Planetary Protection Implementation and Verification Approach for the Mars 2020 Mission.

The Mars 2020 Flight System comprises a Cruise Stage; Aeroshell; Entry, Descent, and Landing system; Perseverance rover; and the Ingenuity helicopter. The Perseverance rover was successfully delivered to Jezero Crater on February 18, 2021. Among its science objectives, Perseverance is meant to search for rocks that are capable of preserving chemical traces of ancient life, if it existed, and to core and cache rock and regolith samples. The Perseverance rover is gathering samples for potential return to Earth as part of a Mars Sample Return campaign. Thus, controlling the presence of Earth-sourced biological contamination is important to protect the integrity of the scientific results as well as to comply with international treaty and NASA requirements governing Planetary Protection prior to launch. An unprecedented campaign of sampling and environmental monitoring occurred, which resulted in over 16,000 biological samples collected throughout spacecraft assembly. Engineering design, microbial reduction measures, monitoring, and process controls enabled the mission to limit the total spore bioburden to 3.73 × 105 spores, which provided 25.4% margin against the required limit. Furthermore, the total spore bioburden of all landed hardware was 3.86 × 104, which provided 87% margin against the required limit. This manuscript outlines the Planetary Protection implementation approach and verification methodologies applied to the Mars 2020 flight system and its surrounding environments.

Qualification of Membrane Filtration for Planetary Protection Flight Implementation.

Planetary protection is the practice of preventing forward and backward contamination of solar system bodies. Spacecraft and associated surfaces are sampled to ensure compliance with bioburden requirements. Current planetary protection sampling and processing methodologies consist of extracting microbial cells from wipe or swab samples through a procedure (NASA Standard Assay) that includes sonication, heat shock, and pour-plate steps. The pour-plate steps are laborious and prolonged. Moreover, results can be imprecise because only a fraction of the sample fluid is plated for CFU enumeration (80% for swabs and 25% for wipes). Thus, analysis requires that a pour fraction extrapolation factor be applied to CFU counts to account for bioburden in the remaining sample volume that is not plated. This extrapolation results in large variances for data, decreasing the accuracy of spore bioburden estimation of spacecraft hardware. In this study, we investigated the use of membrane filtration as an alternative method to pour-plate processing. Membrane filtration is an appealing methodology for planetary protection because it can process greater sample volumes and reduces the data variance for bioburden enumeration. A pour fraction extrapolation factor is still applied for both swabs and wipes (92%), however, it is a greater pour fraction than the pour-plate method. Here we present data collected by the Jet Propulsion Laboratory and the Applied Physics Laboratory to experimentally determine the equivalency of membrane filtration to pour-plate methodology for implementation during the NASA Standard Assay. Additionally, we outline the planned procedures for two membrane filtration systems: Pall® Laboratory Manifold system and Milliflex® Plus Vacuum Pump System. Both systems demonstrated equivalence of the membrane filtration method to the pour-plate method.

(p)ppGpp modulates cell size and the initiation of DNA replication in Caulobacter crescentus in response to a block in lipid biosynthesis.

Stress conditions, such as a block in fatty acid synthesis, signal bacterial cells to exit the cell cycle. Caulobacter crescentus FabH is a cell-cycle-regulated ß-ketoacyl-acyl carrier protein synthase that initiates lipid biosynthesis and is essential for growth in rich media. To explore how C. crescentus responds to a block in lipid biosynthesis, we created a FabH-depletion strain. We found that FabH depletion blocks lipid biosynthesis in rich media and causes a cell cycle arrest that requires the alarmone (p)ppGpp for adaptation. Notably, basal levels of (p)ppGpp coordinate both a reduction in cell volume and a block in the over-initiation of DNA replication in response to FabH depletion. The gene ctrA encodes a master transcription factor that directly regulates 95 cell-cycle-controlled genes while also functioning to inhibit the initiation of DNA replication. Here, we demonstrate that ctrA transcription is (p)ppGpp-dependent during fatty acid starvation. CtrA fails to accumulate when FabH is depleted in the absence of (p)ppGpp due to a substantial reduction in ctrA transcription. The (p)ppGpp-dependent maintenance of ctrA transcription during fatty acid starvation initiated from only one of the two ctrA promoters. In the absence of (p)ppGpp, the majority of FabH-depleted cells enter a viable but non-culturable state, with multiple chromosomes, and are unable to recover from the miscoordination of cell cycle events. Thus, basal levels of (p)ppGpp facilitate C. crescentus' re-entry into the cell cycle after termination of fatty acid starvation.