THERMOREGULATION OF ESCHERICHIA-COLI PAP TRANSCRIPTION - H-NS IS A TEMPERATURE-DEPENDENT DNA METHYLATION BLOCKING FASTER

The expression of Pap pill that facilitate the attachment of Escherich ia coil to uroepithelial cells is shut off outside the host at tempera tures below 26 degrees C. Ribonuclease protection analysis showed that this thermoregulatory response was rapid as evidenced by the absence of papBA transcripts, coding for Pap pilin, after only one generation of growth at 23 degrees C, The histone-like nucleoid structuring prote in H-NS and DNA sequences within papB were required for thermoregulati on, but the papa and Papl regulatory proteins were not. In vivo analys is of pap DNA methylation patterns indicated that H-NS or a factor reg ulated by H-NS bound within the pap regulatory region at 23 degrees C but not at 37 degrees C, as evidenced by H-NS-dependent inhibition of methylation of the pap GATC sites designated GATC-I and GATC-II. These GATC sites lie upstream of the papBAp promoter and have been shown pr eviously to play a role in controlling Pap pill expression by regulati ng the binding of Lrp, a global regulator that is essential for activa ting papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H-NS bound specifically to a pap DNA fragment con taining the GATC-I and GATC-II sites. Moreover, H-NS blocked methylati on of these pap GATC sites in vitro : H-NS blocked pap GATC methylatio n at 1,4 mu M but was unable to do so at higher concentrations at whic h non-specific binding occurred. Thus, non-specific binding of H-NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sit es. These results suggest that the ability of H-NS to act as a methyla tion blocking factor is dependent upon the formation of a specific com plex of H-NS with pap regulatory DNA, We hypothesize that a function o f H-NS such as oligomerization was altered at 23 degrees C, which enab led H-NS to repress pap gene expression through the formation of a spe cific nucleoprotein complex.