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Journal of Bacteriology, December 2005, p. 8256-8266, Vol. 187, No. 24
0021-9193/05/$08.00+0     doi:10.1128/JB.187.24.8256-8266.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of the Detachable Rho-Dependent Transcription Terminator of the fimE Gene in Escherichia coli K-12

Paul Hinde, Padraig Deighan, and Charles J. Dorman^*

Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland

Received 27 June 2005/ Accepted 19 September 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The fim genetic switch in the chromosome of Escherichia coli K-12 is an invertible DNA element that harbors the promoter for transcription of the downstream fim structural genes and a transcription terminator that acts on the upstream fimE regulatory gene. Switches oriented appropriately for structural gene transcription also allow fimE mRNA to read through, whereas those in the opposite orientation terminate the fimE message. We show here that termination is Rho dependent and is suppressed in a rho mutant or by bicyclomycin treatment when fimE mRNA is expressed by the fimE gene, either from a multicopy recombinant plasmid or in its native chromosomal location. Two cis-acting elements within the central portion of the 314-bp invertible DNA switch were identified as contributors to Rho-dependent termination and dissected. These fim sequence elements show similarities to well-characterized Rho utilization (rut) sites and consist of a boxA motif and a C-rich and G-poor region of approximately 40 bp. Deletion of the boxA motif alone had only a subtle negative effect on Rho function. However, when this element was deleted in combination with the C-rich, G-poor region, Rho function was considerably decreased. Altering the C-to-G ratio in favor of G in this portion of the switch also strongly attenuated transcription termination. The implications of the existence of a fimE-specific Rho-dependent terminator within the invertible switch are discussed in the context of the fim regulatory circuit.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Escherichia coli and other bacteria end the process of transcript elongation by employing one of the following two types of transcription terminator: intrinsic terminators and factor-dependent terminators (36, 40, 43, 58). Intrinsic terminators typically consist of an approximately 20-bp stretch of DNA that is composed of a G+C region of hyphenated dyad symmetry followed by a run of T residues. This specifies in the RNA transcript a stable stem-loop structure that is followed by up to eight U residues (8, 31, 50). These U residues base pair weakly with the corresponding A residues in the template DNA strand, and in combination with the formation of the RNA stem-loop structure, lead to destabilization of the RNA polymerase-template-transcript elongation complex and the consequent termination event (43, 61).

Factor-dependent terminators utilize the hexameric ring-shaped Rho protein to facilitate transcript release from regions of the template that are not characterized by intrinsic RNA-DNA hybrid helix instability (5, 7, 37, 40, 47). Rho-dependent terminators lack the obvious structural features that are associated with intrinsic terminators (7). In general, they consist of a bipartite structure that extends for up to 150 bp of DNA and have a high proportion of C relative to G residues (2, 14, 20, 33, 39, 44, 60, 62). The Rho utilization (rut) site of a Rho-dependent terminator is a site on the RNA transcript which generally has little secondary structure and is recognized by the RNA-binding Rho factor, while the transcription stop point (tsp) is the region within which Rho enhances transcription termination of paused RNA polymerase complexes (6, 17, 35, 45).

The Rho monomer is composed of two domains, both of which bind RNA. The N-terminal domain binds to a ribosome-free region of the nascent transcript at a rut site, and the C-terminal domain uses energy from the hydrolysis of ATP to translocate towards the elongating RNA polymerase (11, 15, 33, 34, 44, 53, 56, 59). Following engagement with the polymerase, Rho acts as a helicase, using ATP binding and hydrolysis to unwind the RNA-DNA hybrid, and presumably pulls the transcript out of the RNA polymerase RNA exit channel, thereby terminating transcription (39, 41, 42, 52). Unlike intrinsic terminators, for which the termination points can be localized to 1 to 3 bp, termination sites within Rho-dependent terminators can be distributed over as much as 100 bp of DNA (28, 43).

Both factor-dependent and intrinsic terminators of transcription are normally associated permanently with the transcripts that they regulate. In contrast, this study describes the dissection of a Rho-dependent terminator in the fim gene cluster of E. coli K-12 that is alternately connected to and disconnected from the gene that it regulates by site-specific DNA inversion.

The fim genes encode type 1 fimbriae, which are stiff, rod-like, proteinaceous appendages that protrude from the bacterial cell surface and are composed mainly of the FimA subunit protein (24), with a fibrillum at the tip that is made up of the FimH adhesin and the FimF and FimG adaptor proteins (19, 22). The FimH protein mediates lectin-like binding to D-mannosides (27), contributing to attachment of the bacteria to host surfaces during colonization. The promoter used for transcription of the fimA gene is located within a 314-bp invertible DNA sequence known as the fim switch, or fimS (1, 10). Site-specific recombination between 9-bp inverted repeats that flank fimS inverts the switch. The result is a process of phase variation in which the fimA gene is alternately connected to (phase ON) and disconnected from (phase OFF) its promoter. DNA inversion is catalyzed by the tyrosine integrase site-specific recombinases FimB and FimE (9, 25). These highly homologous proteins have distinct DNA inversion activities at fimS. FimB inverts the switch in the ON-to-OFF and OFF-to-ON directions with approximately equal efficiencies, whereas FimE shows a strong bias for inverting fimS in the ON-to-OFF direction (13, 32, 54). When FimB and FimE are coexpressed, the ON-to-OFF bias imposed by FimE predominates. However, the orientation of the switch feeds back into the DNA inversion mechanism. Called orientational control (55), this process includes a role for phase-variable termination of the fimE mRNA (23). With fimS in the ON orientation, fimE transcription traverses the switch to produce a stable transcript of 1,800 bases. When fimS is in the OFF orientation, fimE transcription terminates within the switch, resulting in an unstable truncated transcript of only 900 bases (Fig. 1). The instability of the truncated fimE transcript is responsible for a strong reduction in FimE recombinase levels, allowing FimB to reset fimS to the ON orientation (23). Therefore, fimS is a bifunctional invertible genetic element: it is responsible for phase-variable transcription initiation at the fimA structural gene and for phase-variable transcription termination at the fimE regulatory gene. We investigated the structure of the fimS terminator to discover the basis of its phase-variable transcription termination activity.

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FIG. 1. Structure of the fim operon and location of the features associated with Rho-dependent termination of fimE transcription. (A) The segment of the horizontal line that is bracketed by filled triangles represents the invertible fim switch (fimS). The angled arrow at fimE represents the promoter (PfimE) of that gene and shows the direction of transcription. In the ON phase, the transcript extends through the fim switch, whereas in the OFF phase it terminates in fimS. The angled arrow within fimS represents the fimA promoter (PfimA). In the ON phase (upper diagram), this arrow is directed towards fimA, and it is directed away in the OFF phase (lower diagram). Switching between the ON and OFF phases involves site-specific recombination between the inverted repeats IRL and IRR (filled triangles). The sequences of the 9-bp inverted repeats are shown in the filled rectangles. (B) Expanded view of fimS in the OFF orientation. The 3' end of the fimE gene is shown on the left side of the diagram, and the positions of the three binding sites for Lrp (filled ovals with Roman numerals) and the boxA sequence (filled box) are indicated. The 52-nucleotide sequence associated with the Rho-dependent transcription termination rut site is given at the bottom of the figure. The overlined portion is the 8-bp fimS sequence that shows the most identity to other boxA elements. C residues that were converted to G by SDM are shown in bold and underlined. The identified fimS Rho-dependent terminator (accession number X03923) (24) is aligned with the corresponding parts of the Rho-dependent terminator from bacteriophage lambda ( tR1; the rutA element is shown in bold; accession number J02460) (38), the boxA motif and part of the rut site of the E. coli tna operon leader region (accession number M11990) (57), and the boxA sequence of the E. coli ribosomal operon rrnG (accession number V00350) (51).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and growth conditions. All of the bacterial strains used in this study were derivatives of E. coli K-12 and are described in Table 1, together with details of the plasmids used in this work. Bacteria were cultured routinely at 37°C in Luria-Bertani (LB) broth or on LB agar plates. The growth medium was supplemented where required with the following antibiotics: carbenicillin (100 µg ml^–1), chloramphenicol (30 µg ml^–1), and kanamycin (50 µg ml^–1). Bicyclomycin was a generous gift from Fujisawa Pharmaceutical (Osaka, Japan) and was used at a final concentration of 50 µM.

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TABLE 1. Bacterial strains and plasmids used for this study

DNA manipulations and plasmid construction. Standard procedures were used for DNA manipulations (49). Plasmid pSAJ109 contains the fimE-fimS portion of the fim operon cloned in the expression plasmid pBAD24 (Table 1). In this plasmid, the arabinose-inducible P_BAD promoter drives fimE transcription (23). This plasmid was modified to lock the fim switch permanently in the OFF orientation so that the effect of Rho on fimE transcription termination could be studied in a population of exclusively OFF-phase fimS elements. This was achieved by altering the left-hand inverted repeat (IRL) from 5'-TTGGGGCCAA-3' to 5'-TTGGGGGGTT-3' by site-directed mutagenesis (SDM) to produce plasmid pPH100 (Table 1).

The fimS boxA element (5'-CGCATCTT-3') at the 5' end of the fimS rut region (Fig. 1B) shares a high degree of sequence homology (7/8 bp match) with the boxA sequence at the 5' end of the rutA element of the lambda tR1 terminator and with the boxA sequence of the Rho-dependent tna operon leader region terminator (6/8 bp; Fig. 1B). This element was deleted from the fimS locked-OFF plasmid pPH100 by inverse PCR using primers INV_FOR_BOXA and INV_REV (Table 2), generating plasmid pPH101 (Table 1). Next, 22 bases of the C-rich, G-poor rut region located immediately 3' of the boxA deletion in pPH101 were removed (30 base pairs deleted from pPH100 in total) to create plasmid pPH102 (Table 1). This was also performed by inverse PCR, and the reaction was primed with oligonucleotides INV_FOR_B+22 and IN_REV (Table 2). A further 22 bases located immediately 3' of the deletion in pPH102 were removed by inverse PCR (52 base pairs deleted from pPH100 in total) to produce plasmid pPH103 (Table 1). The oligonucleotide primers used were INV_FOR_B+44 and INV_REV (Table 2). The eight bases of the boxA element (5'-CGCATCTT-3') of plasmid pPH100 were also mutated such that each purine was replaced by a pyrimidine and vice versa. The resulting plasmid was named pPH104 (Table 1), and the base changes were made by SDM using primers BOXA_SCR_TOP and BOXA_SCR_BTM (Table 2).

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TABLE 2. Oligonucleotides used for this study

Plasmid pSAJ112 is derived from pSAJ109 and has the fimS element locked permanently in the ON phase (23). The boxA element of the fimS rut region (5'-CGCATCTT-3') was inverted, bringing it into the path of the RNA polymerase, which initiated the transcription of fimE as it traversed the phase ON switch. This allowed this component of the fimS terminator to be tested in isolation for its effects on fimE transcription termination. The resulting pSAJ112 derivative was named pPH105 (Table 1) and was constructed by SDM using primers ON_BOXA-ENG_BTM and ON_BOXA_ENG_TOP (Table 2). A portion of the C-rich fimS rut region 3' of the boxA component (Fig. 1B) was mutated by converting the C residues to G's by SDM. Using plasmid pPH100 as the template, four C residues were altered to G residues by using primers CMUT1_TOP and CMUT1_BTM (Table 2; C1 modification), generating plasmid pPH106 (Table 1). Plasmid pPH107 (Table 1) was derived from pPH106 such that the next four C residues were mutated to G residues by using primers CMUT2_TOP and CMUT2_BTM (Table 2; C2 modification), resulting in eight C-to-G substitutions in total. None of the genetic manipulations described in this section resulted in a detectable change in plasmid copy number.

The integrity of each new construct was confirmed by DNA sequencing. The orientation of the fimS switch was confirmed by a PCR-based assay in which the oligonucleotide primers OL4 and OL20 (Table 2) were used to amplify the switch DNA and its ON/OFF status was then assessed by restriction digestion analysis (54).

RNA manipulations. Cultures of strain AAEC178 harboring pBAD24 derivative plasmids pPH100 to pPH107 were grown in LB at 37°C in the absence of arabinose. At the mid-exponential phase of growth (optical density at 600 nm, 0.5), transcription from the P_BAD promoter was induced by the addition of arabinose to a final concentration of 0.2%. Cells were harvested after another 60 min of growth, washed in phosphate-buffered saline, and lysed for RNA extraction (see below). In experiments where Rho was inhibited by bicyclomycin treatment, the antibiotic and arabinose inducer were added to the culture at the same time. A similar protocol was used for experiments with the temperature-sensitive rho mutant CJD3000 (Table 1), except that the bacteria were grown at 30°C until the mid-exponential phase of growth and were then shifted to 42°C for 60 min following the addition of arabinose. This heat shock procedure inhibits Rho factor production. Growth at 42°C does not influence fimE transcription termination or message stability in a wild-type strain (23). RNAs were extracted using the TRI (Sigma) procedure according to the kit maker's instructions. DNAs were removed from RNA samples with an Ambion DNA-free kit. The purified RNAs were subjected to reverse transcription-PCR (RT-PCR) using a QIAGEN OneStep RT-PCR kit in compliance with the manufacturer's directions. RNA quantity and quality were determined by measuring the absorbance of the sample (A₂₆₀/A₂₈₀) in triplicate. All RT-PCRs were performed within the linear range of a logarithmic scale, as determined by performing trial RT-PCRs with twofold serial dilutions of DNA-free RNA as the template, with a fixed number of amplification cycles. Under our experimental conditions, 200 ng of purified DNA-free RNA was used as the template in reactions that underwent 18 amplification cycles. The amount of RNA loaded and its integrity were monitored by agarose gel electrophoresis. In all cases, the amplimer sizes (measured electrophoretically) were as expected. RT-PCR DNA bands were quantified by densitometry using AlphaEaseRC software, version 3.2.1 (Alpha Innotech). Bands were recognized and quantified automatically from unsaturated images using the analysis tool suite. The number given for the total area of each band was converted to a percentage relative to the area of a control band (usually the corresponding band obtained from the wild type). To ensure that there were no variations in priming efficiency among the different reverse oligonucleotides, control reactions were done with the forward primer (RT-fwd) in combination with each of the reverse primers (RT-a, RT-b, RT-c, RT-d, and RT-e), using a fixed quantity of DNA as the template. The experiments were performed at least three times, and typical data from one experiment are shown.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of a Rho-dependent terminator in fimS. Previous work had shown that fimS contains a transcription terminator that terminates fimE transcription when fimS is in the OFF, but not the ON, orientation (23). RNA polymerase reads through fimS in the OFF orientation in a rho-15 mutant background, indicating that the fimS terminator requires the Rho protein for function. Measuring the length of the fimE transcript by Northern blot analysis with fimS in the OFF orientation allowed us to estimate that the terminator was located at approximately the midpoint of the switch (23). Precise identification of a potential Rho terminator by sequence analysis alone is difficult because of the absence of readily identifiable features such as inverted repeats. We decided to determine the likely location of the terminator using an approach based on RT-PCR. For this assay, oligonucleotides were designed to prime reverse transcription of the fimE transcript from different positions within OFF-phase fimS (Fig. 2). It was anticipated that the terminator would strongly reduce the level of transcript that would be available for use as a template for RT-PCR for positions lying 3' of the terminator. It was also expected that the presence of the rho-15 mutation or the presence of bicyclomycin (an inhibitor of Rho) in the culture would overcome this reduction, confirming that a Rho-dependent termination event was responsible. Due to the low level of fimE transcription in its native chromosomal location (23), we utilized pBAD24-derived plasmids, by which the termination of fimE transcripts initiating at an arabinose-inducible promoter could be monitored in the bacterial strain AAEE178 (Table 1), from whose chromosome the entire fim gene cluster has been deleted.

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FIG. 2. Effect of a rho mutation and bicyclomycin on fimE readthrough of the locked OFF-phase fim switch. The RT-PCR strategy used to detect the fimE transcript at different positions along the switch is summarized at the top of panel A. Reverse primers RT-a and RT-b detected the transcript at locations 5' of the putative Rho-dependent terminator, while detection at 3' positions was performed using primers RT-c, RT-d, and RT-e. The level of transcript detected with each primer from constructs harboring wild-type fimS in untreated wild-type bacteria was normalized to the value for RT-a (100%) and is shown in parentheses below each primer name. Termination was also assayed in three mutant derivatives of the locked OFF switch. These had just the 8-bp boxA sequence deleted (B) or were deleted for the boxA sequence together with the following 22 bases (B + 22) or the following 44 bases (B + 44). In the lower three panels (B, C, and D), RT-PCR data are shown for the locked OFF switch (WT) and its three deletion mutant derivatives in wild-type E. coli strain AAEC178 (B), a rho-15 mutant (C), and strain AAEC178 treated with the Rho-inhibiting antibiotic bicyclomycin (D). In all cases, expression of the fimE transcript was from the PBAD promoter induced with arabinose. The name of the reverse primer used for the RT-PCR experiment is given to the left of each set of four RT-PCR products. The intensity of each product band was quantified by densitometry and expressed as a percentage of that obtained with the undeleted construct (WT; 100%). The ethidium bromide-stained 16S rRNA shown at the bottom of panels B, C, and D indicates that equivalent amounts of total RNA were used in the PCRs.

The fim switch is rich in regulatory features concerned with the efficient operation of the DNA inversion mechanism. These include three binding sites for the leucine-responsive regulatory protein (Lrp), which is required for normal inversion of fimS (12, 48). Based on our measurements of the length of the truncated fimE transcript that is associated with OFF-phase fimS and our knowledge of the fimE transcription initiation site, we estimated that the most likely location for the DNA sequence specifying the terminator would be at or near Lrp binding site III (23). With this in mind, a forward primer (RT-fwd) was designed to prime DNA synthesis from a cDNA template encompassing fimE-fimS that was generated from fimE mRNA by reverse transcription primed with one of a series of oligonucleotides that primed from different positions within the fimS element, on either side of the Lrp-III site (RT-a, RT-b, RT-c, RT-d, and RT-e) (Fig. 2).

Transcripts crossing the wild-type phase OFF switch were detected readily using primer RT-a or RT-b with the wild-type strain, whereas amplimers generated using any of the primers (RT-c, RT-d, and RT-e) that bound to the 3' side of the Lrp-III site were approximately sixfold less abundant (Fig. 2A, percentages shown in parentheses, and B, wild-type column). These data were consistent with termination of fimE transcription at a location between those bound by primers RT-b and RT-e. When the experiment was repeated with a rho-15 strain, the fimE transcript was readily detected using all five reverse primers (RT-a, RT-b, RT-c, RT-d, and RT-e) (Fig. 2C, wild-type column). Similarly, when the experiment was performed with the wild-type strain in the presence of the Rho-inhibiting antibiotic bicyclomycin, all five reverse primers readily amplified the fimE transcript (Fig. 2D, wild-type column). Moreover, Northern blot analysis showed that the inactivation of Rho function by bicyclomycin treatment resulted in a full-length (1,800-nucleotide) stable fimE message (data not shown), a finding that was consistent with data obtained previously with the rho-15 mutant (23). Taken together, these results support the hypothesis that a Rho-dependent terminator is located within fimS and suggest that the majority of termination stop points occur within the region between the sites targeted by reverse primers RT-b and RT-e. Moreover, these results validated the use of the RT-PCR-based assay as an approach suitable for identifying the cis-acting elements required for the fimE mRNA Rho-dependent termination event.

Identification of sequences required for Rho-dependent termination. An inspection of the fimS sequence (OFF orientation) in the region suggested by RT-PCR analysis to contain the Rho-dependent terminator led to the identification of a section capable of specifying a rut region. This 52-bp portion of the fimE transcript was abnormally rich in pyrimidine residues, with 19 C and 15 T residues out of 52 total (Fig. 1B). The same sequence contained only two G residues. The presence of such a high concentration of C relative to G residues is a feature of several well-characterized rut sites, as Rho interacts preferentially with C-rich, G-poor RNA (2, 14, 20, 33, 39, 46, 60, 62). Moreover, while Rho-dependent terminators may have poor sequence homology with one another, it was evident that this region of fimS aligned with other rut elements, in particular with the boxA and rutA regions of lambda tR1 (13/16 bp identity; Fig. 1B) (16, 17), the boxA sequence of the rrnG operon (6/8 bp identity) (51), and the boxA element from the leader region of the tna operon (6/8 bp identity; Fig. 1B) (26).

To assess the importance of the fimS rut region, derivatives of the switch were constructed from which appropriate rut DNA elements were deleted. The deletions removed just the 8 bases of the boxA element (5'-CGCATCTT-3') (removing 3 C nucleotides), boxA plus 22 bases of the C-rich region (removing 12 C residues in total), or boxA plus 44 bases of the C-rich region (removing 19 C residues in total). The ability of the mutant switches to terminate fimE transcription was monitored in wild-type cells, rho-15 mutant cells, and wild-type cells treated with the Rho-inhibiting antibiotic bicyclomycin. The results showed that the 8-bp boxA deletion alone had a subtle but detectable effect on the wild type. Up to twofold more fimE transcript was detectable using reverse primer RT-c, RT-d, or RT-e, which primes cDNA production from sites that are distal to the putative terminator stop points, than with primer RT-a or RT-b, which primes from positions proximal to the terminator (Fig. 2B). However, the removal of 22 or 44 bases of the C-rich sequence in addition to the boxA element resulted in a progressive increase in detection of the fimE transcript by the reverse primers RT-c, RT-d, and RT-e. In the case of the 44-bp deletion (52 bp deleted in total, including 19 C residues), increases of up to sixfold were detected with RT-e compared with the level detected with RT-a. This was consistent with a significant weakening of the effect of the Rho-dependent terminator, presumably due to the loss of the rut site. No increase in fimE transcript level was detected using the RT-c, RT-d, or RT-e primer with any of the constructs when mRNA was assayed in the rho-15 mutant or the wild-type strain treated with bicyclomycin (Fig. 2C and D). This was consistent with the Rho protein requiring the fimS rut site for function. These data showed that the Rho-dependent terminator within fimS is located in the region of Lrp site III and consists of the boxA-like element and the C-rich, G-poor sequences located immediately 3' of it (Fig. 1B).

The fimS boxA element alone is not essential for termination. The deletion analysis data suggested that removal of the boxA element alone resulted in only a modest effect on fimE transcription termination. In order to investigate this further, we altered the residues of fimS boxA (5'-CGCATCTT-3') by site-directed mutagenesis such that each purine was replaced by a pyrimidine and vice versa (Fig. 3). This removed any similarity to the fimS boxA element (or the tR1 or tna boxA elements) at the level of the nucleotide sequence without changing the length of the fimE transcript. In this case, the altered fimS gene still terminated fimE transcription, as measured by the RT-PCR assay (Fig. 3).

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FIG. 3. Altering the 8-bp boxA component of the fimS rut sequence does not affect transcription termination. The RT-PCR strategy used to detect the fimE transcript at different positions along the locked OFF switch is summarized at the top of the figure. Reverse primers RT-a and RT-b detected the transcript at locations 5' of the putative Rho-dependent terminator, while detection at 3' positions was performed using primers RT-c, RT-d, and RT-e. RT-PCR products are shown at the bottom of the figure, labeled with the reverse primers used to detect them. A reduction in transcript levels was recorded at all three positions 3' of the putative terminator with the altered nucleotide sequence, a pattern that was strongly reminiscent of that seen for the fim switch with an unmutated terminator (Fig. 2A).

We next took the native fimS boxA element and inverted it in the switch. No other sequence was altered. This modification placed the fimS OFF boxA element within the fimE transcript when the switch was in the ON orientation (Fig. 4). RT-PCR analysis of fimE transcription across the ON-phase switch showed that the presence of the boxA element had no negative effect on readthrough (Fig. 4). These data confirmed that the presence in the fimE transcript of this sequence motif (in either the ON or OFF orientation) did not in itself lead to a strong effect on termination, consistent with the deletion analysis presented in Fig. 2.

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FIG. 4. The 8-bp boxA component of the fimS rut sequence cannot terminate transcription in the ON phase. The RT-PCR strategy used to detect the fimE transcript at different positions along the locked ON switch is summarized at the top of the figure. Reverse primer RT-ON a detected the transcript at a location 5' of the putative Rho-dependent terminator, while detection at 3' positions was performed using primers RT-ON b and RT-ON c. RT-PCR products generated from transcripts specified by the phase ON switch without or with the 8-bp boxA element are shown at the bottom of the figure, labeled with the reverse primers used to detect them.

The C-rich sequence motif is essential for Rho-dependent transcription termination. Our deletion analysis indicated that within the fimS rut region, the C-rich, G-poor sequences located 3' of the boxA element were important for Rho-mediated transcription termination (Fig. 2). We decided to characterize this region further by making a series of targeted mutations changing C residues to G residues without altering the length of the transcript. The nucleotide sequence of the 5' portion of the putative rut site in fimS is given in Fig. 1B, where the C residues targeted for substitution are shown in bold and are underlined. The four C residues located immediately 3' of the boxA element were altered to G by SDM (construct C1; Fig. 5A). The next four C residues were also altered to G (construct C2; Fig. 5A). The latter alteration reduced the total number of C residues in the 44 bases located 3' of the boxA element from 16 to 8. The effect of the base substitutions on Rho-dependent termination was measured by the RT-PCR assay. The results showed that the fimE transcript now read through the region in the OFF-phase fim switch beyond the point where the majority of the transcript had previously terminated. Moreover, the efficiency of readthrough correlated with the number of C-to-G substitutions (Fig. 5B, panel RT-e). In a control experiment where cultures harboring the wild-type terminator or its C1 or C2 derivative were treated with bicyclomycin, readthrough measured by all primer sets occurred to similar levels (Fig. 5C). This confirmed that the rut sequence rich in C residues was needed for efficient Rho-dependent termination, a finding that was in keeping with descriptions of other Rho-dependent terminators (2, 14, 17, 20, 26, 33, 39, 46, 60, 62).

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FIG. 5. Converting C residues to G abolishes terminator activity in fimS. RT-PCR analysis was used to measure fimE transcript levels at positions 5' and 3' of the putative Rho-dependent terminator in fimS in constructs with a wild-type terminator sequence (WT) or with four C-to-G substitutions (C1) or eight C-to-G substitutions (C2) in the terminator. The RT-PCR strategy used to detect the fimE transcript at different positions along the locked OFF switch is summarized in panel A. Reverse primers RT-a and RT-b detected the transcript at locations 5' of the putative Rho-dependent terminator, while detection at 3' positions was performed using primers RT-c, RT-d, and RT-e. The experiment was performed in the absence (B) or presence (C) of the Rho-inhibiting antibiotic bicyclomycin. Band intensities were measured by densitometry and expressed as percentages of the wild-type value (100%) for each reverse primer reaction. The ethidium bromide-stained 16S rRNA shown at the bottom of panels B and C indicates that equivalent amounts of total RNA were used in the PCRs.

The terminator is effective in its native chromosomal location. The experiments described in the preceding sections were performed using recombinant plasmids in which fimE was transcribed from the arabinose-inducible P_BAD promoter. To assess Rho-dependent fimE transcription termination in a more physiologically relevant setting, it was examined in the chromosomally located fim gene cluster in three bacterial strains, namely, W3110, AAEC198A, and MM1 (Table 1). Each strain had a wild-type chromosomal copy of the fimE gene, from which transcription was expressed from the native promoter. The presence of the FimE recombinase kept the fim switch in the OFF phase in these bacteria (data not shown). An RT-PCR assay was designed in which an oligonucleotide primer (RT-e) was used to report on fimE readthrough of the fimS-located Rho-dependent terminator (Fig. 6). Low levels of fimE transcript were detected downstream of the terminator only after more than 25 PCR amplification cycles for all three strains, consistent with the presence of a low-abundance mRNA that was extended to this point (23). However, treatment with the Rho-inactivating antibiotic bicyclomycin allowed transcription readthrough to be detected readily at only 20 cycles using RT-e (Fig. 6). These results demonstrated that the Rho-dependent terminator within OFF-phase fimS functions when the fim operon is in its native location in the chromosome.

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FIG. 6. The fimE transcript is subject to Rho-dependent termination within fimS on the E. coli chromosome. Transcripts expressed from the bacterial chromosome were detected by RT-PCR using reverse primer RT-e, specific for transcripts that traverse a position 3' of the terminator (summarized in panel A), after 20, 25, or 30 PCR cycles in the absence (BY–) or presence (BY+) of the Rho-inhibiting antibiotic bicyclomycin (B). The experiment was performed with three distinct bacterial strains, all of which have fimS predominantly in the OFF orientation and one of which (MM1) lacks an active fimB gene with which to invert the switch back to the ON phase.

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The 295 bp of the fimS element that lie between the IRL and IRR inverted repeats are 70% A+T, which is abnormally high for E. coli (3). Moreover, the ON and OFF orientations of the switch potentially attach sequences with different amounts of C to the fimE transcript. In the ON orientation, the transcript specified by the switch region is 10% C, whereas in the OFF orientation it is 20% C. In the OFF orientation, the C residues are not randomly distributed but cluster in a region extending approximately from the center to the IRR portion of the switch (Fig. 1). In the present report, we extend our analysis of the fimE mRNA Rho-dependent termination event that occurs specifically when the fimS switch is in the OFF orientation by identifying and dissecting the fimS Rho utilization (rut) element. Consistent with other rut sites, such as those of tR1 and the tna region, the fimS rut site consists of a boxA element followed by a sequence rich in C residues (Fig. 1B). Using an RT-PCR-based approach, we showed that when the fimS switch was in the OFF orientation, transcription from the upstream fimE mRNA terminated within the switch (Fig. 2). This termination event required the Rho factor, as the fimE transcript extended through the switch in a rho-15 genetic background or when Rho was inhibited by bicyclomycin treatment (Fig. 2). In order to understand the contribution of the different components of the fimS rut site, we made a series of progressive deletions of the rut site and tested the effects of these on Rho-dependent fimE termination. Our data show that removing just the 8-bp boxA feature has only a modest effect on fimE termination (Fig. 2). Moreover, scrambling its sequence in the OFF orientation did not decrease Rho-dependent termination events (Fig. 3), and presenting it to RNA polymerase traversing an ON-phase switch, and thereby having it incorporated into the fimE ON-phase mRNA transcript, did not induce Rho termination (Fig. 4). However, when boxA was deleted in combination with the C-rich, G-poor region of the fimS rut site (progressively removing a total of 8 bp [3 C residues], 30 bp [12 C residues], or 52 bp [19 C residues]), up to sixfold more amplimer was returned using primers designed to amplify cDNA from mRNA that extended distal to the terminator (Fig. 2). Additionally, mutating the 52-bp fimS rut site (contains 19 C's) such that the first four C residues or the first eight C residues were replaced by G residues caused a significant (up to 12-fold) decrease in fimE mRNA termination within the switch (Fig. 5), confirming that this component of fimS rut is crucially important for Rho action. Finally, we showed that the fimE gene is subject to Rho-dependent termination within fimS in the context of the E. coli chromosome when expressed from its native promoter (Fig. 6) as well as in recombinant plasmids when expressed from an ectopic promoter (Fig. 2). Together, these data on the effect of Rho on the fimS rut site (23; this work) provide another example of the Rho protein's preference for interacting with RNAs that are low in secondary structure and rich in C residues (2, 14, 16, 20, 33, 39, 62).

It can be argued that a Rho-dependent terminator is more suitable than an intrinsic terminator for use in controlling transcript traffic through the invertible fim switch. This is because the "visibility" of the factor-dependent terminator is altered more radically by DNA inversion. Essentially, the fimS terminator becomes invisible to Rho when the switch is in the ON orientation, allowing fimE transcription to traverse the switch, with a concomitant and significant positive effect on message stability (23). On the other hand, an intrinsic terminator with its requirement for a stable stem-loop structure that is specified by inverted repeats in the DNA template would display this palindromic feature regardless of the DNA template orientation.

It is interesting that in the extant fim switch, there is considerable overlap between the C-rich region of the Rho-dependent terminator and Lrp binding site III (Fig. 1). This may represent an influence for the preservation of both, since any sequence divergence would be expected to alter Lrp binding, and therefore the switching efficiency, and our data show that deletions or C-to-G substitutions have a negative impact on fimE termination (Fig. 5), something that is known to feed back into the switch inversion mechanism (23). Bacterial mutants unable to invert the switch at an optimal rate may be at a competitive disadvantage leading to their disappearance from the population. Similar arguments can be advanced concerning conservative influences associated with overlapping regulatory features in other invertible DNA elements. For example, the binding sites for the factor for inversion stimulation in the invertible switch that is responsible for phase-variable expression of flagellar genes in Salmonella are located within the coding sequence of the gene that specifies the Hin site-specific recombinase (21). Consequently, bacteria that acquire mutations in the factor for inversion stimulation binding site may also be expected to lose the ability to express the wild-type recombinase, and the associated loss of competitiveness may cause them to be eliminated from the population.

The possibility that Lrp protein binding to Lrp binding site III influences fimE transcription termination remains an open question. However, this seems unlikely since under the growth conditions used in our experiments, the Lrp protein is not expected to occupy Lrp site III (48). Lrp may influence the system at other levels. For example, inactivation of the lrp gene causes a 50% reduction in fimE transcription from its weak native promoter (4). However, in our experiments fimE was expressed from the arabinose-inducible and Lrp-independent P_BAD promoter. While it is possible that Rho may directly or indirectly influence Lrp expression at some level, the inhibition of Rho function by bicyclomycin treatment does not alter lrp gene expression, at least at the level of transcription (our unpublished data).

Differential transcription termination has been described for other systems. The phase-variable expression of capsular polysaccharide in Neisseria meningitidis is modulated by premature transcription termination at a cryptic Rho-dependent site in the siaD gene coding for polysialyltransferase (29), and the E. coli clpP-clpX operon expresses transcripts of different lengths when bacteria undergo carbon starvation (30). However, the E. coli K-12 fimS Rho-dependent terminator differs from other factor-dependent transcription terminators in being specified by an invertible DNA element such that it is alternately attached to and detached from the transcript that it regulates. This provides the fimE gene with a valuable element of posttranscriptional control through differential mRNA stability that influences the level of the FimE recombinase in the cell, and thus the orientation of the fim switch (23). Thus, fimS is a sophisticated genetic element that integrates several distinct biological functions. These include site-specific recombination, in which the trans-acting FimB and FimE tyrosine recombinases act on the cis-acting inverted repeats to set the orientation of fimS in the chromosome. This in turn determines whether or not the P_fimA promoter is attached to or detached from the fimA gene coding for the major fimbrial subunit protein. Switch orientation also determines whether or not fimE transcription is abbreviated, leading to a decline in FimE protein levels (23), or extended such that the level of this OFF-biased recombinase is enhanced (Fig. 7). In this way, the orientation of the switch is self-regulating, and the Rho-dependent fimE terminator plays a pivotal role in this homeostatic process.

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FIG. 7. The orientation of the fim switch controls the expression of the FimE recombinase. The fimB and fimE genes are transcribed from independent promoters. Their transcripts are translated into recombinases that invert the switch without bias (FimB) or with a strong ON-to-OFF bias. The phase ON switch does not terminate fimE transcription, and the extended message is stable (23). Bacterial populations coexpressing FimB and FimE have the switch predominantly in an OFF orientation (13). In this case, Rho-dependent termination at the terminator (T) produces a truncated fimE message that is unstable (23). This instability (represented by a dotted arrow) results in a strong reduction in FimE protein levels, giving FimB an opportunity to invert the switch back to the ON phase.

 
This work was supported by grant 061796 from the Wellcome Trust.

We thank Nicola Holden and David Gally for bacterial strains, Michael Mangan for assistance with imaging software, and Matthew McCusker and Stephen Smith for helpful discussions. We are very grateful to Fujisawa Pharmaceutical, Osaka, Japan, for samples of bicyclomycin.

 
* Corresponding author. Mailing address: Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland. Phone: 353 1 608 2013. Fax: 353 1 679 9294. E-mail: cjdorman{at}tcd.ie.

Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, December 2005, p. 8256-8266, Vol. 187, No. 24
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