The regulation of pyelonephritis‐associated pili (pap) pilin gene transcription has been examined using two operons (pap‐17 and pap‐21) isolated from the pyelonephritogenic Escherichia coli strain C1212. DNA sequence analysis and E. coli minicell analysis were used to map two genes (papB and papI) within the pilin regulatory regions of both pap‐17 and pap‐21, and the protein products of these genes were identified. Pilin transcription, initiated at the papBA promoter, was monitored by constructing single copy operon fusions with lacZYA in E. coli K‐12. Inoculation of E. coli (pap'‐lac) strains onto solid M9 minimal medium containing glycerol and the Lac indicator X‐gal (M9‐Glycerol) yielded both Lac+ and Lac‐ colony phenotypes. The Lac+ (‘phase on’) and Lac‐ (‘phase off’) phenotypes were heritable since reinoculation of M9‐Glycerol with bacteria picked from Lac+ colonies gave rise to a much higher fraction of Lac+ colonies than reinoculation of M9‐Glycerol with bacteria picked from Lac‐ colonies. Measurement of phase transition rates for E. coli (pap17′‐lac) inoculated onto M9‐Glycerol showed that the Lac(‐)––Lac+ transition frequency (1.57 X 10(‐4)/cell/generation) was reduced 35‐fold when cells were inoculated onto minimal medium containing glucose (M9‐Glucose). However, the Lac+––Lac‐transition frequency obtained using M9‐Glycerol (2.60 X 10(‐2)/cell/generation) was 1.4‐fold lower compared to results obtained with M9‐Glucose. In contrast, lowering the incubation temperature of E. coli (pap17′‐lac) cultures from 37 degrees C to 23 degrees C caused all cells to shift to the Lac‐ state.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature serves as a cue to regulate gene expression in Escherichia coli and other bacteria. Using DNA microarrays, we identified 297 genes whose expression is increased at 23 6C compared to 37 6C in E. coli K-12. Of these genes, 122 are RpoS-controlled, confirming genome-wide the model that low temperature serves as a primary cue to trigger the general stress response. Several genes expressed at 23 6C overlap with the cold-shock response, suggesting that strategies used to adapt to sudden shifts in temperature also mediate long-term growth at 23 6C. Another category of genes more highly expressed at 23 6C are associated with biofilm development, implicating temperature as an important cue influencing this developmental pathway. In a candidate set of genes tested, the biofilm genes (adrA, bolA, mlrA, nhaR, csgA, yceP/bssS) and cold-shock genes (otsA, yceP/bssS) were found to be RpoS-and DsrAdependent for their transcription at 23 6C. In contrast, transcription of three genes (ycgZ, dps and ymgB) was either partially or fully independent of these regulators, signifying there is an alternative thermoregulatory mechanism(s) that increases gene expression at 23 6C. Increased expression at 23 6C compared to 37 6C is retained in various media tested for most of the genes, supporting the relative importance of this cue in adaptation to changing environments. Both the RpoS-dependent gene otsA and the RpoS-independent gene ymgB demonstrated increased expression levels within 1 h after a shift from 37 to 23 6C, indicating a rapid response to this environmental cue. Despite changes in gene expression for many RpoS-dependent genes, experiments assessing growth rate at 23 6C and viability at 4 6C did not demonstrate significant impairment in rpoS : : Tn10 or dsrA : : cat mutant strains in comparison to the wild-type strain. Biofilm formation was favoured at low temperature and is moderately impaired in both the rpoS : : Tn10 and dsrA : : cat mutants at 23 6C, suggesting genes controlled by these regulators play a role necessary for optimal biofilm formation at 23 6C. Taken together, our data demonstrate that a large number of genes are increased in expression at 23 6C to globally respond to this environmental change and that at least two thermoregulatory pathways are involved in co-ordinating this response -the RpoS/DsrA pathway and an alternative thermoregulatory pathway, independent of these regulators.

SummaryThe expression of Pap pili that facilitate the attachment of Escherichia coli to uroepithelial cells is shut off outside the host at temperatures below 26ЊC. 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 23ЊC. The histone-like nucleoid structuring protein H-NS and DNA sequences within papB were required for thermoregulation, but the PapB and PapI regulatory proteins were not. In vivo analysis of pap DNA methylation patterns indicated that H-NS or a factor regulated by H-NS bound within the pap regulatory region at 23ЊC but not at 37ЊC, 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 previously to play a role in controlling Pap pili expression by regulating the binding of Lrp, a global regulator that is essential for activating papBAp transcription. Competitive electrophoretic mobility shift analysis showed that H-NS bound specifically to a pap DNA fragment containing the GATC-I and GATC-II sites. Moreover, H-NS blocked methylation of these pap GATC sites in vitro : H-NS blocked pap GATC methylation at 1.4 M but was unable to do so at higher concentrations at which non-specific binding occurred. Thus, non-specific binding of H-NS to pap DNA was not sufficient to inhibit methylation of the pap GATC sites. These results suggest that the ability of H-NS to act as a methylation blocking factor is dependent upon the formation of a specific complex of H-NS with pap regulatory DNA. We hypothesize that a function of H-NS such as oligomerization was altered at 23ЊC, which enabled H-NS to repress pap gene expression through the formation of a specific nucleoprotein complex.

DNA microarrays demonstrate that H-NS controls 69% of the temperature regulated genes in Escherichia coli K-12. H-NS is shown to be a common regulator of multiple iron and other nutrient acquisition systems preferentially expressed at 37 degrees C and of general stress response, biofilm formation, and cold shock genes highly expressed at 23 degrees C.

In uropathogenic Escherichia coli, P pili (Pap) facilitate binding to host epithelial cells and subsequent colonization. Whereas P pili can be produced at 37°C, the expression of these fimbriae is suppressed at 23°C. Previously, insertion mutations in rimJ, a gene encoding the N-terminal acetyltransferase of ribosomal protein S5, were shown to disrupt this thermoregulatory response, allowing papBA transcription at low temperature. In this study, we created an in-frame deletion of rimJ. This deletion relieved the repressive effects not only of low temperature but also of rich (Luria-Bertani [LB]) medium and glucose on papBA transcription, indicating that RimJ modulates papBA transcription in response to multiple environmental stimuli. papI transcription was also shown to be regulated by RimJ. papBA transcription is also controlled by a phase variation mechanism. We demonstrated that the regulators necessary to establish a phase ON state-PapI, PapB, Dam, Lrp, and cyclic AMP-CAP-are still required for papBA transcription in a rimJ mutant strain. rimJ mutations increase the rate at which bacteria transition into the phase ON state, indicating that RimJ inhibits the phase OFF3ON transition. A ⌬rimJ hns651 mutant is viable on LB medium but not on minimal medium. This synthetic lethality, along with transcriptional analyses, indicates that RimJ and H-NS work through separate pathways to control papBA transcription. Mutations in rimJ do not greatly influence the transcription of the fan, daa, or fim operon, suggesting that RimJ may be a pap-specific regulator. Overexpression of rimJ under conditions repressive for papBA transcription complements the ⌬rimJ mutation but has little effect on transcription under activating conditions, indicating that the ability of RimJ to regulate transcription is environmentally controlled.