scholarly journals Random mutagenesis of the gene for the β-subunit of F1-ATPase from Escherichia coli

1989 ◽  
Vol 259 (2) ◽  
pp. 421-426 ◽  
Author(s):  
F A Kironde ◽  
D Parsonage ◽  
A E Senior
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Catalytic Mechanism ◽  
Random Mutagenesis ◽  
Proton Pumping ◽  
Beta Subunit ◽  
Phenotypic Screening ◽  
Haploid Strain ◽  
F1 Atpase

ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.

scholarly journals Lysine 155 in beta-subunit is a catalytic residue of Escherichia coli F1 ATPase.

1993 ◽  
Vol 268 (10) ◽  
pp. 6989-6994
Author(s):  
A.E. Senior ◽  
S. Wilke-Mounts ◽  
M.K. al-Shawi
Keyword(s):  
Escherichia Coli ◽  
Beta Subunit ◽  
Catalytic Residue ◽  
F1 Atpase

scholarly journals An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 381-393
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Strand Break ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Chromatid Cohesion ◽  
K 12 ◽  
Structural Maintenance Of Chromosomes ◽  
Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals Dra/AfaE Adhesin of Uropathogenic Dr/Afa+Escherichia coli Mediates Mortality in Pregnant Rats

2005 ◽  
Vol 73 (11) ◽  
pp. 7597-7601 ◽  
Author(s):  
K. Wroblewska-Seniuk ◽  
R. Selvarangan ◽  
A. Hart ◽  
R. Pladzyk ◽  
P. Goluszko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Maternal Mortality ◽  
Double Mutant ◽  
Lethal Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
Pregnant Rats ◽  
E Coli

ABSTRACT Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE + afaD and afaE afaD + mutants. The clinical E. coli strain (afaE + afaD +) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE + afaD + strain, followed by the group infected with the afaE + afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE + strains were the most invasive (afaE + afaD strain > afaE + afaD + strain), while the afaE mutant strains (afaE afaD + and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE + E. coli strains in vitro.

scholarly journals The amino terminus of the yeast F1-ATPase beta-subunit precursor functions as a mitochondrial import signal.

1986 ◽  
Vol 102 (2) ◽  
pp. 523-533 ◽  
Author(s):  
S D Emr ◽  
A Vassarotti ◽  
J Garrett ◽  
B L Geller ◽  
M Takeda ◽  
...  
Keyword(s):  
Beta Subunit ◽  
Lacz Gene ◽  
Gene Fusions ◽  
Yeast Mitochondria ◽  
Subunit Protein ◽  
F1 Atpase ◽  
Amino Terminal ◽  
Hybrid Proteins ◽  
Suc2 Gene

The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.

scholarly journals Multiple Genetic Pathways for Restarting DNA Replication Forks in Escherichia coli K-12

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 487-497
Author(s):  
Steven J Sandler
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Double Mutant ◽  
Synthetic Lethality ◽  
Replication Forks ◽  
Viability Assay ◽  
Replication Restart ◽  
K 12 ◽  
Redundant Functions

Abstract In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Δrep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3′-5′ helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.

scholarly journals Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli.

1987 ◽  
Vol 262 (17) ◽  
pp. 8022-8026 ◽  
Author(s):  
D Parsonage ◽  
S Wilke-Mounts ◽  
A E Senior
Keyword(s):  
Escherichia Coli ◽  
Beta Subunit ◽  
Directed Mutagenesis ◽  
F1 Atpase
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