title: Non-invasive in situ monitoring and quantification of TOL plasmid segregational loss within Pseudomonas putida biofilms.
abstract: Methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and PCR. However, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. We have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. The probability of plasmid segregational loss in planktonic and biofilm cultures of Pseudomonas putida carrying the TOL plasmid (pWWO::gfpmut3b) was determined directly in situ, in the absence of any applied selection pressure. Compared to suspended liquid culture, we report that the biofilm mode of growth enhances plasmid segregational loss. Results based on a biofilm-averaged analysis reveal that the probability of plasmid loss in biofilm cultures (0.016 ± 0.004) was significantly greater than that determined in planktonic cultures (0.0052 ± 0.0011). Non-invasive assessments showed that probabilities of plasmid segregational loss at different locations in a biofilm increased dramatically from 0.1% at the substratum surface to 8% at outside layers of biofilm. Results suggest that higher nutrient concentrations and subsequentially higher growth rates resulted in higher probability of plasmid segregational loss at the outer layers of the biofilm.
authors: Department of Bioengineering, University of Washington, Seattle, Washington, 98195.
authors: Hongyan
authors: H
authors: Ma
authors: Kristy N
authors: KN
authors: Katzenmeyer
authors: James D
authors: JD
authors: Bryers
mesh_terms: D018441:Biofilms / Q000254:growth & development*; D005827:Genetics, Microbial / Q000379:methods; D042822:Genomic Instability; D008853:Microscopy / Q000379:methods; D010957:Plasmids / Q000032:analysis*; D016958:Pseudomonas putida / Q000235:genetics* / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods
keywords: biofilm plasmid loss; mathematical modeling; non-invasive biofilm diagnostics
references: Biotechnol Bioeng. 1994 Jul;44(3):329-36
references: 18618749
references: Math Biosci. 2005 Feb;193(2):183-204
references: 15748729
references: Appl Environ Microbiol. 1998 Jun;64(6):2247-55
references: 9603843
references: J Gen Microbiol. 1983 Mar;129(3):755-70
references: 6348208
references: Biotechnol Bioeng. 1989 Oct 5;34(7):902-8
references: 18588181
references: FEMS Microbiol Ecol. 2002 Nov 1;42(2):261-8
references: 19709286
references: Appl Environ Microbiol. 2005 Jan;71(1):51-7
references: 15640169
references: FEMS Microbiol Ecol. 2003 May 1;44(1):67-78
references: 19719652
references: Appl Environ Microbiol. 1990 Mar;56(3):782-7
references: 2317046
references: Appl Environ Microbiol. 2003 Aug;69(8):4846-52
references: 12902279
references: Appl Microbiol Biotechnol. 2013 Jan;97(1):317-28
references: 22669634
references: J Microbiol. 2005 Apr;43(2):196-203
references: 15880097
references: Biotechnol Bioeng. 1985 Feb;27(2):156-66
references: 18553650
references: Gene. 1979 Jun;6(2):91-106
references: 383579
references: Trends Biotechnol. 1991 Aug;9(8):273-8
references: 1367567
references: Appl Environ Microbiol. 1996 Jul;62(7):2521-6
references: 8779592
references: Cell. 1993 Jun 4;73(5):873-85
references: 8500179
references: Biotechnol Bioeng. 1987 May;29(7):859-72
references: 18576531
references: Genetics. 2002 Dec;162(4):1525-32
references: 12524329
references: Science. 1987 Jan 30;235(4788):593-6
references: 3468623
references: Biotechnol Bioeng. 1982 Nov;24(11):2583-6
references: 18546225
references: J Bacteriol. 1978 Feb;133(2):527-35
references: 415040
references: Annu Rev Microbiol. 1986;40:211-35
references: 3535646
references: Mol Gen Genet. 1978 Oct 4;165(2):167-79
references: 366376
references: Plasmid. 1981 Jul;6(1):99-111
references: 6269132
references: Appl Environ Microbiol. 1993 Jun;59(6):1735-41
references: 8328798
references: Appl Environ Microbiol. 2005 Sep;71(9):5309-17
references: 16151119
references: Biotechnol Bioeng. 1991 Aug 5;38(4):397-412
references: 18600776
references: J Mol Biol. 1982 Sep 25;160(3):411-30
references: 6759658
references: Biotechnol Bioeng. 1993 Jan 20;41(2):211-20
references: 18609540
references: J Ind Microbiol Biotechnol. 2010 Oct;37(10):1081-9
references: 20552252
references: BMC Biotechnol. 2011 Mar 01;11:18
references: 21362179
references: Appl Environ Microbiol. 2003 Sep;69(9):5536-42
references: 12957943
references: Cell. 1984 Apr;36(4):1097-103
references: 6323019
references: Ann N Y Acad Sci. 1981;369:1-14
references: 7020540
references: Curr Opin Biotechnol. 2003 Jun;14(3):255-61
references: 12849777
references: J Antimicrob Chemother. 1997 Sep;40(3):415-7
references: 9338495
references: J Gen Microbiol. 1992 Jan;138(1):17-21
references: 1556548
references: Biotechnol Bioeng. 1995 Dec 20;48(6):737-44
references: 18623544
references: Biotechnol Bioeng. 2007 Jun 15;97(3):439-46
references: 17058286
references: Microbiology (Reading). 1999 Sep;145 ( Pt 9):2615-2622
references: 10517615
references: Appl Environ Microbiol. 2000 Aug;66(8):3297-304
references: 10919784
references: Annu Rev Microbiol. 1977;31:63-87
references: 334047
references: Water Sci Technol. 2004;49(11-12):327-36
references: 15303758
references: Microbiology (Reading). 2000 Jun;146 ( Pt 6):1256-1257
references: 10846203
references: Methods Enzymol. 2001;336:129-43
references: 11398394
references: Microbiol Rev. 1995 Mar;59(1):143-69
references: 7535888
references: Water Sci Technol. 2004;49(11-12):337-44
references: 15303759
references: Biotechnol Bioeng. 1998 Aug 5;59(3):318-27
references: 10099343