Mutational analysis of the Yersinia enterocolitica virC operon: characterization of yscE, F, G, I, J, K required for Yop secretion and yscH encoding YopR

Abstract

Pathogenic yersiniae secrete the Yop anti-host proteins using a type-III secretion pathway. The components of the secretion machinery are encoded by three loci on the pYV plasmid: virA, virB, and virC. In this paper we describe the characterization of eight non-polar mutants of the virC locus, constructed by allelic exchange. The yscE, FG, I, J and K mutants were defective in Yop secretion and independent of Ca2+ (CI) for their growth at 37 degrees C. Substitution of the 12 N-terminal amino-acid residues of YscF impaired secretion of YopB and YopD only and led also to a CI phenotype. The culture supernatant of the yscH mutant contained all the Yops except the 18 kDa YopR. Complementation experiments and an immunoblot analysis confirmed that YopR is encoded by the yscH gene. The LD50 for the mouse of the yscH mutant was 10-fold higher than that of the parental strain indicating that YopR is involved in pathogenesis. The phenotype of the yscM mutant was similar to that of the wild-type strain. However, overproduction of YscM from a multicopy plasmid in wild-type Yersinia enterocolitica prevented Yop secretion and synthesis. A hybrid YopH-LacZ' protein, encoded by a gene transcribed from the lac promoter, was secreted by a strain overexpressing YscM, showing that the secretion machinery was still functional. These results indicate that YscM plays a role in the feedback inhibition of Yop synthesis when secretion is compromised by acting as a negative regulator of Yop synthesis.

YscM1 and YscM2, two Yersinia enterocolitica proteins causing downregulation of yop transcription

“…Overexpression of yscM inhibits Yop synthesis, like overexpression of lcrQ, but the yscM mutant, unlike the lcrQ mutant, does not show any sign of deregulation of Yop synthesis (Allaoui et al, 1995).…”

“…The yscM1 allele, mutated by insertion of the aphA-3 cassette (Allaoui et al, 1995), was introduced by allelic exchange in pSI2271, a pYV plasmid already mutated in yscM2. (pAA125) yopH-cat þþ , yscM þþþ , all pYV genes (*) 0 a. Chloramphenicol acetyltransferase (CAT) activity was measured in extracts of bacteria grown for 2 h at 25ЊC and for 2 h at 37ЊC and expressed in units per optical density of bacterial suspension at 600 nm.…”

“…To test this hypothesis, we constructed a strain mutated in the two copies of yscM as well as in yscN, a gene governing an early step of Yop secretion . We constructed the triple yscN, yscM1, yscM2 mutant called KNG22703 (pSI2274) by the introduction of the yscM2 mutated allele into the yscN, yscM1 mutant (pAA213) constructed previously (Allaoui et al, 1995). We monitored the amount of intrabacterial YopD and YopE in this triple mutant by Western blotting and observed an accumulation (Fig.…”

Functional analysis of ssaJ and the ssaK/U operon, 13 genes encoding components of the type III secretion apparatus of Salmonella Pathogenicity Island 2

“…and S. flexneri, invG and mxiD (homologues of ssaC ) are separated by one gene from invA and mxiA, respectively, whereas the yscC gene of Yersinia spp. is part of the 13-gene virC operon (Allaoui et al, 1995). In SPI2, ssaC is separated from ssaV by more than 13 kb (Shea et al, 1996), and appears be one of a number of genes arranged in a manner similar to that of the virC locus of Yersinia spp.…”

Reduced polysialic acid capsule expression in Escherichia coli K1 mutants with chromosomal defects in kpsF

“…Allaoui et al, 1995). Using methods similar to those described previously for the mutagenesis of kpsF with the strongly polar aphT cassette (Cieslewicz and Vimr, 1996), the non-polar aph3 Km r cassette (A. Honeyman, personal communication) was inserted into the HindIII site of kpsF in pSX383.…”