An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

Thermophilic archaeon orchestrates temporal expression of GDGT ring synthases in response to temperature and acidity stress.

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are unique archaeal membrane-spanning lipids with 0-8 cyclopentane rings on the biphytanyl chains. The cyclization pattern of GDGTs is affected by many environmental factors, such as temperature and pH, but the underlying molecular mechanism remains elusive. Here, we find that the expression regulation of GDGT ring synthase genes grsA and grsB in thermophilic archaeon Sulfolobus acidocaldarius is temperature- and pH-dependent. Moreover, the presence of functional GrsA protein, or more likely its products cyclic GDGTs rather than the accumulation of GrsA protein itself, is required to induce grsB expression, resulting in temporal regulation of grsA and grsB expression. Our findings establish a molecular model of GDGT cyclization regulated by environment factors in a thermophilic ecosystem, which could be also relevant to that in mesophilic marine archaea. Our study will help better understand the biological basis for GDGT-based paleoclimate proxies. Archaea inhabit a wide range of terrestrial and marine environments. In response to environment fluctuations, archaea modulate their unique membrane GDGTs lipid composition with different strategies, in particular GDGTs cyclization significantly alters membrane permeability. However, the regulation details of archaeal GDGTs cyclization in response to different environmental factor changes remain unknown. We demonstrated, for the first time, thermophilic archaea orchestrate the temporal expression of GDGT ring synthases, leading to delicate control of GDGTs cyclization to respond environmental temperature and acidity stress. Our study provides insight into the regulation of archaea membrane plasticity, and the survival strategy of archaea in fluctuating environments.

Duplication of leucyl-tRNA synthetase in an archaeal extremophile may play a role in adaptation to variable environmental conditions.

Aminoacyl-tRNA synthetases (aaRSs) are ancient enzymes that play a fundamental role in protein synthesis. They catalyze the esterification of specific amino acids to the 3'-end of their cognate tRNAs and therefore play a pivotal role in protein synthesis. Although previous studies suggest that aaRS-dependent errors in protein synthesis can be beneficial to some microbial species, evidence that reduced aaRS fidelity can be adaptive is limited. Using bioinformatics analyses, we identified two distinct leucyl-tRNA synthetase (LeuRS) genes within all genomes of the archaeal family Sulfolobaceae. Remarkably, one copy, designated LeuRS-I, had key amino acid substitutions within its editing domain that would be expected to disrupt hydrolytic editing of mischarged tRNALeu and to result in variation within the proteome of these extremophiles. We found that another copy, LeuRS-F, contains canonical active sites for aminoacylation and editing. Biochemical and genetic analyses of the paralogs within Sulfolobus islandicus supported the hypothesis that LeuRS-F, but not LeuRS-I, functions as an essential tRNA synthetase that accurately charges leucine to tRNALeu for protein translation. Although LeuRS-I was not essential, its expression clearly supported optimal S. islandicus growth. We conclude that LeuRS-I may have evolved to confer a selective advantage under the extreme and fluctuating environmental conditions characteristic of the volcanic hot springs in which these archaeal extremophiles reside.

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus.

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus-host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus-host interactions in the archaeal domain.

Association Between Body Mass Index (BMI) and Brachial-Ankle Pulse Wave Velocity (baPWV) in Males with Hypertension: A Community-Based Cross-Section Study in North China.

BACKGROUND The aim of this study was to investigate the association between body mass index (BMI) and brachial-ankle pulse wave velocity (baPWV) in hypertensive males. MATERIAL AND METHODS Altogether, 14 866 male hypertensive participants were included in the analysis. Participants were divided into 3 groups: low BMI group (BMI <24 kg/m²), moderate BMI group (24 kg/m² ≤BMI <28 kg/m²), and high BMI group (BMI ≥28 kg/m²). According to baPWV values, arteriosclerosis (AS) was set as 3 degrees: mild AS (baPWV ≥1400 cm/s), moderate AS (baPWV ≥1700 cm/s) and severe AS (baPWV ≥2000 cm/s). Multivariate logistic regression analysis was used to explore the effect of different BMI groups on different degrees of AS. The multivariate linear regression analysis was used to explore the relationship between BMI and baPWV. RESULTS Among low BMI, moderate BMI, and high BMI groups, the average baPWV values were 1824±401 cm/s, 1758±363 cm/s, and 1686±341 cm/s, respectively. Prevalence in the mild, moderate and high BMI groups were 91.0%, 87.8%, 81.5%, respectively for mild AS; 55.3%, 48.8%, and 40.0% respectively for moderate AS; and 25.9%, 20.2%, and 14.9% respectively for severe AS. Compared to the low BMI group, multivariate logistic regression analysis showed that odds ratio (OR) and 95% confidence intervals (95%CI) in the moderate BMI group and the high BMI were 0.71 (95%Cl, 0.62-0.80), 0.43 (95%Cl, 0.38-0.50) for mild AS; and similar trends were shown for moderate AS and severe AS. Based on age-stratification, a negative relationship remained for 35-55 years old participants for different degrees of AS among the moderate BMI group and the high BMI group. A negative relationship was detected between BMI and baPWV in total and different age-stages. CONCLUSIONS Among male hypertension participants in this study, there was a negative relationship between BMI and baPWV. High BMI was found to be a protective factor for AS especially in the age range of 35-55 years.

Microhomology-Mediated High-Throughput Gene Inactivation Strategy for the Hyperthermophilic Crenarchaeon Sulfolobus islandicus.

Sulfolobus islandicus is rapidly emerging as a model system for studying the biology and evolution within the TACK lineage of the archaeal domain. As the tree of life grows, identifying the cellular functions of genes within this lineage will have significant impacts on our understanding of the evolution of the last archaeal eukaryote common ancestor (LEACA) and the differentiation of archaea from eukaryotes during the evolution of the modern-day cell. To increase our understanding of this key archaeal organism, we report a novel high-throughput method for targeted gene inactivation in S. islandicus through one-step microhomology-directed homologous recombination (HR). We validated the efficacy of this approach by systematically deleting 21 individual toxin-antitoxin gene pairs and its application to delete chromosomal regions as large as 50 kb. Sequence analysis of 96 ArgD+ transformants showed that S. islandicus can effectively incorporate donor markers as short segments through HR in a continuous or discontinuous manner. We determined that the minimal size of homology allowing native argD marker replacement was as few as 10 bp, whereas argD marker replacement was frequently observed when increasing the size of homology to 30 to 50 bp. The microhomology-mediated gene inactivation system developed here will greatly facilitate isolation of S. islandicus gene deletion strains, making generation of a collection of genome-wide targeted mutants feasible and providing a tool to investigate homologous recombination in this organism.IMPORTANCE Current procedures for the construction of deletion mutants of S. islandicus are still tedious and time-consuming. We developed a novel procedure based on microhomology-mediated HR, allowing for rapid and efficient removal for genetic regions as large as 50 kb. Our work will greatly facilitate functional genomic studies in this promising model organism. Additionally, we developed a quantitative genetic assay to measure HR properties in S. islandicus, providing evidence that the ability to incorporate short, mismatched donor DNA into the genome through HR was probably a common trait for members of the Sulfolobus genus that are recombinogenic.

The apt/6-Methylpurine Counterselection System and Its Applications in Genetic Studies of the Hyperthermophilic Archaeon Sulfolobus islandicus.

UNLABELLED: Sulfolobus islandicus serves as a model for studying archaeal biology as well as linking novel biology to evolutionary ecology using functional population genomics. In the present study, we developed a new counterselectable genetic marker in S. islandicus to expand the genetic toolbox for this species. We show that resistance to the purine analog 6-methylpurine (6-MP) in S. islandicus M.16.4 is due to the inactivation of a putative adenine phosphoribosyltransferase encoded by M164_0158 (apt). The application of the apt gene as a novel counterselectable marker was first illustrated by constructing an unmarked α-amylase deletion mutant. Furthermore, the 6-MP counterselection feature was employed in a forward (loss-of-function) mutation assay to reveal the profile of spontaneous mutations in S. islandicus M.16.4 at the apt locus. Moreover, the general conservation of apt genes in the crenarchaea suggests that the same strategy can be broadly applied to other crenarchaeal model organisms. These results demonstrate that the apt locus represents a new tool for genetic manipulation and sequence analysis of the hyperthermophilic crenarchaeon S. islandicus IMPORTANCE: Currently, the pyrEF/5-fluoroorotic acid (5-FOA) counterselection system remains the sole counterselection marker in crenarchaeal genetics. Since most Sulfolobus mutants constructed by the research community were derived from genetic hosts lacking the pyrEF genes, the pyrEF/5-FOA system is no longer available for use in forward mutation assays. Demonstration of the apt/6-MP counterselection system for the Sulfolobus model renders it possible to again study the mutation profiles in mutants that have already been constructed by the use of strains with a pyrEF-deficient background. Furthermore, additional counterselectable markers will allow us to conduct more sophisticated genetic studies, i.e., investigate mechanisms of chromosomal DNA transfer and quantify recombination frequencies among S. islandicus strains.

Integration host factor is required for the induction of acid resistance in Escherichia coli.

Integration host factor (IHF) is a heterodimeric histone-like DNA-binding protein that participates in many cellular functions. Many systems and global regulators of acid resistance (AR) under strongly acidic conditions have been reported, but the role of IHF has not been examined. In the present study, we report that IHF is necessary for the induction of AR in Escherichia coli. At acidic pH, a ∆ihfA∆ifhB-mutant strain was found to have significantly depressed levels of transcription of the arginine decarboxylase gene (adiA) and of translation of the lysine/cadaverine antiporter gene (cadB), when compared with wild-type strain. Thus, IHF induces the arginine- and lysine-dependent AR. These results indicate that in E. coli, by combined transcriptional and translational controls of gene expression, IHF activates expression of a specific set of genes required for survival at extremely acidic pH.

Distinct metabolic responses to thermal stress between invasive freshwater turtle Trachemys scripta elegans and native freshwater turtles in China.

Different responses or tolerance to thermal stress between invasive and native species can affect the outcome of interactions between climate change and biological invasion. However, knowledge about the physiological mechanisms that modulate the interspecific differences in thermal tolerance is limited. The present study analyzes the metabolic responses to thermal stress by the globally invasive turtle, Trachemys scripta elegans, as compared with two co-occurring native turtle species in China, Pelodiscus sinensis and Mauremys reevesii. Changes in metabolite contents and the expression or enzyme activities of genes involved in energy sensing, glucose metabolism, lipid metabolism, and tricarboxylic acid (TCA) cycle after exposure to gradient temperatures were assessed in turtle juveniles. Invasive and native turtles showed distinct metabolic responses to thermal stress. T. scripta elegans showed greater transcriptional regulation of energy sensors than the native turtles. Enhanced anaerobic metabolism was needed by all three species under extreme heat conditions, but phosphoenolpyruvate carboxykinase and lactate dehydrogenase in the invader showed stronger upregulation or stable responses than the native species, which showed inhibition by high temperatures. These contrasts were pronounced in the muscles of the three species. Regulation of lipid metabolism was observed in both T. scripta elegans and P. sinensis but not in M. reevesii under thermal stress. Thermal stress did not inhibit the TCA cycle in turtles. Different metabolic responses to thermal stress may contribute to interspecific differences in thermal tolerance. Overall, our study further suggested the potential role of physiological differences in mediating interactions between climate change and biological invasion.

Computational analysis of genes with lethal knockout phenotype and prediction of essential genes in archaea.

The identification of microbial genes essential for survival as those with lethal knockout phenotype (LKP) is a common strategy for functional interrogation of genomes. However, interpretation of the LKP is complicated because a substantial fraction of the genes with this phenotype remains poorly functionally characterized. Furthermore, many genes can exhibit LKP not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes (conditionally essential genes). We analyzed the sets of LKP genes for two archaea, Methanococcus maripaludis and Sulfolobus islandicus, using a variety of computational approaches aiming to differentiate between essential and conditionally essential genes and to predict at least a general function for as many of the proteins encoded by these genes as possible. This analysis allowed us to predict the functions of several LKP genes including previously uncharacterized subunit of the GINS protein complex with an essential function in genome replication and of the KEOPS complex that is responsible for an essential tRNA modification as well as GRP protease implicated in protein quality control. Additionally, several novel antitoxins (conditionally essential genes) were predicted, and this prediction was experimentally validated by showing that the deletion of these genes together with the adjacent genes apparently encoding the cognate toxins caused no growth defect. We applied principal component analysis based on sequence and comparative genomic features showing that this approach can separate essential genes from conditionally essential ones and used it to predict essential genes in other archaeal genomes.IMPORTANCEOnly a relatively small fraction of the genes in any bacterium or archaeon is essential for survival as demonstrated by the lethal effect of their disruption. The identification of essential genes and their functions is crucial for understanding fundamental cell biology. However, many of the genes with a lethal knockout phenotype remain poorly functionally characterized, and furthermore, many genes can exhibit this phenotype not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes. We applied state-of-the-art computational methods to predict the functions of a number of uncharacterized genes with the lethal knockout phenotype in two archaeal species and developed a computational approach to predict genes involved in essential functions. These findings advance the current understanding of key functionalities of archaeal cells.

Association between milk consumption and kidney stones in U.S. adults: results from NHANES 2007-2018.

Background: Dietary strategies play a crucial role in the prevention of kidney stones. While milk is known for its rich nutritional content, its impact on kidney stone formation remains unclear. This study aimed to examine the relationship between milk consumption and the risk of kidney stones among U.S. adults. Methods: We included 24,620 participants aged 20 and older from the National Health and Nutrition Examination Survey (2007-2018). Milk consumption was defined based on each participant's response to the questionnaire item on "Past 30 day milk product consumption." Kidney stones history was self-reported by participants. The analysis employed weighted multivariate logistic regression models, followed by subgroup analyses for result validation, and explored the age-related dynamics of milk consumption's effect on kidney stone risk using a restricted cubic spline model. Results: Adjusted findings revealed that higher milk intake was associated with a decreased risk of kidney stones (odds ratio [OR] = 0.90, 95% confidence interval [CI] 0.85-0.96), notably among women (OR = 0.86, 95% CI 0.80-0.92) but not significantly in men (OR = 0.94, 95% CI 0.86-1.02). Smoothed curves across all ages showed that women consuming milk had a lower incidence of kidney stones than those who did not, particularly with regular consumption. Conclusion: This study uncovered that across all age groups, higher frequency of milk consumption in women is associated with a reduced risk of kidney stones. However, further prospective cohort studies are needed to confirm this finding.

Magnolol-driven microbiota modulation elicits changes in tryptophan metabolism resulting in reduced skatole formation in pigs.

Skatole of gut origin has garnered significant attention as a malodorous pollutant due to its escalating emissions, recalcitrance to biodegradation and harm to animal and human health. Magnolol is a health-promoting polyphenol with potential to considerably mitigate the skatole production in the intestines. To investigate the impact of magnolol and its underlying mechanism on the skatole formation, in vivo and in vitro experiments were conducted in pigs. Our results revealed that skatole concentrations in the cecum, colon, and faeces decreased by 58.24% (P = 0.088), 44.98% (P < 0.05) and 43.52% (P < 0.05), respectively, following magnolol supplementation. Magnolol supplementation significantly decreased the abundance of Lachnospira, Faecalibacterium, Paramuribaculum, Faecalimonas, Desulfovibrio, Bariatricus, and Mogibacterium within the colon (P < 0.05). Moreover, a strong positive correlation (P < 0.05) between skatole concentration and Desulfovibrio abundance was observed. Subsequent in silico studies showed that magnolol could dock well with indolepyruvate decarboxylase (IPDC) within Desulfovibrio. Further in vitro investigation unveiled that magnolol addition led to less indole-3-pyruvate diverted towards the oxidative skatole pathway by the potential docking of magnolol towards IPDC, thereby diminishing the conversion of substrate into skatole. Our findings offer novel targets and strategies for mitigating skatole emission from the source.

The multi-protective effect of IL-37-Smad3 against ox-LDL induced dysfunction of endothelial cells.

Atherosclerosis is a lipid-driven inflammatory arterial disease, with one crucial factor is oxidized low-density lipoprotein (ox-LDL), which can induce endothelial dysfunction through endoplasmic reticulum stress (ERS). Interleukin-37 (IL-37) exerts vascular protective functions. This study aims to investigates whether IL-37 can alleviate ERS and autophagy induced by ox-LDL, therely potentialy treating atherosclerosis. We found that ox-LDL enhances the wound healing rate in Rat Coronary Artery Endothelial Cells (RCAECs) and IL-37 reduce the ox-LDL-induced pro-osteogenic response, ERS, and autophagy by binding to Smad3. In RCAECs treated with ox-LDL and recombinant human IL-37, the wound healing rate was mitigated. The expression of osteogenic transcription factors and proteins involved in the ERS pathway was reduced in the group pretreated with IL-37 and ox-LDL. However, these responses were not alleviated when Smads silenced. Electron microscopy revealed that the IL-37/Smad3 complex could suppress endoplasmic reticulum autophagy under ox-LDL stimulation. Thus, IL-37 might treat atherosclerosis through its multi-protective effect by binding Smad3.

Sulfolobus islandicus: a model system for evolutionary genomics.

Sulfolobus islandicus has been developed as a model system for combining approaches of evolutionary and molecular biology in Archaea. We describe how the application of this interdisciplinary approach can lead to novel hypotheses derived from patterns of natural variation that can be tested in the laboratory when combined with a diversity of natural variants and versatile genetic markers. We review how this approach has highlighted the importance of recombination as an evolutionary parameter and provided insight into a molecular mechanism of recombination that may be unique in the archaeal domain. We review the development and improvement of the model system S. islandicus that will enable us to study the mechanism and genomic architecture of recombination guided by evolutionary genomic analysis of Nature's ongoing experiments in wild populations.

Genetic manipulation in Sulfolobus islandicus and functional analysis of DNA repair genes.

Recently, a novel gene-deletion method was developed for the crenarchaeal model Sulfolobus islandicus, which is a suitable tool for addressing gene essentiality in depth. Using this technique, we have investigated functions of putative DNA repair genes by constructing deletion mutants and studying their phenotype. We found that this archaeon may not encode a eukarya-type of NER (nucleotide excision repair) pathway because depleting each of the eukaryal NER homologues XPD, XPB and XPF did not impair the DNA repair capacity in their mutants. However, among seven homologous recombination proteins, including RadA, Hel308/Hjm, Rad50, Mre11, HerA, NurA and Hjc, only the Hjc nuclease is dispensable for cell viability. Sulfolobus encodes redundant BER (base excision repair) enzymes such as two uracil DNA glycosylases and two putative apurinic/apyrimidinic lyases, but inactivation of one of the redundant enzymes already impaired cell growth, highlighting their important roles in archaeal DNA repair. Systematically characterizing these mutants and generating mutants lacking two or more DNA repair genes will yield further insights into the genetic mechanisms of DNA repair in this model organism.

Augmenting the genetic toolbox for Sulfolobus islandicus with a stringent positive selectable marker for agmatine prototrophy.

Sulfolobus species have become the model organisms for studying the unique biology of the crenarchaeal division of the archaeal domain. In particular, Sulfolobus islandicus provides a powerful opportunity to explore natural variation via experimental functional genomics. To support these efforts, we further expanded genetic tools for S. islandicus by developing a stringent positive selection for agmatine prototrophs in strains in which the argD gene, encoding arginine decarboxylase, has been deleted. Strains with deletions in argD were shown to be auxotrophic for agmatine even in nutrient-rich medium, but growth could be restored by either supplementation of exogenous agmatine or reintroduction of a functional copy of the argD gene from S. solfataricus P2 into the ΔargD host. Using this stringent selection, a robust targeted gene knockout system was established via an improved next generation of the MID (marker insertion and unmarked target gene deletion) method. Application of this novel system was validated by targeted knockout of the upsEF genes involved in UV-inducible cell aggregation formation.

Integrated conjugative plasmid drives high frequency chromosomal gene transfer in Sulfolobus islandicus.

Gene transfer in crenarchaea has been observed within natural and experimental populations of Sulfolobus. However, the molecular factors that govern how gene transfer and recombination manifest themselves in these populations is still unknown. In this study, we examine a plasmid-mediated mechanism of gene transfer in S. islandicus that results in localized high frequency recombination within the chromosome. Through chromosomal marker exchange assays with defined donors and recipients, we find that while bidirectional exchange occurs among all cells, those possessing the integrated conjugative plasmid, pM164, mobilize a nearby locus at a significantly higher frequency when compared to a more distal marker. We establish that traG is essential for this phenotype and that high frequency recombination can be replicated in transconjugants after plasmid transfer. Mapping recombinants through genomic analysis, we establish the distribution of recombinant tracts with decreasing frequency at increasing distance from pM164. We suggest the bias in transfer is a result of an Hfr (high frequency recombination)-like conjugation mechanism in this strain. In addition, we find recombinants containing distal non-selected recombination events, potentially mediated by a different host-encoded marker exchange (ME) mechanism.

Isolation of archaeal viruses with lipid membrane from Tengchong acidic hot springs.

Archaeal viruses are one of the most mysterious parts of the virosphere because of their diverse morphologies and unique genome contents. The crenarchaeal viruses are commonly found in high temperature and acidic hot springs, and the number of identified crenarchaeal viruses is being rapidly increased in recent two decades. Over fifty viruses infecting the members of the order Sulfolobales have been identified, most of which are from hot springs distributed in the United States, Russia, Iceland, Japan, and Italy. To further expand the reservoir of viruses infecting strains of Sulfolobaceae, we investigated virus diversity through cultivation-dependent approaches in hot springs in Tengchong, Yunnan, China. Eight different virus-like particles were detected in enrichment cultures, among which five new archaeal viruses were isolated and characterized. We showed that these viruses can infect acidophilic hyperthermophiles belonging to three different genera of the family Sulfolobaceae, namely, Saccharolobus, Sulfolobus, and Metallosphaera. We also compared the lipid compositions of the viral and cellular membranes and found that the lipid composition of some viral envelopes was very different from that of the host membrane. Collectively, our results showed that the Tengchong hot springs harbor highly diverse viruses, providing excellent models for archaeal virus-host studies.

A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection.

Sulfolobus species have been developed as excellent model organisms to address fundamental questions of archaeal biology. Interesting patterns of natural variation among Sulfolobus islandicus strains have been identified through genome sequencing. Experimentally testing hypotheses about the biological causes and consequences of this natural variation requires genetic tools that apply to a diversity of strains. Previously, a genetic transformation system for S. islandicus was reported, in which overexpression of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene on the shuttle vector pSSR allowed the selection of transformants resistant to high concentrations of the thermostable antibiotic simvastatin. Here, we developed a novel gene knockout system based on simvastatin resistance. With this system, we created via homologous recombination an in-frame, markerless deletion of the intact S. islandicus M.16.4 pyrEF genes encoding orotidine-5'-monophosphate pyrophosphorylase (OPRTase) and orotidine-5'-monophosphate decarboxylase (OMPdecase), and a disruption of the lacS gene encoding ß-galactosidase. Phenotypic analyses of the mutants revealed that the pyrEF deletion mutant lost the ability to synthesize uracil, and the lacS deletion mutants exhibited a white colour after X-Gal staining, demonstrating that the ß-galactosidase function was inactivated. Our data demonstrate efficient tools to generate gene knockouts in a broad range of wild-type Sulfolobus strains.

Development of a simvastatin selection marker for a hyperthermophilic acidophile, Sulfolobus islandicus.

We report here a novel selectable marker for the hyperthermophilic crenarchaeon Sulfolobus islandicus. The marker cassette is composed of the sac7d promoter and the hmg gene coding for the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (P(sac7d)-hmg), which confers simvastatin resistance to this crenarchaeon. The basic plasmid vector pSSR was constructed by substituting the pyrEF gene of the expression vector pSeSD for P(sac7d)-hmg with which the Sulfolobus expression plasmids pSSRlacS, pSSRAherA, and pSSRNherA were constructed. Characterization of Sulfolobus transformants carrying pSSRlacS indicated that the plasmid was properly maintained under selection. High-level expression of the His(6)-tagged HerA helicase was obtained with the cells harboring pSSRAherA. The establishment of two efficient selectable markers (pyrEF and hmg) was subsequently exploited for genetic analysis. A herA merodiploid strain of S. islandicus was constructed using pyrEF marker and used as the host to obtain pSSRNherA transformant with simvastatin selection. While the gene knockout (ΔherA) cells generated from the herA merodiploid cells failed to form colonies in the presence of 5-fluoroorotic acid (5-FOA), the mutant cells could be rescued by expression of the gene from a plasmid (pSSRNherA), because their transformants formed colonies on a solid medium containing 5-FOA and simvastatin. This demonstrates that HerA is essential for cell viability of S. islandicus. To our knowledge, this is the first application of an antibiotic selectable marker in genetic study for a hyperthermophilic acidophile and in the crenarchaeal lineage.