Spontaneous plasmid transformation of Escherichia coli is independent of the DNA uptake machinery for single-stranded DNA (ssDNA) entry. The one-hit kinetic pattern of plasmid transformation indicates that double-stranded DNA (dsDNA) enters E. coli cells on agar plates. However, DNA uptake and transformation regulation remain unclear in this new type of plasmid transformation. In this study, we developed our previous plasmid transformation system and induced competence at early stationary phase. Despite of inoculum size, the development of competence was determined by optical cell density. DNase I interruption experiment showed that DNA was taken up exponentially within the initial 2 minutes and most transforming DNA entered E. coli cells within 10 minutes on LB-agar plates. A half-order kinetics between recipient cells and transformants was identified when cell density was high on plates. To determine whether the stationary phase master regulator RpoS plays roles in plasmid transformation, we investigated the effects of inactivating and over-expressing its encoding gene rpoS on plasmid transformation. The inactivation of rpoS systematically reduced transformation frequency, while over-expressing rpoS increased plasmid transformation. Normally, RpoS recognizes promoters by its lysine 173 (K173). We found that the K173E mutation caused RpoS unable to promote plasmid transformation, further confirming a role of RpoS in regulating plasmid transformation. In classical transformation, DNA was transferred across membranes by DNA uptake proteins and integrated by DNA processing proteins. At stationary growth phase, RpoS regulates some genes encoding membrane/periplasmic proteins and DNA processing proteins. We quantified transcription of 22 of them and found that transcription of only 4 genes (osmC, yqjC, ygiW and ugpC) encoding membrane/periplasmic proteins showed significant differential expression when wildtype RpoS and RpoSK173E mutant were expressed. Further investigation showed that inactivation of any one of these genes did not significantly reduce transformation, suggesting that RpoS may regulate plasmid transformation through other/multiple target genes.
References
[1]
Nikaido H (2009) Multidrug resistance in bacteria. Annu Rev Biochem 78: 119–146.
Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, et al. (2009) Characterization of a new metallo-beta-lactamase gene, bla(NDM-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 53: 5046–5054.
[4]
Solomon JM, Grossman AD (1996) Who's competent and when: regulation of natural genetic competence in bacteria. Trends Genet 12: 150–155.
[5]
Claverys JP, Martin B (2003) Bacterial “competence” genes: signatures of active transformation, or only remnants? Trends Microbiol 11: 161–165.
[6]
Sinha S, Cameron AD, Redfield RJ (2009) Sxy induces a CRP-S regulon in Escherichia coli. J Bacteriol 191: 5180–5195.
[7]
Maeda S, Sawamura A, Matsuda A (2004) Transformation of colonial Escherichia coli on solid media. FEMS Microbiol Lett 236: 61–64.
[8]
Sun D, Zhang Y, Mei Y, Jiang H, Xie Z, et al. (2006) Escherichia coli is naturally transformable in a novel transformation system. FEMS Microbiol Lett 265: 249–255.
[9]
Tsen SD, Fang SS, Chen MJ, Chien JY, Lee CC, et al. (2002) Natural plasmid transformation in Escherichia coli. J Biomed Sci 9: 246–252.
[10]
Sun D, Zhang X, Wang L, Prudhomme M, Xie Z, et al. (2009) Transforming DNA uptake gene orthologs do not mediate spontaneous plasmid transformation in Escherichia coli. J Bacteriol 191: 713–719.
[11]
Chen I, Dubnau D (2004) DNA uptake during bacterial transformation. Nat Rev Microbiol 2: 241–249.
[12]
Mortier-Barriere I, Velten M, Dupaigne P, Mirouze N, Pietrement O, et al. (2007) A key presynaptic role in transformation for a widespread bacterial protein: DprA conveys incoming ssDNA to RecA. Cell 130: 824–836.
[13]
Saunders CW, Guild WR (1981) Monomer plasmid DNA transforms Streptococcus pneumoniae. Mol Gen Genet 181: 57–62.
[14]
Hengge R (2009) Proteolysis of sigmaS (RpoS) and the general stress response in Escherichia coli. Res Microbiol 160: 667–676.
[15]
Battesti A, Majdalani N, Gottesman S (2011) The RpoS-mediated general stress response in Escherichia coli. Annu Rev Microbiol 65: 189–213.
[16]
Lacour S, Landini P (2004) SigmaS-dependent gene expression at the onset of stationary phase in Escherichia coli: function of sigmaS-dependent genes and identification of their promoter sequences. J Bacteriol 186: 7186–7195.
[17]
Patten CL, Kirchhof MG, Schertzberg MR, Morton RA, Schellhorn HE (2004) Microarray analysis of RpoS-mediated gene expression in Escherichia coli K-12. Mol Genet Genomics 272: 580–591.
[18]
Vijayakumar SR, Kirchhof MG, Patten CL, Schellhorn HE (2004) RpoS-regulated genes of Escherichia coli identified by random lacZ fusion mutagenesis. J Bacteriol 186: 8499–8507.
[19]
Weber H, Polen T, Heuveling J, Wendisch VF, Hengge R (2005) Genome-wide analysis of the general stress response network in Escherichia coli: sigmaS-dependent genes, promoters, and sigma factor selectivity. J Bacteriol 187: 1591–1603.
[20]
Sledjeski DD, Gupta A, Gottesman S (1996) The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli. EMBO J 15: 3993–4000.
[21]
Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, et al. (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2: 2006 0008.
[22]
Becker G, Hengge-Aronis R (2001) What makes an Escherichia coli promoter sigmaS dependent? Role of the ？13/？14 nucleotide promoter positions and region 2.5 of sigmaS. Mol Microbiol 39: 1153–1165.
[23]
Smith HO, Danner DB, Deich RA (1981) Genetic transformation. Annu Rev Biochem 50: 41–68.
[24]
Claverys JP, Prudhomme M, Martin B (2006) Induction of competence regulons as a general response to stress in gram-positive bacteria. Annu Rev Microbiol 60: 451–475.
[25]
Dubnau D (1991) Genetic competence in Bacillus subtilis. Microbiol Rev 55: 395–424.
Redfield RJ, Cameron AD, Qian Q, Hinds J, Ali TR, et al. (2005) A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae. J Mol Biol 347: 735–747.
[28]
Etchuuya R, Ito M, Kitano S, Shigi F, Sobue R, et al. (2011) Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone. PLoS One 6: e16355.
[29]
Havarstein LS, Coomaraswamy G, Morrison DA (1995) An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae. Proc Natl Acad Sci USA 92: 11140–11144.
[30]
Pestova EV, Havarstein LS, Morrison DA (1996) Regulation of competence for genetic transformation in Streptococcus pneumoniae by an auto-induced peptide pheromone and a two-component regulatory system. Mol Microbiol 21: 853–862.
Wang X, Li M, Yan Q, Chen X, Geng J, et al. (2007) Across genus plasmid transformation between Bacillus subtilis and Escherichia coli and the effect of Escherichia coli on the transforming ability of free plasmid DNA. Curr Microbiol 54: 450–456.
[33]
Zhang Y, Li F, Sun D, Liu J, Liu N, et al. (2011) Molecular analysis shows differential expression of R-spondin1 in zebrafish (Danio rerio) gonads. Mol Biol Rep 38: 275–282.
[34]
Finkel SE, Kolter R (2001) DNA as a nutrient: novel role for bacterial competence gene homologs. J Bacteriol 183: 6288–6293.
[35]
Sauvonnet N, Gounon P, Pugsley AP (2000) PpdD type IV pilin of Escherichia coli K-12 can be assembled into pili in Pseudomonas aeruginosa. J Bacteriol 182: 848–854.
[36]
Mulvey MR, Loewen PC (1989) Nucleotide sequence of katF of Escherichia coli suggests KatF protein is a novel sigma transcription factor. Nucleic Acids Res 17: 9979–9991.
[37]
Subbarayan PR, Sarkar M (2004) A comparative study of variation in codon 33 of the rpoS gene in Escherichia coli K12 stocks: implications for the synthesis of sigmaS. Mol Genet Genomics 270: 533–538.
[38]
Becker G, Klauck E, Hengge-Aronis R (1999) Regulation of RpoS proteolysis in Escherichia coli: the response regulator RssB is a recognition factor that interacts with the turnover element in RpoS. Proc Natl Acad Sci USA 96: 6439–6444.
