YscO of Yersinia pestis is a mobile core component of the Yop secretion system

Abstract

The Yersinia pestis low-Ca2+ response stimulon is responsible for the temperature- and Ca2+-regulated expression and secretion of plasmid pCD1-encoded antihost proteins (V antigen and Yops). We have previously shown that lcrD, yscC, yscD, yscG, and yscR encode proteins that are essential for high-level expression and secretion of V antigen and Yops at 37 degreesC in the absence of Ca2+. In this study, we characterized yscO of the Yop secretion (ysc) operon that contains yscN through yscU by determining the localization of its gene product and the phenotype of an in-frame deletion. The yscO mutant grew and expressed the same levels of Yops as the parent at 37 degreesC in the presence of Ca2+. In the absence of Ca2+, the mutant grew independently of Ca2+, expressed only basal levels of V antigen and Yops, and failed to secrete these. These defects could be partially complemented by providing yscO in trans in the yscO mutant. Overexpression of YopM and V antigen in the mutant failed to restore the export of either protein, showing that the mutation had a direct effect on secretion. These results indicated that the yscO gene product is required for high-level expression and secretion of V antigen and Yops. YscO was found by immunoblot analysis in the soluble and membrane fractions of bacteria growing at 37 degreesC irrespective of the presence of Ca2+ and in the culture medium in the absence of Ca2+. YscO is the only mobile protein identified so far in the Yersinia species that is required for secretion of V antigen and Yops.

FIG. 1

Physical and genetic map of the region of pCD1 that encompasses yscO and yscP. (A) Coding regions for yscO and yscP and parts of yscN and yscQ carried on pYscOP.2, as well as selected restriction sites. Asterisks denote restriction sites introduced by site-directed mutagenesis. (B) Regions included in selected clones.

FIG. 2

Growth of Y. pestis KIM5-3001 (wt); KIM5-3001.16, the yscO mutant (ΔyscO); and the mutant carrying yscO in trans (ΔyscO/O) and (ΔyscO/OP). Y. pestis strains were grown at 37°C in the presence or absence of Ca²⁺ in TMH defined medium. The temperature was shifted from 26 to 37°C (temperature shifts are denoted by arrowheads). Symbols: •, +Ca²⁺; ▩, −Ca²⁺.

FIG. 3

Secretion profile of ΔyscO Y. pestis grown in the presence or absence of Ca²⁺. Shown is an immunoblot analysis of proteins expressed and secreted from Y. pestis KIM5-3001 (wt); KIM5-3001.16, the yscO mutant (ΔyscO); and the mutant carrying pYscO.2 or pYscOP.2 in trans (ΔyscO/O and ΔyscO/OP). Bacteria were grown in TMH with (+) or without (−) Ca²⁺, and proteins from bacterial fractions were separated by SDS-PAGE (A, B, and D, 12% [wt/vol] acrylamide; C, 15% [wt/vol] acrylamide). Proteins from soluble (s), membrane (m), whole-cell (c), and culture medium (e) fractions were visualized with polyclonal antibodies specific to YopE, YopM, V antigen, and YscP and with antibody raised to a mixture of extracellular Yersinia proteins (ECP). The secondary antibody used was conjugated to alkaline phosphatase. Arrows denote the positions of proteins. (A) YopE was visualized with mouse anti-YopE (YopE and its Pla-generated degradation products are enclosed in brackets). (B) YopE was visualized with rabbit anti-YopE. (C) The complexity of the protein pattern reflects degradation products from multiple Yops due to the Pla protease. (D) YscP was visualized with antibody to a GST-YscP fusion protein. Molecular masses (in kilodaltons) of prestained molecular mass standards (Bio-Rad) are denoted to the right in panels A and C.

FIG. 4

Immunoblot analysis of YopM, V antigen, and HTV in the soluble (s), membrane (m), and culture medium (e) fractions from Y. pestis KIM5-3001 (wt) and KIM5-3001.16 (ΔyscO) with (+) or without (−) pHTV or pTRCM.2. The Y. pestis strains were grown at 37°C in the absence of Ca²⁺. Expression of HTV from pHTV and YopM from pTRCM.2 was induced by the addition of IPTG to 1 mM 5 h prior to harvest. The proteins were separated by SDS-PAGE (12% [wt/vol] acrylamide). Polyclonal antibody to HTV or YopM was used to detect HTV and V antigen, and YopM, respectively. Secondary antibody was conjugated to alkaline phosphatase. In the lower panel, YopM was detected as two closely migrating species.

FIG. 5

Detection of LcrD and YscD in ΔyscO Y. pestis. Y. pestis strains were grown in TMH at 37°C in the absence of Ca²⁺. The proteins in membrane fractions were separated by SDS-PAGE (12% [wt/vol] acrylamide), transferred to Immobilon P, and analyzed by immunoanalysis with antibodies specific to LcrD or YscD. (A) Y. pestis KIM5-3001 (wt) and Y. pestis KIM5-3001.16 (ΔyscO) were analyzed with anti-LcrD. (B) Y. pestis KIM8-3002 (wt Pla⁻) and Y. pestis KIM8-3002.3 (ΔyscO Pla⁻) were analyzed with anti-YscD. The secondary antibody used was conjugated to alkaline phosphatase. Arrows indicate each protein.

FIG. 6

Localization of YscO by immunoblot analysis. Y. pestis KIM5-3001 (wt), KIM5-3001.16 (ΔyscO), KIM8-3002 (wt Pla⁻), and KIM8-3002.3 (ΔyscO Pla⁻) are shown. Strains carrying plasmids are denoted /O or /OP for pYscO.2 or pYscOP.2, respectively. Bacteria were grown at 37°C in TMH with (+) or without (−) Ca²⁺ for 5 h prior to harvest. Proteins from bacterial fractions were separated by SDS-PAGE (15% [wt/vol] acrylamide). Antibody raised against a GST-YscO fusion protein was used to detect YscO in soluble (s), total membrane (m), whole-cell (wc), or culture medium (e) proteins. All panels were analyzed with alkaline phosphatase. Arrows indicate the various proteins that were potential candidates for YscO.