The prokaryotic community composition, species diversity and the distribution patterns at various taxonomic levels in a salt lake (Chaka salt lake), solar salterns (Taipei saltworks and Dongfang saltworks), and salt mines (Yuanyongjing salt mine, Xiangyan salt mine, and Dinyuan salt mine) were investigated using clone library or Illumina MiSeq sequencing. The clone library approach revealed that the salt lake harbors low species diversity (H’ = 0.98) as compared to the solar saltern (H’ = 4.36) and salt mine (H’ = 3.57). The dominant group in the salt lake is a species from the genus Haloparvum which constitutes about 85% of the total sequences analyzed. The species diversities in salt salterns and salt mines are richer than in the salt lake, and the dominant group is less significant in terms of total percentage. High-throughput sequencing showed that geographical isolation greatly impacted on the microbial community (phyla level) and species diversity (operational taxonomic units (OTUs) level) of salt mines. Species of the genus Natronomonas are found in all three types of environments investigated. In addition, the microbial community and species diversity of different stratums of the salt mine are very similar. Furthermore, species of the genus Halorubrum flourish in the newest stratum of salt mine and have become the dominant group. This study provides some new knowledge on the species diversity and prokaryotic community composition of three typical hypersaline environments.

Arbuscular mycorrhizal fungi (AMF) could establish symbiosis with plant roots, which enhances plant resistance to various stresses, including drought stress and salt stress. Besides AMF, chemical stimulants such as trehalose (Tre) can also play an important role in helping plants alleviate damage of adversity. However, the mechanism of the effect of AMF combined with chemicals on plant stress resistance is unclear. The objective of this study was to explore the synergistic effects of Claroideoglomus etunicatum AMF and exogenous Tre on the antioxidant system, osmoregulation, and resistance-protective substance in plants in response to salt stress. Tomato seedlings were inoculated with Claroideoglomus etunicatum and combined with exogenous Tre in a greenhouse aseptic soil cultivation experiment. We measured the arbuscular mycorrhizal symbiont development, organic matter content, and antioxidant enzyme activity in tomato seedlings. Both AMF and Tre improved the synthesis of chlorophyll content in tomato seedlings; regulated the osmotic substance including soluble sugars, soluble protein, and proline of plants; and increased the activity of superoxide dismutase, peroxidase, and catalase. The combination of AMF and Tre also reduced the accumulation of malondialdehyde and alleviated the damage of harmful substances to plant cells in tomato seedlings. We studied the effects of AMF combined with extraneous Tre on salt tolerance in tomato seedlings, and the results showed that the synergistic treatment of AMF and Tre was more efficient than the effects of AMF inoculation or Tre spraying separately by regulating host substance synthesis, osmosis, and antioxidant enzymes. Our results indicated that the synergistic effects of AMF and Tre increased the plant adaptability against salt damage by enhancing cell osmotic protection and cell antioxidant capacity. IMPORTANCE: AMF improve the plant adaptability to salt resistance by increasing mineral absorption and reducing the damage of saline soil. Trehalose plays an important role in plant response to salt damage by regulating osmotic pressure. Together, the use of AMF and trehalose in tomato seedlings proved efficient in regulating host substance synthesis, osmosis, and antioxidant enzymes. These synergistic effects significantly improved seedling adaptability to salt stress by enhancing cell osmotic protection and cell antioxidant capacity, ultimately reducing losses to crops grown on land where salinization has occurred.

Halocins are antimicrobial peptides or proteins that are produced by halophilic archaea. Although their function in inhibiting the growth of closely related haloarchaeal strains is well known, other physiological functions of halocins have also been proposed in recent years. To unveil the possible function and mechanism of halocins in DNA uptake, the halocin H4 producing strain Haloferax mediterranei DF50-ΔEPS (incapable of EPS production) was used in this study. We found that deletion of the halH4 resulted in the strain DF50-ΔEPSΔhalH4 which exhibited loss of natural DNA uptake ability. Moreover, supernatants of the halocin producing strain were capable of inducing the ability to uptake DNA. Obviously, halocin is likely responsible for inducing DNA uptake. Cell surface ultrastructures of these strains are varied from strains DF50-ΔEPS to DF50-ΔEPSΔhalH4. The cell surface of strain DF50-ΔEPS is rough due to numerous pinholes, while that of the strain DF50-ΔEPSΔhalH4 is smooth without visible pinholes. The morphology of the halH4 complemented strain, DF50-ΔEPSΔhalH4::H4, shows an intermediate phenotype between strains DF50-ΔEPS and DF50-ΔEPSΔhalH4. We speculate that halocin H4 may accelerate DNA uptake by perforating the cell surface ultrastructure. The halocin H4 may represent a novel inducer or activator of DNA uptake in Hfx. mediterranei. IMPORTANCE Halocin H4 (HalH4) secreted by Haloferax mediterranei has always been taken as a typical antimicrobial protein. In this work, we found that deletion of the halocin H4-encoding gene, halH4, blocks the DNA uptake in Hfx. mediterranei by changing the cell surface ultrastructure. It is the first study on the function of halocins in natural environments, substantially contributing to new knowledge in this domain.

Halolysins, which are subtilisin-like serine proteases of haloarchaea, are usually secreted into the extracellular matrix via the twin-arginine translocation pathway. A small number of activated molecules can greatly affect cell growth owing to their proteolytic activity. It is, however, unclear as to whether this proteolysis-based growth inhibition by halolysins conveys antagonistic or defensive effects against other resident abd potentially competitive microorganisms. Here, we report that halolysin R4 (HlyR4), encoded by the hlyR4 gene, is the key enzyme in the initial steps of extracellular protein utilization in Haloferax mediterranei. HlyR4 shows significant antagonistic activity against other haloarchaeal strains. Deletion of hlyR4 completely halts the inhibition activity of Hfx. mediterranei towards other haloarchaea, while correspondingly, complementation of hlyR4 almost completely restores the inhibition activity. Furthermore, Hfx. mediterranei strains containing hlyR4 showed a certain amount of resistance to halocins and halolysins in milieu, and this function of hlyR4 is reproducible in Haloarcula hispanica. The versatility of HlyR4 enables its host to outcompete other haloarchaea living in the same hypersaline environment. Intriguingly, unlike the growth phase-dependent halolysins SptA and Nep, it is likely that HlyR4 may be secreted independent of growth phase. This study provides a new peptide antibiotics candidate in haloarchaea, as well as new insight towards a better understanding of the ecological roles of halolysins. Importance: This study shows that halolysin R4 from Haloferax mediterranei provides its host antagonistic and defensive activities against other haloarchaea, which expands our knowledge on the traditional function of haloarchaeal extracellular proteases. Haloarchaeal extracellular serine proteases have been previously discussed as growth-phase-dependent proteins, whereas our study reports constitutive expression of halolysin R4. This work also clearly reveals a hidden diversity of extracellular proteases from haloarchaea. Studies on multifunctional halolysins reveal that they play an important ecological role in shaping microbial community composition and provide a new perspective towards understanding the intricate interactions between haloarchaeal cells in hypersaline environments. HlyR4 can lyse competing cells living in the same environment, and the cell debris may probably be utilized as nutrients, which may constitute an important part of nutrient cycling in extremely hypersaline environments.

Halophilic microorganisms abound in numerous hypersaline environments, such as salt lakes, salt mines, solar salterns, and salted seafood. In the northwest of Dingbian county (Shaanxi province, China), there exists a belt of hypersaline habitats extending from the west to the north consisting of saline-alkali soil and salt lakes. Theoretically, such a hypersaline environment has a high probability of containing abundant halophilic archaea communities. Nevertheless, there is nearly no systematic research on halophilic archaea in this area. Here, we employed a combination of culture-dependent and culture-independent methods to analyze the collected samples. The high-throughput sequencing results of the archaeal 16S rRNA gene indicated that the richness of halophilic archaea in saline-alkali soils was significantly higher than that in salt lakes. In saline-alkali soils, the Natronomonas genus of archaea was more predominant compared to other genera, while in salt lakes, the Halonotius, Halorubrum, and Haloarcula genera of archaea had relatively higher abundances. However, the dominant families of halophilic archaea in both environments were mainly Haloferacaceae (30.96-72%), Halomicrobiaceae (17-53.19%) and Nanosalinaceae (1-19.08%). Based on the outcomes of pure culture experiments, a total of 26 genera and 98 strains were identified. Among the identified halophilic microorganisms, the predominant species were Halorubrum and Fodinibius, accounting for 33.67% and 13.27%, respectively. The remainder were mostly low-abundance groups within the community, and 22 potential novel taxa were discovered. Additionally, metagenomic technology was employed in our research. The analysis results demonstrated that the microorganisms in this area possess metabolic pathways capable of degrading various pollutants such as atrazine, methane, and dioxins, suggesting that some microorganisms in this area play a positive role in environmental remediation. This study roughly reveals the diversity composition and dominant species of halophilic archaea in these hypersaline environments and provides a scientific basis for the possible ecological functions of microorganisms in this area during long-term survival. It also offers scientific evidence for the development and utilization of halophilic microbial resources and ecological protection.

A halophilic archaeon named strain LR21^T was isolated from a salt mine in Yunnan Province, PR China. Cells were spherical, Gram-stain-negative and motile. Strain LR21^T grew at 20–50 °C (optimum, 42 °C), with 8–30 % (w/v) NaCl (optimum, 23 %) and at pH 5.5–9.5 (optimum, pH 7.5–8.5). Mg²⁺ was not required for growth. The major polar lipid profile comprised phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. Strain LR21^T had two dissimilar 16S rRNA genes (rrnA and rrnB) and they were closely related to Halomicroarcula limicola YGHS32^T, Hma. pellucida BNERC31^T and Hma. salina YGHS18^T with sequence similarities of 95.3–99.0, 93.0–96.2 and 93.2–95.9 %, respectively, and much lower values to other members. The rpoB′ gene sequence similarities between strain LR21^T and Hma. limicola YGHS32^T, Hma. pellucida BNERC31^T and Hma. salina YGHS18^T were 95.2, 91.2 and 91.2 % respectively. The values of average nucleotide identity (ANI) and average amino-acid identity (AAI) between strain LR21^T and Hma. limicola YGHS32^T, were 89.0 and 90.1 %, respectively. DNA relatedness between strains LR21^T and Hma. limicola YGHS32^T determined by in silico DNA–DNA hybridization was 36.8 %. Values of ANI and AAI between strain LR21^T and other members in the genus Halomicroarcula were far below 95 % and the DNA–DNA relatedness values between strain LR21^T and its close relatives were much lower than 70 %, which is far below the boundary for delineation of a new species prokaryote. The DNA G+C content of strain LR21^T was 62.0 mol% (genome). The results suggested that strain LR21^T represents a novel species of the genus Halomicroarcula , for which the name Halomicroarcula amylolytica sp. nov. is proposed. The type strain is LR21^T (=CGMCC 1.13611^T=NBRC 113588^T).