Changyi Zhang

Members of the order Sulfolobales are known to thrive in acidic, high-temperature hot springs. Here, we report the sequencing and complete genome assembly of a hyperthermophilic archaeon Saccharolobus islandicus Y08.82.36, isolated from a hot spring in the Norris Geyser Basin, Yellowstone National Park, USA, in 2008.

Microbial communities are often studied by measuring gene expression (mRNA levels), but translating these data into functional insights is challenging because mRNA abundance does not always predict protein levels. Here, we present a strategy to bridge this gap by deriving gene-specific RNA-to-protein conversion factors that improve the prediction of protein abundance from transcriptomic data. Using paired mRNA–protein data sets from seven bacteria and one archaeon, we identified orthologous genes where mRNA levels poorly predicted protein abundance, yet each gene’s protein-to-RNA ratio was consistent across these diverse organisms. Applying the resulting conversion factors to mRNA levels dramatically improved protein abundance predictions, even when the conversion factors were obtained from distantly related species. Remarkably, conversion factors derived from bacteria also enhanced protein prediction in an archaeon, demonstrating the robustness of this approach. This cross-domain framework enables more accurate functional inference in microbiomes without requiring organism-specific proteomic data, offering a powerful new tool for microbial ecology, systems biology, and functional genomics. IMPORTANCE: Deciphering the biology of natural microbial communities is limited by the lack of functional data. While transcriptomics enables gene expression profiling, mRNA levels often fail to predict protein abundance, the primary indicator of microbial function. Prior studies addressed this by calculating RNA-to-protein (RTP) conversion factors using conserved protein-to-RNA (ptr) ratios across bacterial strains, but their cross-species and cross-domain utility remained unknown. We generated comprehensive transcriptomic and proteomic data sets from seven bacteria and one archaeon spanning diverse metabolisms and ecological niches. We identified orthologous genes with conserved ptr ratios, enabling the discovery of RTP conversion factors that significantly improved protein prediction from mRNA, even between distant species and domains. This reveals previously unrecognized conservation in ptr ratios across domains and eliminates the need for paired proteomic data in many cases. Our approach offers a broadly applicable framework to enhance functional prediction in microbiomes using only transcriptomic data.

Sulfolobus islandicus , an emerging crenarchaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has considerably limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs (crRNAs) targeting eight chromosomal integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. By leveraging the endogenous CRISPR-Cas systems, we integrated the reporter gene lacS into these sites and validated heterologous gene expression through a β -galactosidase reporter assay, which revealed significant positional effects on expression levels. As a proof of concept, we utilized these characterized sites to genetically manipulate lipid ether composition by overexpressing GDGT (glycerol dibiphytanyl glycerol tetraether) ring synthase B (GrsB) in S. islandicus , a key enzyme in GDGT biosynthesis. This study expands the genetic toolbox for S. islandicus and highlights a concept that could be widely applicable to other Sulfolobales , advancing their potential as robust platforms for archaeal synthetic biology and industrial biotechnology.

Sinorhizobium meliloti is noted for its exceptional capacity to produce unsaturated fatty acids (UFAs). Earlier studies have indicated that S. meliloti primarily employs the FabA-FabB pathway for UFA synthesis, however, the mechanisms remain elusive. This study was conducted to elucidate these mechanisms responsible for the significant UFA production in S. meliloti. The genes encoding enoyl-acyl carrier protein (ACP) reductase (ENR) were disrupted using the suicide plasmid pK18mobsacB, followed by the creation of single-crossover and double-crossover mutants. The ENR proteins were expressed in Escherichia coli BL21(DE3) strains and subsequently purified. Their enzymatic activities were assessed through gel electrophoresis and NADH oxidation assays. Additionally, the fatty acid composition was determined using gas chromatography-mass spectrometry (GC-MS) and thin-layer chromatography. Our findings demonstrate that the heterologous expression of fabI2 in a temperature-sensitive E. coli fabI mutant results in a significant enhancement of UFA production. Genetic analyses confirmed that fabI2 is an indispensable gene in the S. meliloti genome, as it cannot be disrupted. Interestingly, we observed that fabI2 could only be functionally replaced by the Enterococcus faecalis fabI gene and not by the homologous fabI1 from S. meliloti, E. coli fabI, or Pseudomonas aeruginosa fabV. Furthermore, we validated that the deletion of fabI1 in S. meliloti triggered an increase in UFA production compared to the wild-type strain Rm1021. In this study, we identified the ENR, encoded by the S. meliloti SMc00326 gene (fabI2), as playing a pivotal role in the biosynthesis of UFAs. Additionally, the FabI1 enzyme, encoded by SMc00005, was found to modulate the fatty acid composition within S. meliloti. Together, these discoveries establish a foundation for the development of a model that explains the significant contribution of FabI2 to the robust synthesis of UFAs in S. meliloti.

Only 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.

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.

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.