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Background Abundant populations of bacteria have been observed on Mir and the International Space Station. While some experiments have shown that bacteria cultured during spaceflight exhibit a range of potentially troublesome characteristics, including increases in growth, antibiotic resistance and virulence, other studies have shown minimal differences when cells were cultured during spaceflight or on Earth. Although the final cell density of bacteria grown during spaceflight has been reported for several species, we are not yet able to predict how different microorganisms will respond to the microgravity environment. In order to build our understanding of how spaceflight affects bacterial final cell densities, additional studies are needed to determine whether the observed differences are due to varied methods, experimental conditions, or organism specific responses. Results Here, we have explored how phosphate concentration, carbon source, oxygen availability, and motility affect the growth of Pseudomonas aeruginosa in modified artificial urine media during spaceflight. We observed that P. aeruginosa grown during spaceflight exhibited increased final cell density relative to normal gravity controls when low concentrations of phosphate in the media were combined with decreased oxygen availability. In contrast, when the availability of either phosphate or oxygen was increased, no difference in final cell density was observed between spaceflight and normal gravity. Because motility has been suggested to affect how microbes respond to microgravity, we compared the growth of wild-type P. aeruginosa to a ΔmotABCD mutant deficient in swimming motility. However, the final cell densities observed with the motility mutant were consistent with those observed with wild type for all conditions tested. Conclusions These results indicate that differences in bacterial final cell densities observed between spaceflight and normal gravity are due to an interplay between microgravity conditions and the availability of substrates essential for growth. Further, our results suggest that microbes grown under nutrient-limiting conditions are likely to reach higher cell densities under microgravity conditions than they would on Earth. Considering that the majority of bacteria inhabiting spacecrafts and space stations are likely to live under nutrient limitations, our findings highlight the need to explore the impact microgravity and other aspects of the spaceflight environment have on microbial growth and physiology.

We have constructed and characterized two synthetic AND-gate promoters that require both a quorum-sensing (QS) signal and an exogenously added inducer to turn on gene expression. The engineered promoters, LEE and TTE, contain binding sites for the QS-dependent repressor, EsaR, and either LacI or TetR, and they are induced by an acyl-homoserine lactone (AHL) signal and IPTG or aTc. Although repression of both LEE and TTE by wild-type EsaR was observed, induction of gene expression at physiologically relevant concentrations of AHL required the use of an EsaR variant with higher signal sensitivity. Gene expression from both LEE and TTE was shown to require both signal molecules, and gene expression above background levels was not observed with either signal alone. We added endogenous production of AHL to evaluate the ability of the promoters to function in a QS-dependent manner and observed that gene expression increased as a function of cell density only in the presence of exogenously added IPTG or aTc. Cell-cell communication-dependent AND-gate behaviors were demonstrated using an agar plate assay, where cells containing the engineered promoters were shown to respond to AHL produced by a second E. coli strain only in the presence of exogenously added IPTG or aTc. The promoters described in this work demonstrate that EsaR and its target DNA sequence can be used to engineer new promoters to respond to cell density or cell-cell communication. Further, the AND-gate promoters described here may serve as a template for new regulatory systems that integrate QS and the presence of key metabolites or other environmental cues to enable dynamic changes in gene expression for metabolic engineering applications.

Quorum sensing (QS) systems enable bacteria to coordinate their behavior as a function of local population density and are often used in synthetic systems that require cell−cell communication. We have engineered the esaR promoter, P(esaR), which is repressed by the QS regulator E(saR). E(saR)-dependent gene expression from P(esaR) is induced by 3-oxo-hexanoyl-homoserine lactone (3OC6HSL). Here, we report a set of modified P(esaR) promoters that contain a second E(saR) binding site. We observed changes in gene expression levels, regulatory range, 3OC6HSL sensitivity, and the regulatory role of E(saR) that are dependent on the position of the second binding site. Combining the new promoters with endogenous 3OC6HSL production led to QS-dependent systems that exhibit a range of expression levels and timing. These promoters represent a new set of tools for modulating QS-dependent gene expression and may be used to tune the regulation of multiple genes in response to a single QS signal.

The use of cell-cell communication or "quorum sensing (QS)" elements from Gram-negative Proteobacteria has enabled synthetic biologists to begin engineering systems composed of multiple interacting organisms. However, additional tools are necessary if we are to progress toward synthetic microbial consortia that exhibit more complex, dynamic behaviors. EsaR from Pantoea stewartii subsp. stewartii is a QS regulator that binds to DNA as an apoprotein and releases the DNA when it binds to its cognate signal molecule, 3-oxohexanoyl-homoserine lactone (3OC6HSL). In the absence of 3OC6HSL, EsaR binds to DNA and can act as either an activator or a repressor of transcription. Gene expression from P(esaR), which is repressed by wild-type EsaR, requires 100- to 1000-fold higher concentrations of signal than commonly used QS activators, such as LuxR and LasR. Here we have identified EsaR variants with increased sensitivity to 3OC6HSL using directed evolution and a dual ON/OFF screening strategy. Although we targeted EsaR-dependent derepression of P(esaR), our EsaR variants also showed increased 3OC6HSL sensitivity at a second promoter, P(esaS), which is activated by EsaR in the absence of 3OC6HSL. Here, the increase in AHL sensitivity led to gene expression being turned off at lower concentrations of 3OC6HSL. Overall, we have increased the signal sensitivity of EsaR more than 70-fold and generated a set of EsaR variants that recognize 3OC6HSL concentrations ranging over 4 orders of magnitude. QS-dependent transcriptional regulators that bind to DNA and are active in the absence of a QS signal represent a new set of tools for engineering cell-cell communication-dependent gene expression.

Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.