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Journal of Bacteriology, December 2008, p. 7709-7718, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.00995-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of Conserved Bases in 4.5S RNA of Escherichia coli by Construction of New F' Factors{triangledown}

James M. Peterson and Gregory J. Phillips*

Department of Veterinary Microbiology, Iowa State University, Ames, Iowa 50011

Received 18 July 2008/ Accepted 6 September 2008


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ABSTRACT
 
To more clearly understand the function of conserved bases of 4.5S RNA, the product of the essential ffs gene of Escherichia coli, and to address conflicting results reported in other studies, we have developed a new genetic system to characterize ffs mutants. Multiple ffs alleles were generated by altering positions that correspond to the region of the RNA molecule that interacts directly with Ffh in assembly of the signal recognition particle. To facilitate characterization of the ffs mutations with minimal manipulation, recombineering was used to construct new F' factors to easily move each allele into different genetic backgrounds for expression in single copy. In combination with plasmids that expressed ffs in multiple copy numbers, the F' factors provided an accurate assessment of the ability of the different 4.5S RNA mutants to function in vivo. Consistent with structural analysis of the signal recognition particle (SRP), highly conserved bases in 4.5S RNA are important for binding Ffh. Despite the high degree of conservation, however, only a single base (C62) was indispensable for RNA function under all conditions tested. To quantify the interaction between 4.5S RNA and Ffh, an assay was developed to measure the ability of mutant 4.5S RNA molecules to copurify with Ffh. Defects in Ffh binding correlated with loss of SRP-dependent protein localization. Real-time quantitative PCR was also used to measure the levels of wild-type and mutant 4.5S RNA expressed in vivo. These results clarify inconsistencies from prior studies and yielded a convenient method to study the function of multiple alleles.


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INTRODUCTION
 
The signal recognition particle (SRP) is an RNA-protein complex that functions in all three domains of life to localize proteins outside of the cytoplasmic compartment. The SRP functions early in the targeting pathway by binding to hydrophobic signal sequences or transmembrane domains as they emerge from the ribosome. The nascent polypeptide/ribosome/SRP complex subsequently interacts with a membrane-bound receptor to facilitate cotranslational insertion of the polypeptide into or through the membrane (17-19, 27). Mechanistically, the process of targeting proteins either into or across the endoplasmic reticulum in eukaryotes is equivalent to localization to the cytoplasmic membrane of bacteria.

In higher eukaryotes the SRP consists of a 7S RNA species in association with six distinct proteins (48), while in Escherichia coli the SRP includes a single protein (Ffh) in a complex with 4.5S RNA (39, 41). Genetic analysis in E. coli has shown that the genes encoding both Ffh and 4.5S RNA (ffs) are essential for viability (9, 38), and depletion of either component results in a defect in localization of proteins to the cytoplasmic membrane (15). The relative simplicity of the E. coli SRP, coupled with its conserved cellular function, makes it an attractive system to study the fundamental components of membrane protein targeting.

Biochemical studies have shown that the SRP is a multifunctional ribonucleoprotein complex. It functions to bind and release hydrophobic polypeptides, interacts with ribosomes and the SRP receptor (FtsY in E. coli), and is a GTPase (28, 29, 35, 40, 43, 50). While all of these activities can be attributed directly to the multidomain Ffh protein, the role of 4.5S RNA in SRP function is less clear. In vitro 4.5S RNA has been shown to stabilize Ffh upon binding hydrophobic polypeptides (12, 52). Kinetic analysis of Ffh/FtsY interaction revealed that 4.5S RNA also enhances association and stability of the complex (34, 35). The RNA also plays a role in stabilizing Ffh when bound to membrane proteins (12, 52) and may also participate in interaction with membrane proteins targeted by the SRP (4). Prior to its discovery as a component of the SRP, 4.5S RNA was proposed to function in protein synthesis (7, 8). Clearly, more studies are required to understand the role of 4.5S RNA in E. coli physiology.

Genetic approaches, including characterization of multiple mutant ffs alleles, have been used to better identify how different regions of the molecule contribute to its function. For example, deletion mutants of ffs (2, 32) confirmed that the region absolutely required for 4.5S RNA function includes the highly conserved helix 8 (25), which makes direct contact with Ffh (2). Other studies have focused on the four-base tetraloop structure, which apparently is important for stimulating GTP hydrolysis of the SRP-FtsY complex (22, 45).

Mutant alleles have also been constructed to identify individual bases within helix 8 that are essential for 4.5S RNA function both in vivo and in vitro (31, 51). In reviewing the results of these studies, however, we identified limitations of the genetic systems used that made interpretation of the data difficult. For example, these studies used mutants in which the sole functional copy of ffs was under the control of the tac promoter for complementation tests (31, 51). However, expression of ffs from this construct is not fully repressed in the absence of the inducer isopropyl-β-D-thiogalactopyranoside (IPTG), as evidenced by the ability of the strains to yield suppressor mutations that allowed E. coli to survive with low levels of 4.5S RNA (7, 8). This feature, although useful for isolation of suppressor mutants, indicates that sufficient levels of 4.5S RNA are produced that could complicate the interpretation of complementation tests.

We also questioned the sensitivity of the methods for complementation tests since the ffs alleles were expressed from multiple-copy-number, pBR322-derivative plasmids. Also, the wild-type ffs promoter was not used for expression in these constructs (31, 51). As observed previously (51), and as confirmed by the results presented here, expression of mutant 4.5S RNA at sufficiently high levels can mask functional defects.

In addition to methodological concerns, we noted that the results of these prior studies did not always match expectations. For example, Wood et al. (51) showed that all of the ffs mutants, with the exception of ffs carrying the mutation C62G [ffs(C62G)], were able to complement a mutant in which the wild-type copy of ffs was expressed at sufficiently high levels. However, binding of selected mutant RNAs to Ffh was not detected either by in vitro binding assays or in vivo by Northern blot analysis of RNA recovered by immunoprecipitation of Ffh. Since the SRP is required for E. coli viability, it is difficult to understand how a mutant 4.5S RNA that no longer bound Ffh could also support growth. Nakamura et al. conducted a similar study of ffs mutants (31). Although this study was designed to examine the interaction between 4.5S RNA and elongation factor G (EF-G), complementation tests using a similar conditional ffs mutant revealed that alleles G64U and A67C failed to support growth when expressed from recombinant plasmids. In contrast, the G48C allele complemented as well as wild-type ffs. Inspection of the E. coli SRP crystal structure shows that while bases A47, G48, and C62 make significant contacts with Ffh, bases G64 and A67 do not make direct contacts (2, 3). It is unclear why the complementation tests did not reflect the predicted importance of the bases as revealed by the SRP structure. Although Wood et al. (51) showed that G48U failed to complement under some growth conditions, they did not test mutants altered at positions G64 and A67.

Measurements of the levels of 4.5S RNA expressed in the cell have also resulted in varied conclusions. The levels of 4.5S RNA have been reported as high as 20% of the value of 5S rRNA (20, 26), while Jensen and Pedersen reported levels significantly lower (24). Determining the intracellular levels of 4.5S RNA has implications for understanding how the RNA functions in E. coli other than as a component of the SRP.

To clarify the results of previous studies and to unambiguously identify the bases within helix 8 that are important for 4.5S RNA function, we developed a genetic system to express ffs alleles in single copy. Although there are multiple ways to express genes in single copy, including low-copy-number plasmids or integration of gene constructs into the bacterial chromosome (reviewed in reference 37), we used recombineering, i.e., bacteriophage {lambda} Red recombination (13), to construct new F' factors for this purpose. This approach allowed us to construct and transfer multiple ffs alleles, with minimal manipulation, to a strain engineered specifically to perform complementation tests in haploid colonies. Historically, F' plasmids have been instrumental in understanding gene function and regulation in E. coli (37) but have largely been replaced by recombinant DNA. The use of the recombineering strategy described here reintroduces F' factors as useful tools for bacterial genetics. To more thoroughly test the ability of the mutant 4.5S RNA to interact with Ffh, we also developed an assay to measure this association in vivo. These assays clearly identified the bases of helix 8 essential for SRP function and Ffh binding, thus clarifying inconsistencies found in prior studies. In addition, we provide an independent measurement of the levels of 4.5S RNA in E. coli.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and medium. Strains and plasmids used in this study are shown in Table 1. Medium was prepared as described by Miller (30). Antibiotics and other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Enzymes for cloning were obtained from New England Biolabs (Ispsich, MA) and Fermentas Life Sciences (Glen Burnie, MD).


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  		TABLE 1. Strains and plasmids used in this study

Construction of ffs alleles. PCR mutagenesis was used to construct the ffs alleles used in this study. PCR primers are shown in Table 2 and were obtained from Integrated DNA Technologies (Coralville, IA). pSB832 (Table 1) was used as a template for all PCRs. PCR was performed using rTaq Mastermix (Takara Bio, Inc., Otsu, Japan) using a PTC-100 thermocycler (MJ Research, Inc., Waltham, MA) with the following cycling conditions: 5 min at 95°; 25 cycles of 30 s at 95°, 30 s at 55°, and 1 min at 72°; and 10 min at 72°. The A39C, C40G, C41G, A42U, C46G, A47C, A47G, G48C, G48U, and G49C mutant products were produced using their respective primers with the EcoRI-40s-S primer (Table 2). The G58A, A60U, G61U, C62G, A63C, G64U, C65G, A67C, A67G, and A67U mutant products were produced using their respective primers with the 832.AS primer (Table 2).


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  		TABLE 2. PCR primers used in this study

PCR products were purified from 0.8% agarose gels using a Qiaex II elution kit (Qiagen, Valencia, CA) and dissolved in sterile, nanopure water. The purified products representing alleles A39C, C40G, C41G, A42U, C46G, A47C, A47G, G48C, G48U, and G49C were digested with BspEI and EcoRI and cloned into gel-purified pSB832 digested with the same enzymes. Products representing alleles G58A, A60U, G61U, C62G, A63C, G64U, C65G, A67C, A67G, and A67U were digested with BspEI and SalI and cloned into pSB832. All constructs were confirmed by DNA sequencing at the DNA Sequencing and Synthesis Facility of Iowa State University.

Construction of new F' plasmids. Derivatives of F' lac proA+B+ were constructed using the recombineering method described by Warming et al. (49) for modification of bacterial artificial chromosomes. Initially, F' lac proA+B+ was moved from CSH100 into SW102 by conjugation with selection for Lac+. To modify the F' by recombineering, 0.5 ml of overnight cultures of SW102 F' lac proA+B+ were transferred to 50 ml of fresh LB medium and grown at 30°C with vigorous shaking until an optical density at 600 nm (OD600) of between 0.5 to 0.6 was reached. The culture was shifted to 42°C with continued shaking for exactly 15 min to induce the Red recombinase genes. Cultures were immediately placed in an ice water slurry with gentle agitation for 5 min. Cells were pelleted at 4°C and washed twice with ice-cold, sterile, nanopure water before being resuspended in 500 µl of water. To provide a convenient selectable marker, cat, encoding chloramphenicol resistance (Camr), was first introduced to F' lac proA+B+ by recombination into cynX, carried by the F'. The cat cassette was amplified from pKD3 with primers cynX-KD3.S and cynX-KD3.AS (Table 2) using the following cycling conditions: 5 min at 95°; 25 cycles of 30 s at 95°, 1 min at 55°, and 30 s at 72°; and 10 min at 72°. The PCR product was gel purified, and ~1 µg of DNA was electroporated into SW102 F' lac proA+B+. Camr recombinants were selected at 30°C on LB medium plus Cam (12.5 µg/ml).

As summarized in Fig. 1, galK+ was introduced to F' lac proA+B+ at lacA using a similar strategy. galK was amplified from pGalK (49) using primers lacA-galK.S and lacA-galK.AS (Table 2). As described, recombinants were selected on galactose minimal medium at 30°C for 48 h and then tested for sensitivity to 2-deoxy-galactose (2-DOG) (49). A second round of recombineering was used to replace galK at the lacA target site with each ffs allele. PCR was used to amplify the ffs alleles from each of the pSB832-derivtive plasmids described above using primers 832-lacA.S and 832-lacA.AS (Table 2). Purified products were electroporated into SW102 F' lac proA+B+ and plated on 2-DOG minimal medium and incubated 48 h at 30°C. Several of the resulting colonies were picked and screened by PCR to identify the correct recombinants. For this, total cellular DNA was purified from recombinants using a Masterpure Complete DNA and RNA Purification Kit (Epicentre, Madison, WI). The DNA was probed using primers lacA-diag.S and lacA-diag.AS (Table 2) and the following cycling conditions: 5 min at 95°; 25 cycles of 30 s at 95°, 1 min at 55°, and 1 min at 72°; and 10 min at 72°. Desired recombinants were identified by detection of a ~0.4-kb product, in contrast to the ~1.4-kb product yielded by the galK+ parent.


Figure 1
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  		FIG. 1. Summary of the genetic system used for characterizing ffs mutants. (A) Recombineering was used to construct a series of F' factors expressing different ffs alleles. As explained in Materials and Methods, galK+ was first incorporated into the F' plasmid by recombineering using homology to lacA carried by F' lac+ pro+. Next, individual ffs alleles are introduced to the F' elements by selecting against galK+ by resistance to 2-DOG. (B) JP2005 has a deletion of the chromosomal copy of ffs and carries a complementing copy of ffs on a plasmid that is temperature sensitive for replication. F' plasmids or ColE1-derivative plasmids bearing mutant alleles of ffs were introduced by conjugation or transformation, respectively. The ability of the ffs alleles to support growth of JP2005 was determined by comparing the efficiency of plating at 30°C and 42°C, the nonpermissive temperature of the temperature-sensitive plasmid.

Complementation tests. JP2005 was constructed by transforming CSH109 with plasmid pffsTS-Spc, a spectinomycin resistant (Spcr) derivative of a pSC101 cloning vector with a temperature-sensitive origin of replication (36) and expressing wild-type 4.5S RNA. Transformations were performed as described by Inoue et al. (21). Transformants were then transduced to ffs::kan-591 using a bacteriophage P1 lysate prepared on S1693 (Table 1) (30). Kanamycin-resistant (Kanr) transductants were selected at 30°C and tested for temperature sensitivity at 42°C. The resulting strain, JP2005, was used as the recipient in conjugation reactions with each of the SW102 F' donors carrying wild-type and mutant ffs alleles. Donors and recipient were patched together on an LB agar plate and incubated at 30°C for 12 h. Transconjugants were selected by streaking cells onto LB agar plates supplemented with Cam (12.5 µg/ml) and Spc (100 µg/ml) (for counter-selection). To express the mutant alleles in multiple copies, each of the pSB832-derivative plasmids described above was used to transform JP2005.

Efficiency of plating was used to assess the ability of the ffs alleles to complement ffs::kan-591 in JP2005. Transconjugants or transformants were grown overnight at 30°C, and then serial dilutions were plated in duplicate at 30°C and 42°C, the nonpermissive temperature for plasmid pffsTS-Spc. Single colonies were counted after overnight incubation, and the results are reported as the number of colonies formed at 42°C divided by the colonies formed at 30°C. The colonies that formed at 42°C were also replica plated onto LB medium supplemented with Spc to confirm the loss of pffsTS-Spc. Spc-sensitive JP2005 derivatives were saved to further characterize the ffs alleles in haploid colonies.

To compare growth of the mutants in haploid colonies, overnight cultures of JP2005 transformants were serially diluted, and ~5 µl was spotted onto 100-mm2 LB agar plates. The plates were incubated at 42°C for 48 h, and images of the colonies were taken with a digital camera (Nikon CoolPix 4500). The plates were incubated for an additional 48 h at 30°C, and images of the same colonies were again captured.

Determination of 4.5S RNA levels by RT-qPCR. Reverse transcriptase real-time quantitative PCR (RT-qPCR) was used to measure the amount of 4.5S RNA in E. coli in comparison to 5S rRNA. Total cellular RNA was extracted from overnight cultures using a MasterPure Complete DNA and RNA Purification Kit (Epicentre, Madison, WI) following the manufacturer's protocol for RNA purification from gram-negative bacteria. Samples were probed for both 4.5S RNA and 5S RNA using a Bio-Rad iCycler with an MyiQ detection system (Bio-Rad, Hercules CA). RT-qPCR reactions were performed by adding 1 µl of a 10 µM solution of each primer, RTffs-for and RTffs-rev (Table 2) for 4.5S RNA or RTrrf-for and RTrrf-rev (Table 2) for 5S RNA, 1 µl of purified total RNA (~20 pg), and 9 µl of PCR-grade water. Samples were incubated at 85°C for 3 min to denature the RNA and then placed on ice for 1 min before the addition of 13 µl of prereaction mix, which included 12.5 µl of RT-qPCR with Sybr Green Master Mix (Bio-Rad, Hercules, CA) and 0.5 µl of Reverse Transcriptase Solution (Bio-Rad, Hercules, CA). Amplification conditions were the following: 10 min at 52°C, 5 min at 95°C, and 40 cycles of 30 s at 95°C followed by 30 s at 55°C. Measurements were recorded by the instrument at the 55°C incubation step of each cycle.

The specificity of the RT-qPCR products was confirmed by determining melting curves to measure the change in fluorescence as the PCR amplicons denatured. Melting curves were performed by incubating the PCR products for 1 min at 95°C, followed by a return to 55°C. The temperature was then increased in 0.5°C increments every 30 s for 40 min, and readings were taken at each step.

MyiQ software (Bio-Rad, Hercules CA) was used to determine the threshold cycle (CT) values and melting curve peaks. The CTvalue represents the cycle number where the signal for a sample passes a specific threshold above background established by the software to obtain the most precise values. The MyiQ software then generated the melting curves by plotting the change in fluorescent signal versus temperature. Detection of a single product or peak on the graph confirmed that the RT-qPCR reaction yielded an accurate measurement of input RNA concentration.

The ratio of 4.5S RNA to 5S RNA in each sample was calculated by first establishing a standard curve correlating RNA quantity to the CTvalue. RNA was produced by in vitro transcription of both ffs and rrfG (encoding 5S rRNA) under the control of the T7 promoter on the plasmid pT7T3ffs (51) or plasmid pZERO-T7rrfG using an AmpliScribe T7-Flash Transcription Kit (Epicentre Biotechnologies, Madison, WI). The T7 promoter-rrfG construct was synthesized (Integrated DNA Technologies, Coralville, IA) using the sequence information of a similar construct (16) and carried by the vector pZERO (Invitrogen, Carlsbad, CA). Dilutions of the in vitro transcripts were analyzed by RT-qPCR to establish standard curves for both RNA species. RT-qPCR was then performed on total cellular RNA to determine the CTvalues for both 4.5S RNA and 5S RNA. The resulting CT values were all within the range of the standard curve (data not shown). The absolute values determined from the standard curve for 4.5S RNA were divided by the same values for 5S RNA to determine the number of 4.5S RNA molecules per 5S RNA.

To test the levels of expression of the ffs alleles that failed to complement in single copy, we introduced a plasmid (pSB1334) expressing an ffs orthologue from Micrococcus lysodeikticus (ffsMl) (8). While ffsMl complements an E. coli ffs mutant, it is sufficiently distinct so as not to yield a signal in the RT-qPCR reactions used here.

Assay of 4.5S/Ffh interaction in vivo. Ffh was purified from JP2005 derivatives transformed with pBADffh-6XHis, a plasmid that expresses Ffh tagged at the C terminus with a hexahistidine epitope. Fifty-milliliter cultures were grown in LB medium at 37°C until cells reached mid-exponential growth phase (~3 h). Fifty-milliliters of 10% L-arabinose was then added to induce ffh expression. After ~30 min, cells were pelleted and resuspended in 3 ml of binding buffer (Zymo Research, Orange, CA). Lysozyme (1,000 U) was added, and the suspensions were placed on ice for 30 min. Cells were maintained in an ice slurry and lysed by sonication using a Vibra Cell machine (Sonics and Materials Inc., Newtown, CT) at 20% power for 5 min using 10-s intervals. The lysates were centrifuged, and 300 µl of the supernatant was added to a spin column for purification of Ffh using a His-spin Purification Kit (Zymo Research, Orange, CA).

RT-qPCR was used to quantify the amount of 4.5S RNA that copurified with Ffh. RT-qPCR reactions were performed using 1 µl of purified protein (200 ng) under the conditions described in the previous section. The specificity of the RT-qPCR products was again confirmed by performing melting curves to measure the change in fluorescence as the PCR amplicons denature. The relative signals for the test samples were calculated by comparing the CT values of each sample with a standard curve generated using serially diluted 4.5S RNA transcribed in vitro. The CT values for these samples were within the range of the standard curve (data not shown). The signal-to-protein value was calculated by dividing the relative signal from the RT-qPCR by the protein concentration of each sample. Protein concentrations were measured using a Quant-iT Protein Assay Kit (Invitrogen, Carlsbad, CA), and readings were taken on a NanoDrop ND-3300 Fluorospectrometer (NanoDrop Technologies, Wilmington, DE) using a Quant-iT protein module in the NanoDrop ND-3300 software (version 2.6). Concentrations of purified Ffh were determined by comparison with the linear portion of a standard curve generated using a Quant-iT Protein Assay Kit.

FtsQ localization assay. The biotinylation assay described previously was used to monitor localization of the SRP-dependent, cytoplasmic membrane protein, FtsQ (33, 47). The plasmid pBADftsQ-V5-PSBT was constructed using the pBADtopo cloning system (Invitrogen, Carslbad, CA) to encode FtsQ fused to the V5 epitope and the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (23). Selected ffs mutants were transformed with this plasmid and grown to mid-logarithmic growth phase before induction with arabinose. After cells reached an OD600 of ~0.6, proteins were prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and resolved on 12% polyacrylamide gels.

Proteins were transferred to a polyvinylidene difluoride membrane and probed for FtsQ using goat anti-V5 antibody (Bethyl Inc., Montgomery, TX) and rabbit anti-goat-horseradish peroxidase conjugate (Bio-Rad, Hercules, CA). Biotinylated FtsQ was detected using streptavidin-horseradish peroxidase conjugate (Sigma Chemical Co., St. Louis, MO), as described previously (33). Proteins were visualized using a SuperSignal West Femto substrate kit (Pierce, Rockford, IL), imaged using a MultiImage II light cabinet (DE-500) (Alpha Innotech Corporation, San Leandro, CA), and analyzed with FluorChem 8000 advanced fluorescence, chemiluminescence, and visible light imaging software (Alpha Innotech Corporation, San Leandro, CA).


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RESULTS
 
A new genetic system to study ffs. To better understand how conserved bases within helix 8 of 4.5S RNA contribute to its function, we constructed JP2005, an E. coli strain that allowed complementation tests of multiple ffs alleles to be performed in haploid. As shown in Fig. 1, the ffs::kan-591 allele in JP2005 is complemented by ffs+ carried on a plasmid that is temperature sensitive for replication. As a result, JP2005 grows normally at 30°C but grows at 42°C only if a functional copy of ffs is provided.

Figure 2 summarizes the mutations made for this study and depicts the consensus sequence of helix 8 compiled from prokaryotic, eukaryotic, and archaeal sources (1). As described in Materials and Methods, we used PCR to construct several ffs alleles on the pBR322-derivative plasmid, pSB832. Since one of our goals was to reevaluate the results of previous studies, we made ffs mutations based largely on the changes reported previously (31, 51). This collection included bases known to be contacted directly by Ffh, as well as bases representing various degrees of evolutionary conservation. The bases of the tetraloop at the end of the hairpin structure of the 4.5S RNA molecule have been shown to be dispensable for Ffh binding (22, 45) and were not included in this study.


Figure 2
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  		FIG. 2. Summary of 4.5S RNA mutants. The primary sequence and predicted secondary structure of 4.5S RNA is shown at the top. Helix 8, the region bound by Ffh, is underlined. The mutations generated in this study are indicated by arrows. The degree of conservation of each base among SRP RNAs from all three domains of life is represented by the size of the base, with the largest sizes representing the most highly conserved. The consensus sequence of SRP RNA is shown at the bottom, where the largest-sized bases are also most highly conserved. The numbers represent the positions of the bases in E. coli 4.5S RNA. The degree of base conservation and the consensus sequence were from the SRP database (1).

We next developed a convenient way to test the ability of the ffs alleles to complement ffs::kan-591 in JP2005 when expressed in single copy. Since F' factors offer several advantages for complementation tests, we used the recombineering method described by Warming et al. (49), originally designed for modification of bacterial artificial chromosomes, to construct a series of F' plasmids to introduce the ffs alleles in single copy. For this, a derivative of strain SW102 expressing {Delta}galK {lambda}-Red (49) was made by introducing F' lac+ pro+ by conjugation. A PCR product in which galK+ was flanked by 45-bp sequences with homology to lacA was generated and introduced to SW102 F' lac+ pro+ by selection for Gal+.

Using plasmids as templates, PCR products representing each ffs allele shown in Fig. 2 were generated with the same 45-bp extensions homologous to lacA. Since Gal+ E. coli is sensitive to 2-DOG, colonies that grew in the presence of this galactose analog were selected and largely represented recombinants where galK+ had been replaced by ffs (Fig. 1). Individual colonies were tested by PCR to confirm the replacement of galK for ffs (data not shown). The recombinant F' factors were introduced to JP2005 by conjugation, and the resulting strains were used in complementation tests.

Complementation tests with ffs mutants. The ability of the ffs alleles to complement ffs::kan-591 was determined by comparing the efficiency of plating at 30°C and 42°C, the nonpermissive temperature for replication of the ffs+ plasmid. The results, summarized in Table 3, showed that six of the mutants (A47G, G48C, G48U, G58A, G61U, and C62G) failed to form single-copy colonies at 42°C. Four additional mutants (A39C, A42U, G49C, and A63C) showed a small, but consistent, decrease in efficiency of plating, while the remaining mutants were indistinguishable from the wild type. All of the colonies that grew at 42°C became Spc sensitive, indicating loss of the temperature-sensitive ffs+ plasmid.


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  		TABLE 3. Efficiency of plating results for ffs alleles expressed from F' factors or from multiple-copy-number plasmids

Efficiency of plating was also determined using the multiple-copy-number, pSB832-derivative plasmids to express the ffs alleles (Table 3). In contrast to the results from the single-copy experiments, with one exception, all of the ffs mutations complemented ffs::kan-591. Only the C62G mutation failed to support growth at 42°C.

In the course of conducting these experiments, we observed that colony sizes of the different mutants, when expressed in either single copy or from multiple-copy-number plasmids, varied significantly, indicating that the mutants grew at different rates. To directly compare the growth characteristics of the mutants, cultures were spotted onto LB plates, and colony size was monitored after growth at 42°C for 48 h (Fig. 3). While the efficiency of plating of all of these mutants was near unity when expressed from multiple-copy-number plasmids (Table 3), mutants A47G, G48C, G48U, and G61U grew poorly in comparison to the wild type following incubation at 42°C (Fig. 3). We further observed that colonies of some of the mutants resumed normal growth when incubated below 42°C. To characterize this observation further, we grew cultures at 42°C on solid medium for 24 h and then shifted the plates to 30°C for an additional 48-h incubation. Figure 3 shows that each of the mutants that grew poorly at 42°C formed colonies essentially the same size as the wild type at 30°C, confirming the temperature sensitivity of the mutants. These results also showed that mutants that exhibited only a small decrease in efficiency of plating (Table 3) had no significant difference in colony size compared to the wild type at 30°C or 42°C (Fig. 3).


Figure 3
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  		FIG. 3. Growth of ffs mutants. Cultures of JP2005 transformed with plasmids expressing different ffs alleles in multiple copy numbers were grown as described in Materials and Methods. Each ffs allele is specified in the columns on the left. Images represent identical colonies formed after an initial incubation at 42°C followed by further incubation at 30°C, as indicated on the right of each column.

Levels of 4.5S RNA expressed in vivo. Since the function of mutant RNAs is directly related to their expression levels, we used RT-qPCR to measure the amounts of 4.5S RNA in comparison to 5S rRNA, an RNA species of similar size. RT-qPCR yielded CT values, defined as the fractional cycle number at which the fluorescence signal from the specific product passes a fixed threshold that is significantly above background levels. Since CT values typically vary between different RNA targets and reaction conditions, we first established standard curves for both 4.5S RNA and 5S RNA to correlate the CTvalue with moles of RNA. We then compared the amounts of the two RNA species based on the common absolute value of moles rather than CT.

Using this method, 4.5S RNA was found at 3 to 4% of the levels of 5S rRNA when expressed from either an F' or the chromosome (Table 4). The levels of 4.5S RNA increased ~10-fold, however, when the ffs alleles were expressed on multiple-copy-number plasmids. Also, the levels of the different mutant RNAs did not vary significantly from one another when they were expressed from either single- or multiple-copy-number vectors (Table 4).


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  		TABLE 4. Levels of 4.5S RNA in strains expressing ffs from F' or ColE1 plasmids

In vivo assay for Ffh-4.5S RNA interaction. To characterize the interaction between 4.5S RNA and Ffh in vivo, we used RT-qPCR to determine the amount of 4.5S RNA found associated with purified Ffh protein. A subset of the ffs mutants, representing mutants whose growth ranged from poor to nearly wild-type levels (Fig. 3), were tested for their ability to bind Ffh in vivo. Since all of the ffs alleles, with the exception of C62G, supported growth of JP2005 when expressed from multiple-copy-number plasmids, the assays were performed with pSB832-derivative transformants. The results are summarized in Table 5. A strong correlation was observed between how well an individual mutant RNA associated with Ffh and its ability to support growth of JP2005. For example, mutants C40G, A42U, and A67U supported growth as well as ffs+ at all temperatures (Fig. 3) and associated with Ffh nearly as well as wild-type RNA (Table 5).


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  		TABLE 5. Copurification of 4.5S RNA mutants with Ffh

Mutants A39C, A47C, and A63C showed a more significant reduction in Ffh copurification compared to wild-type RNA (Table 5). While all of the mutants showed only a modest decrease in efficiency of plating in single copy, A47C also showed a growth defect at 42°C (Fig. 3). Mutant G58A appeared to be an exception. Although it displayed a similar decrease in Ffh binding (Table 5), it failed to complement in single copy. Also, while growth of the G58A mutant was not temperature sensitive, it formed heterogeneously sized colonies at both 42°C and 30°C. The behavior of G58A indicates that it is defective in a function of 4.5S RNA in addition to impaired Ffh binding.

Mutants A47G, G48U, G48C, and G61U all showed the most significant reduction in Ffh copurification (Table 5), and none supported growth in single copy or grew well at 42°C in multiple copy numbers (Fig. 3). We were unable to test the C62G mutant in the Ffh binding assay since it did not complement JP2005 even when expressed from a multiple-copy-number plasmid.

Effect of 4.5S RNA mutations on SRP function. We predicted that the ffs mutants that copurified with Ffh at the lowest levels would also exhibit the most distinct defects in SRP function. To test this, we monitored the localization of FtsQ, an E. coli cell division protein positioned in the inner membrane with the N terminus in the cytoplasm and the C terminus in the periplasmic space (11) and whose targeting is SRP dependent. For these assays, pBADftsQ-V5-PSBT (23) was constructed. When expressed in E. coli, the FtsQ protein fused with the P. shermanii transcarboxylase is biotinylated only if SRP function is impaired (33, 46, 47). Biotinylated FtsQ in ffs mutants is shown in the top panel of Fig. 4. To confirm that FtsQ was expressed in each mutant, Western blot analysis was performed using antibody against the V5 epitope, as shown in the bottom panel of Fig. 4.


Figure 4
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  		FIG. 4. Detection of biotinylated FtsQ. The ffs mutants shown were tested to determine the extent of biotinylation of the SRP-dependent protein FtsQ. (Top) Biotinylated FtsQ as detected by decoration with streptavidin-horseradish peroxidase conjugate. (Bottom) FtsQ as detected by Western blot analysis using antibody specific for the V5 epitope. A plus sign indicates ffs+, and other alleles are as designated.

FtsQ localization was monitored in ffs mutants that represented slight (C40G and A67U), modest (A39C, A47C, and G58A), and severe (G48C, G48U, and G61U) defects in Ffh copurification. A direct correlation was observed between the levels of Ffh copurification and SRP function. Specifically, mutants G48C, G48U, and G61U showed the greatest defects in FtsQ localization, as evidenced by detection of biotinylated protein (Fig. 4, lanes 5, 6, and 8) and also copurified poorly with Ffh (Table 3). Relatively minor localization defects were observed in mutants A39C and A47C (lanes 2 and 4), corresponding to their higher levels of Ffh copurification. Mutants C40G and A67U copurified with Ffh at levels near that of the wild type (Table 3) and also showed no defects in FtsQ localization (Fig. 4, lanes 3 and 9). Mutant G58A was an exception, however, as it showed a significant SRP defect (lane 7) but only a modest defect in copurification with Ffh (Table 3). As anticipated, biotinylated FtsQ was not detected in cells expressing wild-type 4.5S RNA (lanes 1 and 10). Most samples showed that similar amounts of FtsQ were expressed in each mutant (Fig. 4, bottom panel). Mutant G58A (lane 7) showed the lowest levels of FtsQ, which correlated with its relatively poor growth.


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DISCUSSION
 
To dissect the diverse functions of 4.5S RNA, we have developed a new genetic system to characterize ffs mutants altered primarily at highly conserved residues (Fig. 1). Although an alternative genetic system has been reported where wild-type ffs is carried on a bacteriophage {lambda}cI857 prophage (10), it has not been widely used. While this strain was used to clone ffs homologues from different sources (6, 10), it requires multiple manipulations to be performed carefully since the phage remains fully virulent.

Alleles were constructed that corresponded to mutations that had been characterized previously using more limited genetic systems (31, 51). Our studies, in combination with a previous report (51), indicated that the phenotypes of ffs mutants are masked by elevated expression of mutant RNA. To express the ffs alleles in single copy and to facilitate manipulation of the multiple alleles, we incorporated several ffs alleles (Fig. 2) onto F' lac+ pro+ using recombineering. In the experiments reported here, we also used recombineering to introduce a Camr marker to the F' lac+ pro+ in order to facilitate selection of exconjugants; however, in other experiments we have also selected Lac+.

RT-qPCR confirmed that each allele was expressed at nearly wild-type levels when expressed from an F' plasmid and showed ~10-fold higher levels when expressed from ColE1 plasmids (Table 4). This analysis also allowed us to measure the amount of 4.5S RNA expressed from wild-type E. coli. Our results showed that 4.5S RNA was present at only 3% of the levels of 5S rRNA (Table 4). This value agrees closely with the results of Jensen and Pedersen (24), who used autoradiography to show that 4.5S RNA was present at 4% of the levels of 5S RNA. In contrast, other studies had reported that 4.5S RNA was more abundant, at up to 20% of the levels of 5S rRNA (20, 26). As pointed out by Jensen and Pedersen (24), this discrepancy may be due to different methods used to subtract background radioactivity from autoradiograms (20) or to growth under phosphate starvation conditions that may have led to a reduction of the 5S RNA signal (26). In any case, the use of RT-qPCR represents an independent method to measure 4.5S RNA levels and indicates that the RNA is not highly abundant.

Comparison of mutants expressing ffs alleles from F' plasmids versus multiple-copy-number plasmids confirmed the importance of performing complementation tests in single copy. All of the ffs mutants, with the exception of C62G, complemented JP2005 when expressed from ColE1 plasmids. Consistent with structural studies, C62 occupies a key position in assembly of the SRP (2). These results are also consistent with the study by Wood et al. (51), who also showed that C62G destroyed 4.5S RNA function, while none of the other mutations tested completely failed to support growth of an E. coli ffs mutant. However, the ability of ffs alleles to complement a true ffs knockout mutation when expressed in single copy was not tested in this study (51).

In general, mutants altered at highly conserved bases of 4.5S RNA known to interact directly with Ffh did not support growth of JP2005 in single copy. Figure 2 shows eight positions in 4.5S RNA that are nearly universally conserved in all three kingdoms of life (1). Of these, bases at positions A39, A47, G48, G61, C62, and A63 make direct contact with specific amino acids of Ffh (2, 3). With the exception of A39 and A63, mutations at these positions failed to complement JP2005 when expressed in single copy. Also, although alleles A47G, G48C, G48U, and G61U were able to support growth of JP2005 when expressed from multiple-copy-number plasmids, growth was poor at 42°C (Fig. 3). The growth defects showed allele specificity at position A47. Although A47C grew at a slower rate than the wild type at both 30°C and 42°C (data not shown), A47G failed to complement in single copy and grew poorly at 42°C in multiple copy numbers (Fig. 3).

We observed that a mutation at A39, also a highly conserved position, did not yield a significant growth defect even in single copy. Although efficiency of plating of the A39C mutant was consistently half that of the wild type (Table 3), no other growth defects were observed (Fig. 3). Ffh also contacts the ribose or phosphodiester backbone at positions G49, A60, and A63 (2, 3), and all of the mutants altered at these positions complemented in single copy. Of the remaining mutations, only G58A failed to complement in JP2005 in single copy.

Using a semiquantitative assay to measure the amount of 4.5S RNA that copurified with Ffh, we were able to distinguish between different 4.5S RNA mutants with respect to growth and SRP function. Mutants G48C, G48U, and G61U, all severely reduced in Ffh copurification (Table 5), had significant growth defects (Fig. 3) and reduced SRP function, as evidenced by biotinylated FtsQ (Fig. 4, lanes 5, 6, and 8). Conversely, mutants A39C and A67U both copurified with Ffh at levels only slightly lower than the wild type (Table 5) and supported growth at nearly wild-type levels (Fig. 3) with only minor SRP defects (Fig. 4, lanes 2 and 9).

Previously, Wood et al. (51) used Northern blot analysis of Ffh recovered by immunoprecipitation to detect and quantify the amount of 4.5S RNA in an SRP complex. In contrast to our results, this method found that A39C bound Ffh at a level similar to that of the negative control. Given our current understanding of the essential role of the SRP in E. coli (15, 38), it is difficult to understand how the A39C RNA could fail to bind Ffh and yet support growth at nearly wild-type levels. Wood et al. proposed that the immunoprecipitation assay used may not have been sensitive enough to detect small differences in binding efficiency of the mutant RNAs or that the conditions used for immunoprecipitation disrupted the weak association between 4.5S RNA and Ffh (51). This explanation appears valid since the alternative approach used here using qPCR was sufficiently sensitive to detect an interaction between Ffh and the A39C mutant.

A distinction was also noted between the two A47 alleles. As noted above, while the A47C mutant grew when expressed in single copy, A47G was nonviable with single-copy expression and was temperature sensitive when expressed in multiple copy numbers (Fig. 3). Table 5 shows that while both A47C and A47G showed a significant reduction in Ffh copurification when expressed at similar levels (Table 4), A47C apparently exceeds a threshold of Ffh binding that is necessary to support growth. In contrast, Wood et al. (51) reported that A47C showed a severe growth defect under all conditions tested. Wood et al. also failed to detect binding of A47C to Ffh, while we were able to show that the RNA copurified with Ffh above the levels observed with other mutants that failed to complement (A47G, G48C, and G61U). This difference is most likely due to the lower sensitivity of the method used to detect 4.5S RNA (51). The inconsistencies notwithstanding, Wood et al. (51) observed that, in general, the highly evolutionarily conserved bases of 4.5S RNA are required for efficient binding of Ffh. Our results are in agreement with this conclusion.

The results with G58A suggest that it is defective in a function in addition to Ffh binding. Although the G58A RNA was only modestly reduced in Ffh copurification, (Table 3), it caused a severe SRP defect (Fig. 4, lane 7) and failed to support growth when expressed in single copy (Table 3). The G58A mutant was also not temperature sensitive although colony size was heterogeneous at all growth temperatures (Fig. 3). While G58 is not a highly conserved base, its position between the tetraloop region and the symmetrical loop of 4.5S RNA (Fig. 2) likely explains its importance. The tetraloop region has been shown to be important for GTPase activation of the SRP/receptor complex (34, 35), and mutations within the region can disrupt SRP function (22, 45). Interestingly, Tian and Beckwith isolated a similar ffs mutant in their search for mutants defective in localization of cytoplasmic membrane proteins (46). Although a double G58A G57A mutant was viable, it was reported to have significant growth defects (46). Although the mutant was not studied further, it is likely that it was also altered in tetraloop structure. In general, while the Ffh copurification assay was only semiquantitative, in part since the amount of disassociation of the Ffh and 4.5S RNA complex during purification is unknown, it nonetheless provided a sensitive assay to measure the extend of protein-RNA interaction in vivo.

4.5S RNA has also been shown to interact with EF-G in vivo in a role that is likely independent of its function as a component of the SRP (42, 44). To characterize this interaction, Nakumura et al. generated a set of ffs mutants and tested their ability to bind EF-G in vitro and to support growth of a strain where expression of ffs was under the control of the LacI repressor (31). Surprisingly, when the alleles were expressed from multiple-copy-number plasmids, mutants G48C and G49C were viable in the absence of IPTG while mutants C62G, G64U, and A67C failed to grow. While our results agree with C62G, we found G48C to be a highly defective mutant. Also, we observed no significant growth defects with G64U and A67C, even in single copy (Table 3). To confirm these results, we also constructed and tested mutants A67U and A67G, both of which also complemented JP2005 at wild-type levels. One difference between the results of this study and those of Nakumura et al. (31) that may explain the different findings is that the authors of this latter study performed complementation tests by monitoring change in OD over a period of 10 h in the absence of IPTG. The use of JP2005 required the ffs mutants to form colonies in haploid and in single copy, which represents a more rigorous test of gene function.

Our results suggest that the primary function of 4.5S RNA in E. coli is as a component of the SRP while its putative role in translation is secondary, as has been suggested by others (42). Although the A67 mutants were previously found to be defective in binding EF-G in vitro (32), we observed that they manifested essentially no growth defects, a result incompatible with an essential role of 4.5S RNA outside of the SRP. Our results are also consistent with the structural data that indicate that A67 does not participate in direct interaction with Ffh (2, 3). Finally, our confirmation that 4.5S RNA is expressed at only a fraction of the level of 5S RNA is also inconsistent with a significant role of the RNA in protein synthesis.

In addition to clarifying the function of specific bases in binding Ffh, the genetic system reported here should prove useful to better define the role of other features of 4.5S RNA in SRP function and in protein synthesis. The construction of new F' factors should also be useful to characterize any number of genes where multiple alleles exist.


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ACKNOWLEDGMENTS
 
Funding for this research was from the National Institutes of Health grant R01 GM069628.

We thank Les Miller for helping with the calculations involving the RT-qPCR results. We also thank the reviewers of the manuscript for their helpful suggestions.


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FOOTNOTES
 
* Corresponding author. Mailing address: Veterinary Medical Research Institute, Iowa State University, 1802 University Boulevard, Building 6, Ames, IA 50011. Phone: (515) 294-1525. Fax: (515) 294-1401. E-mail: gregory{at}iastate.edu Back

{triangledown} Published ahead of print on 19 September 2008. Back


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Journal of Bacteriology, December 2008, p. 7709-7718, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.00995-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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