Analysis of Bacillus subtilis spore germination and outgrowth in high-salinity environments

Upon nutrient depletion, the soil bacterium Bacillus subtilis can form highly resistant, metabolically dormant spores. Spores consist of a dehydrated core (harboring the spore genome) enveloped in an inner spore membrane, a peptidoglycan germ cell wall and cortex, an outer spore membrane, and a proteinaceous coat. When specific nutrients (‘germinants’) become available again, they can bind to germinant receptors in the inner spore membrane and induce spore revival, consisting of a germination and an outgrowth phase. During germination, spores lose their resistance, release ions and Ca2+-dipicolinate (Ca2+-DPA) from the core in exchange for water (‘core rehydration’), and hydrolyze their cortex. When core rehydration is sufficient to allow enzymatic activity, metabolism is re-activated. This hallmarks the beginning of the outgrowth phase, during which spores undergo molecular reorganization and elongate. The effects of high salt concentrations and osmotic stress on spore revival were previously poorly investigated, although this topic is relevant for basic research, food microbiology, soil ecology, and astrobiology. Therefore, in this doctoral thesis, the impact of high salinity on Bacillus spore revival was examined, primarily focusing on B. subtilis spore germination in the presence of high NaCl concentrations. In general, increasing salt concentrations exerted increasingly detrimental effects on germination, although some spores initiated germination despite very high salinities. In the presence of high NaCl concentrations (≥ 1.2 mol/L), B. subtilis spore germination was delayed, slower, more heterogeneous, and less efficient. Other salts also inhibited germination, although their inhibitory strength varied depending on ion concentrations, ionic species (and their combination), and the chemical properties of the salt. Although ionic stress was indeed an important factor, high concentrations of non-ionic osmotic solutes had similar inhibitory strengths as iso-osmotic NaCl concentrations, suggesting that osmotic stress plays a decisive role in NaCl-inhibition. Strikingly, spores having strong coat defects showed exacerbated inhibition by NaCl but not by non-ionic solutes, indicating an important role of the spore coat (possibly in combination with the outer spore membrane) in protecting the subjacent inner spore structures (i.e. cortex, germ cell wall, germination apparatus, and inner spore membrane) from ionic stress. Based on these findings, a first mechanistic model for germination inhibition by high salinity is proposed. In this model, ionic interactions with the germinant and/or spore coat slow germinant passage to the germinant receptors, thereby delaying germination initiation. Subsequently, osmotic inhibition of core rehydration and concomitant Ca2+-DPA release slows germination after its initiation. While metabolic reactivation was observable at up to 4.8 mol/L NaCl, successful outgrowth in terms of elongation was observable at up to 2.4 mol/L NaCl, but only under nutrient-rich conditions. Transcriptomic analyses of salt-stressed outgrowing spores indicated many similarities to vegetative cells exposed to sustained high salinity, including the induction of B. subtilis’ complete genetic repertoire of osmoprotectant uptake and compatible solute synthesis. Taken together, this doctoral thesis yielded the first mechanistic model for inhibitory high-salinity effects on B. subtilis spore germination as well as the first comprehensive transcriptomics study of the salt stress response of outgrowing B. subtilis spores. These results contribute to the basic understanding of the influence of salt on B. subtilis’ life cycle, and are valuable for the aforementioned applied research fields as well

Identification of Differentially Expressed Genes during Bacillus subtilis Spore Outgrowth in High-Salinity Environments Using RNA Sequencing

In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study, we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σB-dependent general stress response typically triggered by salt shocks was not induced, whereas the σW regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes (“ripening”) of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism

Experimental studies addressing the longevity of Bacillus subtilis spores - The first data from a 500-year experiment

The ability to form endospores allows certain Gram-positive bacteria (e.g. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Despite thorough study of spore resistance to external stresses, precisely how long B. subtilis spores can lie dormant while remaining viable, a period that potentially far exceeds the human lifespan; is not known although convincing examples of long term spore survival have been recorded. In this study, we report the first data from a 500-year microbial experiment, which started in 2014 and will finish in 2514. A set of vials containing a defined concentration of desiccated B. subtilis spores is opened and tested for viability every two years for the first 24 years and then every 25 years until experiment completion. Desiccated baseline spore samples were also exposed to environmental stresses, including X-rays, 254 nm UV-C, 10% H2O2, dry heat (120°C) and wet heat (100°C) to investigate how desiccated spores respond to harsh environmental conditions after long periods of storage. Data from the first 2 years of storage show no significant decrease in spore viability. Additionally, spores of B. subtilis were subjected to various short-term storage experiments, revealing that space-like vacuum and high NaCl concentration negatively affected spore viability.Peer Reviewe

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Investigation of Bacillus subtilis spore germination at high salinity

The effects of high salt concentrations on the germination of Bacillus subtilis endospores are barely investigated. This thesis addressed questions concerning high-salinity effects on spore germination, the roles of cellular components in germination under high salt conditions and salt-dependent alterations in gene expression during spore outgrowth. B. subtilis wild type spores are generally able to germinate despite the presence of very high salt concentrations and osmolalities (up to 3.6M and 7.2 osmol/kg, respectively). However, elevated salt concentrations exert inhibitory effects on germination, leading to a delay of germination onset and decreased germination efficiency. Four major factors were identified to differentially affect spore germination at high salinity: the germination medium, the employed germination trigger, the type of ionic species and the ion concentration. Cationic and anionic species, valence and general chemical properties seem to influence the inhibitory features of a salt, whereas the overall osmolality of the germination medium is apparently less important. Salt inhibition seems to have several targets, at least one being related to nutrient germination receptor (GR) functionality. In fact, the different GRs were found to be unequally affected by different salts. However, high salinity can also inhibit non-nutrient germination that does not involve GRs, indicating at least one additional, GR-independent inhibition target. The germination analyses of mutant spores provided interesting information on the involvement of different cellular components within the germination process. The anionic phospholipid cardiolipin (CL) is not essential, but beneficial for successful nutrient germination in the absence of salt. Yet, CL is highly important for germination in a high-salinity environment, possibly due to stabilizing effects on the GRs. The K⁺-transport systems KtrAB and KtrCD are not essential for germination, but KtrAB seems to play a role for germination in the presence of high NaCl concentrations. Regardless of the salinity of the germination medium, the five osmoprotectant uptake systems (OpuA to OpuE) are not involved in germination, although they generally seem to facilitate outgrowth and subsequent growth

Translating physics to microbiology: spore resistance to terrestrial and extraterrestrial extremes

Spore-forming bacteria are of particular concern in the context of planetary protection because their tough endospores are capable of withstanding certain sterilization procedures as well as harsh environments. Spores of Bacillus subtilis have been shown to be suitable dosimeters for probing extreme terrestrial and extraterrestrial environmental conditions in astrobiological and environmental studies. During dormancy spores are metabolically inactive; thus substantial DNA, protein, tRNA and ribosome damage can accumulate while the spores are incapable of repairing and/or degrading damaged DNA and proteins. Consequently, damage to essential components of spores poses a unique problem, since damage repair does not occur until the processes of spore revival. Spores appear to have two possible ways to minimize deleterious effects of environmental extremes: (i) by protecting dormant spore macromolecules (in particular the spore DNA) from damage in the first place and (ii) by ensuring repair of damage during spore outgrowth. In our research, we used spores of different genotypes of B. subtilis to study the effects of various extraterrestrial conditions (e.g., planetary conditions as present on Mars or low Earth orbit (LEO)) for astrobiological purposes. Spores of wild-type and mutant B. subtilis strains lacking various structural components were exposed to simulated Martian atmospheric, galactic cosmic and UV irradiation conditions. Spore survival was strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid (DPA) as key protectants. In addition, the interaction of several DNA repair mechanisms (e.g., non-homologous end joining (NHEJ) and spore photoproduct (SP) lyase) was identified as crucial for surviving environmental extremes in space or Martian surface (i.e., exposure to solar UV and galactic cosmic radiation. The ultimate goal is to obtain a complete model describing spore persistence and longevity in harsh environments

Germination of Spores of Astrobiologically Relevant Bacillus Species in High-Salinity Environments

In times of increasing space exploration and search for extraterrestrial life, new questions and challenges for planetary protection, aiming to avoid forward contamination of different planets or moons with terrestrial life, are emerging. Spore-forming bacteria such as Bacillus species have a high contamination potential due to their spores’ extreme resistance, enabling them to withstand space conditions. Spores require liquid water for their conversion into a growing cell (i.e., spore germination and subsequent growth). If present, water on extraterrestrial planets or moons is likely to be closely associated with salts (e.g., in salty oceans or brines), thus constituting high-salinity environments. Spores of Bacillus subtilis can germinate despite very high salt concentrations, although salt stress does exert negative effects on this process. In this study, germination and metabolic reactivation (‘‘outgrowth’’) of spores of five astrobiologically relevant Bacillus species (B. megaterium, B. pumilus SAFR-032, B. nealsonii, B. mojavensis, and B. vallismortis) in high salinity (≤3.6 M NaCl) were investigated. Spores of different species exhibited different germination and outgrowth capabilities in high salinity, which strongly depended on germination conditions, especially the exact composition of the medium. In this context, a new ‘‘universal’’ germination trigger for Bacillus spores, named KAGE (KCl, L-alanine, D-glucose, ectoine), was identified, which will be very useful for future comparative germination and outgrowth studies on different Bacillus species. Overall, this study yielded interesting new insights on salt stress effects on spore germination and points out the difficulty of predicting the potential of spores to contaminate salty environments on extraterrestrial celestial bodies

Analysis of Bacillus subtilis spore germination and outgrowth in high-salinity environments

Upon nutrient depletion, the soil bacterium Bacillus subtilis can form highly resistant, metabolically dormant spores. Spores consist of a dehydrated core (harboring the spore genome) enveloped in an inner spore membrane, a peptidoglycan germ cell wall and cortex, an outer spore membrane, and a proteinaceous coat. When specific nutrients (‘germinants’) become available again, they can bind to germinant receptors in the inner spore membrane and induce spore revival, consisting of a germination and an outgrowth phase. During germination, spores lose their resistance, release ions and Ca2+-dipicolinate (Ca2+-DPA) from the core in exchange for water (‘core rehydration’), and hydrolyze their cortex. When core rehydration is sufficient to allow enzymatic activity, metabolism is re-activated. This hallmarks the beginning of the outgrowth phase, during which spores undergo molecular reorganization and elongate. The effects of high salt concentrations and osmotic stress on spore revival were previously poorly investigated, although this topic is relevant for basic research, food microbiology, soil ecology, and astrobiology. Therefore, in this doctoral thesis, the impact of high salinity on Bacillus spore revival was examined, primarily focusing on B. subtilis spore germination in the presence of high NaCl concentrations. In general, increasing salt concentrations exerted increasingly detrimental effects on germination, although some spores initiated germination despite very high salinities. In the presence of high NaCl concentrations (≥ 1.2 mol/L), B. subtilis spore germination was delayed, slower, more heterogeneous, and less efficient. Other salts also inhibited germination, although their inhibitory strength varied depending on ion concentrations, ionic species (and their combination), and the chemical properties of the salt. Although ionic stress was indeed an important factor, high concentrations of non-ionic osmotic solutes had similar inhibitory strengths as iso-osmotic NaCl concentrations, suggesting that osmotic stress plays a decisive role in NaCl-inhibition. Strikingly, spores having strong coat defects showed exacerbated inhibition by NaCl but not by non-ionic solutes, indicating an important role of the spore coat (possibly in combination with the outer spore membrane) in protecting the subjacent inner spore structures (i.e. cortex, germ cell wall, germination apparatus, and inner spore membrane) from ionic stress. Based on these findings, a first mechanistic model for germination inhibition by high salinity is proposed. In this model, ionic interactions with the germinant and/or spore coat slow germinant passage to the germinant receptors, thereby delaying germination initiation. Subsequently, osmotic inhibition of core rehydration and concomitant Ca2+-DPA release slows germination after its initiation. While metabolic reactivation was observable at up to 4.8 mol/L NaCl, successful outgrowth in terms of elongation was observable at up to 2.4 mol/L NaCl, but only under nutrient-rich conditions. Transcriptomic analyses of salt-stressed outgrowing spores indicated many similarities to vegetative cells exposed to sustained high salinity, including the induction of B. subtilis’ complete genetic repertoire of osmoprotectant uptake and compatible solute synthesis. Taken together, this doctoral thesis yielded the first mechanistic model for inhibitory high-salinity effects on B. subtilis spore germination as well as the first comprehensive transcriptomics study of the salt stress response of outgrowing B. subtilis spores. These results contribute to the basic understanding of the influence of salt on B. subtilis’ life cycle, and are valuable for the aforementioned applied research fields as well

Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

Bacillus subtilis is one of the bacterial members provided with a nonhomologous end joining (NHEJ) system constituted by the DNA-binding Ku homodimer that recruits the ATP-dependent DNA Ligase D (BsuLigD) to the double-stranded DNA breaks (DSBs) ends. BsuLigD has inherent polymerization and ligase activities that allow it to fill the short gaps that can arise after realignment of the broken ends and to seal the resulting nicks, contributing to genome stability during the stationary phase and germination of spores. Here we show that BsuLigD also has an intrinsic 5'-2-deoxyribose-5-phosphate (dRP) lyase activity located at the N-terminal ligase domain that in coordination with the polymerization and ligase activities allows efficient repairing of 2'- deoxyuridine-containing DNA in an in vitro reconstituted Base Excision Repair (BER) reaction. The requirement of a polymerization, a dRP removal and a final sealing step in BER, together with the joint participation of BsuLigD with the spore specific AP endonuclease in conferring spore resistance to ultrahigh vacuum desiccation suggest that BsuLigD could actively participate in this pathway.We demonstrate the presence of the dRP lyase activity also in the homolog protein from the distantly related bacterium Pseudomonas aeruginosa, allowing us to expand our results to other bacterial LigDs