Genetic Study of Four Candidate Holliday Junction Processing Proteins in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

Homologous recombination (HR) is thought to be important for the repair of stalled replication forks in hyperthermophilic archaea. Previous biochemical studies identified two branch migration helicases (Hjm and PINA) and two Holliday junction (HJ) resolvases (Hjc and Hje) as HJ-processing proteins; however, due to the lack of genetic evidence, it is still unclear whether these proteins are actually involved in HR in vivo and how their functional relation is associated with the process. To address the above questions, we constructed hjc-, hje-, hjm-, and pina single-knockout strains and double-knockout strains of the thermophilic crenarchaeon Sulfolobus acidocaldarius and characterized the mutant phenotypes. Notably, we succeeded in isolating the hjm- and/or pina-deleted strains, suggesting that the functions of Hjm and PINA are not essential for cellular growth in this archaeon, as they were previously thought to be essential. Growth retardation in Δpina was observed at low temperatures (cold sensitivity). When deletion of the HJ resolvase genes was combined, Δpina Δhjc and Δpina Δhje exhibited severe cold sensitivity. Δhjm exhibited severe sensitivity to interstrand crosslinkers, suggesting that Hjm is involved in repairing stalled replication forks, as previously demonstrated in euryarchaea. Our findings suggest that the function of PINA and HJ resolvases is functionally related at lower temperatures to support robust cellular growth, and Hjm is important for the repair of stalled replication forks in vivo.

Genetic and Biochemical Characterizations of aLhr1 Helicase in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

Homologous recombination (HR) refers to the process of information exchange between homologous DNA duplexes and is composed of four main steps: end resection, strand invasion and formation of a Holliday junction (HJ), branch migration, and resolution of the HJ. Within each step of HR in Archaea, the helicase-promoting branch migration is not fully understood. Previous biochemical studies identified three candidates for archaeal helicase promoting branch migration in vitro: Hjm/Hel308, PINA, and archaeal long helicase related (aLhr) 2. However, there is no direct evidence of their involvement in HR in vivo. Here, we identified a novel helicase encoded by Saci_0814, isolated from the thermophilic crenarchaeon Sulfolobus acidocaldarius; the helicase dissociated a synthetic HJ. Notably, HR frequency in the Saci_0814-deleted strain was lower than that of the parent strain (5-fold decrease), indicating that Saci_0814 may be involved in HR in vivo. Saci_0814 is classified as an aLhr1 under superfamily 2 helicases; its homologs are conserved among Archaea. Purified protein produced in Escherichia coli showed branch migration activity in vitro. Based on both genetic and biochemical evidence, we suggest that aLhr1 is involved in HR and may function as a branch migration helicase in S. acidocaldarius.

Certain, but Not All, Tetraether Lipids from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Can Form Black Lipid Membranes with Remarkable Stability and Exhibiting Mthk Channel Activity with Unusually High Ca2+ Sensitivity

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLMPLFE can be attributed to strong PLFE–PLFE and PLFE–substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLMPLFE to values recorded in BLMSa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLMPLFE and exhibit channel activity, but not in BLMSa-MV. Additionally, the opening/closing of the MthK in BLMPLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLMPLFE and BLMSa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

Putative Nucleotide-Based Second Messengers in the Archaeal Model Organisms Haloferax volcanii and Sulfolobus acidocaldarius

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3′,5′-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3′,5′-cAMP, cyclic guanosine monophosphate (3′,5′-cGMP), 5′-phosphoadenylyl-3′,5′-adenosine (5′-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2′,3′-cyclic isomers of all four RNA building blocks (2′,3′-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3′,5′-cCMP), cyclic uridine monophosphate (3′,5′-cUMP) and cyclic diadenosine monophosphate (3′,5′-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3′,5′-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

Machine Learning Uncovers a Data-Driven Transcriptional Regulatory Network for the Crenarchaeal Thermoacidophile Sulfolobus acidocaldarius

Dynamic cellular responses to environmental constraints are coordinated by the transcriptional regulatory network (TRN), which modulates gene expression. This network controls most fundamental cellular responses, including metabolism, motility, and stress responses. Here, we apply independent component analysis, an unsupervised machine learning approach, to 95 high-quality Sulfolobus acidocaldarius RNA-seq datasets and extract 45 independently modulated gene sets, or iModulons. Together, these iModulons contain 755 genes (32% of the genes identified on the genome) and explain over 70% of the variance in the expression compendium. We show that five modules represent the effects of known transcriptional regulators, and hypothesize that most of the remaining modules represent the effects of uncharacterized regulators. Further analysis of these gene sets results in: (1) the prediction of a DNA export system composed of five uncharacterized genes, (2) expansion of the LysM regulon, and (3) evidence for an as-yet-undiscovered global regulon. Our approach allows for a mechanistic, systems-level elucidation of an extremophile’s responses to biological perturbations, which could inform research on gene-regulator interactions and facilitate regulator discovery in S. acidocaldarius. We also provide the first global TRN for S. acidocaldarius. Collectively, these results provide a roadmap toward regulatory network discovery in archaea.

High-Temperature Live-Cell Imaging of Cytokinesis, Cell Motility, and Cell-Cell Interactions in the Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

Significant technical challenges have limited the study of extremophile cell biology. Here we describe a system for imaging samples at 75°C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius with unprecedented resolution. In addition, we observed previously undescribed dynamic cell shape changes, cell motility, and cell-cell interactions, shedding significant new light on the high-temperature lifestyle of this organism.

Machine learning uncovers a data-driven transcriptional regulatory network for the Crenarchaeal thermoacidophile Sulfolobus acidocaldarius

Dynamic cellular responses to environmental constraints are coordinated by the transcriptional regulatory network (TRN), which modulates gene expression. This network controls most fundamental cellular responses, including metabolism, motility, and stress responses. Here, we apply independent component analysis, an unsupervised machine learning approach, to 95 high-quality Sulfolobus acidocaldarius RNA-seq datasets and extract 45 independently modulated gene sets, or iModulons. Together, these iModulons contain 755 genes (32% of the genes identified on the genome) and explain over 70% of the variance in the expression compendium. We show that 5 modules represent the effects of known transcriptional regulators, and hypothesize that most of the remaining modules represent the effects of uncharacterized regulators. Further analysis of these gene sets results in: (1) the prediction of a DNA export system composed of 5 uncharacterized genes, (2) expansion of the LysM regulon, and (3) evidence for an as-yet-undiscovered global regulon. Our approach allows for a mechanistic, systems-level elucidation of an extremophile's responses to biological perturbations, which could inform research on gene-regulator interactions and facilitate regulator discovery in S. acidocaldarius. We also provide the first global TRN for S. acidocaldarius. Collectively, these results provide a roadmap towards regulatory network discovery in archaea.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Response of the thermoacidophilic Archaeon Sulfolobus acidocaldarius to solvent stress exemplified by 1-butanol exposure

Sulfolobus acidocaldarius is a thermoacidophilic crenarchaeon with optimal growth at 80 °C and pH 2 - 3. Due to its unique physiological properties allowing life at environmental extremes and recent availability of genetic tools, this extremophile receives increasing interest for biotechnological application. In order to elucidate the potential of tolerating process-related stress conditions, we investigated the response of S. acidocaldarius towards the industrially relevant organic solvent 1-butanol. In response to butanol exposure, biofilm formation of S. acidocaldarius was enhanced and occurred up to 1.5% (v/v) 1-butanol, while planktonic growth was only observed up to 1% (v/v) 1-butanol. Confocal laser scanning microscopy revealed that biofilm architecture changed with the formation of denser and higher tower-like structures. Concomitantly, changes in the extracellular polymeric substances with enhanced carbohydrate and protein content were determined in 1-butanol-exposed biofilms. Using scanning electron microscopy three different cell morphotypes were observed in response to 1-butanol. Transcriptome and proteome analyses were performed comparing the response of planktonic and biofilm cells in absence and presence of 1-butanol. In response to 1% (v/v) 1-butanol transcript levels of genes encoding motility and cell envelope structures as well as membrane proteins were reduced. Cell division and/or vesicle formation was upregulated. Furthermore, changes of immune and defence systems, as well as metabolism and general stress response were observed. Our findings show that the extreme lifestyle of S. acidocaldarius coincided with a high tolerance to organic solvents. This study provides first insights into biofilm formation and membrane/cell stress caused by organic solvents in S. acidocaldarius. Importance Archaea are unique in terms of metabolic and cellular processes as well as the adaptation to extreme environments. In the past few years, the development of genetic systems and biochemical, genetic and poly-omics studies have provided deep insights into the physiology of some archaeal model organisms. In this study, we used S. acidocaldarius adapted to two extremes, low pH and high temperature, to study its tolerance and robustness as well as its global cellular response towards organic solvents exemplified by 1-butanol. We were able to identify biofilm formation as primary cellular response to 1-butanol. Furthermore, the triggered cell/membrane stress led to significant changes in culture heterogeneity accompanied by changes in central cellular processes such as cell division and cellular defense systems, thus suggesting a global response for the protection at population level.

DNA Polymerase B1 Binding Protein 1 Is Important for DNA Repair by Holoenzyme PolB1 in the Extremely Thermophilic Crenarchaeon Sulfolobus acidocaldarius

DNA polymerase B1 (PolB1) is a member of the B-family DNA polymerase family and is a replicative DNA polymerase in Crenarchaea. PolB1 is responsible for the DNA replication of both the leading and lagging strands in the thermophilic crenarchaeon Sulfolobus acidocaldarius. Recently, two subunits, PolB1-binding protein (PBP)1 and PBP2, were identified in Saccharolobus solfataricus. Previous in vitro studies suggested that PBP1 and PBP2 influence the core activity of apoenzyme PolB1 (apo-PolB1). PBP1 contains a C-terminal acidic tail and modulates the strand-displacement synthesis activity of PolB1 during the synthesis of Okazaki fragments. PBP2 modestly enhances the DNA polymerase activity of apo-PolB1. These subunits are present in Sulfolobales, Acidilobales, and Desulfurococcales, which belong to Crenarchaea. However, it has not been determined whether these subunits are essential for the activity of apo-PolB1. In this study, we constructed a pbp1 deletion strain in S. acidocaldarius and characterized its phenotypes. However, a pbp2 deletion strain was not obtained, indicating that PBP2 is essential for replication by holoenzyme PolB1. A pbp1 deletion strain was sensitive to various types of DNA damage and exhibited an increased mutation rate, suggesting that PBP1 contribute to the repair or tolerance of DNA damage by holoenzyme PolB1. The results of our study suggest that PBP1 is important for DNA repair by holoenzyme PolB1 in S. acidocaldarius.