Leucine‐responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli

The Escherichia coli operons daa and sfa encode F1845 and S pill, respectively. In this paper we show that the expression of these operons is under phase variation control at a transcriptional level. The transcription of both operons is dependent on the global regulator leucine‐responsive regulatory protein (Lrp) and deoxyadenosine methylase (Dam). Lrp is required for methylation protection of two GATC sites located within conserved DNA sequences in the regulatory regions of these operons. These GATC sites are differentially methylated, establishing a methylation pattern which is characteristic of either the phase ON or phase OFF state. We also show that Lrp binds to the daa and sfa regulatory regions and that this binding is modulated by the methylation of the GATC sites. These results indicate that the phase variation of the daa and sfa operons is regulated by a mechanism involving differential binding of Lrp owing to methylation of GATC sites in the regulatory region, which is similar to the mechanism that controls phase variation of the pap operon.

[1]  L. Blyn,et al.  Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli , 1991, Journal of bacteriology.

[2]  J. Calvo,et al.  Lrp, a global regulatory protein of Escherichia coli, binds co-operatively to multiple sites and activates transcription of ilvIH. , 1993, Journal of molecular biology.

[3]  S. Ringquist,et al.  The Escherichia coli chromosome contains specific, unmethylated dam and dcm sites. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[4]  B. Nowicki,et al.  Role of type 1 and S fimbriae in the pathogenesis of Escherichia coli O18:K1 bacteremia and meningitis in the infant rat , 1988, Infection and immunity.

[5]  L. Blyn,et al.  Regulation of pap pilin phase variation by a mechanism involving differential dam methylation states. , 1990, The EMBO journal.

[6]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[7]  L. Blyn,et al.  Phase‐variation of pyelonephritis‐associated pili in Escherichia coli: evidence for transcriptional regulation. , 1989, The EMBO journal.

[8]  S. Moseley,et al.  Transcriptional organization of the F1845 fimbrial adhesin determinant of Escherichia coli , 1993, Molecular microbiology.

[9]  M. Casadaban,et al.  Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. , 1976, Journal of molecular biology.

[10]  R. Simons,et al.  Improved single and multicopy lac-based cloning vectors for protein and operon fusions. , 1987, Gene.

[11]  J. Calvo,et al.  Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher‐order nucleoprotein structure. , 1993, The EMBO journal.

[12]  R. D'ari,et al.  The leucine-responsive regulatory protein: more than a regulator? , 1993, Trends in biochemical sciences.

[13]  E. Southern.  Detection of specific sequences among DNA fragments separated by gel electrophoresis. , 1975, Journal of molecular biology.

[14]  J. Calvo,et al.  The ilvIH operon of Escherichia coli is positively regulated , 1990, Journal of bacteriology.

[15]  G. Church,et al.  A whole genome approach to in vivo DNA-protein interactions in E. coli , 1992, Nature.

[16]  S. Moseley,et al.  Molecular characterization of a fimbrial adhesin, F1845, mediating diffuse adherence of diarrhea-associated Escherichia coli to HEp-2 cells , 1989, Journal of bacteriology.

[17]  H. Sambrook.  Molecular cloning : a laboratory manual. Cold Spring Harbor, NY , 1989 .

[18]  C. Richardson,et al.  A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Ezio Ricca,et al.  Organization of Lrp‐binding sites upstream of ilvlH in Salmonella typhimurium , 1993, Molecular microbiology.

[20]  F. Mooi,et al.  K88ab gene of Escherichia coli encodes a fimbria-like protein distinct from the K88ab fimbrial adhesin , 1984, Journal of bacteriology.

[21]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[22]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[23]  J. Hacker,et al.  Complete genetic organization and functional aspects of the Escherichia coli S fimbrial adhesion determinant: nucleotide sequence of the genes sfa B, C, D, E, F. , 1990, Microbial pathogenesis.

[24]  M. Friedrich,et al.  Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbriai biosynthetic genes , 1993, Molecular microbiology.

[25]  R. D'ari,et al.  The leucine-Lrp regulon in E. coli: A global response in search of a raison d'Être , 1992, Cell.

[26]  M. W. Woude,et al.  Evidence for global regulatory control of pilus expression in Escherichia coli by Lrp and DNA methylation: model building based on analysis of pap , 1992, Molecular microbiology.

[27]  M. S. McClain,et al.  Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12 , 1993, Journal of bacteriology.

[28]  G. Nossal,et al.  The molecular and cellular basis of affinity maturation in the antibody response , 1992, Cell.

[29]  A. Hochschild.  Detecting cooperative protein-DNA interactions and DNA loop formation by footprinting. , 1991, Methods in enzymology.

[30]  Xiangwu Nou,et al.  Regulation of pyelonephritis‐associated pili phase‐variation in Escherichia coli: binding of the Papl and the Lrp regulatory proteins is controlled by DNA methylation , 1993, Molecular microbiology.

[31]  L. Enquist,et al.  Experiments With Gene Fusions , 1984 .

[32]  D. Galas,et al.  DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. , 1978, Nucleic acids research.

[33]  J. Calvo,et al.  Leucine-responsive regulatory protein controls the expression of both the pap and fan pili operons in Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.