Environmental Dependence of Competitive Fitness in Rifampin-Resistant rpoB Mutants of Bacillus subtilis.

RNA polymerase (RNAP) is a highly conserved macromolecular machine that contributes to the flow of genetic information from genotype to phenotype. In Bacillus subtilis, mutations in the rpoB gene encoding the ß-subunit of RNAP have been shown to alter a number of global phenotypes, including growth, utilization of unusual nutrient sources, sporulation, germination, and production of secondary metabolites. In addition, the spectrum of mutations in rpoB leading to rifampin resistance (Rifr) can change dramatically depending upon the environment to which B. subtilis cells or spores are exposed. RifrrpoB mutations have historically been associated with slower growth and reduced fitness; however, these assessments of fitness were conducted on limited collections of mutants in rich laboratory media that poorly reflect natural environments typically inhabited by B. subtilis. Using a novel deep-sequencing approach in addition to traditional measurements of growth rate, lag time, and pairwise competitions, we demonstrated that the competitive advantages of specific rpoB alleles differ depending on the growth environment in which they are determined. IMPORTANCE Microbial resistance to antibiotics is a growing threat to public health across the world. Historically, resistance to antibiotics has been associated with reduced fitness. A growing body of evidence indicates that resistance to rifampin, a frontline antibiotic used to treat mycobacterial and biofilm-associated infections, may increase fitness given an appropriate environment even in the absence of the selective antibiotic. Here, we experimentally confirm this phenomenon by directly comparing the fitness of multiple rifampin-resistant mutants of Bacillus subtilis in rich LB medium and an asparagine minimal medium. Our research demonstrates that the fitness cost of rifampin resistance can vary greatly depending upon the environment. This has important implications for understanding how microbes develop antimicrobial resistance in the absence of antibiotic selection.

The Spectrum of Spontaneous Rifampin Resistance Mutations in the Bacillus subtilis rpoB Gene Depends on the Growth Environment.

Results from previous investigations into spontaneous rifampin resistance (Rifr) mutations in the Bacillus subtilis rpoB gene suggested that the spectrum of mutations depends on the growth environment. However, these studies were limited by low sample numbers, allowing for the potential distortion of the data by the presence of "jackpot" mutations that may have arisen early in the growth of a population. Here, we addressed this issue by performing fluctuation analyses to assess both the rate and spectrum of Rifr mutations in two distinct media: LB, a complete laboratory medium, and SMMAsn, a minimal medium utilizing l-asparagine as the sole carbon source. We cultivated 60 separate populations under each growth condition and determined the mutation rate to Rifr to be slightly but significantly higher in LB cultures. We then sequenced the relevant regions of rpoB to map the spectrum of Rifr mutations under each growth condition. We found a distinct spectrum of mutations in each medium; LB cultures were dominated by the H482Y mutation (27/53 or 51%), whereas SMMAsn cultures were dominated by the S487L mutation (24/51 or 47%). Furthermore, we found through competition experiments that the relative fitness of the S487L mutant was significantly higher in SMMAsn than in LB medium. We therefore conclude that both the spectrum of Rifr mutations in the B. subtilis rpoB gene and the fitness of resulting mutants are influenced by the growth environment. IMPORTANCE The rpoB gene encodes the beta subunit of RNA polymerase, and mutations in rpoB are key determinants of resistance to the clinically important antibiotic rifampin. We show here that the spectrum of mutations in Bacillus subtilis rpoB depends on the medium in which the cells are cultivated. The results show that the growth environment not only plays a role in natural selection and fitness but also influences the probability of mutation at particular bases within the target gene.

Experimental evolution of Bacillus subtilis.

The endospore-forming bacteria have persisted on earth perhaps 3Ga, leveraging the flexibility of their distinctive lifestyle to adapt to a remarkably wide range of environments. This process of adaptation can be investigated through the simple but powerful technique of laboratory evolution. Evolved strains can be analyzed by whole genome sequencing and an array of omics technologies. The intensively studied, genetically tractable endospore-former, Bacillus subtilis, is an ideal subject for laboratory evolution experiments. Here, we describe the use of the B. subtilis model system to study the adaptation of these bacteria to reduced and stringent selection for endospore formation, as well as to novel environmental challenges of low atmospheric pressure, high ultraviolet radiation, and unfavourable growth temperatures. In combination with other approaches, including comparative genomics and environmental field work, laboratory evolution may help elucidate how these bacteria have so successfully adapted to life on earth, and perhaps beyond.

Cultivation in Space Flight Produces Minimal Alterations in the Susceptibility of Bacillus subtilis Cells to 72 Different Antibiotics and Growth-Inhibiting Compounds.

Past results have suggested that bacterial antibiotic susceptibility is altered during space flight. To test this notion, Bacillus subtilis cells were cultivated in matched hardware, medium, and environmental conditions either in space flight microgravity on the International Space Station, termed flight (FL) samples, or at Earth-normal gravity, termed ground control (GC) samples. The susceptibility of FL and GC samples was compared to 72 antibiotics and growth-inhibitory compounds using the Omnilog phenotype microarray (PM) system. Only 9 compounds were identified by PM screening as exhibiting significant differences (P < 0.05, Student's t test) in FL versus GC samples: 6-mercaptopurine, cesium chloride, enoxacin, lomefloxacin, manganese(II) chloride, nalidixic acid, penimepicycline, rolitetracycline, and trifluoperazine. Testing of the same compounds by standard broth dilution assay did not reveal statistically significant differences in the 50% inhibitory concentrations (IC50s) between FL and GC samples. The results indicate that the susceptibility of B. subtilis cells to a wide range of antibiotics and growth inhibitors is not dramatically altered by space flight.IMPORTANCE This study addresses a major concern of mission planners for human space flight, that bacteria accompanying astronauts on long-duration missions might develop a higher level of resistance to antibiotics due to exposure to the space flight environment. The results of this study do not support that notion.

Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli.

To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl ß-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals.

Growth of Carnobacterium spp. from permafrost under low pressure, temperature, and anoxic atmosphere has implications for Earth microbes on Mars.

The ability of terrestrial microorganisms to grow in the near-surface environment of Mars is of importance to the search for life and protection of that planet from forward contamination by human and robotic exploration. Because most water on present-day Mars is frozen in the regolith, permafrosts are considered to be terrestrial analogs of the martian subsurface environment. Six bacterial isolates were obtained from a permafrost borehole in northeastern Siberia capable of growth under conditions of low temperature (0 °C), low pressure (7 mbar), and a CO(2)-enriched anoxic atmosphere. By 16S ribosomal DNA analysis, all six permafrost isolates were identified as species of the genus Carnobacterium, most closely related to C. inhibens (five isolates) and C. viridans (one isolate). Quantitative growth assays demonstrated that the six permafrost isolates, as well as nine type species of Carnobacterium (C. alterfunditum, C. divergens, C. funditum, C. gallinarum, C. inhibens, C. maltaromaticum, C. mobile, C. pleistocenium, and C. viridans) were all capable of growth under cold, low-pressure, anoxic conditions, thus extending the low-pressure extreme at which life can function.

Experimental evolution of enhanced growth by Bacillus subtilis at low atmospheric pressure: genomic changes revealed by whole-genome sequencing.

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.

Proposal to rename Carnobacterium inhibens as Carnobacterium inhibens subsp. inhibens subsp. nov. and description of Carnobacterium inhibens subsp. gilichinskyi subsp. nov., a psychrotolerant bacterium isolated from Siberian permafrost.

A novel, psychrotolerant facultative anaerobe, strain WN1359(T), was isolated from a permafrost borehole sample collected at the right bank of the Kolyma River in Siberia, Russia. Gram-positive-staining, non-motile, rod-shaped cells were observed with sizes of 1-2 µm long and 0.4-0.5 µm wide. Growth occurred in the range of pH 5.8-9.0 with optimal growth at pH 7.8-8.6 (pH optimum 8.2). The novel isolate grew at temperatures from 0-37 °C and optimal growth occurred at 25 °C. The novel isolate does not require NaCl; growth was observed between 0 and 8.8 % (1.5 M) NaCl with optimal growth at 0.5 % (w/v) NaCl. The isolate was a catalase-negative, facultatively anaerobic chemo-organoheterotroph that used sugars but not several single amino acids or dipeptides as substrates. The major metabolic end-product was lactic acid in the ratio of 86 % l-lactate : 14 % d-lactate. Strain WN1359(T) was sensitive to ampicillin, chloramphenicol, fusidic acid, lincomycin, monocycline, rifampicin, rifamycin SV, spectinomycin, streptomycin, troleandomycin and vancomycin, and resistant to nalidixic acid and aztreonam. The fatty acid content was predominantly unsaturated (70.2 %), branched-chain unsaturated (11.7 %) and saturated (12.5 %). The DNA G+C content was 35.3 mol% by whole genome sequence analysis. 16S rRNA gene sequence analysis showed 98.7 % sequence identity between strain WN1359(T) and Carnobacterium inhibens. Genome relatedness was computed using both Genome-to-Genome Distance Analysis (GGDA) and Average Nucleotide Identity (ANI), which both strongly supported strain WN1359(T) belonging to the species C. inhibens. On the basis of these results, the permafrost isolate WN1359(T) represents a novel subspecies of C. inhibens, for which the name Carnobacterium inhibens subsp. gilichinskyi subsp. nov. is proposed. The type strain is WN1359(T) ( = ATCC BAA-2557(T) = DSM 27470(T)). The subspecies Carnobacterium inhibens subsp. inhibens subsp. nov. is created automatically. An emended description of C. inhibens is also provided.

Exposure of Bacillus subtilis to low pressure (5 kilopascals) induces several global regulons, including those involved in the SigB-mediated general stress response.

Studies of how microorganisms respond to pressure have been limited mostly to the extreme high pressures of the deep sea (i.e., the piezosphere). In contrast, despite the fact that the growth of most bacteria is inhibited at pressures below ∼2.5 kPa, little is known of microbial responses to low pressure (LP). To study the global LP response, we performed transcription microarrays on Bacillus subtilis cells grown under normal atmospheric pressure (∼101 kPa) and a nearly inhibitory LP (5 kPa), equivalent to the pressure found at an altitude of ∼20 km. Microarray analysis revealed altered levels of 363 transcripts belonging to several global regulons (AbrB, CcpA, CodY, Fur, IolR, ResD, Rok, SigH, Spo0A). Notably, the highest number of upregulated genes, 86, belonged to the SigB-mediated general stress response (GSR) regulon. Upregulation of the GSR by LP was confirmed by monitoring the expression of the SigB-dependent ctc-lacZ reporter fusion. Measuring transcriptome changes resulting from exposure of bacterial cells to LP reveals insights into cellular processes that may respond to LP exposure.

Resistance of Bacillus subtilis spore DNA to lethal ionizing radiation damage relies primarily on spore core components and DNA repair, with minor effects of oxygen radical detoxification.

The roles of various core components, including α/ß/γ-type small acid-soluble spore proteins (SASP), dipicolinic acid (DPA), core water content, and DNA repair by apurinic/apyrimidinic (AP) endonucleases or nonhomologous end joining (NHEJ), in Bacillus subtilis spore resistance to different types of ionizing radiation including X rays, protons, and high-energy charged iron ions have been studied. Spores deficient in DNA repair by NHEJ or AP endonucleases, the oxidative stress response, or protection by major α/ß-type SASP, DPA, and decreased core water content were significantly more sensitive to ionizing radiation than wild-type spores, with highest sensitivity to high-energy-charged iron ions. DNA repair via NHEJ and AP endonucleases appears to be the most important mechanism for spore resistance to ionizing radiation, whereas oxygen radical detoxification via the MrgA-mediated oxidative stress response or KatX catalase activity plays only a very minor role. Synergistic radioprotective effects of α/ß-type but not γ-type SASP were also identified, indicating that α/ß-type SASP's binding to spore DNA is important in preventing DNA damage due to reactive oxygen species generated by ionizing radiation.

The LysR-type transcriptional regulator (LTTR) AlsR indirectly regulates expression of the Bacillus subtilis bdhA gene encoding 2,3-butanediol dehydrogenase.

Bacillus subtilis ferments pyruvate to 2,3-butanediol via α-acetolactate synthase, α-acetolactate decarboxylase, and butanediol dehydrogenase (BDH), encoded by the alsSD operon and the unlinked monocistronic bdhA gene, respectively. Upstream and divergent from alsSD is the alsR gene that encodes AlsR, a member of the LysR-type transcriptional regulator family. AlsR directly stimulates alsSD transcription by binding to characteristic sites preceding the alsS promoter, but its effect on bdhA expression was unknown. The effect of AlsR on bdhA expression was assessed in a wild-type strain and a congenic strain carrying an alsR::spc knockout mutation by measuring: (a) expression of a transcriptional bdhA-lacZ fusion; (b) bdhA mRNA steady-state levels by quantitative reverse transcriptase PCR; and (c) expression of BDH enzymatic activity. Activation of bdhA expression occurred in early stationary phase, and expression was lowered, but not abolished, in the alsR::spc mutant. Mapping the transcriptional start site of bdhA by primer extension revealed a 268-nucleotide 5'-untranslated region preceding the bdhA initiation methionine codon. Transcription initiation was not reduced in the alsR::spc mutant, and by electrophoretic mobility shift assay, purified AlsR protein did not bind to the bdhA promoter region, suggesting that bdhA expression is indirectly under AlsR transcriptional control.

Carnobacterium Species Capable of Growth at Pressures Ranging Over 5 Orders of Magnitude, from the Surface of Mars (103 Pa) to Deep Oceans (107 Pa) in the Solar System.

Several permanently cold solar system bodies are being investigated with regard to their potential habitability, including Mars and icy moons. In such locations, microbial life would have to cope with low temperatures and both high and low pressures, ranging from ∼102 to 103 Pa on the surface of Mars to upward of ∼108-109 Pa in the subsurface oceans of icy moons. The bacterial genus Carnobacterium consists of species that were previously shown to be capable of growth in the absence of oxygen at low temperatures and at either low pressure or high pressure, but to date the entire pressure range of the genus has not been explored. In the present study, we subjected 14 Carnobacterium strains representing 11 species to cultivation in a complex liquid medium under anaerobic conditions at 2°C and at a range of pressures spanning 5 orders of magnitude, from 103 to 107 Pa. Eleven of the 14 strains showed measurable growth rates at all pressures tested, representing the first demonstration of terrestrial life forms capable of growth under such a wide range of pressures. These findings expand the physical boundaries of the capabilities of life to occur in extreme extraterrestrial environments.

Increased competitive fitness of Bacillus subtilis under nonsporulating conditions via inactivation of pleiotropic regulators AlsR, SigD, and SigW.

Previous studies implicated loss of motility and mutations of the alsR and sigW regulatory genes in enhanced fitness of the Bacillus subtilis evolved strain WN716 over that of its ancestral strain WN624. The fitness of strains carrying knockout mutations alsR::spc, sigD::kan, and/or sigW::erm was measured and compared to that of the congenic ancestral strain by competition experiments.

Protective role of spore structural components in determining Bacillus subtilis spore resistance to simulated mars surface conditions.

Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.

Role of altered rpoB alleles in Bacillus subtilis sporulation and spore resistance to heat, hydrogen peroxide, formaldehyde, and glutaraldehyde.

Mutations in the RNA polymerase ß-subunit gene rpoB causing resistance to rifampicin (Rif(R)) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control. To better understand the influence of rpoB mutations on sporulation and spore resistance to heat and chemicals, cells and spores of the wild-type and twelve distinct congenic Rif(R) mutant strains of B. subtilis were tested. Different levels of glucose catabolite repression during sporulation and spore resistance to heat and chemicals were observed in the Rif(R) mutants, indicating the important role played by the RNA polymerase ß-subunit, not only in the catalytic aspect of transcription, but also in the initiation of sporulation and in the spore resistance properties of B. subtilis.

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in radiation resistance and radiation-induced mutagenesis of Bacillus subtilis spores.

The roles of DNA repair by apurinic/apyrimidinic (AP) endonucleases alone, and together with DNA protection by α/ß-type small acid-soluble spore proteins (SASP), in Bacillus subtilis spore resistance to different types of radiation have been studied. Spores lacking both AP endonucleases (Nfo and ExoA) and major SASP were significantly more sensitive to 254-nm UV-C, environmental UV (>280 nm), X-ray exposure, and high-energy charged (HZE)-particle bombardment and had elevated mutation frequencies compared to those of wild-type spores and spores lacking only one or both AP endonucleases or major SASP. These findings further implicate AP endonucleases and α/ß-type SASP in repair and protection, respectively, of spore DNA against effects of UV and ionizing radiation.

Increased fitness and alteration of metabolic pathways during Bacillus subtilis evolution in the laboratory.

Five batch cultures of Bacillus subtilis were subjected to evolution in the laboratory for 6,000 generations under conditions repressing sporulation in complex liquid medium containing glucose. Between generations 1,000 and 2,000, variants with a distinct small-colony morphology arose and swept through four of the five populations that had been previously noted for their loss of sporulation (H. Maughan et al., Genetics 177:937-948, 2007). To better understand the nature of adaptation in these variants, individual strains were isolated from one population before (WN715) and after (WN716) the sweep. In addition to colony morphology, strains WN715 and WN716 differed in their motility, aerotaxis, and cell morphology. Competition experiments showed that strain WN716 had evolved a distinct fitness advantage over the ancestral strain and strain WN715 during growth and the transition to the postexponential growth phase, which was more pronounced when WN715 was present in the coculture. Microarray analyses revealed candidate genes in which mutations may have produced some of the observed phenotypes. For example, loss of motility in WN716 was accompanied by decreased transcription of all flagellar, motility, and chemotaxis genes on the microarray. Transcription of alsS and alsD was also lower in strain WN716, and the predicted loss of acetoin production and enhanced acetate production was confirmed by high-performance liquid chromatography (HPLC) analysis. The results suggested that the derived colony morphology of strain WN716 was associated with increased fitness, the alteration of several metabolic pathways, and the loss of a typical postexponential-phase response.

Whole-genome sequencing and phenotypic analysis of Bacillus subtilis mutants following evolution under conditions of relaxed selection for sporulation.

Little is known about how genetic variation at the nucleotide level contributes to competitive fitness within species. During a 6,000-generation study of Bacillus subtilis evolved under relaxed selection for sporulation, a new strain, designated WN716, emerged with significantly different colony and cell morphologies; loss of sporulation, competence, acetoin production, and motility; multiple auxotrophies; and increased competitive fitness (H. Maughan and W. L. Nicholson, Appl. Environ. Microbiol. 77:4105-4118, 2011). The genome of WN716 was analyzed by OpGen optical mapping, whole-genome 454 pyrosequencing, and the CLC Genomics Workbench. No large chromosomal rearrangements were found; however, 34 single-nucleotide polymorphisms (SNPs) and +1 frameshifts were identified in WN716 that resulted in amino acid changes in coding sequences of annotated genes, and 11 SNPs were located in intergenic regions. Several classes of genes were affected, including biosynthetic pathways, sporulation, competence, and DNA repair. In several cases, attempts were made to link observed phenotypes of WN716 with the discovered mutations, with various degrees of success. For example, a +1 frameshift was identified at codon 13 of sigW, the product of which (SigW) controls a regulon of genes involved in resistance to bacteriocins and membrane-damaging antibiotics. Consistent with this finding, WN716 exhibited sensitivity to fosfomycin and to a bacteriocin produced by B. subtilis subsp. spizizenii and exhibited downregulation of SigW-dependent genes on a transcriptional microarray, consistent with WN716 carrying a knockout of sigW. The results suggest that propagation of B. subtilis for less than 2,000 generations in a nutrient-rich environment where sporulation is suppressed led to rapid initiation of genomic erosion.

Shelf Life and Simulated Gastrointestinal Tract Survival of Selected Commercial Probiotics During a Simulated Round-Trip Journey to Mars.

To enhance the gastrointestinal health of astronauts, probiotic microorganisms are being considered for inclusion on long-duration human missions to the Moon and Mars. Here we tested three commercial probiotics-Bifidobacterium longum strain BB536, Lactobacillus acidophilus strain DDS-1, and spores of Bacillus subtilis strain HU58-for their survival to some of the conditions expected to be encountered during a 3-year, round trip voyage to Mars. All probiotics were supplied as freeze-dried cells in capsules at a titer of >109 colony forming units per capsule. Parameters tested were survival to: (i) long-term storage at ambient conditions, (ii) simulated Galactic Cosmic Radiation and Solar Particle Event radiation provided by the NASA Space Radiation Laboratory, (iii) exposure to simulated gastric fluid, and (iv) exposure to simulated intestinal fluid. We found that radiation exposure produced minimal effects on the probiotic strains. However, we found that that the shelf-lives of the three strains, and their survival during passage through simulations of the upper GI tract, differed dramatically. We observed that only spores of B. subtilis were capable of surviving all conditions and maintaining a titer of >109 spores per capsule. The results indicate that probiotics consisting of bacterial spores could be a viable option for long-duration human space travel.

Mechanotransduction in Prokaryotes: A Possible Mechanism of Spaceflight Adaptation.

Our understanding of the mechanisms of microgravity perception and response in prokaryotes (Bacteria and Archaea) lag behind those which have been elucidated in eukaryotic organisms. In this hypothesis paper, we: (i) review how eukaryotic cells sense and respond to microgravity using various pathways responsive to unloading of mechanical stress; (ii) we observe that prokaryotic cells possess many structures analogous to mechanosensitive structures in eukaryotes; (iii) we review current evidence indicating that prokaryotes also possess active mechanosensing and mechanotransduction mechanisms; and (iv) we propose a complete mechanotransduction model including mechanisms by which mechanical signals may be transduced to the gene expression apparatus through alterations in bacterial nucleoid architecture, DNA supercoiling, and epigenetic pathways.