A Molecular Genetic Basis Explaining Altered Bacterial Behavior in Space

Abstract

Bacteria behave differently in space, as indicated by reports of reduced lag phase, higher final cell counts, enhanced biofilm formation, increased virulence, and reduced susceptibility to antibiotics. These phenomena are theorized, at least in part, to result from reduced mass transport in the local extracellular environment, where movement of molecules consumed and excreted by the cell is limited to diffusion in the absence of gravity-dependent convection. However, to date neither empirical nor computational approaches have been able to provide sufficient evidence to confirm this explanation. Molecular genetic analysis findings, conducted as part of a recent spaceflight investigation, support the proposed model. This investigation indicated an overexpression of genes associated with starvation, the search for alternative energy sources, increased metabolism, enhanced acetate production, and other systematic responses to acidity-all of which can be associated with reduced extracellular mass transport.

Fig 1. Spearman correlation.

The hierarchical clustering of Spearman correlation coefficients showing pairwise comparisons of effect of Gentamicin Sulfate exposure at various concentrations to E. coli grown in space and on Earth. This observation indicates that transcriptome-wide RNA expression profiles may be used to distinguish and characterize the response of E.coli to antibiotic concentration and gravitational conditions at molecular levels.

Fig 2. Differential Gene Expression.

Scatter plots showing averaged gene expression (log₁₀ scale) in E.coli as detected by RNA-seq between space (y-axis) and Earth (x-axis) at various Gentamicin Sulfate concentrations: 25 μg/mL (A); 50 μg/mL plot (B); and 75 μg/mL (C). Red dots indicate overexpression, blue non-differential expression, and green underexpression of genes in space with respect to Earth (1g) controls. Pink triangles show genes associated with metabolism but not directly involved in the glucose catabolism pathways (listed in S6 Table); white circles indicate the expression of genes associated with acid resistance; yellow diamonds indicate the genes associated with acetate production from glucose (listed in S7 Table); and purple squares represent the genes induced by acetate but not by acidity (listed in S8 Table).

Fig 3. Genes involved in the TCA and Glyoxylate Cycles.

E. coli metabolic pathways, compiled from ref. [45] and [46]. Most of the genes associated with these metabolic pathways were overexpressed in the spaceflight samples with respect to their matched Earth (1g) controls. ^⬇Underexpressed in one of the three groups;^⬆overexpressed in one group; ^⬆⬆overexpressed in two groups; ^⬆⬆⬆overexpressed in all three groups.

Fig 4. Altered Extracellular Model.

Biomolecular model based on the gene expression data analyses support the reduction of glucose molecules (blue gradient) and acid buildup (gold gradient) proposed to occur in the boundary layer around the cell. This altered extracellular environment has been hypothesized to result as an effect of reduced gravity-driven forces acting on the cell-fluid system and has been put forth as the biophysical mechanism governing bacterial behavior in space. Blue circles indicate overexpression of genes associated with metabolism, while gold circles represent the overexpression of acidic condition genes.

Fig 5. Fluid Processing Apparatus (FPA).

BioServe’s Fluid Processing Apparatus (FPA) loaded with colored solutions to best describe the actual contents per chamber of the AES-1 configuration: A– 2.75 mL of sterile growth medium with glucose; B– 0.50 mL of inoculum in minimal medium without glucose; C– 0.25 mL of antibiotic solution; D– 2.10 mL of fixative (either paraformaldehyde or RNA Later II). In this figure, pushing from right to left would move the septum separating chambers A and B into the bypass, thus allowing for the solution in chamber B to be transferred and mixed into chamber A, and likewise to later add the contents of C and D. The actual solutions as flown in AES-1 were all colorless.

Fig 6. AES-1 operation in space.

Astronaut Mike Hopkins operating one of the GAPs containing the AES-1 experiment onboard ISS (Photo credit: NASA). The individual in this manuscript has given written informed consent (as outlined in PLOS consent form) to publish these case details.