PMID:17892462

Citation	Lithgow, JK, Haider, F, Roberts, IS and Green, J (2007) Alternate SlyA and H-NS nucleoprotein complexes control hlyE expression in Escherichia coli K-12. Mol. Microbiol. 66:685-98
Abstract	Haemolysin E is a cytolytic pore-forming toxin found in several Escherichia coli and Salmonella enterica strains. Expression of hlyE is repressed by the global regulator H-NS (histone-like nucleoid structuring protein), but can be activated by the regulator SlyA. Expression of a chromosomal hlyE-lacZ fusion in an E. coli slyA mutant was reduced to 60% of the wild-type level confirming a positive role for SlyA. DNase I footprint analysis revealed the presence of two separate SlyA binding sites, one located upstream, the other downstream of the hlyE transcriptional start site. These sites overlap AT-rich H-NS binding sites. Footprint and gel shift data showed that whereas H-NS prevented binding of RNA polymerase (RNAP) at the hlyE promoter (PhlyE), SlyA allowed binding of RNAP, but inhibited binding of H-NS. Accordingly, in vitro transcription analyses showed that addition of SlyA protein relieved H-NS-mediated repression of hlyE. Based on these observations a model for SlyA/H-NS regulation of hlyE expression is proposed in which the relative concentrations of SlyA and H-NS govern the nature of the nucleoprotein complexes formed at PhlyE. When H-NS is dominant RNAP binding is inhibited and hlyE expression is silenced; when SlyA is dominant H-NS binding is inhibited allowing RNAP access to the promoter facilitating hlyE transcription.
Links	PubMed PMC2156107 Online version:10.1111/j.1365-2958.2007.05950.x
Keywords	Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Electrophoretic Mobility Shift Assay; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Gene Expression Regulation, Bacterial; Hemolysin Proteins/genetics; Hemolysin Proteins/metabolism; Models, Molecular; Molecular Sequence Data; Mutation; Promoter Regions, Genetic/genetics; Protein Binding; Transcription Factors/genetics; Transcription Factors/metabolism; Transcription, Genetic
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
ECOLI:SLYA		GO:0045893: positive regulation of transcription, DNA-templated	ECO:0001204: in vitro transcription assay evidence used in manual assertion		P		Figure 5 shows the results of an In vitro transcription assay in which 20 ng of linearized pGS1886 DNA was incubated with RNA polymerase (RNAP) and differing amounts of H-NS and SlyA. H-NS is shown to inhibit transcription of hlyE in Figure 5C through use of a PCR-amplified DNA fragment extending from −474 to +222 that includes the hlyE promoter, seemingly completely inhibiting it at H-NS concentration of 4.0 μM. Figure 5D shows that transcription can be restored by increasing concentrations of SlyA through using the same In vitro transcription assay process and 2.0 μM of H-NS. At about 1.0 μM of SlyA, hlyE appears to be transcribed at similar levels to the control, containing no H-NS. From this it can be concluded that H-NS represses hlyE transcription, and SlyA derepresses hlyE transcription. Therefore, SlyA positively regulates transcription of hlyE.	complete CACAO 13659

References

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