Cnu (an OriC-binding nucleoid protein) associates with H-NS. A variant of Cnu was identified as a key factor for filamentous growth of a wild-type Escherichia coli strain at 37°C. This variant (CnuK9E) bears a substitution of a lysine to glutamic acid, causing a charge reversal in the first helix. The temperature-dependent filamentous growth of E. coli bearing CnuK9E could be reversed by either lowering the temperature to 25°C or lowering the CnuK9E concentration in the cell. Gene expression analysis suggested that downregulation of dicA by CnuK9E causes a burst of dicB transcription, which, in turn, elicits filamentous growth. In vivo assays indicated that DicA transcriptionally activates its own gene, by binding to its operator in a temperature-dependent manner. The antagonizing effect of CnuK9E with H-NS on DNA-binding activity of DicA was stronger at 37°C, presumably due to the lower operator binding of DicA at 37°C. These data suggest that the temperature-dependent negative effect of CnuK9E on DicA binding plays a major role in filamentous growth. The C-terminus of DicA shows significant amino acid sequence similarity to the DNA-binding domains of RovA and SlyA, regulators of pathogenic genes in Yersinia and Salmonella, respectively, which also show better DNA-binding activity at 25°C.
References
[1]
Kim MS, Bae SH, Yun SH, Lee HJ, Ji SC, et al. (2005) Cnu, a novel oriC-binding protein of Escherichia coli. J Bacteriol 187: 6998–7008.
[2]
Nieto JM, Mourino M, Balsalobre C, Madrid C, Prenafeta A, et al. (1997) Construction of a double hha hns mutant of Escherichia coli: effect on DNA supercoiling and alpha-haemolysin production. FEMS Microbiol Lett 155: 39–44.
[3]
Paytubi S, Madrid C, Forns N, Nieto JM, Balsalobre C, et al. (2004) YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA. Mol Microbiol 54: 251–263.
[4]
Nieto JM, Madrid C, Miquelay E, Parra JL, Rodriguez S, et al. (2002) Evidence for direct protein-protein interaction between members of the enterobacterial Hha/YmoA and H-NS families of proteins. J Bacteriol 184: 629–635.
[5]
Madrid C, Garcia J, Pons M, Juarez A (2007) Molecular evolution of the H-NS protein: interaction with Hha-like proteins is restricted to enterobacteriaceae. Journal of bacteriology 189: 265–268.
[6]
Lucht JM, Dersch P, Kempf B, Bremer E (1994) Interactions of the nucleoid-associated DNA-binding protein H-NS with the regulatory region of the osmotically controlled proU operon of Escherichia coli. J Biol Chem 269: 6578–6578.
[7]
Prosseda G, Falconi M, Giangrossi M, Gualerzi CO, Micheli G, et al. (2004) The virF promoter in Shigella: more than just a curved DNA stretch. Molecular microbiology 51: 523–537.
[8]
Falconi M, Colonna B, Prosseda G, Micheli G, Gualerzi CO (1998) Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. The EMBO journal 17: 7033–7043.
[9]
Bae SH, Liu D, Lim HM, Lee Y, Choi BS (2008) Structure of the nucleoid-associated protein Cnu reveals common binding sites for H-NS in Cnu and Hha. Biochemistry 47: 1993–2001.
[10]
Yee A, Chang X, Pineda-Lucena A, Wu B, Semesi A, et al. (2002) An NMR approach to structural proteomics. Proc Natl Acad Sci U S A 99: 1825–1830.
[11]
Shindo H, Iwaki T, Ieda R, Kurumizaka H, Ueguchi C, et al. (1995) Solution structure of the DNA binding domain of a nucleoid-associated protein, H-NS, from Escherichia coli. FEBS letters 360: 125–131.
[12]
Esposito D, Petrovic A, Harris R, Ono S, Eccleston JF, et al. (2002) H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein. Journal of molecular biology 324: 841–850.
[13]
Bloch V, Yang Y, Margeat E, Chavanieu A, Auge MT, et al. (2003) The H-NS dimerization domain defines a new fold contributing to DNA recognition. Nature structural biology 10: 212–218.
[14]
Arold ST, Leonard PG, Parkinson GN, Ladbury JE (2010) H-NS forms a superhelical protein scaffold for DNA condensation. Proc Natl Acad Sci U S A 107: 15728–15732.
[15]
Wang WQ, Li GW, Chen CY, Xie XS, Zhuang XW (2011) Chromosome Organization by a Nucleoid-Associated Protein in Live Bacteria. Science 333: 1445–1449.
[16]
Bejar S, Bouche F, Bouche JP (1988) Cell division inhibition gene dicB is regulated by a locus similar to lambdoid bacteriophage immunity loci. Mol Gen Genet 212: 11–19.
[17]
Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, et al. (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 97: 5978–5983.
[18]
Bertani G (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. Journal of bacteriology 186: 595–600.
[19]
Lee SY, Lee HJ, Lee H, Kim S, Cho EH, et al. (1998) In vivo assay of protein-protein interactions in Hin-mediated DNA inversion. J Bacteriol 180: 5954–5960.
[20]
Ji SC, Wang X, Jeon HJ, Yun SH, Lee HJ, et al. (2010) Quantification of the galactose-operon mRNAs 5 bases different in their 5′-ends. BMB Rep 43: 474–479.
[21]
Park JW, Jung Y, Lee SJ, Jin DJ, Lee Y (2002) Alteration of stringent response of the Escherichia coli rnpB promoter by mutations in the ？35 region. Biochemical and biophysical research communications 290: 1183–1187.
[22]
Yun SH, Ji SC, Jeon HJ, Wang X, Lee Y, et al. (2012) A mutational study of Cnu reveals attractive forces between Cnu and H-NS. Mol Cells 33: 211–216.
[23]
Zhang A, Belfort M (1992) Nucleotide sequence of a newly-identified Escherichia coli gene, stpA, encoding an H-NS-like protein. Nucleic Acids Res 20: 6735.
[24]
Lutkenhaus J (2007) Assembly dynamics of the bacterial MinCDE system and spatial regulation of the Z ring. Annu Rev Biochem 76: 539–562.
[25]
Labie C, Bouche F, Bouche JP (1990) Minicell-forming mutants of Escherichia coli: suppression of both DicB- and MinD-dependent division inhibition by inactivation of the minC gene product. J Bacteriol 172: 5852–5855.
[26]
Bejar S, Bouche JP (1985) A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli. Mol Gen Genet 201: 146–150.
[27]
Bejar S, Cam K, Bouche JP (1986) Control of cell division in Escherichia coli. DNA sequence of dicA and of a second gene complementing mutation dicA1, dicC. Nucleic Acids Res 14: 6821–6833.
[28]
Cam K, Bejar S, Gil D, Bouche JP (1988) Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start. Nucleic acids research 16: 6327–6338.
[29]
Labie C, Bouche F, Bouche JP (1989) Isolation and mapping of Escherichia coli mutations conferring resistance to division inhibition protein DicB. Journal of bacteriology 171: 4315–4319.
[30]
Tetart F, Bouche JP (1992) Regulation of the expression of the cell-cycle gene ftsZ by DicF antisense RNA. Division does not require a fixed number of FtsZ molecules. Mol Microbiol 6: 615–620.
[31]
Lim HM, Lee HJ, Roy S, Adhya S (2001) A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding. Proceedings of the National Academy of Sciences of the United States of America 98: 14849–14852.
[32]
Johnson AD, Poteete AR, Lauer G, Sauer RT, Ackers GK, et al. (1981) lambda Repressor and cro–components of an efficient molecular switch. Nature 294: 217–223.
[33]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research 25: 3389–3402.
[34]
Wu RY, Zhang RG, Zagnitko O, Dementieva I, Maltzev N, et al. (2003) Crystal structure of Enterococcus faecalis SlyA-like transcriptional factor. The Journal of biological chemistry 278: 20240–20244.
[35]
Tran HJ, Heroven AK, Winkler L, Spreter T, Beatrix B, et al. (2005) Analysis of RovA, a transcriptional regulator of Yersinia pseudotuberculosis virulence that acts through antirepression and direct transcriptional activation. The Journal of biological chemistry 280: 42423–42432.
[36]
Dolan KT, Duguid EM, He C (2011) Crystal structures of SlyA protein, a master virulence regulator of Salmonella, in free and DNA-bound states. The Journal of biological chemistry 286: 22178–22185.
[37]
Ellison DW, Miller VL (2006) Regulation of virulence by members of the MarR/SlyA family. Current opinion in microbiology 9: 153–159.
[38]
Herbst K, Bujara M, Heroven AK, Opitz W, Weichert M, et al. (2009) Intrinsic thermal sensing controls proteolysis of Yersinia virulence regulator RovA. PLoS pathogens 5: e1000435.
[39]
Heroven AK, Nagel G, Tran HJ, Parr S, Dersch P (2004) RovA is autoregulated and antagonizes H-NS-mediated silencing of invasin and rovA expression in Yersinia pseudotuberculosis. Molecular microbiology 53: 871–888.
[40]
Perez JC, Latifi T, Groisman EA (2008) Overcoming H-NS-mediated transcriptional silencing of horizontally acquired genes by the PhoP and SlyA proteins in Salmonella enterica. The Journal of biological chemistry 283: 10773–10783.
