Antisense RNAs (asRNAs) pair to RNAs expressed from the complementary strand, and their functions are thought to depend on nucleotide overlap with genes on the opposite strand. There is little information on the roles and mechanisms of asRNAs. We show that a cis asRNA acts in trans, using a domain outside its target complementary sequence. SprA1 small regulatory RNA (sRNA) and SprA1(AS) asRNA are concomitantly expressed in S. aureus. SprA1(AS) forms a complex with SprA1, preventing translation of the SprA1-encoded open reading frame by occluding translation initiation signals through pairing interactions. The SprA1 peptide sequence is within two RNA pseudoknots. SprA1(AS) represses production of the SprA1-encoded cytolytic peptide in trans, as its overlapping region is dispensable for regulation. These findings demonstrate that sometimes asRNA functional domains are not their gene-target complementary sequences, suggesting there is a need for mechanistic re-evaluation of asRNAs expressed in prokaryotes and eukaryotes.

The pleiotropic post-transcriptional regulator Hfq is an RNA chaperone that facilitates pairing interactions between small regulatory RNAs (sRNAs) and their mRNA targets in several bacteria. However, this classical pattern, derived from the Escherichia coli model, is not applicable to the whole bacterial kingdom. In this article we discuss the facultative requirement for Hfq for sRNA-mRNA duplex formation among bacteria and the specific features of the Hfq protein and RNA duplexes that might account for the dispensability or requirement of the chaperone. Apparent links between the need for Hfq, the GC content of bacterial genomes and the free energy of experimentally validated sRNA-mRNA pairing interactions are presented.

Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology. Peptidoglycan (PGN) synthesis is an essential process that is both spatially and temporally regulated to ensure that the bacterial cell shape is maintained 1 . Rod-shaped bacteria elongate by synthesising PGN along the length of the cell in a process directed by the cytoskeletal protein MreB 2 . In Escherichia coli and Bacillus subtilis, this protein polymerises into short filaments that move processively around the cell diameter, and organise a multi-protein machinery, including PGN synthesis proteins, referred to as the elongasome or the Rod system [3][4][5] . Cell division is dependent on another cytoskeletal protein, FtsZ, which polymerises to form the Z-ring and recruits a multi-protein complex responsible for septum synthesis, known as the divisome 6,7 . This complex directs PGN incorporation to the midcell, resulting in inward PGN synthesis, and eventually bisects the mother cell, leading to daughter cell separation.Ovococci such as Streptococcus pneumoniae and Lactococcus lactis lack MreB, and FtsZ is proposed to coordinate both elongation and septation 8,9 . In these organisms PGN is

ABSTRACTUnderstanding in detail the factors which permitStaphylococcus aureusto counteract cell wall-active antibiotics is a prerequisite to elaborating effective strategies to prolong the usefulness of these drugs and define new targets for pharmacological intervention. Methicillin-resistantS. aureus(MRSA) strains are major pathogens of hospital-acquired and community-acquired infections and are most often treated with glycopeptides (vancomycin and teicoplanin) because of their resistance to most penicillins and a limited arsenal of clinically proven alternatives. In this study, we examined PrsA, a lipid-anchored protein of the parvulin PPIase family (peptidyl-prolylcis/transisomerase) found ubiquitously in all Gram-positive species, in which it assists posttranslocational folding at the outer surface of the cytoplasmic membrane. We show by both genetic and biochemical assays thatprsAis directly regulated by the VraRS two-component sentinel system of cell wall stress. Disruption ofprsAis tolerated byS. aureus, and its loss results in no detectable overt macroscopic changes in cell wall architecture or growth rate under nonstressed growth conditions. Disruption ofprsAleads, however, to notable alterations in the sensitivity to glycopeptides and dramatically decreases the resistance of COL (MRSA) to oxacillin. Quantitative transcriptional analysis reveals thatprsAandvraRare coordinately upregulated in a panel of stable laboratory and clinical glycopeptide-intermediateS. aureus(GISA) strains compared to their susceptible parents. Collectively, our results point to a role forprsAas a facultative facilitator of protein secretion or extracellular folding and provide a framework for understanding whyprsAis a key element of the VraRS-mediated cell wall stress response.

The precise mechanisms leading to the emergence of low-level glycopeptide resistance in Staphylococcus aureus are poorly understood. In this study, we used whole genome deep sequencing to detect differences between two isogenic strains: a parental strain and a stable derivative selected stepwise for survival on 4 µg/ml teicoplanin, but which grows at higher drug concentrations (MIC 8 µg/ml). We uncovered only three single nucleotide changes in the selected strain. Nonsense mutations occurred in stp1, encoding a serine/threonine phosphatase, and in yjbH, encoding a post-transcriptional negative regulator of the redox/thiol stress sensor and global transcriptional regulator, Spx. A missense mutation (G45R) occurred in the histidine kinase sensor of cell wall stress, VraS. Using genetic methods, all single, pairwise combinations, and a fully reconstructed triple mutant were evaluated for their contribution to low-level glycopeptide resistance. We found a synergistic cooperation between dual phospho-signalling systems and a subtle contribution from YjbH, suggesting the activation of oxidative stress defences via Spx. To our knowledge, this is the first genetic demonstration of multiple sensor and stress pathways contributing simultaneously to glycopeptide resistance development. The multifactorial nature of glycopeptide resistance in this strain suggests a complex reprogramming of cell physiology to survive in the face of drug challenge.

Expression of the methicillin-resistant S. aureus (MRSA) phenotype results from the expression of the extra penicillin-binding protein 2A (PBP2A), which is encoded by mecA and acquired horizontally on part of the SCCmec cassette. PBP2A can catalyze DD-transpeptidation of peptidoglycan (PG) because of its low affinity for ␤-lactam antibiotics and can functionally cooperate with the PBP2 transglycosylase in the biosynthesis of PG. Here, we focus upon the role of the membrane-bound PrsA foldase protein as a regulator of ␤-lactam resistance expression. Deletion of prsA altered oxacillin resistance in three different SCCmec backgrounds and, more importantly, caused a decrease in PBP2A membrane amounts without affecting mecA mRNA levels. The N-and C-terminal domains of PrsA were found to be critical features for PBP2A protein membrane levels and oxacillin resistance. We propose that PrsA has a role in posttranscriptional maturation of PBP2A, possibly in the export and/or folding of newly synthesized PBP2A. This additional level of control in the expression of the mecA-dependent MRSA phenotype constitutes an opportunity to expand the strategies to design anti-infective agents.

Staphylococcus aureus is a major pathogen worldwide and can provoke a range of diseases that range from relatively minor to life-threatening. It is the most common etiological agent of surgical site infections and ventilator-associated pneumonia. Of particular concern are infections arising from encounters with antibiotic-resistant strains, such as methicillin-resistant S. aureus (MRSA). Methicillin resistance in S. aureus is dependent upon the acquisition of the mecA gene, which encodes penicillin-binding protein 2A (PBP2A), which is refractory to inhibition by methicillin and numerous other ␤-lactam antibiotics (1).MRSA is widely recognized as a serious threat to public health, and new antibiotics are urgently needed (2). Penicillin-binding proteins (PBPs), which are bacterial enzymes catalyzing the last steps of cell wall biosynthesis, have long been used as lethal targets for ␤-lactam antibiotics. Ceftaroline, a novel ␤-lactam broadspectrum cephalosporin, also binds to PBPs (3); however, an exceptional characteristic of ceftaroline is that it also binds to and inhibits PBP2A, effectively blocking the principal ␤-lactam resistance determinant of MRSA strains. Accordingly, numerous studies have attested that ceftaroline shows robust in vitro activity against MRSA strains (4-8).Neither clinical nor in vitro studies have detected high percentages or rapid development of ceftaroline resistance (9). Nevertheless, some rare resistant strains have been reported (10, 11) (EUCAST MICs, Ͼ1 g/ml or CLSI MIC, Ն4 g/ml). To our knowledge, only a few studies have been conducted that identified PBP2A mutations as being correlated with a reduction in sensitivity to ceftaroline (11,12). Some of these PBP2A mutations (N146K and E150K) correlated with decreased affinity to ceftaroline (13) and were found to be located in the recently discovered allosteric site that is considerably distant from the transpeptidase active site domain (13,14). Interestingly, as shown by Otero and coworkers (14) using X-ray crystallographic analysis, ceftaroline is capable of binding both the PBP2A allosteric and the DD-transpeptidase domains. In the proposed model, noncovalent ceftaroline binding to the allosteric site might influence accessibility to the PBP2A active site by a second ceftaroline molecule over a considerable spatial distance by an intricate network of salt bridges and conformational changes. Consequently, mutations in the PBP2A alloste-

β-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of β-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of β-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with β-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by β-lactams or by inhibiting an early step in WTA biosynthesis. The finding that β-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that β-lactams do not kill S. aureus simply by weakening the cell wall.