The transposon TnphoA was used for construction of gene fusions and for studies of gene regulation in an enteroinvasive strain of Escherichia coli. Several plasmid-encoded virulence genes (e.g., the ipaB and virG operons) of such enteroinvasive strains are subject to coordinated thermoregulation involving both operon-specific (the VirB and VirF activators) and global regulators. The nucleoid-associated E. coli protein H-NS was shown to be a negative regulator as judged by studies using H-NS gene deletion mutations and by increasing the level of H-NS protein in the cells. An increased gene dosage of H-NS led to enhanced repression of the ipa and virG operons, particularly at low (30 degrees C) growth temperature. The cyclic AMP receptor protein complex, which is another global transcriptional regulator in E. coli, was not required for the regulation of ipa and virG expression. The virG operon was expressed in an activator-independent manner in cells lacking H-NS protein. We suggest that the role of the VirF activator is to counteract the silencing effect of H-NS.