INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The rho gene codes for transcription termination factor Rho (19) and is essential for the viability of Escherichia coli (5). The function of Rho is to catalyze the release of RNA from a transcription complex after a Rho-dependent terminator sequence has been transcribed. Rho has an RNA-dependent NTPase activity, which is required for transcript release (10, 12), and an RNA-DNA helicase activity (1). Several Rho-dependent transcription terminators in Escherichia coli and coliphage  are known (4, 20).

Rho is a hexamer of identical 47-kDa monomers (2, 9, 11, 17). Its ability to bind RNA is thought to be conferred by residues 22 to 116 of the 419-amino-acid protein (14). The ATP binding domain is located between residues 167 and 319 (7). No activity has been assigned to the essential C terminus of Rho, although it has been speculated as being involved in subunit interactions (1, 6, 8).

The antibiotic bicyclomycin inhibits Rho (23). With the exception of Micrococcus luteus (15, 16), gram-positive bacteria are resistant to bicyclomycin, and rho is not essential in the gram-positive bacterium Bacillus subtilis (13, 21). This study reports the sequence and characterization of the rho gene in the clinically important gram-positive pathogen Staphylococcus aureus.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacteria and growth conditions. Staphylococcus aureus was propagated in either tryptic soy broth (DIFCO) or Luria broth (LB). E. coli was grown in LB. For auxotrophy measurements with S. aureus, M9 minimal medium was supplemented with arginine, cysteine, glutamate, glycine, isoleucine, leucine, methionine, proline, and valine, all at 20 µg/ml. Nicotinate and thiamine were added at 0.2 µg/ml. All cultures were routinely incubated at 37°C. All strains used in this study are described in Table 1.

Identification of the S. aureus rho gene. The rho gene from Staphylococcus aureus was identified from an S. aureus genomic sequence database by homology with E. coli rho. The plasmid pAU2442 used in this study is from a genomic library used in the sequencing project.

Deletion mutagenesis. A rho deletion was introduced into S. aureus RN4220. This was achieved by electroporating S. aureus RN4220 to tetracycline resistance (Tc^r) with pRW101, which has the rho gene with 500 bp of flanking sequence interrupted by a tetracyline resistance marker that replaces the rho gene from codon 1 to codon 313 (Fig. 1). The resulting Tc^r colonies were then screened for erythromycin resistance (Em^r), which is carried elsewhere on pRW101. Tc^r Em^s colonies indicated a double-crossover event occurred. The deletion of rho was confirmed by PCR analysis with primers flanking the Tc^r marker. To construct a rho strain in a pathogenic background, the rho::tet mutation was moved into the S. aureus clinical isolate WCUH29 by bacteriophage 11 transduction. The resulting strain, RSW101, was used in virulence testing as described below.

View larger version (64K): [in this window] [in a new window]   FIG. 1.   Sequence of S. aureus rho and flanking sequences used in this report. Shown is the DNA sequence of the region of the S. aureus chromosome that contains rho. The rho gene product is shown below its coding sequence. DNA sequence with a strikethrough line depicts the sequence deleted in the rho deletion-insertion strain described in this paper.

Complemention of E. coli rho. E. coli rho was replaced with S. aureus rho by introducing the S. aureus rho-containing plasmid pAU2442 into the E. coli rho deletion strain BLS107(pPMrho). This strain has a deletion-insertion (rho::kan) mutation in the chromosomal copy of rho. A wild-type copy of rho is carried on the plasmid pPMrho, which has a temperature-sensitive replicon. This strain fails to grow at 42°C due to the loss of rho at this temperature (22). pAU2442 was introduced into BLS107(pPMrho) by electroporation and was plated at 30, 37, and 42°C. As a control, the parent strain was also plated at the different temperatures. While the parent strain failed to form isolated colonies at 37 and 42°C, the strain containing pAU2442 grew well and formed isolated colonies at all temperatures. The loss of pPMrho was confirmed by scoring for loss of chloramphenicol resistance, which is encoded by this plasmid. To determine if the S. aureus Rho protein is sensitive to bicyclomycin, strain BLS107(pAU2442) was tested for sensitivity by MIC determination. Liquid MICs were determined by a serial dilution method. Strain BLS107 was found to have an MIC of bicyclomycin of 125 µg/ml. This is twofold lower than that of the wild-type E. coli strain MG1655 (250 µg/ml). The MIC for the S. aureus strain RN4220 was found to be >2.5 mg/ml.

Virulence testing. The rho deletion strain RSW101 and its wild-type parent, WCUH29, were tested for virulence with two mouse S. aureus infection models. The surgical wound model measures the ability to grow on sutures sewn in the skin of healthy mice, while the hematogenous pyelonephritis model measures the ability of the strains to cause kidney infections following parenteral infection with the bacteria.

Surgical wound infections. The mice used for these infections were 4- to 6-week-old male CD-1 mice. Approximately 15-cm sutures were soaked in overnight cultures of WCUH29 and RSW101 for 30 min at room temperature. The backs of the mice were shaved, the animals were anesthetized with isoflurane, and an ~2-cm-long incision was made along the animals' backs. The infected sutures were secured at one end of the wound and tacked once midway along the wound on the underside of the skin. The suture was then secured at the other end of the wound, and the wound was closed with a single staple. After 5 days, the mice were sacrificed by CO₂ overdose. The skin surrounding the wound was excised and then homogenized in 1 ml of phosphate-buffered saline (PBS) in a stomacher, and bacterial viable counts were enumerated. Both infections yielded over 10⁶ bacteria after 5 days, with no significant difference observed between the strains. For the hematogenous pyelonephritis model, bacterial recovery rates (mean ± standard deviation; n = 5) were 5.35 ± 0.41 log₁₀ CFU/ml for strain WCUH29 and 6.44 ± 0.81 log₁₀ CFU/ml for strain WCUH29 rho. For the surgical wound model, the recovery rates were 6.58 ± 0.78 log₁₀ CFU/ml for strain WCUH29 and 7.41 ± 0.57 log₁₀ CFU/ml for strain WCUH29 rho. One hundred colonies isolated from the rho mutant infection were checked for tetracycline resistance, indicative of the presence of the mutation, and all were found to be tetracycline resistant.

Hematogenous pyelonephritis infections. Six- to eight-week-old male CD-1 mice were used for hematogenous pyelonephritis infections. Cultures of WCUH29 and RSW101 cells were adjusted to an A₆₀₀ of 0.6 or 0.3 per ml, and 200 µl was injected into each mouse via a tail vein. Mice were sacrificed on day 5 postinfection by CO₂ overdose, and their kidneys were aseptically removed and then homogenized in 1 ml of PBS in a stomacher, and viable counts were enumerated (described above). The inoculum for both strains was 10⁷ cells. The number of bacteria recovered from the kidneys infected with RSW101 after 5 days was not decreased. One hundred colonies from the RSW101 infection were checked for the tetracycline resistance marker, and 98% were found to be tetracycline resistant.

Nucleotide sequence accession number. The nucleotide sequence of the S. aureus rho gene has been deposited in GenBank under accession no. AF333962.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The S. aureus Rho protein. S. aureus Rho is predicted to code for a 443-amino-acid protein with a predicted molecular mass of 50 kDa. S. aureus Rho is 53% identical and 67% similar to E. coli Rho and 66% identical and 75% similar to B. subtilis Rho (Fig. 2). The predicted ATPase domain (residues 167 to 342 of E. coli Rho) is highly conserved among different bacterial species (18). The predicted ATPase domain of S. aureus Rho (residues 180 to 356) is 80% similar and 70% identical to E. coli Rho and 86% similar and 81% identical to B. subtilis Rho. Finally, the predicted RNA binding domain of S. aureus Rho (residues 40 to 132) has 63% similarity and 41% identity to E. coli Rho and 77% similarity and 60% identity to B. subtilis Rho.

View larger version (85K): [in this window] [in a new window]   FIG. 2.   Alignment of S. aureus Rho with B. subtilis and E. coli Rho. Sequences were aligned by using the MegAlign program of the DNAstar DNA analysis package. Identical residues are shaded.

Complementation. The S. aureus rho gene can complement the lack of growth of an E. coli strain with rho deleted.. This was determined by curing a rho-deleted strain of a plasmid that carries E. coli rho in the presence of S. aureus rho carried on another plasmid. The sole Rho activity in the resulting strain, BLS107(pAU2442), is from S. aureus Rho. Strain BLS107(pAU2442) is sensitive to bicyclomycin, as indicated by the bicyclomycin MIC of 125 µg/ml, indicating that S. aureus Rho is inhibited by bicyclomycin. As further evidence of this, bicyclomycin was found to inhibit the ATPase activity of partially purified S. aureus Rho (data not shown).

The S. aureus rho gene is not essential for in vitro growth. A rho strain, RSW100, was constructed by replacing nearly all of the rho gene with a tetracycline resistance cassette (Fig. 1). The resulting strain is viable and grows normally at 30, 37, 42, and 46°C. RSW100 also grows normally on rich medium (LB or tryptic soy broth) or minimal medium (supplemented M9 medium) and under anaerobic conditions (data not shown).

rho is not required for S. aureus virulence. The rho deletion was transduced into the pathogenic clinical isolate WCUH29, yielding strain RSW101. Cultures of WCUH29 and RSW101 were used in the two in vivo models described in Materials and Methods. S. aureus strains mutated in a variety of different genes affecting cell metabolism or virulence have been examined in our laboratories. These strains have displayed reductions of up to 3 logs in the pyelonephritis model and 1 log in the surgical wound infection. In the experiments described here, the in vivo growth of RSW101 was not impaired compared to that of wild-type WCUH29 in either of the model systems. From these data, we conclude that, as with in vitro growth, rho is not essential for the virulence of the strain tested.

Review of Rho homologs present in public sequence database. By using the S. aureus Rho sequence as a query, we searched the public databases and some proprietary sequence databases for homologs. Rho was found to be highly conserved; BLAST scores were 5 × 10³⁸ or lower for all full-length Rho sequences annotated as such. As indicated previously by Opperman and Richardson (18), Rho is broadly distributed among all the major phyla of the eubacteria (Table 2). A major exception to the universal presence of Rho among the eubacteria are the members of the low-GC, gram-positive bacteria Streptococus pyogenes, Streptococcus pneumoniae, Mycoplasma genitalium, M. pneumoniae, and Ureaplasma urealyticum, all of which do not possess a significant homolog. In the case of S. pneumoniae, we searched several proprietary databases in addition to publicly available databases and failed to identify a homolog. In addition to the mentioned bacteria, Synechocystis sp. strain PCC6803, a cyanobacterium, also apparently lacks a Rho homolog. As indicated previously (18), the ATP binding motif of Rho shares homology with the  and  subunits of F1 ATPases. This homology is conserved among the bacteria as well.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this report, we show that the S. aureus rho gene can complement an E. coli rho deletion strain. The ability of S. aureus rho to replace the essential E. coli rho gene raises the possibility that it performs the same functions in S. aureus and E. coli. rho is not essential in the low-GC, gram-positive bacterium Bacillus subtilis (13, 21), and several gram-positive species, including Staphylococcus aureus, are resistant to the Rho inhibitor bicyclomycin (15). Here we show that rho is not required for viability or virulence in S. aureus. The E. coli strain constructed here, which has had its own rho gene replaced with S. aureus rho, remains sensitive to bicyclomycin. This suggests that the resistance of gram-positive bacteria to bicyclomycin is a result of the nonessentiality of Rho. Supporting this conclusion and extending the lack of essentiality of rho to other genera, we found that the completed genomes of several members of the low-GC, gram-positive bacteria lack a Rho homolog. The cyanobacterium Synechocystis sp. strain PCC6803 was also found to lack a Rho homolog.

An open question is why rho should be essential in E. coli and other gram-negative bacteria, but not be essential in some gram-positive bacteria. It could be that Rho activity is redundant in the bacteria where it is not essential. This possibility cannot be readily discounted, but it is clear that the enzyme responsible for this redundant activity would have to have no sequence homology with Rho for this to be the case. A second possibility, that Rho is essential for organisms with a higher GC content, seems unlikely, given that Synechocystis sp. strain PCC6803 also lacks Rho, and yet its genome is 47% GC, close to that of E. coli. The lack of rho among several members of the low-GC, gram-positive bacteria and a member of the very distantly related cyanobacteria could indicate a trend that the essentiality of rho may be more the exception than the rule. Because of this, a more informative approach to the question is to ask why rho is essential in E. coli and other gram-negative bacteria. There are only a few Rho-dependent terminators in E. coli; the vast majority of E. coli operons contain factor-independent terminators. Furthermore, it is not obvious why inhibition of termination at Rho-dependent terminators would lead to a lethal event. It might be that there is some other function of Rho that better explains its essential nature in some bacteria.