Summary In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate the sensor kinase’s phosphatase activity. Efficient activation of the phosphatase activity required the presence of both SaeP and SaeQ. When SaeP and SaeQ were ectopically expressed, the expression of coagulase, a sae target with low affinity for phosphorylated SaeR, was greatly reduced, while the expression of alpha-hemolysin, a sae target with high affinity for phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by the SaeRS TCS, these results suggest that the SaeRS TCS returns to the pre-activation state by a negative feedback mechanism.

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are predatory bacteria that naturally—and obligately—prey on other Gram-negative bacteria, and their use has been proposed as a potential new approach to control microbial infection. The ability of predatory bacteria to prey on Gram-negative human pathogens in vitro is well documented; however, the in vivo safety and efficacy of predatory bacteria have yet to be fully assessed. In this study, we examined whether predatory bacteria can reduce bacterial burden in the lungs in an in vivo mammalian system. Initial safety studies were performed by intranasal inoculation of rats with predatory bacteria. No adverse effects or lung pathology were observed in rats exposed to high concentrations of predatory bacteria at up to 10 days postinoculation. Enzyme-linked immunosorbent assay (ELISA) of the immune response revealed a slight increase in inflammatory cytokine levels at 1 h postinoculation that was not sustained by 48 h. Additionally, dissemination experiments showed that predators were efficiently cleared from the host by 10 days postinoculation. To measure the ability of predatory bacteria to reduce microbial burden in vivo, we introduced sublethal concentrations of Klebsiella pneumoniae into the lungs of rats via intranasal inoculation and followed with multiple doses of predatory bacteria over 24 h. Predatory bacteria were able to reduce K. pneumoniae bacterial burden, on average, by more than 3.0 log10 in the lungs of most rats as measured by CFU plating. The work presented here provides further support for the idea of developing predatory bacteria as a novel biocontrol agent.

Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negative bacteria, making predatory bacteria potential alternatives to antibiotics for treating multi-drug resistant infections. While the ability of predatory bacteria to control bacterial infections in vitro is well documented, the in vivo effect of predators on a living host has yet to be extensively examined. In this study, respiratory and intravenous inoculations were used to determine the effects of predatory bacteria in mice. We found no reduction in mouse viability after intranasal or intravenous inoculation of B. bacteriovorus 109J, HD100 or M. aeruginosavorus. Introducing predators into the respiratory tract of mice provoked a modest inflammatory response at 1 hour post-exposure, but was not sustained at 24 hours, as measured by RT-qPCR and ELISA. Intravenous injection caused an increase of IL-6 in the kidney and spleen, TNF in the liver and CXCL-1/KC in the blood at 3 hours post-exposure, returning to baseline levels by 18 hours. Histological analysis of tissues showed no pathological changes due to predatory bacteria. Furthermore, qPCR detected predators were cleared from the host quickly and efficiently. This work addresses some of the safety concerns regarding the potential use of predatory bacteria as a live antibiotic.Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are small, highly motile, uniflagellate Gram-negative bacteria that prey naturally on other Gram-negative bacteria 1,2 . Recently, predatory bacteria have been considered as potential alternatives to traditional antibiotics for treating multi-drug resistant (MDR) Gram-negative bacterial infections. B. bacteriovorus have a predatory lifestyle where they attach to and enter the prey periplasm, multiply by exhausting the nutrients, lyse the cell, and then continue to seek out more prey to invade 1,3,4 . Micavibrio spp., in contrast, attach to, grow and kill prey at the surface of the prey cell in a 'vampire'-like fashion 2,5,6 . Bdellovibrio-and-like organisms (BALOs) are a promising potential novel agent against bacterial pathogens and present several advantages when considering their use for controlling infection 7 . Previous studies have confirmed the ability of predatory bacteria to control a broad range of important human pathogens in vitro, including MDR bacteria 8 , grown both planktonically and in biofilms [9][10][11] . In addition,

Sample nucleic acid purification can often be rate-limiting for conventional quantitative PCR (qPCR) workflows. We recently developed high-throughput virus microneutralization assays using an endpoint assessment approach based on reverse transcription qPCR (RT-qPCR). The need for cumbersome RNA purification is circumvented in our assays by making use of a commercial reagent that can easily generate crude cell lysates amenable to direct analysis by one-step RT-qPCR. In the present study, we demonstrate that a simple buffer containing a non-ionic detergent can serve as an inexpensive alternative to commercially available reagents for the purpose of generating RT-qPCR-ready cell lysates from MDCK cells infected with influenza virus. We have found that addition of exogenous RNase inhibitor as a buffer component is not essential in order to maintain RNA integrity, even following stress at 37°C incubation for 1–2 hours, in cell-lysate samples either freshly prepared or previously stored frozen at −80°C.