title: A robust genetic system for producing heterodimeric native and mutant cytochrome bc(1).
abstract: The ubihydroquinone:cytochrome c oxidoreductase, or cytochrome bc1, is central to the production of ATP by oxidative phosphorylation and photophosphorylation in many organisms. Its three-dimensional structure depicts it as a homodimer with each monomer composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits. Recent genetic approaches successfully produced heterodimeric variants of this enzyme, providing insights into its mechanism of function. However, these experimental setups are inherently prone to genetic rearrangements as they carry repeated copies of cytochrome bc1 structural genes. Duplications present on a single replicon (one-plasmid system) or a double replicon (two-plasmid system) could yield heterogeneous populations via homologous recombination or other genetic events at different frequencies, especially under selective growth conditions. In this work, we assessed the origins and frequencies of genetic variations encountered in these systems and describe an improved variant of the two-plasmid system. We found that use of a recombination-deficient background (recA) minimizes spontaneous formation of co-integrant plasmids and renders the homologous recombination within the cytochrome b gene copies inconsequential. On the basis of the data, we conclude that both the newly improved RecA-deficient and the previously used RecA-proficient two-plasmid systems reliably produce native and mutant heterodimeric cytochrome bc1 variants. The two-plasmid system developed here might contribute to the study of "mitochondrial heteroplasmy"-like heterogeneous states in model bacteria (e.g., Rhodobacter species) suitable for bioenergetics studies. In the following paper (DOI 10.1021/bi400561e), we describe the use of the two-plasmid system to produce and characterize, in membranes and in purified states, an active heterodimeric cytochrome bc1 variant with unusual intermonomer electron transfer properties.
authors.affiliation: Department of Biology, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.
authors.forename: Bahia
authors.initials: B
authors.lastname: Khalfaoui-Hassani
authors.forename: Pascal
authors.initials: P
authors.lastname: Lanciano
authors.forename: Fevzi
authors.initials: F
authors.lastname: Daldal
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045303:Cytochromes b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003575:Cytochromes c1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004579:Electron Transport; D014450:Electron Transport Complex III / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005821:Genetic Techniques; D009154:Mutation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016354:Rhodobacter capsulatus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D045303:Cytochromes b; D003575:Cytochromes c1; D014450:Electron Transport Complex III
references.citation: Cell. 1981 Jul;25(1):3-4
references.pmid: 7023698
references.citation: Cell Mol Life Sci. 2009 Apr;66(7):1257-70
references.pmid: 19132290
references.citation: J Biol Chem. 1990 Jul 15;265(20):11409-12
references.pmid: 2164001
references.citation: J Mol Biol. 1979 Jun 25;131(2):157-68
references.pmid: 490646
references.citation: Science. 1989 Oct 27;246(4929):500-3
references.pmid: 2814477
references.citation: Biochim Biophys Acta. 2012 Feb;1817(2):276-86
references.pmid: 22138626
references.citation: Biochemistry. 1992 Apr 7;31(13):3351-8
references.pmid: 1313293
references.citation: FEBS Lett. 1998 Jul 17;431(2):291-6
references.pmid: 9708922
references.citation: J Biol Chem. 2004 Apr 9;279(15):15040-9
references.pmid: 14761953
references.citation: Biochim Biophys Acta. 2013 Nov-Dec;1827(11-12):1332-9
references.pmid: 23542447
references.citation: Photosynth Res. 2004;81(3):251-75
references.pmid: 16034531
references.citation: Proc Natl Acad Sci U S A. 1977 Oct;74(10):4168-72
references.pmid: 270661
references.citation: Biochim Biophys Acta. 2013 Jun;1827(6):751-60
references.pmid: 23428397
references.citation: Biochemistry. 2005 Aug 9;44(31):10520-32
references.pmid: 16060661
references.citation: Science. 1998 Jul 3;281(5373):64-71
references.pmid: 9651245
references.citation: J Biol Chem. 2002 Feb 1;277(5):3471-6
references.pmid: 11707449
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Biochemistry. 2009 Mar 10;48(9):1888-99
references.pmid: 19254042
references.citation: Biochim Biophys Acta. 2002 Sep 10;1555(1-3):166-73
references.pmid: 12206910
references.citation: Mutat Res. 1981 Jul;82(2):239-50
references.pmid: 7022177
references.citation: Nature. 2001 Jun 21;411(6840):909-17
references.pmid: 11418848
references.citation: Proc Natl Acad Sci U S A. 2000 Apr 25;97(9):4567-72
references.pmid: 10781061
references.citation: FEBS Lett. 2012 Mar 9;586(5):617-21
references.pmid: 21878327
references.citation: Biochemistry. 1994 Jan 25;33(3):723-33
references.pmid: 8292600
references.citation: Trends Biochem Sci. 1990 May;15(5):178-80
references.pmid: 2163130
references.citation: Biochim Biophys Acta. 1982 Jul 22;681(1):41-53
references.pmid: 6288082
references.citation: Protein Eng Des Sel. 2012 Jan;25(1):15-25
references.pmid: 22119789
references.citation: Biochim Biophys Acta. 2008 Jul-Aug;1777(7-8):1001-19
references.pmid: 18501698
references.citation: Biochemistry. 2011 Mar 15;50(10):1651-63
references.pmid: 21261281
references.citation: Cell. 1976 Dec;9(4 PT 2):707-16
references.pmid: 797459
references.citation: J Biol Inorg Chem. 2012 Apr;17(4):647-62
references.pmid: 22382353
references.citation: J Biol Chem. 2010 Jan 1;285(1):502-10
references.pmid: 19892700
references.citation: Nature. 1999 Jul 29;400(6743):476-80
references.pmid: 10440380
references.citation: Nature. 1998 Apr 16;392(6677):677-84
references.pmid: 9565029
references.citation: Proc Natl Acad Sci U S A. 1986 Apr;83(7):2012-6
references.pmid: 16593675
references.citation: J Theor Biol. 1976 Oct 21;62(2):327-67
references.pmid: 186667
references.citation: Biochim Biophys Acta. 1983 May 23;723(2):202-218
references.pmid: 21494412
references.citation: Biochemistry. 2012 Jan 31;51(4):829-35
references.pmid: 22233445
references.citation: Science. 2010 Jul 23;329(5990):451-4
references.pmid: 20651150
references.citation: J Biol Chem. 2005 Jun 17;280(24):22732-40
references.pmid: 15833742
references.citation: Plasmid. 1985 Mar;13(2):149-53
references.pmid: 2987994
references.citation: Anal Biochem. 1985 Oct;150(1):76-85
references.pmid: 3843705
references.citation: Biochim Biophys Acta. 2012 Jul;1817(7):1053-62
references.pmid: 22465023
references.citation: Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):492-6
references.pmid: 1846443
references.citation: FEBS Lett. 2007 Apr 17;581(8):1535-41
references.pmid: 17399709
references.citation: Annu Rev Microbiol. 2003;57:551-77
references.pmid: 14527291
references.citation: Science. 1997 Jul 4;277(5322):60-6
references.pmid: 9204897
references.citation: Hum Mol Genet. 2013 Jun 1;22(11):2141-51
references.pmid: 23418307
references.citation: Biochemistry. 1992 Dec 1;31(47):11864-73
references.pmid: 1332776
references.citation: Biochemistry. 1995 Dec 12;34(49):15997-6003
references.pmid: 8519755