Lysine acetylation is a reversible and highly regulated post-translational modification that plays a critical role in regulating many aspects of cellular processes, both in bacteria and in eukaryotes. However, this modification has not been systematically studied in archaea. Herein, we report the lysine acetylome of a model haloarchaeon, Haloferax mediterranei. Using immunoaffinity enrichment and LC–MS/MS analysis, we identified 1017 acetylation sites in 643 proteins, accounting for 17.3% of the total proteins in this haloarchaeon. Bioinformatics analysis indicated that lysine acetylation mainly distributes in cytoplasm (94%) and participates in protein biosynthesis and carbon metabolism. Specifically, the acetylation of key enzymes in PHBV biosynthesis further suggested that acetylation plays a key role in the energy and carbon storage. In addition, a survey of the acetylome revealed a universal rule in acetylated motifs: a positively charged residue (K, R, or H) located downstream of acetylated lysine at the positions +1, +2, or +3. Interestingly, we identified acetylation in several replication initiation proteins Cdc6; mutation on the acetylated site of Cdc6A destroyed the Autonomous Replication Sequence (ARS) activity of its adjacent origin oriC1. Our study indicates that lysine acetylation is an abundant modification in H. mediterranei, and plays key roles in the processes of replication, protein biosynthesis, central metabolism, and carbon storage. This acetylome of H. mediterranei provides opportunities to explore the physiological role of acetylation in halophilic archaea.Keywords: acetylated lysine motif; acetylome; Cdc6; Haloferax mediterranei; interaction network; lysine acetylation; PHBV;

Many haloarchaea are known as polyhydroxyalkanoates (PHAs) producers, but a global and integrated view of the PHA biosynthesis is still lacking in this group of archaea. In this study, a combined proteomic and transcriptomic approach was employed in Haloarcula hispanica, a model haloarchaeon that accumulates poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) under nutrient-limiting conditions with excess carbon source. First, a comprehensive proteome reference map was established for H. hispanica. A total of 936 spots representing 839 unique proteins (21.7% of the predicted proteome) were identified by MALDI-TOF/TOF PMF and MS/MS. The map was further utilized to reconstruct central metabolic pathways to facilitate functional genomic analysis in H. hispanica. The results from the proteomic and transcriptomic analysis indicated that active PHA production coordinated with the TCA cycle to maintain balanced growth in wild-type H. hispanica, which was grown in nutrient-limited medium (PHA-accumulating conditions) versus nutrient-rich medium (non-PHA-accumulating conditions). Under nutrient-limiting conditions with excess carbon source, the PHA biosynthetic genes including phaEC, phaB, and phaP were upregulated at the transcriptional level, whereas the TCA cycle and respiratory chain were downregulated. Thus, acetyl-CoA could be fed into the PHA biosynthetic pathway, leading to the accumulation of PHA granules in the cell. Simultaneously, the large amount of NADPH required during PHA accumulation was likely supplied by the C3 (pyruvate) and C4 (malate) pathway coupled with the urea cycle. When PHA biosynthesis was blocked, that is, in the PHA synthase mutant (ΔphaEC) versus wild type grown in nutrient-limited medium, the mutant might direct additional carbon and energy to the TCA cycle, but without obvious contribution to biomass accumulation. The combined approaches of proteomic and transcriptomic analysis were highly complementary, extending the physiological understanding of PHA biosynthesis and its regulation. This is the first integrated proteome and transcriptome investigation of PHA biosynthesis and regulation in haloarchaea. It has provided basic information for future systemic engineering of haloarchaea to meet industrial needs.Keywords: 2-DE; haloarchaea; polyhydroxyalkanoates; urea cycle;

We previously reported that the tailor-made random poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (R-PHBHV) and higher-order PHBHV (O-PHBHV) produced by haloarchaea possessed unique material properties to meet biomedical application-specific requirements. Here, we further investigated the biocompatibility and biodegradation of these novel materials. Cell biocompatibility of solution-cast films, assessed using rat fibroblast and osteoblast cells, revealed that R-PHBHV and O-PHBHV exhibited better support for cell attachment and proliferation compared with the bacteria-produced poly-3-hydroxybutyrate (PHB) and PHBHV or polylactic acid (PLA). In vitro and in vivo biodegradation of these materials were evaluated in lipase-containing phosphate buffered solution (LPBS) at pH 7.4 and by implantation in the rabbit dorsal subcutis, respectively. As expected, the R-PHBHV and O-PHBHV films degraded much faster in vivo than those observed in vitro, as demonstrated by obvious weight loss, heavy surface erosion, and fast molecular weight drop under implantation condition. These films showed diverse in vivo degradation rates. Among them, the O-PHBHV-1 film degraded fastest and even faster than PLA. Generally, the tissue response was mild for R-PHBHV and O-PHBHV compared with the controls during the implantation period. Taken together, these data revealed that R-PHBHV and O-PHBHV copolyesters had a wild range of biodegradation profiles and excellent biocompatibility. Thus, haloarchaea-produced PHBHV materials would have great potential for use in different biomedical applications.

Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600–800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.

Bioremediation in hypersaline environments is particularly challenging since the microbes that tolerate such harsh environments and degrade pollutants are quite scarce. Haloarchaea, however, due to their inherent ability to grow at high salt concentrations, hold great promise for remediating the contaminated hypersaline sites. This study aimed to isolate and characterize novel haloarchaeal strains with potentials in hydrocarbon degradation. A haloarchaeal strain IM1011 was isolated from Changlu Tanggu saltern near Da Gang Oilfield in Tianjin (China) by enrichment culture in hypersaline medium containing hexadecane. It could degrade 57 ± 5.2% hexadecane (5 g/L) in the presence of 3.6 M NaCl at 37 °C within 24 days. To get further insights into the mechanisms of petroleum hydrocarbon degradation in haloarchaea, complete genome (3,778,989 bp) of IM1011 was sequenced. Phylogenetic analysis of 16S rRNA gene, RNA polymerase beta-subunit (rpoB’) gene and of the complete genome suggested IM1011 to be a new species in Halorientalis genus, and the name Halorientalis hydrocarbonoclasticus sp. nov., is proposed. Notably, with insights from the IM1011 genome sequence, the involvement of diverse alkane hydroxylase enzymes and an intact β-oxidation pathway in hexadecane biodegradation was predicted. This is the first hexadecane-degrading strain from Halorientalis genus, of which the genome sequence information would be helpful for further dissecting the hydrocarbon degradation by haloarchaea and for their application in bioremediation of oil-polluted hypersaline environments.

We report the biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) random copolymers (R-PHBV) or higher-order copolymers (O-PHBV) in Haloferax mediterranei, with adjustable 3-hydroxyvalerate (3HV) incorporation by cofeeding valerate with glucose. Their microchemical structure, molecular weight and its distribution, and thermal and mechanical properties were characterized by NMR, GPC, DSC, TGA, and universal testing machine, respectively. 13C NMR studies showed that O-PHBV copolymers consisted of short segments of PHB and PHV covalently linked together with random PHBV segments. Consistently, two Tg were observed in the DSC curves of O-PHBV. The “blocky” feature of O-PHBV enhanced crystallinity percentages and improved Young’s modulus. Notably, the film of one O-PHBV copolymer, O-PHBV-1, showed unique foveolar cluster-like surface morphology with high hydrophobicity and roughness, as characterized using static contact angle and SEM and AFM analyses. It also exhibited increased platelet adhesion and accelerated blood clotting. The excellent hemostatic properties endow this copolymer with great potential in wound healing.

Halophilic archaea (haloarchaea) inhabit hypersaline environments, tolerating extreme salinity, low oxygen and nutrient availability, and in some cases, high pH (soda lakes) and irradiation (saltern ponds). Membrane-associated proteins of haloarchaea, such as surface layer (S-layer) proteins, transporters, retinal proteins, and internal organellar membrane proteins including intracellular gas vesicle proteins and those associated with polyhydroxyalkanoate (PHA) granules, contribute greatly to their environmental adaptations. This review focuses on these haloarchaeal cellular and organellar membrane-associated proteins, and provides insight into their physiological significance and biotechnological potential.