rho Is Not Essential for Viability or Virulence in Staphylococcus aureus

Abstract

We have identified the gene for transcription termination factor Rho in Staphylococcus aureus. Deletion of rho in S. aureus reveals that it is not essential for viability or virulence. We also searched the available bacterial genomic sequences for homologs of Rho and found that it is broadly distributed and highly conserved. Exceptions include Streptococcus pneumoniae, Streptococcus pyogenes, Mycoplasma genitalium, Mycoplasma pneumoniae, Ureaplasma urealyticum, and Synechocystis sp. strain PCC6803, all of which appear not to possess a Rho homolog. Complementation studies indicate that S. aureus Rho possesses the same activity as Escherichia coli Rho and that the Rho inhibitor bicyclomycin is active against S. aureus Rho. Our results explain the lack of activity of bicyclomycin against many gram-positive bacteria and raise the possibility that the essentiality of rho may be the exception rather than the rule.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 Rhodependent 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 METHODSBacteria 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 g/ml. Nicotinate and thiamine were added at 0.2 g/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 ...

Phyletic distribution and conservation of the bacterial transcription termination factor Rho

“…Species containing rho belong to nearly all bacterial phyla with the noticeable exceptions of Tenericutes and Cyanobacteria (see below), which confirms previous observations (Opperman & Richardson, 1994;Washburn et al, 2001). Analysis of published data also indicates that Rho proteins are expressed in representative species across phyla (Table S2).…”

“…Interestingly, genomes containing rho sequence(s) tend to be larger and GC-richer than rho-less genomes, suggesting a link between Rho function and the complexity of genome structure (or expression) and/or lifestyle of the bacterial species. Overall, our work significantly extends early phylogenetic analyses (Opperman & Richardson, 1994;Washburn et al, 2001) that were performed with limited sets of bacterial sequences and before detailed structural information on Rho became available, thereby providing a comprehensive framework for future analyses of this central regulatory factor.…”

“…Rho is expressed in representative species across phyla (Table S2), yet is dispensable in some of them under laboratory conditions (Quirk et al, 1993;Washburn et al, 2001). Even spurious transcription elicited by rho inactivation is not necessarily catastrophic, as observed for B. subtilis (Nicolas et al, 2012), although condition-dependent or long-term effects may not be readily detected.…”

“…luteus (Nowatzke et al, 1997b;Williams & Durham, 1988). This has been interpreted to suggest that Rho function is important in Gram-negative bacteria, but dispensable in most Gram-positive species (Rabhi et al, 2010b;Washburn et al, 2001). The rationale for this simple rule is, however, not firmly established.…”

“…The rationale for this simple rule is, however, not firmly established. Notably, the phyletic distribution and structural conservation of Rho within living organisms were inferred more than a decade ago from very limited sets of DNA sequences (Opperman & Richardson, 1994;Washburn et al, 2001). Hence, the fractions of bacteria devoid of rho genes or containing Rho factors whose function may be unaffected by BCM are not known.…”