Electron and atomic force microscopy of the trimeric ammonium transporter AmtB

Escherichia coli AmtB is an archetypal member of the ammonium transporter (Amt) family, a family of proteins that are conserved in all domains of life. Reconstitution of AmtB in the presence of lipids produced large, ordered two-dimensional crystals. From these, a 12 A resolution projection map was determined by cryoelectron microscopy, and high-resolution topographs were acquired using atomic force microscopy. Both techniques showed the trimeric structure of AmtB in which each monomer seems to have a pseudo-two-fold symmetry. This arrangement is likely to represent the in vivo structure. This work provides the first views of the structure of any member of the Amt family.     

Genetic Evidence for an Essential Oscillation of Transmembrane-Spanning Segment 5 in the Escherichia coli Ammonium Channel AmtB

Ammonium channels, called Amt or Mep, concentrate against a gradient. Each monomer of the trimer has a pore through which substrate passes and a C-terminal cytoplasmic extension. The importance of the C-terminal extension to AmtB activity remains unclear. We have described lesions in conserved C-terminal residues that inactivate AmtB and here characterize 38 intragenic suppressors upstream of the C terminus (∼1/3 of total suppressors). Three that occurred repeatedly, including the previously characterized W148L at the pore entry, restored growth at low NH₃ to nearly wild-type levels and hence restored high activity. V116L completely restored function to two of the mutant proteins and, when separated from other lesions, did not damage wild-type AmtB. A179E notably altered folding of AmtB, compensated for all inactivating C-terminal lesions, and damaged wild-type AmtB. V116L and A179E lie at the cytoplasmic end of transmembrane-spanning segments (TM) 3 and 5, respectively, and the proximal part of the C-terminal tail makes intimate contacts with the loops following them before crossing to the adjacent monomer. Collectively, the properties of intragenic suppressor strains lead us to postulate that the C-terminal tail facilitates an oscillation of TM 5 that is required for coordinated pore function and high AmtB activity. Movement of TM 5 appears to control the opening of both the periplasmic entry and the cytoplasmic exit to the pore.Amt proteins are trimeric inner membrane channels for the hydrated gas (Andrade and Einsle 2007; Fong et al. 2007; Ludewig et al. 2007). They concentrate their substrate against a gradient (Kleiner and Fitzke 1981; Boussiba et al. 1984) and, to our knowledge, are the only active channels described. Each monomer of the Amt trimer contains a pore through which the substrate passes. Although the substrate for Amt channels appears to be , structural studies and molecular dynamics simulations indicate that neutral NH₃ passes through the pore (Khademi et al. 2004; Zheng et al. 2004; Andrade et al. 2005; Lin et al. 2006; Nygaard et al. 2006; Javelle et al. 2007). Hence the pathway for the proton accompanying NH₃ is not clear, but the two appear to separate during their passage through the channel (Mayer et al. 2006). Despite general similarities in charge and size to , K⁺ is neither a substrate for Amt proteins nor an inhibitor of their function (Fong et al. 2007; Javelle et al. 2008). It is for this reason that we refer to as a hydrated gas.The partly stacked phenyl rings of F107 and F215 block entry of NH₃ into the Amt pore, and F215 appears to play a critical role in deprotonation of (Javelle et al. 2008). It has a high structural temperature (B) factor, indicating that it is mobile. The constriction at the periplasmic opening to the pore has been referred to as the “phe gate,” but we prefer the term “phe flap” because GlnK serves as a gate in the classical sense (Andrade et al. 2005; Javelle and Merrick 2005; Durand and Merrick 2006; Conroy et al. 2007; Gruswitz et al. 2007; and see below) and the two should not be confused. We will use the mechanical analogy that the “phe flap” is “open” as a shorthand way of designating that can somehow enter the channel. Although pores were sterically open when F215 (see discussion and Figure 7) was replaced with A, the channel was inactive (Javelle et al. 2008) and hence the mechanical analogy is not sufficient. Above the phe flap is a collar of residues that appears to bind [at the site designated S1 (Khademi et al. 2004)]. This collar, which includes the aromatic residues W148 and F107, has been proposed to play an essential role in recruitment of by π-cation interactions. However, we have shown that W148 restricts entry of into the channel and have proposed instead that the role of the collar is to restrict movement of through the channel (Fong et al. 2007). As the external concentration declines, increased flexibility of the collar/phe flap may allow more rapid entry of into the channel. At this time, it is not clear how much the functions of the collar and the phe flap overlap or can be distinguished.Open in a separate windowFigure 7.—Three-dimensional locations of intragenic suppressors of AmtB^L394A (A) and AmtB^fs (B). Stick models of AmtB were created using PyMOL as in Figure 1. Sections along the pore are perpendicular to the membrane. The periplasmic entry to the pore, which is marked by W148 in lime green, is at the left, and the cytoplasmic exit, which is marked by R47 of GlnK in red stick, is at the right. The threefold axis of the trimer is at the top and the lipid interface is at the bottom. The twin histidine residues at the center of the pore are in green stick, and there is a dot between them. F107 and F215 at the pore entrance are in green and are marked with asterisks. TM 3 is in cyan and TM 5 is in bright pink. TMs 1, 2, and 4 are in light shades of gray, TMs 6–10 are in dark shades of gray, and TM 11 is in brown. Interior loops are in shades of cyan and exterior loops are in shades of light pink. The C terminus is not in the plane of the section, but the C termini of adjacent monomers are in blue and gold. Positions at which suppressor lesions were obtained are space-filled and numbered in order. (A) Numbers correspond to the following: 1, Y32; 2, G113; 3, V116; 4, A118; 5, L119, 6, W148; 7, G175; and 8, A179. F125 is not visible. (B) Numbers correspond to the following: 1, L35; 2, G113; 3, G117; 4, A120; 5, W148; 6, V166; 7, A179; 8, R185; 9, P199; 10, G211; 11, R307; 12, C312; 13, V314; and 14, I359. Suppressors 2, 3, 9, 10, 12, and 13 are in yellow to make the numbers more visible.In addition to the pore, each monomer of the Amt trimer carries a cytoplasmic C-terminal extension of somewhat mysterious function. The cytoplasmic C-terminal extension of the Escherichia coli AmtB protein is ∼25 residues long and can fold precisely against cytoplasmic loops of the same monomer (loops 3, 5, and 1) and the adjacent monomer (loops 7 and 5) (Andrade et al. 2005; Conroy et al. 2007; Gruswitz et al. 2007) (Figure 1). After crossing between monomers, the distal end of the C-terminal extension completes the cytoplasmic vestibule of the adjacent monomer. Although it is known that C-terminal extensions are required for binding of the regulatory protein GlnK, which gates the channel when sufficient internal glutamine is available, it is not known precisely how the extension contributes to AmtB activity. A protein lacking the entire C-terminal tail (AmtB^ΔC-term) fails to bind GlnK but retains intermediate levels of activity, despite the fact that its three pores must be acting independently of one another (Coutts et al. 2002; Severi et al. 2007; Inwood et al. 2009).Open in a separate windowFigure 1.—View of the cytoplasmic face of AmtB and locations of polar connections at the cytoplasmic pore exit. (A) Space-filling model of the cytoplasmic face of E. coli AmtB. The model was created using PyMOL (Delano 2002) from Protein Data Bank entry 2NUU deposited by Conroy et al. (2007) and is similar to that of Neuhäuser et al. (2007). The cytoplasmic C-terminal tails are brown and the three monomers are in different shades of gray. Loop 5 of a single monomer is in pink, loop 1 is in yellow, and the cytoplasmic pore exit is indicated by a large black dot. Note the contacts between the tail and both the proximal and distal ends of loop 5 within a monomer. The tails cross from one monomer to another (counterclockwise) and make an additional contact with the distal end of loop 5 in the adjacent monomer (Conroy et al. 2007; Gruswitz et al. 2007). (B) Polar connections between cytoplasmic loop residues and R185 at the proximal end of loop 5. The stick representation, which is indicated by a box in A, was created using PyMOL. The C-terminal tail is in aqua, with the exception of L394, which is in gold. Residues making polar connections are numbered, and hydrogen bonds are indicated with dotted yellow lines. The cytoplasmic pore exit is indicated by R47 of GlnK, which is in red. Y404, which contacts the distal end of loop 5 in the monomer to which it is covalently attached, is indicated for all three monomers and marks the threefold symmetry axis for the trimer.Surprisingly, mutations that change residues in a kink about half way through the C-terminal tail inactivate AmtB. The kink is the point at which the tail crosses from one monomer to the next, and altering it even conservatively (e.g., the L394A substitution) inactivates AmtB. We have shown that the damaging effects of mutant C-terminal tails are relieved if the chaperone HflB cannot “tack” them (Inwood et al. 2009). Preventing “tacking,” or abnormal folding, can be effected either by extragenic lesions in the ATPase domain of HflB or by intragenic lesions that shorten the AmtB tail so that HflB cannot bind it. Both types of lesions mimic complete deletion of the tail and yield the intermediate level of activity of AmtB^ΔC-term.In this work, we examine intragenic suppressors of inactivating C-terminal lesions in amtB that affect regions upstream of the C terminus. Those that occur most frequently differ from C-terminal truncations and hflB suppressors in restoring activity toward wild type, i.e., to a much higher level than deletion of the C terminus. They indicate that the proximal portion of the C-terminal tail, which can bind precisely to cytoplasmic loops 3, 5, and 1 of the monomer to which it is covalently attached (Figure 1), plays a central role in channel activity. Analysis of upstream intragenic suppressors leads us to postulate that the proximal region of the tail may facilitate a movement of TM 5 (Andrade et al. 2005) that allows opening of the “phe flap” at the periplasmic entry to the pore. For this communication between the bottom and the top of the pore, the distal portion of the tail need not be normal; its sequence can apparently be randomized as long as it can be “tacked” by HflB to the adjacent monomer (Inwood et al. 2009). Inactivating C-terminal lesions in amtB appear to restrict the oscillation of TM 5, which is essential for channel function.     

Ammonium recruitment and ammonia transport by E. coli ammonia channel AmtB

To investigate substrate recruitment and transport across the Escherichia coli Ammonia transporter B (AmtB) protein, we performed molecular dynamics simulations of the AmtB trimer. We have identified residues important in recruitment of ammonium and intraluminal binding sites selective of ammonium, which provide a means of cation selectivity. Our results indicate that A162 guides translocation of an extraluminal ammonium into the pore lumen. We propose a mechanism for transporting the intraluminally recruited proton back to periplasm. Our mechanism conforms to net transport of ammonia and can explain why ammonia conduction is lost upon mutation of the conserved residue D160. We unify previous suggestions of D160 having either a structural or an ammonium binding function. Finally, our simulations show that the channel lumen is hydrated from the cytoplasmic side via the formation of single file water, while the F107/F215 stack at the inner-most part of the periplasmic vestibule constitutes a hydrophobic filter preventing AmtB from conducting water.     

A stable water chain in the hydrophobic pore of the AmtB ammonium transporter

The accessibility of water molecules to the pore of the AmtB ammonium transporter is studied using molecular dynamics simulations. Free energy calculations show that the so-called hydrophobic pore can stabilize a chain of water molecules in a well of a few kcal/mol, using a favorable electrostatic binding pocket as an anchoring point. Moreover, the structure of the water chain matches precisely the electronic density maxima observed in x-ray diffraction experiments. This result questions the general assumption that the AmtB pore only contains ammonia (NH(3)) molecules diffusing in a single file fashion. The probable presence of water molecules in the pore would influence the relative stability of NH(3) and NH(4)(+), and thus calls for a reassessment of the overall permeation mechanism in ammonium transporters.     

Deprotonation by dehydration: the origin of ammonium sensing in the AmtB channel

The AmtB channel passively allows the transport of NH₄⁺ across the membranes of bacteria via a “gas” NH₃ intermediate and is related by homology (sequentially, structurally, and functionally) to many forms of Rh protein (both erythroid and nonerythroid) found in animals and humans. New structural information on this channel has inspired computational studies aimed at clarifying various aspects of NH₄⁺ recruitment and binding in the periplasm, as well as its deprotonation. However, precise mechanisms for these events are still unknown, and, so far, explanations for subsequent NH₃ translocation and reprotonation at the cytoplasmic end of the channel have not been rigorously addressed. We employ molecular dynamics simulations and free energy methods on a full AmtB trimer system in membrane and bathed in electrolyte. Combining the potential of mean force for NH₄⁺/NH₃ translocation with data from thermodynamic integration calculations allows us to find the apparent pK_a of NH₄⁺ as a function of the transport axis. Our calculations reveal the specific sites at which its deprotonation (at the periplasmic end) and reprotonation (at the cytoplasmic end) occurs. Contrary to most hypotheses, which ascribe a proton-accepting role to various periplasmic or luminal residues of the channel, our results suggest that the most plausible proton donor/acceptor at either of these sites is water. Free-energetic analysis not only verifies crystallographically determined binding sites for NH₄⁺ and NH₃ along the transport axis, but also reveals a previously undetermined binding site for NH₄⁺ at the cytoplasmic end of the channel. Analysis of dynamics and the free energies of all possible loading states for NH₃ inside the channel also reveal that hydrophobic pressure and the free-energetic profile provided by the pore lumen drives this species toward the cytoplasm for protonation just before reaching the newly discovered site.     

Computational study of the binding affinity and selectivity of the bacterial ammonium transporter AmtB

We report results from microscopic molecular dynamics and free energy perturbation simulations of substrate binding and selectivity for the Escherichia coli high-affinity ammonium transporter AmtB. The simulation system consists of the protein embedded in a model membrane/water surrounding. The calculated absolute binding free energies for the external NH(4)(+) ions are between -5.8 and -7.3 kcal/mol and are in close agreement with experimental data. The apparent pK(a) of the bound NH(4)(+) increases by more than 4 units, indicating a preference for binding ammonium ion and not neutral ammonia. The external binding site is also selective for NH(4)(+) toward monovalent metal cations by 2.4-4.4 kcal/mol. The externally bound NH(4)(+) shows strong electrostatic interactions with the proximal buried Asp160, stabilized in the anionic form, whereas the interactions with the aromatic rings of Phe107 and Trp148, lining the binding cavity, are less pronounced. Simulated mutation of the highly conserved Asp160 to Asn reduces the pK(a) of the bound ammonium ion by approximately 7 units and causes loss of its binding. The calculations further predict that the substrate affinity of E. coli AmtB depends on the ionization state of external histidines. The computed free energies of hypothetical intermediate states related to transfer of NH(3), NH(4)(+), or H(2)O from the external binding site to the first position inside the internal channel pore favor permeation of the neutral species through the channel interior. However, the predicted change in the apparent pK(a) of NH(4)(+) upon translocation from the external site, Am1, to the first internal site, Am2, indicates that ammonium ion becomes deprotonated only when it enters the channel interior.     

Membrane sequestration of the signal transduction protein GlnK by the ammonium transporter AmtB

The Amt proteins are ammonium transporters that are conserved throughout all domains of life, being found in bacteria, archaea and eukarya. In bacteria and archaea, the Amt structural genes (amtB) are invariably linked to glnK, which encodes a member of the P(II) signal transduction protein family, proteins that regulate enzyme activity and gene expression in response to the intracellular nitrogen status. We have now shown that in Escherichia coli and Azotobacter vinelandii, GlnK binds to the membrane in an AmtB-dependent manner and that GlnK acts as a negative regulator of the transport activity of AmtB. Membrane binding is dependent on the uridylylation state of GlnK and is modulated according to the cellular nitrogen status such that it is maximal in nitrogen-sufficient situations. The membrane sequestration of GlnK by AmtB represents a novel form of signal transduction in which an integral membrane transport protein functions to link the extracellular ammonium concentration to the intracellular responses to nitrogen status. The results also offer new insights into the evolution of P(II) proteins and a rationale for their trigonal symmetry.     

Role of the Escherichia coli glnALG operon in regulation of ammonium transport.

Escherichia coli expresses a specific ammonium (methylammonium) transport system (Amt) when cultured with glutamate or glutamine as the nitrogen source. Over 95% of this Amt activity is repressed by growth of wild-type cells on media containing ammonia. The control of Amt expression was studied with strains containing specific mutations in the glnALG operon. GlnA- (glutamine synthetase deficient) mutants, which contain polar mutations on glnL and glnG genes and therefore have the Reg- phenotype (fail to turn on nitrogen-regulated operons such as histidase), expressed less than 10% of the Amt activity observed for the parental strain. Similarly, low levels of Amt were found in GlnG mutants having the GlnA+ Reg- phenotype. However, GlnA- RegC mutants (a phenotype constitutive for histidase) contained over 70% of the parental Amt activity. At steady-state levels, GlnA- RegC mutants accumulated chemically unaltered [14C]methylammonium against a 60- to 80-fold concentration gradient, whereas the labeled substrate was trapped within parental cells as gamma-glutamylmethylamide. GlnL Reg- mutants (normal glutamine synthetase regulation) had less than 4% of the Amt activity observed for the parental strain. However, the Amt activity of GlnL RegC mutants was slightly higher than that of the parental strain and was not repressed during growth of cells in media containing ammonia. These findings demonstrate that glutamine synthetase is not required for Amt in E. coli. The loss of Amt in certain GlnA- strains is due to polar effects on glnL and glnG genes, whose products are involved in expression of nitrogen-regulated genes, including that for Amt.     

Periodic formation of the oriC complex of Escherichia coli.

We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.     

Epistatic Effects of the Protease/Chaperone HflB on Some Damaged Forms of the Escherichia coli Ammonium Channel AmtB

The Escherichia coli ammonium channel AmtB is a trimer in which each monomer carries a pore for substrate conduction and a cytoplasmic C-terminal extension of ∼25 residues. Deletion of the entire extension leaves the protein with intermediate activity, but some smaller lesions in this region completely inactivate AmtB, as do some lesions in its cytoplasmic loops. We here provide genetic evidence that inactivation depends on the essential protease HflB, which appears to cause inactivation not as a protease but as a chaperone. Selection for restored function of AmtB is a positive selection for loss of the ATPase/chaperone activity of HflB and reveals that the conditional lethal phenotype for hflB is cold sensitivity. Deletion of only a few residues from the C terminus of damaged AmtB proteins seems to prevent HflB from acting on them. Either yields the intermediate activity of a complete C-terminal deletion. HflB apparently “tacks” damaged AmtB tails to the adjacent monomers. Knowing that HflB has intervened is prerequisite to determining the functional basis for AmtB inactivation.Amt proteins concentrate the hydrated gas NH₄⁺ against a gradient and appear to be “active” channels (Andrade and Einsle 2007; Fong et al. 2007; Ludewig et al. 2007). Each monomer of the trimer carries a pore for substrate conduction and a C-terminal extension of variable length. The ordered C terminus of Escherichia coli AmtB is a long α-helix interrupted in the middle by a sharp kink, a fold very similar to that of the C-terminal region of Amt-1 from Archaeoglobus fulgidis (Andrade et al. 2005). The C terminus binds precisely to short cytoplasmic loop regions within the monomer to which it is covalently attached and also the adjacent monomer. It completes the cytoplasmic vestibule of the adjacent monomer to link the two (Conroy et al. 2007; Gruswitz et al. 2007). Deletion of the entire C terminus of E. coli AmtB yields a trimeric form of the protein with partial activity (Coutts et al. 2002; Severi et al. 2007): Uptake of [¹⁴C]methylammonium by a strain carrying AmtB^ΔC-term is between that of wild type and an amtB null strain.In organisms that are sensitive to the ammonium analog methylammonium, selection for resistance yields lesions in Amt proteins (Monahan et al. 2002; J. Hsu, W. B. Inwood and S. Kustu, unpublished results). These include some lesions that change residues in the C-terminal kink. Hence we introduced changes into the kink of the E. coli AmtB protein. All three that we tried—G393A, L394A, and the combination of the two—completely inactivated the protein, indicating that something had occurred beyond loss of function of the C terminus (Coutts et al. 2002; Severi et al. 2007). Likewise, we changed charged residues in the cytoplasmic loops of AmtB to alanine and this, too, inactivated the protein in several cases. By selecting for growth at NH₃ concentrations <50 nm, where unmediated diffusion is limiting, we isolated strains carrying a large number of mutations that suppressed the growth defect caused by these C-terminal and loop lesions. This work characterizes the intragenic suppressor mutations that affected the C terminus of AmtB, which were about a quarter of the total, and all of the extragenic suppressor mutations, which were ∼40%. It provides genetic evidence that the protease/chaperone HflB attempts to fold damaged C termini unsuccessfully and that this results in loss of AmtB activity. In the absence of intervention by HflB, the mutant C termini mimic a C-terminal delete.HflB (also called FtsH, which is unfortunately a misnomer because the hflB lesion was not responsible for the filamentation that was observed) is a membrane-bound protease that is the only essential ATP-dependent protease in E. coli (Ito and Akiyama 2005). It acts as a processive endopeptidase to release peptides of ∼20 residues. To digest inner-membrane proteins, it requires an N- or C-terminal cytoplasmic extension of about this length. HflB is divided into three regions: an N-terminal membrane-bound region containing two transmembrane segments separated by a large periplasmic loop (residues 1–143), an ATPase segment (AAA⁺ class; residues 144–398), and an unusual metalloprotease segment (residues 399–649) (Krzywda et al. 2002; Ito and Akiyama 2005; Bieniossek et al. 2006). Although the conditional lethal phenotype for hflB was long thought to be heat sensitivity, this has been questioned (Ogura et al. 1999). A deletion of hflB is tolerated in the presence of a suppressor mutation in fabZ that increases FabZ activity and restores the balance between phospholipid and lipopolysaccharide synthesis. The deletion is likewise tolerated in lpxA or lpxD backgrounds that decrease lipopolysacharide synthesis. HflB has been considered by some a charonin (Schumann 1999; Ito and Akiyama 2005), but there are few specific reports of its chaperone activity, and it is known to have difficulty unfolding proteins that are thermodynamically stable (Herman et al. 2003).     

Promoter recognition by Escherichia coli RNA polymerase. Role of the spacer DNA in functional complex formation

The available evidence suggests that during the process of formation of a functional or "open" complex at a promoter, Escherichia coli RNA polymerase transiently realigns the two contacted regions of the promoter, thus stressing the intervening spacer DNA. We tested the possibility that this process plays an active role in the formation of an open complex. Two series of promoters were examined: one with spacer DNAs of 15 to 19 base-pairs and a derivative for which the promoters additionally contained a one-base gap in the spacer, so as to relieve any stress imposed on the DNA. Consistent with an active role for the stressed DNA in driving open complex formation, we have found that for promoters with a 17-base-pair spacer, the presence of a gap leads to a delay in the formation of an open complex, at a step subsequent to the initial binding of RNA polymerase to the promoter. The results with the other gapped promoters rule out direct binding of RNA polymerase to the region of the gap and indicate an increased flexibility in the gapped DNA. As not all observations with the spacer length series of gapped and ungapped promoters can be interpreted in terms of an active role of the spacer DNA without additional assumptions, such a role must still be considered tentative.     

In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate

In Escherichia coli, the ammonia channel AmtB and the P(II) signal transduction protein GlnK constitute an ammonium sensory system that effectively couples the intracellular nitrogen regulation system to external changes in ammonium availability. Binding of GlnK to AmtB apparently inactivates the channel, thereby controlling ammonium influx in response to the intracellular nitrogen status. We designed an N-terminally histidine-tagged version of AmtB with a native C-terminal region in order to purify the AmtB-GlnK complex. Purification revealed a stable and direct interaction between AmtB and GlnK, thereby showing for the first time that stability of the complex does not require other proteins. The stoichiometry of the complex was determined by two independent approaches, both of which indicated a 1:1 ratio of AmtB to GlnK. We also showed by mass spectrometry that only the fully deuridylylated form of GlnK co-purifies with AmtB. The purified complex allowed in vitro studies of dissociation and association of AmtB and GlnK. The interaction of GlnK with AmtB is dependent on ATP and is also sensitive to 2-oxoglutarate. Our in vitro data suggest that in vivo association and dissociation of the complex might not only be dependent on the uridylylation status of GlnK but may also be influenced by intracellular pools of ATP and 2-oxoglutarate.     

Interaction between maltose-binding protein and the membrane-associated maltose transporter complex in Escherichia coli

Active transport of maltose in Escherichia coli requires the presence of both maltose-binding protein (MBP) in the periplasm and a complex of MalF, MalG, and MalK proteins (FGK2) located in the cytoplasmic membrane. Earlier, mutants in malF or malG were isolated that are able to grow on maltose in the complete absence of MBP. When the wild-type malE+ allele, coding for MBP, was introduced into these MBP-independent mutants, they frequently lost their ability to grow on maltose. Furthermore, starting from these Mal- strains, Mal+ secondary mutants that contained suppressor mutations in malE were isolated. In this study, we examined the interaction of wild-type and mutant MBPs with wild-type and mutant FGK2 complexes by using right-side-out membrane vesicles. The vesicles from a MBP-independent mutant (malG511) transported maltose in the absence of MBP, with Km and Vmax values similar to those found in intact cells. However, addition of wild-type MBP to these mutant vesicles produced unexpected responses. Although malE+ malG511 cells could not utilize maltose, wild-type MBP at low concentrations stimulated the maltose uptake by malG511 vesicles. At higher concentrations of the wild-type MBP and maltose, however, maltose transport into malG511 vesicles became severely inhibited. This behaviour of the vesicles was also reflected in the phenotype of malE+ malG511 cells, which were found to be capable of transporting maltose from a low external concentration (1 microM), but apparently not from millimolar concentrations present in maltose minimal medium. We found that the mutant FGK2 complex, containing MalG511, had a much higher apparent affinity towards the wild-type MBP than did the wild-type FGK2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.     

The dnaB-dnaC replication protein complex of Escherichia coli. II. Role of the complex in mobilizing dnaB functions

The dnaC protein of Escherichia coli, by forming a complex with the dnaB protein, facilitates the interactions with single-stranded DNA that enable dnaB to perform its ATPase, helicase, and priming functions. Within the dnaB-dnaC complex, dnaB appears to be inactive but becomes active upon the ATP-dependent release of dnaC from the complex. With adenosine 5'-(gamma-thio)triphosphate substituted for ATP, the dnaB-dnaC complex does not direct dnaB to its targeted actions. Excess dnaC inhibits dna beta actions and augments the ATP gamma S effects. In the dnaA protein-driven initiation of duplex chromosome replication, dnaB is introduced for its essential helicase role via the dnaB-dnaC complex. Similarly, when the dnaA protein interacts nonspecifically with single-stranded DNA, the dnaB-dnaC complex is essential to introduce dnaB for its role in primer formation by primase.     

Role of membrane potential and ATP in complex formation between Escherichia coli male cells and filamentous phage fd

Mutant strains of Escherichia coli male cells defective in Ca2+,Mg2+-dependent ATPase (unc) were constructed and tested for their ability to form a complex between sex pili and the filamentous phage fd under conditions where either the membrane potential or the cellular concentration of ATP was lowered. The uncoupler carbonyl cyanide m-chlorophenylhydrazone and the respiratory inhibitor cyanide, as well as valinomycin-K+ and colicin E1, all markedly diminished complex formation, indicating that the maintenance of a membrane potential, but probably not the pH gradient, is essential for the formation of the complex. Since complex formation with freshly centrifuged cells (which initially lacked sex pili) as well as with preincubated cells (in which pre-existing pili were available for complex formation) was inhibited by exposure to the inhibitors, energy seems to be required for both the reappearance (probably assembly) and the maintenance of sex pili on the cell surface. Brief exposure of freshly centrifuged cells to arsenate resulted in only partial inhibition of complex formation. However, marked inhibition of complex formation was observed following exposure to arsenate of preincubated cells possessing sex pili. This indicates that compounds such as ATP may also be required for maintenance of sex pili on the cell surface.     

Role of the chlC gene in formation of the formate-nitrate reductase pathway in Escherichia coli.

Five temperature-sensitive chlC mutants were isolated from Escherichia coli by the technique of localized mutagenesis. All of the mutants produced severely reduced levels of both nitrate reductase and formate dehydrogenase when grown at 43 degrees C. In three of the mutants, the nitrate reductase activity produced at the permissive temperature was shown to be thermolabile compared with the activity produced by the parent wild-type strain, both in membrane preparations and in preparations released from the membrane by deoxycholate. In each case, formate dehydrogenase activity was similar to the wild-type activity in its stability to heat. It is concluded that the chlC gene codes for at least one of the polypeptide chains of nitrate reductase and that the chlC mutations affect indirectly the formation of formate dehydrogenase.