INTRODUCTION

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Enterococci have emerged as leading causes of nosocomial infection, including urinary tract infections, wound infections, and bacteremia (10, 12, 25, 33, 37, 39, 41, 44). Among these and other types of enterococcal infection, approximately 80% are associated with Enterococcus faecalis (21) and the majority are nosocomial (34). Enterococcal infections are especially troublesome because of the high level of intrinsic antibiotic resistance and because they have acquired resistance determinants capable of rendering the organism completely resistant to all currently approved antibiotics (21).

Because of the emergence of enterococci as leading nosocomial pathogens and the development of broad antimicrobial resistance, particularly to vancomycin, an understanding of host-parasite interactions is a key to the development of new prophylactic and therapeutic strategies. Only a few traits are known to contribute to the pathogenesis of enterococcal infection. The enterococcal cytolysin contributes to toxicity, loss of organ function, and lethality during enterococcal infection (6, 24, 26), and cytolytic strains of E. faecalis have been observed to be enriched among clinical isolates, especially those from the bloodstream (21-23). Enterococcal aggregation substance has been demonstrated to contribute to increased cardiac vegetation size (6) and has been associated with enhanced virulence in a rabbit model of endocarditis (45). Other putative virulence factors include surface carbohydrates (18, 19) and enterococcal lipoteichoic acid (2), but their specific roles in enterococcal infection have yet to be determined. Since enterococci evolved as commensal organisms, the contribution of specific factors to pathogenesis is typically subtle. Thus, sensitive techniques are required for the identification of such factors.

Differential-display PCR (DD-PCR) is a relatively new method for observing the differential expression of genes in comparative studies within a large number of experimental systems (31, 35, 51). The technique was originally developed for use in the study of eukaryotic gene expression, and this continues to be its most common application (31, 35). A derivative of DD-PCR, random arbitrarily primed PCR (RAP-PCR), utilizes an arbitrary primer at a low annealing temperature for both the first- and second-strand cDNA synthesis reactions (51). At such low-stringency temperatures the arbitrary primer is able to bind at random sites within the template that show limited, but not complete, complementarity. Because the same arbitrary primer is used as both the 5' and the 3' primers, the primer sequence is incorporated at both ends of the resulting double-stranded products. The products are amplified by standard PCR by using the arbitrary primer, resolved by polyacrylamide electrophoresis, and visualized by autoradiography. Differences in gene expression can be inferred from the resulting "fingerprint" by observing the presence or absence of specific products between different populations of cells. By eliminating the use of an oligo(dT) primer, the 3' bias toward the polyadenylate tail in eukaryotic mRNA is eliminated. Thus, priming of first-strand synthesis potentially can occur at any point within the RNA. For this reason, RAP-PCR may be used for amplification of RNAs that are not polyadenylated, such as bacterial RNA.

This technology has been applied to only a few prokaryotic systems (1, 52) because of high sample-to-sample variability, as was encountered in initial attempts to study E. faecalis gene expression by using available methods (51, 52). We therefore systematically optimized the RAP-PCR method and identified several key refinements which ultimately allowed the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by E. faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specific reverse transcriptase PCR (RT-PCR).

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. A strain of E. faecalis, MMH594 (23), that caused multiple infections in a hospital ward outbreak was used to study differential gene expression under either aerobic or anaerobic conditions. Escherichia coli XL-1 Blue (Stratagene, La Jolla, Calif.) was used for cloning differentially expressed RAP-PCR products. For aerobically cultured E. faecalis, a 10-ml overnight culture was grown in a 50-ml centrifuge tube for 18 h in brain heart infusion (BHI) broth (Becton Dickinson, Cockeysville, Md.) at 37°C with shaking at 300 rpm. The overnight culture was diluted 1:100 in 10 ml of fresh BHI broth and subcultured in a 50-ml centrifuge tube to mid-log phase (optical density at 560 nm [OD₅₆₀] of 1.0) at 37°C with shaking at 300 rpm for 4 h prior to RNA isolation. For anaerobically cultured E. faecalis, a 10-ml overnight culture was grown in a 50-ml centrifuge tube in BHI broth at 37°C without shaking for 18 h in an anaerobic hood. The overnight culture was diluted 1:100 in 10 ml of fresh BHI broth and subcultured anaerobically in a 50-ml centrifuge tube to mid-log phase (OD₅₆₀ = 1.0) at 37°C without shaking for 3.5 h prior to RNA isolation. E. coli was cultured on plates containing Luria-Bertani (LB) media and 1.5% Bacto-Agar (Difco, Detroit, Mich.), and appropriate transformants were subsequently cultured in LB broth (43). Antibiotics used for selection of transformed E. coli included ampicillin (100 µg/ml) and tetracycline (12.5 µg/ml) (Sigma, St. Louis, Mo.). For detection of insertional inactivation of the lacZ gene contained in the cloning vectors, 50 µM isopropyl--D-thiogalactopyranoside (Sigma) and 0.01% 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (Sigma) were added to the media.

RNA extraction and preparation. Total RNA was isolated as previously described (5) with minor modifications. Briefly, each 10-ml subculture of E. faecalis was centrifuged (2,500 × g for 2 min at 4°C), and the bacterial pellet was resuspended in 1.5 ml of Tri Reagent (Sigma). The cell suspension was immediately transferred to a 2-ml microcentrifuge tube (BioSpec Products, Bartlesville, Okla.) containing 0.5 ml of 100-µm-diameter zirconia-silica beads (BioSpec Products). The tube was immediately placed in a high-speed reciprocating shaker (BioSpec Products) and horizontally shaken at 5,000 rpm for 1 min to lyse the bacterial cells. After lysis, the tube was placed on ice and the beads were allowed to settle out of the lysis mixture. The lysate was clarified by centrifugation (12,000 × g for 10 min at 4°C). The supernatant was recovered and extracted with 300 µl of chloroform. After a manual shaking for 15 s, the mixture was placed on ice for 15 min prior to centrifugation (12,000 × g for 10 min at 4°C). After centrifugation the aqueous phase was recovered, and the RNA was precipitated by adding 750 µl of isopropyl alcohol. After incubation on ice for 10 min, the RNA was pelleted by centrifugation (12,000 × g for 10 min at 4°C). The RNA pellet was washed with 1.7 ml of 75% ethanol, air dried for 5 to 10 min, and resuspended in 200 µl of diethylpyrocarbonate (DEPC)-treated water.

RAP-PCR. To initiate each experimental reaction, 14.5 µl containing 1 µg of total E. faecalis RNA, diluted in DEPC-treated water as needed, was heated in a 0.65-ml thin-wall PCR tube to 70°C for 10 min and immediately placed on ice. After 1 min of incubation on ice and brief centrifugation pulse to collect contents, 5 µl of buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, a 1.25 µM concentration of an arbitrarily chosen primer (Stratagene), 1.25 mM concentrations of each deoxynucleoside triphosphate (dNTP), and 40 U of RNase Block RNase inhibitor (Stratagene) was added. Contents were mixed, collected by brief centrifugation, and allowed to equilibrate to 37°C for 5 min. After equilibration, 25 U of Moloney murine leukemia virus (MMLV) RT (Stratagene) was added for a final volume of 20 µl. The reaction was incubated at 37°C for 1 h. Upon completion of first-strand cDNA synthesis, the reaction was heated to 90°C for 5 min to inactivate the RT and then immediately placed on ice for 10 min. The reaction was diluted 1:10 in sterile water and either used immediately in a second-strand synthesis reaction or stored at 20°C.

Isolation, cloning, and sequencing of RAP-PCR products. The RAP-PCR gel and the autoradiogram were aligned by using radioactive ink marks placed on the gel prior to autoradiography. By using the autoradiogram as a template, individual bands representing differentially expressed products were cut and removed from the gel with a sterile scalpel (30, 52). Each isolated piece of acrylamide gel with filter paper was cut into smaller pieces with a sterile scalpel, and the pieces were collected in a microcentrifuge tube. Fifty microliters of elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA [pH 8.0], 0.1% sodium dodecyl sulfate) (1, 43) was added to the tube, and the mixture was heated to 100°C for 30 min. The eluate from the reaction was collected, and the DNA was precipitated by the addition of 0.1 volume of 3 M sodium acetate (pH 5.17) and 2.5 volumes of 100% ethanol followed by incubation at 70°C for 2 h. DNA was pelleted by centrifugation at 12,000 × g for 30 min at 4°C. The DNA pellet was washed with 2.5 volumes of 75% ethanol, air dried for 5 min, and resuspended in 10 µl of sterile water.

Confirmation of differential gene expression. Sequence data from each differentially expressed RAP-PCR product was used to develop specific primers for use in specifically primed, quantitative RT-PCR to confirm differential gene expression. In each reaction, 1 µg of total RNA, diluted in DEPC-treated water as needed, was incubated at 85°C for 3 min and immediately placed on ice. After a 1-min incubation on ice and brief centrifugation to collect contents, 8 µl of a standard RT-PCR reaction mixture was added in a final reaction volume of 20 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 500 µM concentrations of each dNTP, 50 pmol of the specific reverse primer, 10 U of RNase inhibitor (Ambion, Austin, Tex.), and 100 U of MMLV RT (Ambion). After gentle mixing and brief centrifugation, each reaction was incubated at 42°C for 60 min followed by a 10-min incubation at 92°C. The cDNA from each reaction was either used immediately in PCR or stored at 20°C.

	RESULTS

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RNA isolation. Cells of E. faecalis MMH594 were cultured either aerobically or anaerobically to mid-log phase in BHI broth, isolated, and resuspended in a chaotropic RNA extraction reagent. Cell lysis was achieved by horizontally shaking the cells mixed with zirconia-silica beads on a reciprocating high-speed shaking device, and RNA was isolated as described. RNA was isolated from six independent aerobic cultures and six independent anaerobic E. faecalis cultures. After electrophoresis, bands indicating the 23S, 16S, and 5S rRNA species were clearly identifiable (data not shown). Each band was well defined with no smearing, indicating that no overt degradation of the RNA occurred during isolation. The A₂₆₀/A₂₈₀ ratios ranged from 1.88 to 2.00 for the 12 RNA preparations.

RAP-PCR. To assess the effect of RNA concentration in RAP-PCR, various amounts of total RNA (0.125, 0.25, 0.5, 1, and 2 µg) were used in the first-strand synthesis reactions in a series of amplifications. For standardization, the same template RNA was used in each independent amplification. The results of the titration experiments are shown in Fig. 1. Although broadly similar from lane to lane, there were RNA-dependent differences in the number of RAP-PCR products detected. As the amount of RNA in the first-strand reaction increased from 0.125 to 2 µg, the aggregate number of RAP-PCR products increased, with a plateau in the number of well resolved and easily detected products beginning at ca. 1 µg of RNA in the first-strand reaction. Therefore, 1 µg of RNA was selected for use in subsequent first-strand reactions.

View larger version (40K): [in this window] [in a new window]   FIG. 1.   Effects of RNA concentration on RAP-PCR. Different amounts of the same RNA were amplified by RAP-PCR. In each lane the following amounts of total RNA were used during first-strand synthesis: lane 1, 125 ng; lane 2, 250 ng; lane 3, 500 ng; lane 4, 1 µg; and lane 5, 2 µg. A paired control reaction without RT showed no products derived from genomic DNA contamination.

View larger version (34K): [in this window] [in a new window]   FIG. 2.   Effects of annealing temperatures during second-strand synthesis on RAP-PCR. For each reaction, 1 µg of the same total RNA was amplified by RAP-PCR. In each lane the following annealing temperatures were used during second-strand synthesis: lane 1, 20°C; lane 2, 25°C; lane 3, 30°C; lane 4, 36°C; and lane 5, 40°C. A paired control reaction without RT showed no products derived from genomic DNA contamination.

View larger version (49K): [in this window] [in a new window]   FIG. 3.   Effects of temperature equilibration during first- and second-strand synthesis on reproducibility of RAP-PCR amplification. Total RNA from three independent aerobic cultures of E. faecalis was amplified by RAP-PCR either without (lanes 1 to 3) or with (lanes 4 to 6) extended equilibration during first- and second-strand synthesis prior to the addition of the appropriate enzymes for DNA synthesis. Arrows indicate areas of irreproducibility in the reactions performed without extended equilibration.

View larger version (50K): [in this window] [in a new window]   FIG. 4.   RAP-PCR products derived from samples of total RNA from aerobically and anaerobically cultured E. faecalis. Lanes: 1 to 6, aerobic samples 1 to 6; 7 to 12, anaerobic samples 1 to 6. Arrows indicate differentially amplified products. Primer 2 was used for amplification. For each experimental reaction, a paired negative control reaction lacking RT during first-strand synthesis was performed. These controls were used to qualitatively assess the levels of genomic DNA contamination of each RNA sample. No products derived from residual genomic DNA contamination were identified in any of the negative control reactions.

Sequence identification of differentially expressed RAP-PCR products. To identify genes within the emerging E. faecalis genome database that are differentially expressed in response to changes in oxygen tension, the differentially amplified RAP-PCR products were recovered and reamplified by standard PCR with the same arbitrary primer used in RAP-PCR. After reamplification, the products were gel purified and ligated into a TA cloning vector for transformation of E. coli. Plasmids were isolated, and the inserts were subjected to automated sequence analysis. Specific primer pairs for each RAP-PCR product were designed by using the sequence data and then used in RT-PCR to confirm differential gene expression (Fig. 5).

View larger version (37K): [in this window] [in a new window]   FIG. 5.   RT-PCR analysis to confirm differential gene expression. RT-PCR was performed with both aerobic (A) and anaerobic (An) RNA with primers specific for each product. Product numbers correspond to those given in Table 1. Relative quantities of each RT-PCR product amplified from both aerobic and anaerobic RNA were analyzed after normalization to the internal control. Paired control reactions lacking RT showed no products derived from genomic DNA.

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	DISCUSSION

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In the present study we optimized a technique that has been applied broadly to analyze patterns of eukaryotic gene expression in order to permit its reproducible use in studies of gene expression by a gram-positive bacterium. Initial efforts to use existing protocols for RAP-PCR in a prokaryotic genetic background (1, 52) highlighted several extant pitfalls, most notably a lack of experimental reproducibility. More specifically, we were unable to generate similar RAP-PCR product patterns upon repeated amplification from either the same RNA sample or the same cDNA sample. Moreover, RAP-PCR amplification of RNA from bacteria cultured independently under identical conditions yielded widely different product patterns. Therefore, several modifications, which are detailed in the preceding sections, were made to the RAP-PCR protocol that ultimately allowed the technique to be used in a reproducible manner in the study of differential enterococcal gene expression. Although the optimized RAP-PCR protocol was optimized while studying differential gene expression in E. faecalis, the same protocol has been used by others in our laboratory to reproducibly identify products from differentially expressed genes by Streptococcus gordonii and Actinomyces naeslundii. Additionally, the modifications included in the optimized RAP-PCR protocol may serve to guide others who are interested in applying this technology to other bacteria but who are unable to apply the precise parameters we used.

Of the changes made to previously defined protocols for use of RAP-PCR to study bacterial genetics, the most significant modifications were made to the first-strand and second-strand synthesis reactions. Both steps involve random priming by an arbitrary primer at a low annealing temperature. We found that extended equilibration of each reaction to the low annealing temperature prior to the addition of the appropriate enzyme was essential for achieving reproducibility among similar RNA samples and upon repeated amplifications of the same sample. Thus, during first-strand synthesis, each reaction is allowed to equilibrate to 37°C for 5 min prior to the addition of RT. Similarly, during second-strand synthesis, each reaction is allowed to equilibrate to 36°C for 15 min prior to the addition of Taq polymerase. It was not until these subtle yet significant changes were made that reproducibility in both reactions was achieved. Upon RAP-PCR amplification with the optimized protocol, each of the six aerobic RNA preparations produced similar product patterns, as did each of the six anaerobic RNA preparations (Fig. 4). Thus, there is reproducibility in RAP-PCR amplification of independent samples of RNA from E. faecalis cultured in a similar manner. Perhaps most importantly, three independent but identical amplifications of each RNA preparation yielded identical product patterns (for example, compare Fig. 4, lane 6, with Fig. 1, lane 4, and Fig. 2, lane 4). Thus, there is reproducibility upon repeated RAP-PCR amplification of a single RNA preparation.

Equilibration to the low annealing temperatures in both first- and second-strand synthesis prior to the addition of the appropriate enzymes is essential for eliminating the variability inherent to random priming. For instance, during second-strand synthesis, the RNA-cDNA duplex is initially denatured. As the reaction is cooled from the denaturation temperature of 96°C to the low-stringency annealing temperature of 36°C, it passes through a range of temperatures at which the random primer can bind. If Taq polymerase were present in the reaction during this period, polymerization would begin at various points within the temperature ramp. This introduces experimental variability, because the time course of cycling to the low annealing temperature may be different between independent amplifications of the same sample or between simultaneous amplifications of independent samples. In standard PCR this variability is eliminated by using a specific primer at an annealing temperature at which it must recognize its complement for priming to occur. However, in RAP-PCR such subtle experimental variability is potentiated by using a random primer at low stringency. Equilibration of each reaction to 36°C prior to the addition of Taq polymerase eliminates this variability. By "clamping" each reaction at the low annealing temperature before synthesis, time is allowed for the primer to recognize all potential binding sites. Thus, each sample is randomly primed essentially to completeness prior to the initiation of polymerization, and polymerization is initiated under identical conditions. The same rationale applies for equilibration of the first-strand synthesis reaction to 37°C prior to the addition of RT.

Using the optimized protocol detailed in the Materials and Methods section, RAP-PCR was used to identify products from genes differentially expressed when E. faecalis was cultured in an aerobic versus an anaerobic environment. Additionally, sequence data generated after RAP-PCR was used to both confirm and quantitate, by specific RT-PCR, differences in expression for genes coding for the RAP-PCR products. Several of the RAP-PCR products that were cloned and sequenced demonstrated significant levels of similarity to known sequences in current databases, including several involved in the respiration of B. subtilis. The role of these genes in enterococcal biology in either an aerobic or anaerobic environment is unexplored. However, the data show that the RAP-PCR protocol may be utilized to reproducibly identify products that derive from differentially expressed genes and that the RAP-PCR products may ultimately be used to identify these specific differentially expressed genes.

In the present work, 22% of the RAP-PCR products demonstrated no similarity to sequences in the databases. Recent sequencing of other bacterial genomes has demonstrated that many of the inferred proteins show no homologies to known proteins. For example, 38% of the E. coli K-12 genome (3) and 42% of the Haemophilus influenzae Rd genome (14) code for proteins with no known homologies. Similarly, sensitive techniques for the identification of bacterial genes whose expression is induced by or within host cells have produced similar data (4, 20, 36, 48). The detection of unique enterococcal genes most likely reflects gaps in the understanding of E. faecalis genetics and physiology.

Our current research is directed toward the application of RAP-PCR to studies of enterococcal gene expression in in vitro and in vivo models of enterococcal disease. Additionally, we are employing RAP-PCR in initial studies of gene expression patterns in and among streptococci and actinomyces and are achieving the same level of reproducibility and quality. Thus, the refinements detailed in the present work allow the RAP-PCR protocol to serve as a general and highly reproducible technique for the study of prokaryotic gene expression.