Alanine Tails Signal Proteolysis in Bacterial Ribosome-Associated Quality Control.

In ribosome-associated quality control (RQC), Rqc2/NEMF closely supports the E3 ligase Ltn1/listerin in promoting ubiquitylation and degradation of aberrant nascent-chains obstructing large (60S) ribosomal subunits-products of ribosome stalling during translation. However, while Ltn1 is eukaryote-specific, Rqc2 homologs are also found in bacteria and archaea; whether prokaryotic Rqc2 has an RQC-related function has remained unknown. Here, we show that, as in eukaryotes, a bacterial Rqc2 homolog (RqcH) recognizes obstructed 50S subunits and promotes nascent-chain proteolysis. Unexpectedly, RqcH marks nascent-chains for degradation in a direct manner, by appending C-terminal poly-alanine tails that act as degrons recognized by the ClpXP protease. Furthermore, RqcH acts redundantly with tmRNA/ssrA and protects cells against translational and environmental stresses. Our results uncover a proteolytic-tagging mechanism with implications toward the function of related modifications in eukaryotes and suggest that RQC was already active in the last universal common ancestor (LUCA) to help cope with incomplete translation.

Analysis of the mRNAs in Spores of Bacillus subtilis.

Large-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormant Bacillus subtilis spores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a single B. subtilis spore allowed calculation of the amount of mRNA in an individual spore as ∼106 nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σG, and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival.IMPORTANCE Previous work indicates that dormant Bacillus subtilis spores have many hundreds of mRNAs, some of which are suggested to play roles in spores' "return to life" or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival.

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.

Phenotypic memory in Bacillus subtilis links dormancy entry and exit by a spore quantity-quality tradeoff.

Some bacteria, such as Bacillus subtilis, withstand starvation by forming dormant spores that revive when nutrients become available. Although sporulation and spore revival jointly determine survival in fluctuating environments, the relationship between them has been unclear. Here we show that these two processes are linked by a phenotypic "memory" that arises from a carry-over of molecules from the vegetative cell into the spore. By imaging life histories of individual B. subtilis cells using fluorescent reporters, we demonstrate that sporulation timing controls nutrient-induced spore revival. Alanine dehydrogenase contributes to spore memory and controls alanine-induced outgrowth, thereby coupling a spore's revival capacity to the gene expression and growth history of its progenitors. A theoretical analysis, and experiments with signaling mutants exhibiting altered sporulation timing, support the hypothesis that such an intrinsically generated memory leads to a tradeoff between spore quantity and spore quality, which could drive the emergence of complex microbial traits.

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.

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

UNLABELLED: 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. KEY WORDS: Bacillus species-Spores-Germination-High salinity-Salt stress-NaCl-Inhibition. Astrobiology 16, 500-512.

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.

Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments.

The germination of spore-forming bacteria in high-salinity environments is of applied interest for food microbiology and soil ecology. It has previously been shown that high salt concentrations detrimentally affect Bacillus subtilis spore germination, rendering this process slower and less efficient. The mechanistic details of these salt effects, however, remained obscure. Since initiation of nutrient germination first requires germinant passage through the spores' protective integuments, the aim of this study was to elucidate the role of the proteinaceous spore coat in germination in high-salinity environments. Spores lacking major layers of the coat due to chemical decoating or mutation germinated much worse in the presence of NaCl than untreated wild-type spores at comparable salinities. However, the absence of the crust, the absence of some individual nonmorphogenetic proteins, and the absence of either CwlJ or SleB had no or little effect on germination in high-salinity environments. Although the germination of spores lacking GerP (which is assumed to facilitate germinant flow through the coat) was generally less efficient than the germination of wild-type spores, the presence of up to 2.4 M NaCl enhanced the germination of these mutant spores. Interestingly, nutrient-independent germination by high pressure was also inhibited by NaCl. Taken together, these results suggest that (i) the coat has a protective function during germination in high-salinity environments; (ii) germination inhibition by NaCl is probably not exerted at the level of cortex hydrolysis, germinant accessibility, or germinant-receptor binding; and (iii) the most likely germination processes to be inhibited by NaCl are ion, Ca(2+)-dipicolinic acid, and water fluxes.

Systematic investigation of germination responses of Bacillus subtilis spores in different high-salinity environments.

High-salinity environments play an increasingly important role in ecology regarding soil salinization due to human-induced processes, but also need to be considered in terms of natural soil desiccation and extreme habitats. It has been shown previously that spore germination of the ubiquitous soil bacterium Bacillus subtilis is detrimentally affected by the presence of high NaCl concentrations, but the underlying mechanisms and effects of other salts remained obscure. To address these two points, we performed a systematic analysis with 32 different salts using spectrophotometric and microscopic methods. It could be shown that inhibitory strength varies considerably among different salts. Although osmotic effects seem to play an important role, ionic composition and concentration (especially of the anion) as well as chemical properties seem to be decisive for the extent of germination inhibition. At the current state of knowledge, fluxes of ions, Ca(2+)-DPA and water are likely affected by all salts, whereas the exact inhibition mechanism of each salt might further depend on the respective properties of the involved ions. Hence, the observed inhibition likely is a result of several phenomena interacting with each other. Altogether this study highlights the complex impact of ionic environments on the life cycle of spore formers.

High salinity alters the germination behavior of Bacillus subtilis spores with nutrient and nonnutrient germinants.

The effect of high NaCl concentrations on nutrient and nonnutrient germination of Bacillus subtilis spores was systematically investigated. Under all conditions, increasing NaCl concentrations caused increasing, albeit reversible, inhibition of germination. High salinity delayed and increased the heterogeneity of germination initiation, slowed the germination kinetics of individual spores and the whole spore population, and decreased the overall germination efficiency, as observed by a variety of different analytical techniques. Germination triggered by nutrients which interact with different germinant receptors (GRs) was affected differently by NaCl, suggesting that GRs are targets of NaCl inhibition. However, NaCl also inhibited GR-independent germination, suggesting that there is at least one additional target for NaCl inhibition. Strikingly, a portion of the spore population could initiate germination with l-alanine even at NaCl concentrations near saturation (∼5.4 M), suggesting that spores lack a salt-sensing system preventing them from germinating in a hostile high-salinity environment. Spores that initiated germination at very high NaCl concentrations excreted their large depot of Ca(2+)-pyridine-2,6-dicarboxylic acid and lost their heat resistance, but they remained in a phase-gray state in the phase-contrast microscope, suggesting that there was incomplete germination. However, some metabolic activity could be detected at up to 4.8 M NaCl. Overall, high salinity seems to exert complex effects on spore germination and outgrowth whose detailed elucidation in future investigations could give valuable insights on these processes in general.