Functional role of the MrpA- and MrpD-homologous protein subunits in enzyme complexes evolutionary related to respiratory chain complex I

NADH:quinone oxidoreductase or complex I is a large membrane bound enzyme complex that has evolved from the combination of smaller functional building blocks. Intermediate size enzyme complexes exist in nature that comprise some, but not all of the protein subunits in full size 14-subunit complex I. The membrane spanning complex I subunits NuoL, NuoM and NuoN are homologous to each other and to two proteins from one particular class of Na⁺/H⁺ antiporters, denoted MrpA and MrpD. In complex I, these ion transporter protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Using a model system, consisting of Bacillus subtilis MrpA and MrpD deletion strains and a low copy expression plasmid, it was recently demonstrated that NuoN can rescue the strain deleted for MrpD but not that deleted for MrpA, whereas the opposite tendency was seen for NuoL. This demonstrated that the MrpA-type and MrpD-type proteins have unique functional specializations. In this work, the corresponding antiporter-like protein subunits from the smaller enzymes evolutionarily related to complex I were tested in the same model system. The subunits from 11-subunit complex I from Bacillus cereus behaved essentially as those from full size complex I, corroborating that this enzyme should be regarded as a bona fide complex I. The hydrogenase-3 and hydrogenase-4 antiporter-like proteins on the other hand, could substitute equally well for MrpA or MrpD at pH 7.4, suggesting that these enzymes have intermediate forms of the antiporter-like proteins, which seemingly lack the functional specificity.     

Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex – The evolutionary progenitor of the long horizontal helix in complex I

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required.     

A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli��Expression and characterization of cytochrome-tagged Complex I subunits

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane‐spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C‐terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c₅₅₀. Compared with other available fusion‐protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter‐like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo‐cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c₅₅₀ domain in all the fusion proteins exhibited normal spectra and redox properties, with an E_m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c‐tag. Finally, a his‐tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.     

Phylogenomic analysis of type 1 NADH:Quinone oxidoreductase

We performed phylogenomic analysis of the catalytic core of NADH:quinone oxidoreductases of type 1 (NDH-1). Analysis of phylogenetic trees, as constructed for the core subunits of NDH-1, revealed fundamental differences in their topologies. In the case of four putatively homologous ion-carrying membrane subunits, the trees for the NuoH and NuoN subunits contained separate archaeal clades, whereas subunits NuoL and NuoM were characterized by multiple archaeal clades spread among bacterial branches. Large, separate clades, which united sequences belonging to different archaeal subdomains, were also found for cytoplasmic subunits NuoD and NuoB, homologous to the large and small subunits of nickel-iron hydrogenases. A smaller such clade was also shown for subunit NuoC. Based on these data, we suggest that the ancestral NDH-1 complex could be present already at the stage of the Last Universal Cellular Ancestor (LUCA). Ancestral forms of membrane subunits NuoN and NuoH and cytoplasmic subunits NuoD, NuoB, and, perhaps NuoC, may have formed a membrane complex that operated as an ion-translocating membrane hydrogenase. After the complex attained the ability to reduce membrane quinones, gene duplications could yield the subunits NuoL and NuoM, which enabled translocation of additional ions.     

Structural contribution of C-terminal segments of NuoL (ND5) and NuoM (ND4) subunits of complex I from Escherichia coli

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is a multisubunit enzymatic complex. It has a characteristic L-shaped form with two domains, a hydrophilic peripheral domain and a hydrophobic membrane domain. The membrane domain contains three antiporter-like subunits (NuoL, NuoM, and NuoN, Escherichia coli naming) that are considered to be involved in the proton translocation. Deletion of either NuoL or NuoM resulted in an incomplete assembly of NDH-1 and a total loss of the NADH-quinone oxidoreductase activity. We have truncated the C terminus segments of NuoM and NuoL by introducing STOP codons at different locations using site-directed mutagenesis of chromosomal DNA. Our results suggest an important structural role for the C-terminal segments of both subunits. The data further advocate that the elimination of the last transmembrane helix (TM14) of NuoM and the TM16 (at least C-terminal seven residues) or together with the HL helix and the TM15 of the NuoL subunit lead to reduced stability of the membrane arm and therefore of the whole NDH-1 complex. A region of NuoL critical for stability of NDH-1 architecture has been discussed.     

Transmembrane topology of the NuoL,M and N subunits of NADH:quinone oxidoreductase and their homologues among membrane-bound hydrogenases and bona fide antiporters

Nicotinamide adenine dinucleotide-reduced form (NADH):quinone oxidoreductase (respiratory Complex I), F420H2 oxidoreductase and complex, membrane-bound NiFe-hydrogenase contain protein subunits homologous to a certain type of bona fide antiporters. In Complex I, these polypeptides (NuoL/ND5, NuoM/ND4, NuoN/ND2) are most likely core components of the proton pumping mechanism, and it is thus important to learn more about their structure and function. In this work, we have determined the transmembrane topology of one such polypeptide, and built a 2D structural model of the protein valid for all the homologous polypeptides. The experimentally determined transmembrane topology was different from that predicted by majority vote hydrophobicity analyses of members of the superfamily. A detailed phylogenetic analysis of a large set of primary sequences shed light on the functional relatedness of these polypeptides.     

The location of NuoL and NuoM subunits in the membrane domain of the Escherichia coli complex I: implications for the mechanism of proton pumping

The molecular organization of bacterial NADH: ubiquinone oxidoreductase (complex I or NDH-1) is not established, apart from a rough separation into dehydrogenase, connecting and membrane domains. In this work, complex I was purified from Escherichia coli and fragmented by replacing dodecylmaltoside with other detergents. Exchange into decyl maltoside led to the removal of the hydrophobic subunit NuoL from the otherwise intact complex. Diheptanoyl phosphocholine led to the loss of NuoL and NuoM subunits, whereas other subunits remained in the complex. The presence of N,N-dimethyldodecylamine N-oxide or Triton X-100 led to further disruption of the membrane domain into fragments containing NuoL/M/N, NuoA/K/N, and NuoH/J subunits. Among the hydrophilic subunits, NuoCD was most readily dissociated from the complex, whereas NuoB was partially dissociated from the peripheral arm assembly in N,N-dimethyldodecylamine N-oxide. A model of subunit arrangement in bacterial complex I based on these data is proposed. Subunits NuoL and NuoM, which are homologous to antiporters and are implicated in proton pumping, are located at the distal end of the membrane arm, spatially separated from the redox centers of the peripheral arm. This is consistent with proposals that the mechanism of proton pumping by complex I is likely to involve long range conformational changes.     

Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na(+) and alkali but not cholate resistance

The Bacillus subtilis mrp (multiple resistance and pH) operon supports Na(+) and alkali resistance via an Na(+)/H(+) antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpF mutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans. Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na(+) efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na(+) resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na(+) sensitivity at both pH 7.0 and 8.3. The mrpA and mrpD mutants were particularly sensitive to alkaline pH even without added Na(+). While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships among mrp genes as well. All the mrp mutants, especially the mrpA, -B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type. Expression of an upstream gene, maeN, that encodes an Na(+)/malate symporter, was coordinately regulated with mrp, although it is not part of the operon.     

Energy Transducing Roles of Antiporter-like Subunits in Escherichia coli NDH-1 with Main Focus on Subunit NuoN (ND2)

The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) contains a peripheral and a membrane domain. Three antiporter-like subunits in the membrane domain, NuoL, NuoM, and NuoN (ND5, ND4 and ND2, respectively), are structurally similar. We analyzed the role of NuoN in Escherichia coli NDH-1. The lysine residue at position 395 in NuoN (_NLys³⁹⁵) is conserved in NuoL (_LLys³⁹⁹) but is replaced by glutamic acid (_MGlu⁴⁰⁷) in NuoM. Our mutation study on _NLys³⁹⁵ suggests that this residue participates in the proton translocation. Furthermore, we found that _MGlu⁴⁰⁷ is also essential and most likely interacts with conserved _LArg¹⁷⁵. Glutamic acids, _NGlu¹³³, _MGlu¹⁴⁴, and _LGlu¹⁴⁴, are corresponding residues. Unlike mutants of _MGlu¹⁴⁴ and _LGlu¹⁴⁴, mutation of _NGlu¹³³ scarcely affected the energy-transducing activities. However, a double mutant of _NGlu¹³³ and nearby _KGlu⁷² showed significant inhibition of these activities. This suggests that _NGlu¹³³ bears a functional role similar to _LGlu¹⁴⁴ and _MGlu¹⁴⁴ but its mutation can be partially compensated by the nearby carboxyl residue. Conserved prolines located at loops of discontinuous transmembrane helices of NuoL, NuoM, and NuoN were shown to play a similar role in the energy-transducing activity. It seems likely that NuoL, NuoM, and NuoN pump protons by a similar mechanism. Our data also revealed that _NLys¹⁵⁸ is one of the key interaction points with helix HL in NuoL. A truncation study indicated that the C-terminal amphipathic segments of _NTM14 interacts with the _Mβ sheet located on the opposite side of helix HL. Taken together, the mechanism of H⁺ translocation in NDH-1 is discussed.     

Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.     

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity,Sodium Exclusion,Individual Mrp Protein Levels,or Mrp Complex Formation

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA–MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na⁺(Li⁺)/H⁺ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA–MrpD and MrpE–MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA–MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na⁺]_in. Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na⁺ (Li⁺)/H⁺ antiport.     

A new hypothesis on the simultaneous direct and indirect proton pump mechanisms in NADH-quinone oxidoreductase (complex I)

Recently, Sazanov’s group reported the X-ray structure of whole complex I [Nature, 465, 441 (2010)], which presented a strong clue for a “piston-like” structure as a key element in an “indirect” proton pump. We have studied the NuoL subunit which has a high sequence similarity to Na⁺/H⁺ antiporters, as do the NuoM and N subunits. We constructed 27 site-directed NuoL mutants. Our data suggest that the H⁺/e⁻ stoichiometry seems to have decreased from (4H⁺/2e⁻) in the wild-type to approximately (3H⁺/2e⁻) in NuoL mutants. We propose a revised hypothesis that each of the “direct” and the “indirect” proton pumps transports 2H⁺ per 2e⁻.     

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I)

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.     

Conserved lysine residues of the membrane subunit NuoM are involved in energy conversion by the proton-pumping NADH:ubiquinone oxidoreductase (Complex I)

Analysis of the amino acid sequences of subunits NuoM and NuoN in the membrane domain of Complex I revealed a clear common pattern, including two lysines that are predicted to be located within the membrane, and which are important for quinone reductase activity. Site-directed mutations of the amino acid residues E144, K234, K265 and W243 in this pattern were introduced into the chromosomal gene nuoM of Escherichia coli Complex I. The activity of mutated Complex I was studied in both membranes and in purified Complex I. The quinone reductase activity was practically lost in K234A, K234R and E144A, decreased in W243A and K265A but unchanged in E144D. Complex I from all these mutants contained 1 mol tightly bound ubiquinone per mol FMN like wild type enzyme. The mutant enzymes E144D, W243A and K265A had wild type sensitivity to rolliniastatin and complete proton-pumping efficiency of Complex I. Remarkably, the subunits NuoL and NuoH in the membrane domain also appear to contain conserved lysine residues in transmembrane helices, which may give a clue of the mechanism of proton translocation. A tentative principle of proton translocation by Complex I is suggested based on electrostatic interactions of lysines in the membrane subunits.     

The C-terminally truncated NuoL subunit (ND5 homologue) of the Na+-dependent complex I from Escherichia coli transports Na+

The NADH:quinone oxidoreductase (complex I) from Escherichia coli acts as a primary Na+ pump. Expression of a C-terminally truncated version of the hydrophobic NuoL subunit (ND5 homologue) from E. coli complex I resulted in Na+-dependent growth inhibition of the E. coli host cells. Membrane vesicles containing the truncated NuoL subunit (NuoLN) exhibited 2-4-fold higher Na+ uptake activity than control vesicles without NuoLN. Respiratory proton transport into inverted vesicles containing NuoLN decreased upon addition of Na+, but was not affected by K+, indicating a Na+-dependent increase of proton permeability of membranes in the presence of NuoLN. The His-tagged NuoLN protein was solubilized, enriched by affinity chromatography, and reconstituted into proteoliposomes. Reconstituted His6-NuoLN facilitated the uptake of Na+ into the proteoliposomes along a concentration gradient. This Na+ uptake was prevented by EIPA (5-(N-ethyl-N-isopropyl)-amiloride), which acts as inhibitor against Na+/H+ antiporters.     

Functional involvement of membrane-embedded and conserved acidic residues in the ShaA subunit of the multigene-encoded Na+/H+ antiporter in Bacillus subtilis

ShaA, a member of a multigene-encoded Na+/H+ antiporter in B. subtilis, is a large integral membrane protein consisting of 20 transmembrane helices (TM). Conservation of ShaA-like protein subunits in several cation-coupled enzymes, including the NuoL (ND5) subunit of the H+-translocating complex I, suggests the involvement of ShaA in cation transport. Bacillus subtilis ShaA contains six acidic residues that are conserved in ShaA homologues and are located in putative transmembrane helices. We examined the functional involvement of the six transmembrane acidic residues of ShaA by site-directed mutagenesis. Mutation in glutamate (Glu)-113 in TM-4, Glu-657 in TM-18, aspartate (Asp)-734 and Glu-747 in TM-20 abolished the antiport activity, suggesting that these residues play important roles in the ion transport of Sha. The acidic group was necessary and sufficient in Glu-657 and Asp-743, while it was not true of Glu-113 and Glu-747. Mutation in Asp-103 in TM-3, which is conserved in ShaA-types but not in ShaAB-types, partially affected on the antiport activity. Mutation in Asp-50 in TM-2 resulted in a unexpected phenotype: mutants retained the wild type level of ability to confer NaCl resistance to the Na+/H+ antiporter-deficient E. coli KNabc, but showed a very low antiport activity. The acidic group of Asp-50 and Asp-103 was not essential for the function. Our results suggested that these acidic residues are functionally involved in the ion transport of Sha, and some of them probably in cation binding and/or translocation.     

Projection structure of the membrane domain of Escherichia coli respiratory complex I at 8 A resolution

Respiratory complex I (NADH:ubiquinone oxidoreductase) is an L-shaped multisubunit protein assembly consisting of a hydrophobic membrane arm and a hydrophilic peripheral arm. It catalyses the transfer of two electrons from NADH to quinone coupled to the translocation of four protons across the membrane. Although we have solved recently the crystal structure of the peripheral arm, the structure of the complete enzyme and the coupling mechanism are not yet known. The membrane domain of Escherichia coli complex I consists of seven different subunits with total molecular mass of 258 kDa. It is significantly more stable than the whole enzyme, which allowed us to obtain well-ordered two-dimensional crystals of the domain, belonging to the space group p22(1)2(1). Comparison of the projection map of negatively stained crystals with previously published low-resolution structures indicated that the characteristic curved shape of the membrane domain is remarkably well conserved between bacterial and mitochondrial enzymes, helping us to interpret projection maps in the context of the intact complex. Two pronounced stain-excluding densities at the distal end of the membrane domain are likely to represent the two large antiporter-like subunits NuoL and NuoM. Cryo-electron microscopy on frozen-hydrated crystals allowed us to calculate a projection map at 8 A resolution. About 60 transmembrane alpha-helices, both perpendicular to the membrane plane and tilted, are present within one membrane domain, which is consistent with secondary structure predictions. A possible binding site and access channel for quinone are found at the interface with the peripheral arm. Tentative assignment of individual subunits to the features of the map has been made. The location of subunits NuoL and NuoM at substantial distance from the peripheral arm, which contains all the redox centres of the complex, indicates that conformational changes are likely to play a role in the mechanism of coupling between electron transfer and proton pumping.     

Roles of semiquinone species in proton pumping mechanism by complex I

Complex I (NDH-1) translocates protons across the membrane using electron transfer energy. Two different coupling mechanisms are currently being discussed for complex I: direct (redox-driven) and indirect (conformation-driven). Semiquinone (SQ) intermediates are suggested to be key for the coupling mechanism. Recently, using progressive power saturation and simulation techniques, three distinct SQ species were resolved by EPR analysis of E. coli complex I reconstituted into proteoliposomes. The fast-relaxing SQ (SQ_Nf) signals completely disappeared in the presence of the uncoupler gramicidin D or the potent E. coli complex I inhibitor squamotacin. The slow-relaxing SQ (SQ_Ns) signals were insensitive to gramicidin D, but they were sensitive to squamotacin. The very slow-relaxing SQ (SQ_Nvs) signals were insensitive to both gramicidin D and squamotacin. Interestingly, no SQ_Ns signal was observed in the ΔNuoL mutant, which lacks transporter module subunits NuoL and NuoM. Furthermore, we sought out the effect of using menaquinone (which has a lower redox potential compared to that of ubiquinone) as an electron acceptor on the proton pumping stoichiometry by in vitro reconstitution experiments with ubiquinone-rich or menaquinone-rich double knock-out membrane vesicles, which contain neither complex I nor NDH-2 (non-proton translocating NADH dehydrogenase). No difference in the proton pumping stoichiometry between menaquinone and ubiquinone was observed in the ΔNuoL and D178N mutants, which are considered to lack the indirect proton pumping mechanism. However, the proton pumping stoichiometry with menaquinone decreased by half in the wild-type. The roles and relationships of SQ intermediates in the coupling mechanism of complex I are discussed.     

Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.