H-NS REGULATION OF VIRULENCE GENE-EXPRESSION IN ENTEROINVASIVE ESCHERICHIA-COLI HARBORING THE VIRULENCE PLASMID INTEGRATED INTO THE HOST CHROMOSOME

We have previously shown that integration of the virulence plasmid pIN V into the chromosome of enteroinvasive Escherichia coli and of Shigel la flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B. Colonna, C. Conti, A. Calconi, and M. Nicoletti, Infect. Immun. 59 :792-799, 1991). In this work, we have studied the transcription of th e virulence regulatory genes virB, virF, and hns (virR) in wild-type e nteroinvasive E, coli HN280 and in its pINV-integrated derivative HN28 0/32. While transcription of virF; and of hns is not affected by pINV integration, transcription of virB is severely reduced even if integra tion does not occur within the virB locus. This indicates that VirF ca nnot activate virB transcription when pINV is integrated, and this lac k of expression accounts for the noninvasive phenotype of HN280/32. Vi rulence gene expression in strains HN280 and HN280/32, as well as in d erivatives harboring a mxiC::lacZ operon fusion either on the autonomo usly replicating pINV or on the integrated pINV, aas studied. The effe ct of the introduction of plasmids carrying virB (pBN1) or virF (pHW74 5 and pMYSW6504), and of a Delta hns deletion, in the different strain s was evaluated by measuring beta-galactosidase activity, virB transcr iption, and virB-regulated virulence phenotypes like synthesis of Ipa proteins, contact-mediated hemolysis, and capacity to invade HeLa cell s. The introduction of pBN1 or of the Delta hns deletion in pINV-integ rated strains induces temperature regulated expression or temperature- independent expression, respectively, of beta-galactosidase activity a nd of all virulence phenotypes, while an increase in virF gene dosage does not, in spite of a high-level induction of virB transcription. Mo reover, a wild-type has gene placed in trans fully reversed the induct ion of beta-galactosidase activity due to the Delta hns deletion. Thes e results indicate that virB transcription is negatively regulated by H-NS both at 30 and at 37 degrees C in pINV-integrated strains and tha t there is also a dose-dependent effect of VirF on virB transcription. The negative effect of H-NS on virB transcription at the permissive t emperature of 37 degrees C could be due to changes in the DNA topology occurring upon pINV integration that favor more stable binding of H-N S to the virB promoter DNA region. At 30 degrees C, the introduction o f the high-copy-number plasmid pMYSH6504 (but not of the low copy-numb er pHW745) or of the Delta hns deletion induces, in strains harboring an autonomously replicating pINV, P-galactosidase activity, virB trans cription, and expression of the virulence phenotypes, indicating that, as for HN280/32, the increase in virF gene dosage overcomes the negat ive regulatory effect of H-NS on virB transcription. Moreover, we have found that virF transcription is finely modulated by temperature and, with a call K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us to speculate that, in enteroinvasive bacteria, the leve l of VirF inside the cell controls the temperature-regulated expressio n of invasion genes.