NDLI: A mutant of Salmonella enterica serovar Typhimurium RNA polymerase extracytoplasmic stress response sigma factor σE with altered promoter specificity

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A mutant of Salmonella enterica serovar Typhimurium RNA polymerase extracytoplasmic stress response sigma factor σE with altered promoter specificity

Content Provider	Springer Nature Link
Author	Rezuchova, Bronislava Skovierova, Henrieta Homerova, Dagmar Roberts, Mark Kormanec, Jan
Copyright Year	2009
Abstract	The alternative sigma factor σE is critical for envelope stress response and plays a role in pathogenicity of a variety of different bacteria. We previously identified several critical nucleotides in the Salmonella enterica serovar Typhimurium (S. Typhimurium) σE-dependent rpoEp3 promoter that corresponded to the most conserved nucleotides in the σE consensus sequence of the −10 and −35 promoter elements. In the present study, we exploited a previously established Escherichia coli (E. coli) two-plasmid system with an error-prone PCR mutagenesis to identify mutants in the rpoE gene that suppress the mutation of the most conserved residue A-30G of the rpoEp3 promoter. This analysis identified amino-acid changes in the conserved arginine residue (R171G, R171C) located in the conserved region 4.2 of σE that enabled efficient recognition of the mutated rpoEp3 promoter. However, the change of this conserved arginine to alanine (R171A) resulted in an almost complete loss of σE activity. The activity of the mutant σE factors in directing transcription of the wild-type (WT) and the A-30G mutated rpoEp3 promoters was investigated by S1-nuclease mapping using RNA isolated from the E. coli two-plasmid system. In addition to suppression of the A-30G mutated rpoEp3 promoter, both mutant sigma factors (R171G, R171C) also efficiently directed transcription from the WT rpoEp3 promoter and from the rpoEp3 promoter with other mutations in the −35 element, indicating relaxed recognition of the σE-dependent promoters by both mutants. The activity of both mutant σE factors was confirmed in vivo in S. Typhimurium. In conclusion, replacement of the conserved R171 residue in σE by different amino-acid residues exhibited intriguingly different phenotypes; R171A almost completely abolished sigma factor activity, whereas R171G and R171C impart a relaxed recognition phenotype to σE.
Starting Page	119
Ending Page	129
Page Count	11
File Format	PDF
ISSN	16174615
Journal	Molecular and General Genetics MGG
Volume Number	282
Issue Number	2
e-ISSN	16174623
Language	English
Publisher	Springer-Verlag
Publisher Date	2009-05-05
Publisher Place	Berlin/Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Promoter RNA polymerase RpoE Salmonella Sigma Stress response Animal Genetics and Genomics Microbial Genetics and Genomics Cell Biology Plant Genetics & Genomics Biochemistry
Content Type	Text
Resource Type	Article
Subject	Genetics Medicine Molecular Biology

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