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At groundwater sites contaminated with chlorinated ethenes, fermentable substrates are often added to promote reductive dehalogenation by indigenous or augmented microorganisms. Contemporary bioremediation performance monitoring relies on nucleic acid biomarkers of key organohalide-respiring bacteria, such as Dehalococcoides mccartyi (Dhc). Metagenome sequencing of the commercial, Dhc-containing consortium, SDC-9, identified 12 reductive dehalogenase (RDase) genes, including pceA (two copies), vcrA, and tceA, and allowed for specific detection and quantification of RDase peptides using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Shotgun (i.e., untargeted) proteomics applied to the SDC-9 consortium grown with tetrachloroethene (PCE) and lactate identified 143 RDase peptides, and 36 distinct peptides that covered greater than 99% of the protein-coding sequences of the PceA, TceA, and VcrA RDases. Quantification of RDase peptides using multiple reaction monitoring (MRM) assays with 13C-/15N-labeled peptides determined 1.8 × 103 TceA and 1.2 × 102 VcrA RDase molecules per Dhc cell. The MRM mass spectrometry approach allowed for sensitive detection and accurate quantification of relevant Dhc RDases and has potential utility in bioremediation monitoring regimes.

Small molecules that act through an allosteric mechanism to modulate activity of a protein target are abundantly represented in the pharmacopeia. These allosteric modulators are, however, preeminently therapeutics for eukaryotic diseases rather than therapy for prokaryotic infection. Recent examples of the success of biochemical and computational screening methods, paired with protein structural characterization, underscore the promise of allosteric activity modulation as a new approach for antibacterial discovery. In this review, we show how allostery has been leveraged to this objective. In particular, exploitation of an allosteric site on penicillin-binding protein 2a – the resistance enzyme of methicillin-resistant Staphylococcus aureus – demonstrates both that allosteric-modulating structures may themselves possess antibiotic activity and additionally may act as synergists within multi-drug combinations. Future discovery strategies against both old and new bacterial targets may exploit the opportunities offered by allosteric checkpoints within critical bacterial pathways.

The oxadiazole antibacterials target the bacterial cell wall and are bactericidal. We investigated the synergism of ND-421 with the commonly used β-lactams and non-β-lactam antibiotics by the checkerboard method and by time-kill assays. ND-421 synergizes well with β-lactam antibiotics, and it also exhibits a long postantibiotic effect (4.7 h). We also evaluated the in vivo efficacy of ND-421 in a murine neutropenic thigh infection model alone and in combination with oxacillin. ND-421 has in vivo efficacy by itself in a clinically relevant infection model (1.49 log 10 bacterial reduction for ND-321 versus 0.36 log 10 for linezolid with NRS119) and acts synergistically with β-lactam antibiotics in vitro and in vivo , and the combination of ND-421 with oxacillin is efficacious in a mouse neutropenic thigh methicillin-resistant Staphylococcus aureus (MRSA) infection model (1.60 log 10 bacterial reduction). The activity of oxacillin was potentiated in the presence of ND-421, as the strain would have been resistant to oxacillin otherwise.