title: A novel pair of inducible expression vectors for use in Methylobacterium extorquens.
abstract: BACKGROUND
  Due to the ever increasing use of diverse microbial taxa in basic research and industrial settings, there is a growing need for genetic tools to alter the physiology of these organisms. In particular, there is a dearth of inducible expression systems available for bacteria outside commonly used γ-proteobacteria, such as Escherichia coli or Pseudomonas species. To this end, we have sought to develop a pair of inducible expression vectors for use in the α-proteobacterium Methylobacterium extorquens, a model methylotroph.
  
  
  RESULTS
  We found that the P(R) promoter from rhizobial phage 16-3 was active in M. extorquens and engineered the promoter to be inducible by either p-isopropyl benzoate (cumate) or anhydrotetracycline. These hybrid promoters, P(R/cmtO) and P(R/tetO), were found to have high levels of expression in M. extorquens with a regulatory range of 10-fold and 30-fold, respectively. Compared to an existing cumate-inducible (10-fold range), high-level expression system for M. extorquens, P(R/cmtO) and P(R/tetO) have 33% of the maximal activity but were able to repress gene expression 3 and 8-fold greater, respectively. Both promoters were observed to exhibit homogeneous, titratable activation dynamics rather than on-off, switch-like behavior. The utility of these promoters was further demonstrated by complementing loss of function of ftfL--essential for growth on methanol--where we show P(R/tetO) is capable of not only fully complementing function but also producing a conditional null phenotype. These promoters have been incorporated into a broad-host-range backbone allowing for potential use in a variety of bacterial hosts.
  
  
  CONCLUSIONS
  We have developed two novel expression systems for use in M. extorquens. The expression range of these vectors should allow for increased ability to explore cellular physiology in M. extorquens. Further, the P(R/tetO) promoter is capable of producing conditional null phenotypes, previously unattainable in M. extorquens. As both expression systems rely on the use of membrane permeable inducers, we suspect these expression vectors will be useful for ectopic gene expression in numerous proteobacteria.
authors: Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Ave., Cambridge, MA 02138, USA. cmarx@oeb.harvard.edu
authors: Lon M
authors: LM
authors: Chubiz
authors: Jessica
authors: J
authors: Purswani
authors: Sean Michael
authors: SM
authors: Carroll
authors: Chistopher J
authors: CJ
authors: Marx
mesh_terms: D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005453:Fluorescence; D005822:Genetic Vectors; D020601:Methylobacterium extorquens / Q000235:genetics*; D011401:Promoter Regions, Genetic
chemical_list: D017931:DNA Primers
references: J Bacteriol. 2005 Jul;187(13):4607-14
references: 15968072
references: J Bacteriol. 2012 Sep;194(17):4746-8
references: 22887658
references: Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648-52
references: 377280
references: PLoS One. 2013 Apr 30;8(4):e62957
references: 23646164
references: Evolution. 2009 Nov;63(11):2816-30
references: 19545267
references: Mol Microbiol. 2010 Jan;75(2):513-27
references: 20025666
references: Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547-51
references: 1319065
references: Science. 1995 Jun 23;268(5218):1766-9
references: 7792603
references: Microbiology (Reading). 2004 Jan;150(Pt 1):9-19
references: 14702393
references: Microbiology (Reading). 2005 Nov;151(Pt 11):3723-3728
references: 16272393
references: Science. 1998 Jul 3;281(5373):99-102
references: 9651254
references: Nat Methods. 2009 May;6(5):343-5
references: 19363495
references: Biotechniques. 1992 Aug;13(2):214-20
references: 1327007
references: BMC Bioinformatics. 2009 Apr 09;10:106
references: 19358741
references: Proc Natl Acad Sci U S A. 1988 Dec;85(23):8973-7
references: 3057497
references: Appl Environ Microbiol. 2010 Aug;76(15):5058-66
references: 20562288
references: BMC Res Notes. 2008 Feb 26;1:1
references: 18710539
references: Biotechniques. 2002 Nov;33(5):1062-7
references: 12449384
references: Appl Environ Microbiol. 2006 Dec;72(12):7723-9
references: 17041156
references: Proc Natl Acad Sci U S A. 1957 Jul 15;43(7):553-66
references: 16590055
references: PLoS One. 2009;4(5):e5584
references: 19440302
references: Microbiol Mol Biol Rev. 2005 Jun;69(2):326-56
references: 15944459
references: J Lab Autom. 2013 Apr;18(2):171-7
references: 23032169
references: Nucleic Acids Res. 2008 Jul;36(12):4038-46
references: 18522973
references: Science. 2011 Jun 3;332(6034):1190-2
references: 21636771
references: Nature. 2004 Feb 19;427(6976):737-40
references: 14973486
references: Mol Biol Evol. 2013 Mar;30(3):549-60
references: 23223712
references: Proc Natl Acad Sci U S A. 1997 Jul 22;94(15):8168-72
references: 9223333
references: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8790-5
references: 12084925
references: Microbiology (Reading). 2001 Aug;147(Pt 8):2065-2075
references: 11495985
references: PLoS Genet. 2009 Sep;5(9):e1000652
references: 19763169
references: Mol Biol Evol. 2004 Jul;21(7):1234-41
references: 15014146
references: Cell Rep. 2012 Feb 23;1(2):133-40
references: 22832162
references: J Bacteriol. 2003 Jan;185(2):669-73
references: 12511515
references: Archaea. 2008 Dec;2(3):193-203
references: 19054746
references: Annu Rev Microbiol. 1994;48:345-69
references: 7826010
references: J Bacteriol. 2003 Dec;185(24):7169-75
references: 14645277
references: Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
references: 9092630
references: BMC Biotechnol. 2006 Nov 03;6:43
references: 17083727
references: J Bacteriol. 1994 Jul;176(13):4052-65
references: 8021187