Fig 1.
Yersinia pestis lcrV mutants with the dominant negative low-calcium response (LCR−) phenotype. (A) lcrVD27 was subjected to PCR mutagenesis, cloned under the control of the pHSG576 tac promoter, and electroporated into Y. pestis strain KIM D27. Transformants were quantified after colony formation on TBA supplemented with magnesium oxalate (MgOx) at 26°C or 37°C, at the latter temperature with selection for dominant negative LCR− mutants. Plasmids were isolated and again electroporated into Y. pestis strain KIM D27, growth of cells with the LCR− phenotype was verified, and plasmids were sequenced, which identified 18 mutations with missense mutations in codon 327 (UGA). These mutations extend the lcrV open reading frame by 22 codons (GSKQGGSVSPFFYQYCEYLRP*). (B) Y. pestis strains KIM D27 (wild-type [WT] lcrV) or KLD29 (ΔlcrV) harboring either no plasmid or plcrVD27, plcrV*327W, and plcrV*327R were cultured at 37°C in TMH medium supplemented with either 0 mM or 6.25 mM CaCl2. Growth was recorded as an increase in the optical density at 600 nm (OD600). pyopNF234S, which has been reported to cause a dominant negative LCR− phenotype (34), was used as a control.