Regulation of the Ysa type III secretion system of Yersinia enterocolitica by YsaE/SycB and YsrS/YsrR

Abstract

Yersinia enterocolitica biovar 1B contains two type III secretion systems (TTSSs), the plasmid-encoded Ysc-Yop system and the chromosomally encoded Ysa-Ysp system. Proteins secreted from the Ysa TTSS (Ysps) have only been detected in vitro when cells are cultured at 26 degrees C in a high-NaCl medium. However, the exact role of the Ysa TTSS is unclear. Thus, investigations into the regulation of this system may help elucidate the role of the Ysps during the life cycle of Y. enterocolitica. Here we present evidence that the AraC-like regulator YsaE acts together with the chaperone SycB to regulate transcription of the sycByspBCDA operon, a phenomenon similar to that seen in the closely related Salmonella SPI-1 and Shigella flexneri Mxi-Spa-Ipa TTSSs. Deletion of either sycB or ysaE results in a twofold reduction in the activity of a sycB-lacZ fusion compared to the wild type. In a reconstituted Escherichia coli system, transcription of sycB was activated sixfold only when both YsaE and SycB were present, demonstrating that they are necessary for activation. ysrR and ysrS are located near the ysa genes and encode a putative two-component regulatory system. Mutations in either gene indicated that both YsrR and YsrS were required for secretion of Ysps. In addition, transcription from sycB-lacZ and ysaE-lacZ fusions was decreased 6.5- and 25-fold, respectively, in the ysrS mutant compared to the wild type. Furthermore, in the absence of NaCl, the activity of ysaE-lacZ was reduced 25-fold in the wild-type and DeltaysrS strains, indicating that YsrS is probably required for the salt-dependent expression of the ysa locus. These results suggest that the putative two-component system YsrRS may be a key element in the regulatory cascade for the Ysa TTSS.

FIG. 1.

Organization of the ysa operon. Black arrows indicate putative apparatus genes, gray arrows indicate genes encoding regulators, and white arrows indicate genes encoding secreted proteins. Speckled genes and those labeled with numbers are unique. Open reading frames 8 and 9/10 are believed to encode proteins that are part of the TTSS apparatus (19) and are therefore colored black. sycB and sicA have dual functions as chaperone and regulator. Dotted lines indicate homologous genes between the two systems (only a portion of SPI-1 is shown). The intergenic region between ysaU and sycB is 96 bp; the analogous region in SPI-1 (spaS to sicA) is 137 bp. No terminator structure was predicted to exist in a 300-bp region that includes the ysaU-sycB intergenic region with Mfold (http://www.bioinfo.rpi.edu/applications/mfold/).

FIG. 2.

Strains lacking sycB or ysaE do not secrete Ysps. Proteins were precipitated from culture supernatants and separated by SDS-10% polyacrylamide gel electrophoresis as described in the text. (A) Silver-stained gel showing loss of Ysps from the culture supernatants of ΔsycB and ΔysaE strains. ΔysaC carries a disruption in the ysaC gene and is thought to have a defective apparatus. The culture equivalent of 2 OD units was loaded in each lane. (B) Complementation of the mutant strains as determined by Western blotting with anti-YspC antibody. YspC is indicated by the arrow. The culture equivalent of 1 OD unit was loaded in each lane.

FIG. 3.

Transcription of sycB originates at a promoter upstream of ysaU. (A) Schematic of the ysa locus encompassing the sycB promoter region. The approximate locations of primers KW114 and KW115 are indicated. (B) RT-PCR was performed with primers KW114 and KW115 and cDNA generated from 2 μg of total RNA that was isolated from JB580v as described in the text; 20% of the reaction was loaded on a 1.2% agarose gel and stained with ethidium bromide. Templates for the PCR are listed above each lane and were as follows: gDNA, genomic DNA; +RT, products from cDNA synthesis reaction with Superscript III added; no RT, products from cDNA synthesis reaction with no Superscript III; nt, no DNA or cDNA added.

FIG. 4.

Strains lacking ysrS or ysrR do not secrete Ysps. Proteins were precipitated from culture supernatants and separated by SDS-10% polyacrylamide gel electrophoresis as described in the text. (A) Silver-stained gel showing the loss of all Ysps from the culture supernatants of the ΔysrS and ΔysrR strains. The culture equivalent of 2 OD units was loaded in each lane. (B) Complementation of the mutant strains as determined by Western blotting with anti-YspC antibody. YspC is indicated by the arrow. The culture equivalent of 1 OD unit was loaded in each lane.

FIG. 5.

Model for activation of the ysaE and sycB promoters. YsrS senses NaCl in the culture medium by an unknown mechanism and initiates a phosphorelay that leads to phosphorylation of YsrR. The activated YsrR then stimulates transcription of the ysaE promoter, either directly or indirectly. Once sufficient levels of YsaE and SycB have accumulated, they stimulate transcription of the sycB promoter.