Leucine-responsive regulatory protein, IS 1 insertions, and the negative regulator FaeA control the expression of the fae (K88) operon in Escherichia coli

Nucleotide sequence analysis of the fae operon encoding the biosynthesis of K88 fimbriae revealed the presence of two divergently transcribed regulatory genes, faeA and faeB, separated by two inverted iS 1 insertions. The amino acid sequences of the regulatory proteins FaeA and FaeB show similarity to the primary structure of corresponding regulatory proteins involved in the biosynthesis of Pap and S fimbriae. Expression of faeA is positively controlled by the FaeA protein, whereas K88 fimbriae production is negatively controlled by the co-operative activity of FaeA and the leucine-responsive regulatory protein (Lrp). Exchange of FaeA for Papl, a positive regulator of Pap fimbriae expression, also represses K88 production indicating that the combination Papl/Lrp has opposite effects on fae and pap expression. Mutations in faeB had no effect on the biosynthesis of K88 fimbriae. The presence of the two iS 1 insertions is hypothesized to neutralize part of the repression of K88 biosynthesis by FaeA/Lrp. Like pap, the fae operon does not respond to exogenous leucine.     

Solution structure of Escherichia coli PapI, a key regulator of the pap pili phase variation

Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap is controlled by a phase-variation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. The methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap gene expression in both phase-ON and phase-OFF cells. We used modern NMR techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold resembles closely that of the winged helix-turn-helix family of DNA-binding proteins. We determined that PapI possesses its own DNA-binding activity, albeit non-sequence-specific, independent of Lrp. PapI appears to bind to DNA with a K(d) in the 10 microM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings.     

Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.     

Two regulatory fim genes, fimB and fimE, control the phase variation of type 1 fimbriae in Escherichia coli.

The expression of type 1 fimbriae in Escherichia coli is phase dependent, i.e. a cell is either completely fimbriated or bald. This phenomenon is due to the periodic inversion of a specific 300-bp DNA segment containing the promoter for the fimbrial subunit gene, fimA. The phase switch is controlled by the products of two regulatory genes, fimB and fimE, located upstream of fimA. The fimB and fimE proteins direct the phase switch into the 'on' and 'off' position, respectively. The DNA sequence of a 3000-bp region containing the two genes has been determined. The fimB and fimE proteins exhibit strong homology and have most likely originated by duplication of an ancestral gene. They are highly basic implying that they control the phase switch through interaction at the DNA level.     

Markov Chain modeling of pyelonephritis-associated pili expression in uropathogenic Escherichia coli

Pyelonephritis-associated pili (Pap) expression in uropathogenic Escherichia coli is regulated by a complex phase variation mechanism involving the competition between leucine-responsive regulatory protein (Lrp) and DNA adenine methylase (Dam). Population dynamics of pap gene expression has been studied extensively and the detailed molecular mechanism has been largely elucidated, providing sufficient information for mathematical modeling. Although the Gillespie algorithm is suited for modeling of stochastic systems such as the pap operon, it becomes computationally expensive when detailed molecular steps are explicitly modeled in a population. Here we developed a Markov Chain model to simplify the computation. Our model is analytically derived from the molecular mechanism. The model presented here is able to reproduce results presented using the Gillespie method, but since the regulatory information is incorporated before simulation, our model runs more efficiently and allows investigation of additional regulatory features. The model predictions are consistent with experimental data obtained in this work and in the literature. The results show that pap expression in uropathogenic E. coli is initial-state-dependent, as previously reported. However, without environment stimuli, the pap-expressing fraction in a population will reach an equilibrium level after approximately 50-100 generations. The transient time before reaching equilibrium is determined by PapI stability and Lrp and Dam copy numbers per cell. This work demonstrates that the Markov Chain model captures the essence of the complex molecular mechanism and greatly simplifies the computation.     

Regulation of the L-arabinose transport operons in Escherichia coli

l-Arabinose is transported into Escherichia coli via two independent transport systems, a system possessing relatively low affinity for arabinose, the araE system, and a system of higher affinity for arabinose, the araFG system. In the work reported here we demonstrate that insertion of the Mu-lac bacteriophage isolated by Casadaban &; Cohen (1979) permits a reliable measurement of the expression of these two operons. Using appropriate Mu-lac insertion strains we found that both of the arabinose transport operons can be induced approximately 150-fold by the presence of arabinose, and that induction of both transport operons requires CRP (cyclic AMP receptor protein), but that their catabolite sensitivities differ from one another. In addition, we show that the araC⁺ allele is dominant to the C^c allele in the control of the transport operons, just as is found in the araBAD operon.     

Regulatory cross-talk between adhesin operons in Escherichia coli: inhibition of type 1 fimbriae expression by the PapB protein

Pathogenic Escherichia coli often carry determinants for several different adhesins. We show a direct communication between two adhesin gene clusters in uropathogenic E.coli: type 1 fimbriae (fim) and pyelonephritis-associated pili (pap). A regulator of pap, PapB, is a key factor in this cross-talk. FimB recombinase turns on type 1 fimbrial expression, and PapB inhibited phase transition by FimB in both off-to-on and on-to-off directions. On-to-off switching requiring FimE was increased by PapB. By analysis of FimB- and FimE-LacZ translational fusions it was concluded that the increase in on-to-off transition rates was via an increase in FimE expression. Inhibition of FimB-promoted switching was via a different mechanism: PapB inhibited FimB-promoted in vitro recombination, indicating that FimB activity was blocked at the fim switch. In vitro analyses showed that PapB bound to several DNA regions of the type 1 fimbrial operon, including the fim switch region. These data show that Pap expression turns off type 1 fimbriae expression in the same cell. Such cross-talk between adhesin gene clusters may bring about appropriate expression at the single cell level.     

The frequency of expression of pyelonephritis-associated pili is under regulatory control

The Bscherfchia coli urinary tract Isolate C1212 contains two pyelonephritis-associated pili (pap) DNA sequences designated here as pap-17 and pap-21. Each of these pap sequences encodes antigenically-distinct pilin monomers, pllin-17 and pilin-21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin-21. Only a small fraction (5%) of strain C1212 cells expressed pilin-17. Most of the latter population simultaneously expressed pilin-21, but a low percentage of cells expressed pill composed of pilin-17 alone. In contrast, almost every E. coli K-12 cell containing multicopy pap-17 expressed pilin-17 at the ceil surface. These results Indicated that the regulation of pilin-17 expression observed for strain C1212 was lost when pap-17 was in the multicopy state. Transfer of pap-17 to a single copy vector resulted in a pilin-17 expression frequency lower than strain C1212 (1%). Using E. coli K-12 containing single copy pap-17, we found that the frequency of piiin-17 expression increased about 15-foid when pap-21 was present in multiple copies in trans. Subcloning of pap-21 showed that a 2.2 kilobase-pair DNA sequence adjacent to, but not including, the pilin-21 structural gene was sufficient for activation of pilin-17 expression.     

The frequency of expression of pyelonephritis-associated pili is under regulatory control

The Escherichia coli urinary tract isolate C1212 contains two pyelonephritis-associated pili (pap) DNA sequences designated here as pap-17 and pap-21. Each of these pap sequences encodes antigenically-distinct pilin monomers, pilin-17 and pilin-21, respectively. Most individual strain C1212 cells isolated from a single bacterial colony expressed pilin-21. Only a small fraction (5%) of strain C1212 cells expressed pilin-17. Most of the latter population simultaneously expressed pilin-21, but a low percentage of cells expressed pili composed of pilin-17 alone. In contrast, almost every E. coli K-12 cell containing multicopy pap-17 expressed pilin-17 at the cell surface. These results indicated that the regulation of pilin-17 expression observed for strain C1212 was lost when pap-17 was in the multicopy state. Transfer of pap-17 to a single copy vector resulted in a pilin-17 expression frequency lower than strain C1212 (1%). Using E. coli K-12 containing single copy pap-17, we found that the frequency of pilin-17 expression increased about 15-fold when pap-21 was present in multiple copies in trans. Subcloning of pap-21 showed that a 2.2 kilobase-pair DNA sequence adjacent to, but not including, the pilin-21 structural gene was sufficient for activation of pilin-17 expression.     

Gene expression in the polycistronic operons of Escherichia coli heat-labile toxin and cholera toxin: a new model of translational control

A new model is proposed based on the suggestion that stable local secondary structures of mRNA interfere with ribosome movement on mRNA and consequently reduce the translation rate. This model accounts for a different level of translation for each cistron in the polycistronic mRNA of Escherichia coli heat-labile toxin (LT) and cholera toxin. We also conclude that the mRNA secondary structures have been conserved during the evolution of the toxin genes for its functional importance.