title: New quantitative methods for measuring plasmid loss rates reveal unexpected stability.
abstract: Plasmid loss rate measurements are standard in microbiology and key to understanding plasmid stabilization mechanisms. The conventional assays eliminate selection for plasmids at the beginning of the experiment and screen for the appearance of plasmid-free cells over long-term population growth. However, it has been long appreciated in plasmid biology that the growth rate differential between plasmid-free and plasmid-containing cells at some point overshadows the effect of primary loss events, such that the assays can greatly over-estimate inherent loss rates. The standard solutions to this problem are to either consider the very early phase of loss where the fraction of plasmid-free cells increases linearly, or to measure the growth rate difference either by following the population for longer time or by measuring growth rates separately. Here we mathematically show that in all these cases, seemingly small experimental errors in the growth rate estimates can overshadow the estimates of the loss rates. For many plasmids, loss rates may thus be much lower than previously thought, and for some plasmids, the estimated loss rate may have nothing to do with actual loss rates. We further modify two independent experimental methods to separate inherent losses from growth differences and apply them to the same plasmids. First we use a high-throughput microscopy-based approach to screen for plasmid-free cells at extremely short time scales--tens of minutes rather than tens of generations--and apply it to a par⁻ version of mini-R1. Second we modify a counterselection-based plasmid loss assay inspired by the Luria-Delbrück fluctuation test that completely separates losses from growth, and apply it to various R1 and pSC101 derivatives. Concordant results from the two assays suggest that plasmids are lost at a lower frequency than previously believed. In fact, for par⁻ mini-R1 the observed loss rate of about 10⁻³ per cell and generation seems to be so low as to be inconsistent with what we know about the R1 stabilization mechanisms, suggesting these well characterized plasmids may have some additional and so far unknown stabilization mechanisms, for example improving copy number control or partitioning at cell division.
authors.affiliation: Department of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA.
authors.forename: Billy T C
authors.initials: BT
authors.lastname: Lau
authors.forename: Per
authors.initials: P
authors.lastname: Malkus
authors.forename: Johan
authors.initials: J
authors.lastname: Paulsson
mesh_terms: D001483:Base Sequence; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010957:Plasmids; D012093:Replicon; D017384:Sequence Deletion
keywords: Counterselection; Fluctuation test; Plasmid loss
references.citation: BMC Microbiol. 2006 Apr 28;6:39
references.pmid: 16646962
references.citation: Genetics. 2002 Aug;161(4):1373-84
references.pmid: 12238464
references.citation: Plasmid. 1996 Nov;36(3):153-9
references.pmid: 9007010
references.citation: Annu Rev Genet. 1997;31:1-31
references.pmid: 9442888
references.citation: Curr Protoc Mol Biol. 2007 Apr;Chapter 1:Unit 1.16
references.pmid: 18265390
references.citation: Genetics. 1977 Oct;87(2):209-28
references.pmid: 17248761
references.citation: J Bacteriol. 1987 Oct;169(10):4646-50
references.pmid: 3308847
references.citation: Plasmid. 2012 Mar;67(2):167-73
references.pmid: 22305922
references.citation: Plasmid. 1996 Nov;36(3):161-7
references.pmid: 9007011
references.citation: Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
references.pmid: 9092630
references.citation: Trends Microbiol. 2000 Mar;8(3):128-33
references.pmid: 10707066
references.citation: J Bacteriol. 1994 May;176(10):2781-7
references.pmid: 8188580
references.citation: Biotechnol Bioeng. 1989 Jan 20;33(4):451-9
references.pmid: 18587936
references.citation: Proc Natl Acad Sci U S A. 1986 May;83(10):3116-20
references.pmid: 3517851
references.citation: Genetics. 1980 Feb;94(2):425-43
references.pmid: 6248416
references.citation: Trends Biotechnol. 1991 Aug;9(8):273-8
references.pmid: 1367567
references.citation: Microbiol Mol Biol Rev. 1998 Jun;62(2):434-64
references.pmid: 9618448
references.citation: Curr Protoc Protein Sci. 2001 May;Chapter 5:Unit5.2
references.pmid: 18429183
references.citation: J Mol Biol. 1994 Mar 11;236(5):1289-98
references.pmid: 8126720
references.citation: J Genet. 1949 Dec;49(3):264-85
references.pmid: 24536673
references.citation: Cell. 1984 Aug;38(1):191-201
references.pmid: 6088064
references.citation: Genetics. 1943 Nov;28(6):491-511
references.pmid: 17247100
references.citation: Q Rev Biophys. 2001 Feb;34(1):1-59
references.pmid: 11388089
references.citation: Mol Gen Genet. 1984;197(1):1-7
references.pmid: 6392821
references.citation: J Bacteriol. 2004 Jan;186(1):207-11
references.pmid: 14679240
references.citation: Nat Rev Microbiol. 2010 Oct;8(10):683-92
references.pmid: 20844556
references.citation: EMBO J. 1999 Mar 15;18(6):1712-21
references.pmid: 10075940
references.citation: J Gen Microbiol. 1987 Jul;133(7):1871-80
references.pmid: 3312484
references.citation: Science. 2003 Sep 12;301(5639):1496-9
references.pmid: 12970556
references.citation: J Bacteriol. 1994 Jul;176(14):4306-10
references.pmid: 8021216
references.citation: Nature. 2004 Jan 29;427(6973):415-8
references.pmid: 14749823
references.citation: J Gen Microbiol. 1952 Aug;7(1-2):69-88
references.pmid: 13011259
references.citation: Mol Microbiol. 2000 Aug;37(3):467-76
references.pmid: 10931340
references.citation: Nat Rev Microbiol. 2005 Sep;3(9):711-21
references.pmid: 16138099
references.citation: Nature. 1976 Jan 29;259(5541):285-90
references.pmid: 765836
references.citation: J Antimicrob Chemother. 1977 Nov;3(6):541-53
references.pmid: 340439
references.citation: Infect Immun. 2011 Jul;79(7):2502-9
references.pmid: 21555398
references.citation: Cell. 2010 Jun 11;141(6):927-42
references.pmid: 20550930
references.citation: Plasmid. 2002 Nov;48(3):165-73
references.pmid: 12460531