Genetics of the Adenosine Triphosphatase Complex of Escherichia coli

A strain of Escherichia coli which was derived from a gentamicin-resistant clinical isolate was found to be cross-resistant to neomycin and streptomycin. The molecular nature of the genetic defect was found to be an insertion of two GC base pairs in the uncG gene of the mutant. The insertion led to the production of a truncated -y subunit of 247 amino acids in length instead of the 286 amino acids that are present in the normal y subunit. A plasmid which carried the ATP synthase genes from the mutant produced resistance to aminoglycoside antibiotics when it was introduced into a strain with a chromosomal deletion of the ATP synthase genes. Removal of the genes coding for the and e subunits abolished antibiotic resistance coded by the mutant plasmid. The HfrPOl AuncB-C recAl srl::TnlO thi-J rel-I thi-) Carol Kumamoto; derivative of strain 1100 X1488 F-rpsL hsdR minA minB purE pdxC his ilv met 23 1100HfrPOl thi-l rel-I bglR Laboratory stock was associated with the failure to grow on nonfermentable carbon sources; both were cotransducible with the ilv locus.Other bacterial strains used in this study are given in Table 1. Plasmids. We use a prime symbol (e.g., a') to indicate a truncated polypeptide produced by a partial gene on a plasmid. We used -y'-uncG270 to indicate the mutant -y subunit produced by a plasmid carrying the uncG270 mutation.A plasmid containing the uncG270 mutation was constructed by Hfr-mediated chromosome mobilization (4, 26). This technique relies on the fact that the plasmid-borne chloramphenicol resistance can only be transferred in a mating if the plasmid carrying the resistance element has recombined with the chromosome of the Hfr prior to transfer. When transferred into a recombination-proficient recipient, in some cases, recombinational excision gives rise to a plasmid which has received the chromosomal mutation. pDJK37, which is 8.7 kilobases in length, was derived from plasmid pRPG54 (a, c, b, 8, a, y, 1, e; 12.8 kilobases), but contains a deletion extending from uncH into uncC. Strain RH401 (HfrC uncG270 streptomycin sensitive) was transformed with plasmid pDJK37 (a, c, b). RH401(pDJK37) was mated with strain CK1801 (F-AuncB-C streptomycin resistant) with selection for chloramphenicol resistance and streptomycin resistance. A plasmid DNA preparation was made from the pooled exconjugants which were resistant to both chloramphenicol and streptomycin. A recombinant plasmid that received the mutation contained the uncB through uncC genes and was 12.8 kilobases in length. Uncut plasmid DNA was electrophoresed in a 0.8% agarose gel, with plasmids pRPG54 and pDJK37 used as standards. The region of the gel corresponding in size to pRPG54 was excised, crushed, and soaked in 10 mM Tris hydrochloride (pH 7.8)-i mM EDTA. The eluate was used to transform strain CK1803 (AuncBC recA). Transformants were purified and plasmid DNA was prepared. Plasmid DNA preparations were digested with EcoRI and subjected to agarose gel electrophoresis. Of 12 plasmids tested, 4 had a restriction pattern identical ...

Section: Resultsmentioning

confidence: 99%

“…The gene order is uncIBEF-HAGDC, coding for polypeptides i, a, c, b, 8, a, -y, P, and E, respectively (11,17). The i polypeptide is not known to be present in ATP synthase.…”

mentioning

confidence: 99%

Defective gamma subunit of ATP synthase (F1F0) from Escherichia coli leads to resistance to aminoglycoside antibiotics

Humbert

Altendorf

1989

“…The growth properties and various membrane-bound enzymatic activities of strains carrying the uncC424 allele were described previously (7,9). Since the earlier work, growth conditions and the method of preparing membranes have changed, and for purposes of comparison with revertant strains, some activities have been reassayed (see below).…”

Section: Resultsmentioning

confidence: 99%

“…In this paper we characterize a mutant allele (uncC424) (7,9) which affects the amount of the e subunit formed and four revertant strains which, by various means, have overcome the defect.…”

mentioning

confidence: 99%

Altered translation of the uncC gene coding for the epsilon subunit of the F1F0-ATPase of Escherichia coli

Cox¹,

Webb²,

Hatch³

et al. 1987

The nucleotide sequence of the previously described uncC424 allele was determined and found to be the same as that of a wild-type uncC gene. However, a G-A change occurred 7 nucleotides upstream from the translation start codon, changing the putative Shine-Dalgarno sequence from GAGG to GAAG. Four revertant strains were examined. In one revertant, which had normal growth and membrane properties, a single base deletion had occurred to re-form the Shine-Dalgarno sequence GAGG 1 nucleotide closer to the translation start codon. A second revertant had a single base deletion in the preceding uncD gene, causing an extension of the ,B subunit by 6 amino acids and an increase, presumably by translational coupling, in the amount of e subunit. The third and fourth revertant strains were phenotypically similar and had either C-*T or G-*T changes 18 or 19 nucleotides, respectively, upstream from the translation start codon.The F1FO-ATPase complex of mitochondrial, chloroplast, and bacterial membranes catalyses the terminal reaction in oxidative phosphorylation. This highly conserved complex can be dissociated into a water-soluble portion (F1-ATPase), which catalyzes the hydrolysis of ATP, and an intrinsic membrane portion (FO), which forms a proton pore (23). The genes coding for the subunits of the F1F0-ATPase complex in Escherichia coli make up the unc operon at about 83.5 min on the chromosome, and the complete DNA sequence has been determined (26). The unc operon consists of nine genes in the order IBEFHAGDC corresponding to the subunits i, a, c, b, 5, a, ay, 1, and e. The uncl gene product is thought (12), using site-directed mutagenesis, obtained evidence suggesting that secondary structure in the mRNA and the use of a less efficient initiation codon play a part in restricting translation initiation of the uncF mRNA segment.In this paper we characterize a mutant allele (uncC424) (7, 9) which affects the amount of the e subunit formed and four revertant strains which, by various means, have overcome the defect. MATERIALS AND METHODS Enzymes and chemicals. Restriction endonucleases and T4

“…These represent the hybrid (a-y)1 fusion polypeptide (predicted molecular weight, 81,000) and smaller proteins which are probably either proteolytic products or prematurely terminated proteins. The fusion protein was synthesized very well in vitro compared with the -y subunit, which has been previously shown to be synthesized poorly or not at all in vitro (3,7).…”

Section: Methodsmentioning

confidence: 95%

Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes

Brusilow

1987

To study expression of uncG, the gene coding for the y subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the a subunit, and uncG. Two deletions which fused uncA and uncG coded for a--y fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region. Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E. coli. The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked prqtons. The deleterious effect was eliminated by making a nonpolar deletion in the upstream Fo gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the Fo genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability. A plasmid which carried Fo genes and the gene for the 8 subunit caused deleterious proton leakiness in unc' cells but not in cells from which the unc operon was deleted. The proton leakiness caused by these different plasmids was therefore due to the production of a leaky Fo proton channel and required the presence of F1 genes. The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the Fo proton channel.The unc operon of Escherichia coli is located at 84 min on the E. coli chromosome and consists of nine genes, eight of which code for the subunits of the proton-translocating ATPase. The gene order and gene-polypeptide relationships have been established as uncIBEFHAGDC, coding for, respectively, protein i, a polypeptide of unknown function, and ATPase subunits a, c, b, b, a, -y, 13, and e. Subunits a, b, and c form the Fo sector of the ATPase, an integral membrane sector which conducts protons across the lipid bilayer. Subunits a, 1, -y, 8, and E form the F1 sector which contains the catalytic sites for ATP synthesis and hydrolysis (for a review, see reference 10). Although the genes are present in equal numbers in the operon and the operon is transcribed as a single polycistronic mRNA (13), the polypeptides are present in different numbers in the ATPase complex. The stoichiometry is believed to be al, clO, b2, 81, a3, yl, 13, El (9). The genes have been shown to be differentially expressed both in vitro and in minicells, probably as the result of differential translation of the genes in the mRNA transcript (4, 18).One particularly interesting question involving expression of unc genes concerns control over uncG expression. This gene codes for the -y subunit, which is present in one copy per ATPase, but uncG is situated between uncA and uncD, which code for the a and 13 subunits, respectively, both of which are present in three copies per ATPase. sector in the absence of F1 genes. The study described here was initiated to investigate the role of the nucleotid...