Influence of Na(+), dicarboxylic amino acids, and pH in modulating the low-calcium response of Yersinia pestis

Abstract

The virulence of yersiniae is promoted in part by shared approximately 70-kb plasmids (pCD in Yersinia pestis and pYV in enteropathogenic Yersinia pseudotuberculosis and Yersinia enterocolitica) that mediate a low-calcium response. This phenotype is characterized at 37 degrees C by either bacteriostasis in Ca(2+)-deficient medium with expression of pCD/pYV-encoded virulence effectors (Yops and LcrV) or vegetative growth and repression of Yops and LcrV with > or =2.5 mM Ca(2+) (Lcr(+)). Regulation of Yops and LcrV is well defined but little is known about bacteriostasis other than that Na(+) plus l-glutamate promotes prompt restriction of Y. pestis. As shown here, l-aspartate substituted for l-glutamate in this context but only Na(+) exacerbated the nutritional requirement for Ca(2+). Bacteriostasis of Y. pestis (but not enteropathogenic yersiniae) was abrupt in Ca(2+)-deficient medium at neutral to slightly alkaline pH (7.0 to 8.0), although increasing the pH to 8.5 or 9.0, especially with added Na(+) (but not l-glutamate), facilitated full-scale growth. Added l-glutamate (but not Na(+)) favored Ca(2+)-independent growth at acidic pH (5.0 to 6.5). Yops and LcrV were produced in Ca(2+)-deficient media at pH 6.5 to 9.0 regardless of the presence of added Na(+) or l-glutamate, although their expression at alkaline pH was minimal. Resting Ca(2+)-starved Lcr(+) cells of Y. pestis supplied with l-glutamate first excreted and then destroyed l-aspartate. These findings indicate that expression of Yops and LcrV is necessary but not sufficient for bacteriostasis of Ca(2+)-starved yersiniae and suggest that abrupt restriction of Y. pestis requires Na(+) and the known absence of aspartate ammonia-lyase in this species.

FIG. 1.

Growth of Yersinia enterocolitica strain WA (A), Yersinia pseudotuberculosis strain PB1/+ (B), and Yersinia pestis strain KIM (C) at 37°C in chemically defined medium containing added l-glutamate (25 mM) either with (•) or without (○) added 4.0 mM Ca²⁺ after two prior subcultures at 26°C in the same medium lacking added Ca²⁺. Parallel growth at 37°C of a third culture either lacking (▵) or containing (□) l-glutamate (25 mM) is shown after prior subculture at 26°C for two transfers without added l-glutamate or Ca²⁺. All cultures contained 100 mM Na⁺ and possessed an initial pH of 7.0.

FIG. 2.

Silver-stained preparations following SDS-PAGE of whole cultures of Yersinia enterocolitica strain WA (A), Yersinia pseudotuberculosis strain PB1/+ (B), and Yersinia pestis strain KIM (C) after subculture at 26°C in chemically defined medium containing added 25 mM l-glutamate without Ca²⁺ followed by cultivation at 37°C in the same medium either without (lane 4) or with (lane 1) 4.0 mM Ca²⁺; comparable samples of cells previously subcultured twice at 26°C without Ca²⁺ or l-glutamate and then transferred to third Ca²⁺-deficient cultures at 37°C either lacking (lane 2) or containing (lane 3) l-glutamate are shown. All cultures contained 100 mM Na⁺ and exhibited an initial pH of 7.0. Expression of YopO/YpkA was too faint for reproduction.

FIG. 3.

Growth of Yersinia pestis strain KIM at 37°C in Ca²⁺-deficient chemically defined medium containing (A) constant l-glutamate (25 mM) and 100 mM Na⁺ (○), Li⁺ (▵), K⁺ (□), or Tris⁺ (▿); a parallel control culture containing Na⁺ and Ca²⁺ (4.0 mM) is included (•). Also shown (B) is growth of Y pestis strain KIM at 37°C in Ca²⁺-deficient chemically defined medium containing constant Na⁺ (100 mM) and 25 mM l-glutamate (○), l-aspartate (▵), l-glutamine (□), or l-asparagine (▿) or no added amino acid (⋄); a parallel control culture containing Na⁺ and Ca²⁺ (4.0 mM) is included (•). The initial pH of all cultures was 6.0.

FIG. 4.

Growth of Lcr⁺ cells of Yersinia pestis strain KIM at 37°C in Ca²⁺-deficient chemically defined medium lacking added Na⁺ and l-glutamate (A), containing only 100 mM Na⁺ (B), containing only 25 mM l-glutamate (C), and containing both Na⁺ and l-glutamate (D) at an initial pH of 5.0 (•), 5.5 (○), 6.0 (▪), 6.5 (□), and 7.0 (▴). Dashed lines indicate the extent of growth in control cultures containing 4.0 mM Ca²⁺ at an initial pH of 7.0.

FIG. 5.

Same as Fig. 4 except that the cultures possessed an initial pH of 7.0 (•), 7.5 (○), 8.0 (▪), 8.5 (□), and 9.0 (▴).

FIG. 6.

Immunoblot using mouse monoclonal anti-LcrV following SDS-PAGE of whole cultures of Yersinia pestis strain KIM cultured in Ca²⁺-deficient chemically defined medium lacking both Na⁺ and l-glutamate (A), containing only 100 mM Na⁺ (B), containing only 25 mM l-glutamate (C), and containing both Na⁺ and l-glutamate (D) at an initial pH of 5.0 (lane 1), 5.5 (lane 2), 6.0 (lane 3), 6.5 (lane 4), 7.0 (lane 5), 7.5 (lane 6), 8.0 (lane 7), 8.5 (lane 8), and 9.0 (lane 9); a comparable positive control of a Ca²⁺-deficient culture containing both Na⁺ and l-glutamate is shown in lane 10.

FIG. 7.

Silver-stained preparations following SDS-PAGE of whole cultures of Yersinia pestis strain KIM cultured in Ca²⁺-deficient chemically defined medium lacking both Na⁺ and l-glutamate (A), containing only 100 mM Na⁺ (B), containing only 25 mM l-glutamate (C), and containing both Na⁺ and l-glutamate (D) at an initial pH of 5.0 (lane 2), 5.5 (lane 3), 6.0 (lane 4), 6.5 (lane 5), 7.0 (lane 6), 7.5 (lane 7), 8.0 (lane 8), 8.5 (lane 9), and 9.0 (lane 10); comparable negative (Lcr⁻ cells) and positive (Lcr⁺ cells) controls of Ca²⁺-deficient cultures containing both Na⁺ and l-glutamate are shown in lanes 1 and 11, respectively.

FIG. 8.

Catabolic destruction of l-glutamate (•) with the appearance of metabolic l-aspartate (○) by resting cells of Yersinia pestis strain KIM previously grown at 37°C in synthetic medium containing 25 mM l-glutamate and 100 mM Na⁺. (A) Lcr⁺ cells grown with 4.0 mM Ca²⁺, (B) Lcr⁺ cells grown without added Ca²⁺, (C) Lcr⁻ cells grown with 4.0 mM Ca²⁺, and (D) Lcr⁻ cells grown without added Ca²⁺.