from the cellulosome of Clostridium cellulovorans. In this proposed displaying system, the scaffolding proteins (fusion proteins of Z and Coh) were displayed on the cell surface by fusing with the 3′ half of α-agglutinin, and the target proteins fused with Fc or Dock were secreted. As a target protein, a recombinant Trichoderma reesei endoglucanase II (EGII) was secreted into the medium and immediately displayed on the yeast cell surface via the Z and Fc domains. Display of EGII on the cell surface was confirmed by hydrolysis of β-glucan as a substrate, and EGII activity was detected in the cell pellet fraction. Finally, two enzymes, EGII and Aspergillus aculeatus β-glucosidase 1, were codisplayed on the cell surface via Z-Fc and Dock-Coh interactions, respectively. As a result, the yeast displaying two enzymes hydrolyzed β-glucan to glucose very well. These results strongly indicated that the proposed strategy, the simultaneous display of two enzymes on the yeast cell surface, was accomplished by quantitatively controlling the display system using affinity binding.Microorganisms have been used to enable cell surface display of heterogonous peptides or proteins for various applications (13, 19, 20). For practical purposes, the use of the cell surface display system of the yeast Saccharomyces cerevisiae, which has “generally regarded as safe” status, is suitable and can be employed in many processes, including food and pharmaceutical production. Yeast strains displaying functional peptides or proteins are expected to be used, for instance, as live vaccines (10), matrices for screening of novel proteins from combinatorial libraries (3, 12, 27), whole-cell bioadsorbents (14, 18), reporter substances (23), and whole-cell biocatalysts (8, 9, 11, 16). In yeast-based cell surface display systems, the C-terminal half of α-agglutinin, containing the putative glycosylphosphatidylinositol anchor attachment signal sequence (15), has been used as an anchor protein to successfully display many kinds of proteins on the cell surface. In addition, other surface proteins, such as α-agglutinin (AGA1 and AGA2), Flo1, Sed1, Cwp1, Cwp2, Tip1, and Tir1, were also used for enzyme display (24). To immobilize a target protein at the N terminus, another surface display system has been developed using the flocculation functional domain of Flo1p (16), which is a lectin-like cell wall protein that plays a major role in flocculation (25). These displaying systems could successfully display three kinds of enzymes on the surfaces of yeast cells (8, 9). However, in current yeast-based cell surface display systems, the ratio of the proteins to be displayed on the cell surface cannot be controlled, since the produced target protein attached the protein or cell wall component on the cell surface by only molecular interaction.A large extracellular polysaccharolytic multicomponent complex called the cellulosome provides a model for the concept of a cell surface displaying system. Cellulosomes from anaerobic and cellulolytic bacteria such as Clostridium, Acetivibrio, Bacteroides, and Ruminococcus have been studied in detail (1). The cellulosomes of the thermophilic organism Clostridium thermocellum and of the mesophilic organisms Clostridium cellulovorans and Clostridium cellulolyticum have been extensively discussed (1, 6). The structure of cellulosomes consists of a nonenzymatic scaffolding protein called CbpA (1, 6), CipA (1, 4), or CipC (1) complexed with a number of cellulosomal enzymes. The scaffolding proteins usually contain a number of cohesin (Coh) domains and cellulose binding domains. On the other hand, cellulosomal enzymes contain a Coh binding site called dockerin (Dock), and the Coh-Dock interaction is an important factor in cellulosome assembly. Some cellulosomes are able to anchor to the cell surface through interaction of other scaffolding proteins containing surface layer homology domains. The cellulosome models that include a cell surface anchoring system are similar to our concept of a cell surface display system.Based on this concept, we propose a novel method to control the displaying ratio of enzymes for attaining a complicated reaction such as cellulose degradation. Recently, the display of one kind of protein via Z-Fc interaction on the surfaces of yeast cells was successfully achieved (18). According to this proposed method, we used the following two pairs of proteins for assembling specifically with each other: (i) Z domain and the Fc part of immunoglobulin G (IgG) and (ii) Coh and Dock from cellulosomes. The Z domain, which was derived from the B domain of protein A from Staphylococcus aureus, interacts with IgG from various species (21). Coh is a domain of the scaffolding protein from the cellulase complex (cellulosome) from C. cellulovorans (5). Dock is a domain of the cellulosomal enzymes which binds to Coh of the scaffolding protein on the surface of C. cellulovorans. The secreted enzymes fused with Fc or Dock could be displayed on the cell surface through a ZZ-Coh-Coh scaffolding protein (Fig. ​(Fig.1B1B).Open in a separate windowFIG. 1.Plasmids and proposed protein-protein interaction cell surface display systems with two pairs of proteins. (A) Maps of plasmids used in this study. (B) Sequential degradation of β-glucan by displayed enzymes. The protein pairs used in this study were (i) the Z domain from Staphylococcus aureus protein A and Fc domain from IgG and (ii) the Coh and Dock domains from the C. cellulovorans cellulosome.To carry out the proposed strategy, we constructed the cell surface displaying system with Trichoderma reesei endoglucanase II (EGII) and Aspergillus aculeatus β-glucosidase 1 (BGL1). Furthermore, we demonstrated synergistic saccharification of soluble cellulose by integrating two kinds of cellulase enzymes on the cell surface by using the ZZ-Coh-Coh scaffolding protein as a model for a complex reaction.