Phase-variation of pyelonephritis-associated pili in Escherichia coli: evidence for transcriptional regulation.

Abstract

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)

Evidence for a methylation-blocking factor (mbf) locus involved in pap pilus expression and phase variation in Escherichia coli

“…Previous results have indicated that pap pilin transcription is initiated at the papBA promoter and extends through the papB and papA genes (4,7). Although Northern blot analysis (Fig.…”

“…coli MC1061 MC4100 MC4100 recAl DL379 DL566 DL638 DL832 DL833 DL834 DL835 DL836 DL837 DL838 DL845 DL849 DL850 DL852 DL860 DL875 DL877 DL913 DL1102 MLK782 DL1177 DL1178 DL1200 CAG12112 CAG18528 CAG12130 TABLE 1 Total bacterial RNA was isolated with hot phenol as described previously (30). The RNA was size fractionated on 1.2% formaldehyde-agarose gels as described before (26) and transferred to BioTrace RP nylon membranes in lOX SSC (1.5 M NaCl, 0.15 M sodium citrate [pH 7.0]). The RNA was fixed to the nylon membrane by UV cross-linking with a Stratalinker UV cross-linker (Stratagene, La Jolla, Calif.).…”

“…Pap pilus phase variation, however, is abrogated when multiple copies of pap are present (7,17). Therefore, we determined the effect of mutations within mbf on pap pilin a Where indicated, a single phase-on or phase-off colony was used to inoculate M9-glycerol broth.…”

“…Expression of Pap pili by uropathogenic Escherichia coli is subject to an on/off phase variation regulatory mechanism (7,17). Studies carried out with a single-copy pap-lac operon fusion in E. coli K-12 indicate that alternation between the phase-on and phase-off states occurs at the level ofpap pilin transcription and is affected by both incubation temperature (13,32) and the carbon source used for growth of the bacteria (3,7).…”

“…Studies carried out with a single-copy pap-lac operon fusion in E. coli K-12 indicate that alternation between the phase-on and phase-off states occurs at the level ofpap pilin transcription and is affected by both incubation temperature (13,32) and the carbon source used for growth of the bacteria (3,7). Our recent studies indicate that pap pilin phase variation occurs through a novel mechanism involving the differential methylation of two DNA GATC sites (GATC1028 and GATC1130) near the papBA pilin promoter by deoxyadenosine methylase (Dam) (7). DNA isolated from phase-off E. coli populations contains an unmethylated GATC1130 site.…”