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ABSTRACTThe transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneriand enteroinvasiveEscherichia coli(EIEC) is induced at 37°C and repressed at 30°C. In this work, we report that the O135: K−:H− EIEC strain HN280 and S. flexneriSFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apygene. In addition, the S. flexneristrains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sfgene. Both apyand phoN-Sfare pINV-carried loci whose contribution to the pathogenicity of enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both theapyand the phoN-Sfgenes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virBtranscription) expressed the apygene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Δhnsdeletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virBtranscription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb−) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virFor virBand by the presence of the intact apylocus or intactapyand phoN-Sfloci were detected among Crb−mutants of HN280 and SFZM53, respectively. While all Crb−apy+mutants of HN280 failed to produce apyrase, Crb−apy+phoN-Sf+mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying clonedvirF(pMYSH6504) or virB(pBN1) into Crb−mutants of HN280 and SFZM53 lacking virFor virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apyis another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of enteroinvasive bacteria.