Metabolic fingerprints of Serratia liquefaciens under simulated Martian conditions using Biolog GN2 microarrays

Abstract

Microorganisms growing at atmospheric pressures of 0.7 kPa may have a significant impact on the search for life on Mars. Data on their nutrient requirements in a simulated Martian environment are required to ascertain both the potential risk of forward contamination and the potential of past or present habitability of Mars. Serratia liquefaciens can grow at concomitant conditions of low pressure, low temperature, and anoxic atmosphere. Changes in the metabolic fingerprint of S. liquefaciens grown under varying physical conditions including diverse atmospheric pressures (0.7 kPa to 101.3 kPa), temperatures (30 °C or 0 °C), and atmospheric gas compositions (Earth or CO₂) were investigated using Biolog GN2 assays. Distinct patterns for each condition were observed. Above 10 kPa S. liquefaciens performed similar to Earth-normal pressure conditions (101.3 kPa) whereas below 10 kPa shifts in metabolic patterns were observed. The differences indicated a physiological alteration in which S. liquefaciens lost its ability to metabolize the majority of the provided carbon sources at 0.7 kPa with a significant decrease in the oxidation of amino acids. By measuring the physiological responses to different carbon sources we were able to identify nutritional constraints that support cellular replication under simulated shallow Mars subsurface conditions.

Figure 1

Substrate utilization patterns by Serratia liquefaciens under different pressures using Biolog GN2 microarrays. Ordination plot of metabolic fingerprints for cells grown at 30 °C; Earth-normal atmosphere; and pressures maintained at 2.5, 5.0, 10.0, 20.0, or 101.3 kPa (n = 6).

Figure 2

Overview of the metabolic fingerprints for Serratia liquefaciens cells grown under different atmospheric pressures using Biolog GN2 phenotypic microarrays. (a) The specific numbers of positive wells per substrate group are given for the following categories: alcohols, amides, amino acids, aromatic substrates, brominated substrates, carbohydrates, carboxylic acids, ester, phosphorylated substrates and polymers under the different pressure conditions (2.5, 5.0, 10.0, 20.0, 101.3 kPa) tested. (b) Comparison of OD readings between assays incubated at 2.5, 5.0, or 101.3 kPa. Error bars reflect standard deviations of the means between six independent replicates of the GN2 microarrays.

Figure 3

Detailed results of the organic compounds utilized by Serratia liquefaciens cells for amino acids and carboxylic acids tested at different pressures. (a) The OD colour development per well for each amino acid at 2.5, 5.0, or 101.3 kPa. (b) The OD colour development per well for each carboxylic acid at 2.5, 5, or 101.3 kPa. Error bars are standard deviations of the means between six independent replicates of the Biolog GN2 microarray assays.

Figure 4

Overview of substrate utilization patterns in Serratia liquefaciens under low-PTA conditions and Earth controls at different incubation times. Ordination plot generated by principal component analysis of metabolic fingerprints at (1) low-PTA conditions of 0.7 kPa, 0 °C, and CO₂-enriched anoxic atmosphere; (2) Earth atmospheric pressure, 0 °C, and CO₂; (3) Earth atmospheric pressure, 0 °C, and lab-normal pO₂; (4) Earth atmospheric pressure, 30 °C, and CO₂-enriched anoxic atmosphere; and (5) Earth atmospheric pressure, 30 °C, lab-normal pO₂. Data were taken on 35, 42, or 49 days. The 49 + 2 days treatments refer to Biolog GN2 plates incubated at 30 °C, Earth-normal pressures, and lab-normal atmospheres for an additional 2 d after termination of incubation conditions in (1) through (3) above.

Figure 5

Comparisons of substrate utilization patterns for Serratia liquefaciens cells grown at 0 °C under 0.7 or 101.3 kPa and under pO₂ or pCO₂. The OD readings were taken after 35, 42, and 49 d of incubation. The number at the end of each horizontal bar indicates substrate richness. The assay plates were incubated at (a) 0.7 kPa, CO₂ atmosphere, and 0 °C; (b) 101.3 kPa, CO₂ atmosphere, and 0 °C; or (c) 101.3 kPa, CO₂ atmosphere, and 0 °C. The OD data represent average values of n = 3 replicates.

Figure 6

Venn diagram of the carbon sources utilized by Serratia liquefaciens after 49 d of incubation under three different growth conditions. Comparison of data from GN2 assay plates (n = 3) that were incubated at (1) low-PTA conditions at 0.7 kPa, CO₂ atmosphere, and 0 °C; (2) 101.3 kPa, CO₂ atmosphere, and 0 °C; or (3)101.3 kPa, CO₂ atmosphere, and 0 °C.