The bacterial cell wall is essential for viability, but despite its ability to withstand internal turgor must remain dynamic to permit growth and division. Peptidoglycan is the major cell wall structural polymer, whose synthesis requires multiple interacting components. The human pathogen Staphylococcus aureus is a prolate spheroid that divides in three orthogonal planes. Here, we have integrated cellular morphology during division with molecular level resolution imaging of peptidoglycan synthesis and the components responsible. Synthesis occurs across the developing septal surface in a diffuse pattern, a necessity of the observed septal geometry, that is matched by variegated division component distribution. Synthesis continues after septal annulus completion, where the core division component FtsZ remains. The novel molecular level information requires re-evaluation of the growth and division processes leading to a new conceptual model, whereby the cell cycle is expedited by a set of functionally connected but not regularly distributed components.

The identification of these lineages of E. coli and their shared genetic traits suggest there may be evolutionary events that underpin the emergence of carbapenem resistance plasmid carriage in E. coli.

Antimicrobial resistance is recognized as one of the principal threats to public health worldwide, yet the problem is increasing. Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most difficult to treat in clinical settings due to the resistance of MRSA to nearly all available antibiotics. The cyclic anionic lipopeptide antibiotic daptomycin (DAP) is the clinical mainstay of anti-MRSA therapy. The decreased susceptibility to DAP (DAP resistance [DAP r ]) reported in MRSA is frequently accompanied by a paradoxical decrease in ␤-lactam resistance, a process known as the "seesaw effect." Despite the observed discordance in resistance phenotypes, the combination of DAP and ␤-lactams has been proven to be clinically effective for the prevention and treatment of infections due to DAP r MRSA strains. However, the mechanisms underlying the interactions between DAP and ␤-lactams are largely unknown. In the study described here, we studied the role of mprF with DAP-induced mutations in ␤-lactam sensitization and its involvement in the effective killing by the DAP-oxacillin (OXA) combination. DAP-OXA-mediated effects resulted in cell wall perturbations, including changes in peptidoglycan insertion, penicillin-binding protein 2 (PBP 2) delocalization, and reduced membrane amounts of PBP 2a, despite the increased transcription of mecA through mec regulatory elements. We have found that the VraSR sensor-regulator is a key component of DAP resistance, triggering mutated mprF-mediated cell membrane (CM) modifications that result in impairment of PrsA location and chaperone functions, both of which are essential for PBP 2a maturation, the key determinant of ␤-lactam resistance. These observations provide for the first time evidence that synergistic effects between DAP and ␤-lactams involve PrsA posttranscriptional regulation of CM-associated PBP 2a.KEYWORDS MRSA, daptomycin, seesaw effect, ␤-lactams, PrsA, ␤-lactams S taphylococcus aureus has a proclivity for developing multidrug resistance (e.g., methicillin-resistant S. aureus [MRSA]), and infections with this pathogen result in enhanced attributable mortality (1). Since its FDA approval in 2003, the cyclic anionic lipopeptide antibiotic daptomycin (DAP), produced by Streptomyces roseosporus (2), has become the clinical mainstay of anti-MRSA therapy due to its potent staphylocidal

Carbapenems are potent members of the β-lactam family that inhibit bacterial cell-wall biosynthesis inhibitors . They are highly effective against Gram-negative and Gram-positive drug-resistant infections . As such, carbapenems are typically reserved as an antibiotic of last resort. The WHO lists meropenem as an essential medicine. Nausea and vomiting are reported in ≤20% of carbapenem recipients, with 1.5% suffering seizures. Enzymatic hydrolysis of the β-lactam ring is the main driver of clinical resistance. These enzymes can be classified as Class A, B and D. Classes A and D are serine β-lactamases, whereas Class B rely on metal-mediated hydrolysis, typically through zinc.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.