Schematic of E. coli complex I and the nuo locus. Adapted with permission of the publisher (17, 29, 30, 49). (A) E. coli complex I is comprised of three distinct fragments: the peripheral (light gray), connecting (white), and membrane (dark gray) fragments (17, 29). The peripheral fragment (NDF) is comprised of the nuclear homologs NuoE, -F, and -G and exhibits NADH dehydrogenase activity that oxidizes NADH to NAD⁺; the connecting fragment is comprised of the nuclear homologs NuoB, -C, -D, and -I; and the membrane fragment is comprised of the mitochondrial homologs NuoA, -H, and -J to -N and catalyzes ubiquinone (Q) to its reduced form (QH₂). FMN, flavin mononucleotide. (B) The E. coli nuo locus encodes the 14 Nuo subunits that constitute complex I. The 5′ half of the locus contains a previously identified promoter (nuoP) and the majority of genes that encode the peripheral and connecting subunits (light gray and white, respectively). The 3′ half of the locus contains the majority of the genes encoding the membrane subunits (dark gray). The 3′ end of nuoG encodes a C-Terminal region (CTR) of the NuoG subunit (hatched).

Wild-type nuoG and mutant ΔnuoG1 and nuoG2 alleles. +1, nuo transcription initiation site (49). Restriction enzyme sites: E, EcoRI; P, PstI; S, SalI derived by site-directed mutagenesis to facilitate construction of mutant alleles. Hatched bars, CTR, a 235-bp 3′ region of nuoG present in the wild-type nuoG, deleted in the ΔnuoG1 allele, and duplicated in tandem in the nuoG2 allele. Inverted triangle, location of the CTR deletion. G1 and H1, flanking primers used to amplify the CTR.

Construction of strain AJW1459 by integrational, homologous recombination. White bars, vector-derived nuo sequence; gray bars, chromosome-derived nuo sequence; dotted lines, vector sequence; Ap^R, ampicillin resistance. Other designations are as described in the Fig. 2 legend. The plasmid pAJW105ΔPstI encompasses the 3′ end of nuoF to the 3′ end of nuoL and carries the wild-type nuoG allele. This plasmid was transformed into competent AJW931 [ΔnuoG1 polA(Ts)] cells. Cells in which the plasmid had integrated into the chromosome by homologous recombination were identified by selection of ampicillin resistance at the restrictive temperature, 42°C. Strain AJW932 was constructed similarly (not shown), except that the plasmid pHF17, which carries the ΔnuoG1 allele, was integrated into the Nuo⁺ PolA(Ts) strain CP366. Similarly, strain AJW1472 was constructed by integrating pAJW105ΔPstI into strain AJW1470 [nuoG2 polA(Ts)]. The simultaneous presence of both the wild-type and mutant nuoG alleles in each of these strains was verified by PCR analysis with primers G1 and H1.

Autoradiograph of RNA dot blots. RNA was purified from wild-type or nuo mutant cells, transferred directly to multiple nitrocellulose membrane, and probed with the nuo genes or fragments as indicated on the left. Lanes: 1, wild type (strain CP875); 2, wild type (CP366); 3, nuoB::Km mutant (AJW844); 4, nuoB-C::Cm mutant (AJW853); 5, nuoF::miniTn10Cm mutant (AJW851); 6, Δ(nuoF-L)-1 mutant (CP938); 7, nuoG::Tn10-1 mutant (CP910); 8, ΔnuoG1 mutant (AJW931); 9, nuoH::Km mutant (AJW845); 10, nuoI::Km mutant (AJW846); 11, nuoN::Km mutant (AJW847). The hybridization pattern for the nuoM::miniTn10Cm strain (AJW852) (data not shown) was identical to that for the nuoN::Km strain (AJW847) (lane 11). The hybridization pattern for AJW1516 was identical to that for AJW931 (data not shown).

(A and B) Immunoblot analysis with E. coli anti-complex I polyclonal antibody following SDS-PAGE (7.5% polyacrylamide) and transfer to nitrocellulose. Sizes of molecular mass markers (in kilodaltons) are indicated on the right; the NuoG (G) and NuoCD (D) subunits are identified on the left. (A) Analysis of whole-cell lysates prepared from wild-type or nuo polar mutant cells. Lanes: 1, purified complex I; 2, purified complex I mixed with wild-type lysate (strain CP366); 3, wild type (CP366); 4, wild type (CP875); 5, nuoB::Km mutant (AJW844); 6, nuoB-C::Cm mutant (AJW853); 7, nuoF::miniTn10Cm mutant (AJW851); 8, Δ(nuoF-L)-1 mutant (CP938); 9, nuoG::Tn10-1 mutant (CP910); 10, nuoH::Km mutant (AJW845); 11, nuoI::Km mutant (AJW846); 12, nuoM::miniTn10Cm mutant (AJW852); 13, nuoN::Km mutant (AJW847). Cells were grown with aeration in TB at 32°C until cultures reached mid-exponential phase (OD₆₁₀, 0.35 to 0.4). One hundred micrograms of whole-cell lysate was loaded in each lane. (B) Analysis of whole-cell lysates prepared from wild-type or nuoG mutant cells. Lanes: 1, purified complex I; 2, purified complex I mixed with wild-type whole-cell lysate (CP366); 3, wild type (CP366); 4, Δ(nuoF-L)-1 mutant (CP938); 5, nuoG::Tn10-1 mutant (CP910); 6, ΔnuoG1 mutant (AJW931); 7, nuoG2 mutant (AJW1470). Cells were grown as described for panel A. One hundred micrograms of whole-cell lysate was loaded in each lane. The profile for AJW1516 was identical to that for AJW931, and the profile for AJW1517 was identical to that for AJW1470 (data not shown). (C) Putative NuoG peptides. The lengths and molecular masses (M_r) of translation products of the nuoG alleles as predicted by PeptideSort (20) are shown. aa, amino acid. The CTR of NuoG is hatched in gray. The deletion in ΔnuoG1 shifts the remainder of the NuoG C terminus out of frame (dark gray). The duplication in nuoG2 shifts the duplicated CTR (hatched in black) and the remainder of the NuoG C terminus (dark gray) out of frame.

Effect of nuo mutations on nuo promoter activity. (A) The multicopy nuoPA′::lacZYA transcriptional (operon) reporter fusion, pHF9, constructed from pRS415 (45). The 443-bp nuo insert consists of the nuo promoter (49) and the proximal third of nuoA. Ap^R, ampicillin resistance; T1₄, transcriptional terminator; trpA′, trp operon sequence; lacZYA, lac operon including its translational machinery but missing the lac promoter. (B) β-Galactosidase activity assay. Cells were grown in TB at 32°C to mid-exponential phase (OD₆₁₀, 0.35 to 0.4), harvested, lysed by sonication, and centrifuged to separate the cytoplasmic fraction from the membrane fraction. The β-galactosidase activity of the cytoplasmic fractions was quantified as units per milligram of protein, where 1 U = 1 μmol of o-nitrophenol formed/min. Results are from at least six independent experiments. Error bars indicate standard errors of the mean. Strains: wt, CP875, nuoB::Km, AJW844; Δ(nuoF-L)-1, CP938; nuoG::Tn10-1, CP910; nuoH::Km, AJW845; nuoI::Km, AJW846; nuoM::miniTn10Cm, AJW852; nuoN::Km, AJW847; ΔnuoG1, AJW1516; nuoG2, AJW1517; ΔnuoG1 nuoH::Km, AJW1582; ΔnuoG1 nuoI::Km, AJW1583; nuoG2 nuoH::Km, AJW1584.