title: Ler interdomain linker is essential for anti-silencing activity in enteropathogenic Escherichia coli.
abstract: Enteropathogenic Escherichia coli (EPEC) expresses a type III secretion system (T3SS) required for pathogenesis. Regulation of the genes encoding the T3SS is complex; two major regulators control transcription, the silencer H-NS, and the related H-NS-like protein Ler. Our laboratory is interested in understanding the molecular differences that distinguish the anti-silencer Ler from H-NS, and how Ler differentially regulates EPEC virulence genes. Here, we demonstrate that mutated Ler proteins either containing H-NS alpha-helices 1 and 2, missing from Ler, or truncated for the 11 aa C-terminal extension compared with the related H-NS protein, did not appreciably alter Ler function. In contrast, mutating the proline at position 92 of Ler, in the conserved C-terminal DNA binding motif, eliminated Ler activity. Inserting 11 H-NS-specific amino acids, 11 alanines or 6 alanines into the Ler linker severely impaired the ability of Ler to increase LEE5 transcription. To extend our analysis, we constructed six chimeric proteins containing the N terminus, linker region or C terminus of Ler in different combinations with the complementary domains of H-NS, and monitored their in vivo activities. Replacing the Ler linker domain with that of H-NS, or replacing the Ler C-terminal, DNA binding domain with that of H-NS eliminated the ability of Ler to increase transcription at the LEE5 promoter. Thus, the linker and C-terminal domains of Ler and H-NS are not functionally equivalent. Conversely, replacing the H-NS linker region with that of Ler caused increased transcription at LEE5 in a strain deleted for hns. In summary, the interdomain linker specific to Ler is necessary for anti-silencing activity in EPEC.
authors.affiliation: Biology Department, Reed College, Portland, OR 97202, USA.
authors.forename: Jay L
authors.initials: JL
authors.lastname: Mellies
authors.affiliation: University of Illinois Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Fredrick J
authors.initials: FJ
authors.lastname: Larabee
authors.affiliation: Johns Hopkins University, Baltimore, MD 21202, USA.
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Zarr
authors.affiliation: Oregon Health Sciences University, Portland, OR 97202, USA.
authors.forename: Katy L
authors.initials: KL
authors.lastname: Horback
authors.affiliation: Biology Department, Reed College, Portland, OR 97202, USA.
authors.forename: Emily
authors.initials: E
authors.lastname: Lorenzen
authors.affiliation: Biology Department, Reed College, Portland, OR 97202, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mavor
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054308:Enteropathogenic Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial; D020868:Gene Silencing / Q000187:drug effects; D008969:Molecular Sequence Data; D009154:Mutation; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C031908:H-NS protein, bacteria; C465556:Ler protein, E coli; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D015534:Trans-Activators