Structural and Functional Analysis of the C-terminal DNA Binding Domain of the Salmonella typhimurium SPI-2 Response Regulator SsrB

Abstract

In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB Salmonella infections occur in a wide variety of vertebrate hosts and continue to be a major health problem worldwide for humans. Salmonella infection requires at least two pathogenicity islands, Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), 3 both of which encode type III secretion systems as well as secreted effectors, chaperones, and regulatory proteins (1-3). Genes located on SPI-1 are required for initial adherence to and invasion of intestinal epithelial cells (4). SPI-2 genes are required for intracellular survival, replication, and systemic infection of Salmonella (5, 6). SPI-2 consists of a 40-kb region located at 31 centisomes on the chromosome and contains ϳ32 genes (7). SPI-2 genes are organized into functional clusters encoding regulatory, structural genes, effectors, and chaperones. To date 10 promoters of SPI-2 genes and SPI-2 co-regulated genes have been identified (8 -10). These promoters, located upstream of the ssrA, ssrB, ssaB, sseA, ssaG, ssaM, sseI, srfN, sifA, and sifB genes, transcribe genes either individually or in large operons. Transcription from these promoters is activated by the SPI-2 encoded two-component signal transduction system, SsrA/SsrB (8, 11-13).Two-component signal transduction systems regulate gene expression in response to specific environmental signals (for review see Ref. 14). These systems represent the major paradigm for signal transduction in prokaryotes. They are frequently involved in regulating expression of virulence genes in pathogenic bacteria and are also present in the archaea, lower eukaryotes, and plants. In its simplest form, a two-component system contains a sensor kinase, often a membrane protein that functions in trans-membrane signaling, and a response regulator, usually a DNA-binding protein that regulates transcription. In Salmonella enterica serovar typhimurium, the SsrA/SsrB tw...

Salmonella pathogenicity island 2 expression negatively controlled by EIIA Ntr –SsrB interaction is required for Salmonella virulence

“…1 B and C). Consistent with previous reports (3,6,23), deletion of the ssrB gene markedly impaired transcriptional activation of the ssaG gene at acidic pH (i.e., 11-fold reduction; Fig. 2A).…”

“…EMSA experiments were performed as described previously (23,40). DNA fragments corresponding to the ssaG promoter were amplified by PCR using 32 P-labeled primer EMSA-ssaG-F1 (5′-GGTAGTTTGGGACTACAGCCT-CATTTA-3′) and primer EMSA-ssaG-R1 (5′-AATCATCGATTCTGGGTTGAG-CAAATC-3′) with wild-type Salmonella chromosomal DNA as a template.…”

Salmonella enterica Response Regulator SsrB Relieves H-NS Silencing by Displacing H-NS Bound in Polymerization Mode and Directly Activates Transcription

“…The presence of hypersensitive sites (23), our experiments with magnetic tweezers (Fig. 4), our recent DNA binding studies with SsrB C (35), and the requirement for supercoiled templates for in vitro transcription (Fig. 3) all indicate that SsrB binding distorts and/or bends the DNA both upstream and downstream of its binding sites.…”

Essentiality of DevR/DosR Interaction with SigA for the Dormancy Survival Program in Mycobacterium tuberculosis

“…DevR phosphorylation by phosphotransfer from the sensor kinase DevS 201 (cytoplasmic C-terminal fragment of DevS containing 201 amino acids) was carried out as described previously (25). Briefly, DevS 201 (final concentration, 7.5 M) was incubated in buffer containing 50 mM Tris-Cl (pH 8.0), 50 mM KCl, 10 mM MgCl 2 , 500 M ATP, and 0.1 Ci [␥- 32 P]ATP at 25°C for 60 min. DevR protein (WT or mutant E154A or E178A, 5 M) was added to the reaction mix and incubated for 0.5 and 1 min.…”