Experimental Evolution of Enhanced Growth by Bacillus subtilis at Low Atmospheric Pressure: Genomic Changes Revealed by Whole-Genome Sequencing

Abstract

Knowledge of how microorganisms respond and adapt to low-pressure (LP) environments is limited. Previously, Bacillus subtilis strain WN624 was grown at the near-inhibitory LP of 5 kPa for 1,000 generations and strain WN1106, which exhibited increased relative fitness at 5 kPa, was isolated. Genomic sequence differences between ancestral strain WN624 and LP-evolved strain WN1106 were identified using whole-genome sequencing. LP-evolved strain WN1106 carried amino acid-altering mutations in the coding sequences of only seven genes (fliI, parC, ytoI, bacD, resD, walK, and yvlD) and a single 9-nucleotide in-frame deletion in the rnjB gene that encodes RNase J2, a component of the RNA degradosome. By using a collection of frozen stocks of the LP-evolved culture taken at 50-generation intervals, it was determined that (i) the fitness increase at LP occurred rapidly, while (ii) mutation acquisition exhibited complex kinetics. A knockout mutant of rnjB was shown to increase the competitive fitness of B. subtilis at both LP and standard atmospheric pressure. Microorganisms exhibit an ability to survive and even thrive in a wide range of harsh environments on Earth, which feature extremes of fundamental physical parameters such as temperature and pressure (1). Organisms able to grow at extremely high temperatures (i.e., thermophiles) or low temperatures (i.e., psychrophiles) have been studied extensively (1), as well as organisms capable of growth at high pressure (HP) (i.e., piezophiles) (1, 2). In sharp contrast, our understanding is extremely limited concerning microbial survival, adaptation, and growth in low-pressure (LP) environments. In part, this reflects a relative scarcity of LP environments on the Earth's surface; the atmospheric pressure at sea level averages ϳ101.3 kPa, and the lowest average terrestrial barometric pressure is ϳ34 kPa, atop Mount Everest. However, there has been a recent upsurge of interest in studying the response of microbes to LP exposure. First, Earth's upper atmosphere is a global LP environment that poses unique challenges to microbial survival and growth; for example, 5 kPa of pressure corresponds to an altitude of ϳ19 km, in the lower stratosphere (3-5). Second, man-made LP environments (e.g., hypobaric chambers at pressures of ϳ2 kPa) have proven useful for the long-term storage of high-value agricultural commodities, partly due to LP inhibition of the growth of spoilage microorganisms (6). Third, considerable effort is currently being devoted to the study of the biology of the extraterrestrial environment of Mars, which features an LP atmosphere ranging from ϳ0.1 kPa to ϳ1 kPa; in this context, LP microbiology is important both for life detection and for planetary protection purposes (7).To understand how microbes respond and adapt to LP, we previously reported an evolution experiment in which Bacillus subtilis ancestral strain WN624 was propagated in liquid LB medium for 1,000 generations at an LP of 5 kPa; during this experiment, an enhanced growth capability evolved in the cultur...

Experimental evolution of Bacillus subtilis

“…Changes in colony morphology (110 generations) (Koeppel et al, 2013) Low pressure Increased fitness at low pressure (temperaturedependent) (Waters et al, 2015) High temperature More regular cell morphology near upper limit; altered membrane lipids; deregulated heat shock regulon (Zeigler, unpublished) we failed to find any mutation in the motility-specific sigma factor SigD (Brown et al, 2011;Maughan and Nicholson, 2011). We directly tested whether inactivation of these pleiotropic regulators indeed contributed to the increased fitness of WN716 by constructing in the ancestral strain WN624 insertional knockouts of the alsR, sigW, and sigD genes, singly and in combination.…”

“…Strain WN1106, isolated from the 1000-generation culture, out-competed the ancestral strain at 5 kPa, but not at Earth-normal pressure. Genome sequencing detected only 8 mutations in WN1106: SNPs in the fliI, parC, resD, ytoI, yvlD, bacD and walK genes, and an in-frame, 9-nucleotide deletion in the rnjB gene, rnjBD9 (Waters et al, 2015). The rnjB gene encodes subunit RNase J2 of the RNA degradosome, an enzyme complex that governs global RNA turnover in B. subtilis (Cho, 2017).…”

Airborne Bacteria in Earth's Lower Stratosphere Resemble Taxa Detected in the Troposphere: Results From a New NASA Aircraft Bioaerosol Collector (ABC)

“…Since all marine and surface environments are impacted by winds, the emanation/deposition of airborne microorganisms (hereafter referred to as bioaerosols) contributes to ecosystem dynamics. For instance, airborne microorganisms surviving harsh conditions while aloft, including strong levels of mutagenic ultraviolet radiation, might be altered at the genomic, transcriptomic, and proteomic level upon germination in a new environment (Smith et al, 2011 ; Chudobova et al, 2015 ; Waters et al, 2015 ; Khodadad et al, 2017 ). With hundreds of teragrams of microbe-laden dusts from deserts and agricultural soils moving through Earth's atmosphere each year (Acosta-Martinez et al, 2015 ), additional surveys are needed to better understand the ecological consequences of airborne biomass exchange, including disease dispersal (Brown and Hovmoller, 2002 ; Fröhlich-Nowoisky et al, 2016 ; Mahaffee and Stoll, 2016 ; Van Leuken et al, 2016 ).…”

Genome analysis of a Bacillus subtilis strain reveals genetic mutations determining biocontrol properties

“…Investigation of SNPs also reflects the variability within the cell population at genome sequence level, shows the regions of hypermutation in the genome where the mutations occur with an elevated frequency, and is suitable for the investigation of short-time evolution events (Brown et al 2011 ; Waters et al 2015 ). It has been suggested that the local hypermutation phenomenon in B. subtilis is in connection with the transcription-associated stationary-phase mutagenesis, which is in relation with the high expression level of the target gene and Mfd, the transcription repair coupling factor (Pybus et al 2010 ; Robleto et al 2012 ).…”