Expression, purification, and enzymatic characterization of an extracellular protease from Halococcus salifodinae.

Extracellular proteases from halophilic archaea displays increased enzymatic activities in hypersaline environment. In this study, an extracellular protease-coding gene, hly34, from the haloarchaeal strain Halococcus salifodinae PRR34, was obtained through homologous search. The protease activity produced by this strain at 20% NaCl, 42 °C, and pH 7.0 was 32.5 ± 0.5 (U·mL-1). The codon-optimized hly34 which is specific for Escherichia coli can be expressed in E. coli instead of native hly34. It exhibits proteolytic activity under a wide range of low- or high-salt concentrations, slightly acidic or alkaline conditions, and slightly higher temperatures. The Hly34 presented the highest proteolytic activity at 50 °C, pH 9.0, and 0-1 M NaCl. It was found that the Hly34 showed a higher enzyme activity under low-salt conditions. Hly34 has good stability at different NaCl concentrations (1-4 M) and pH (6.0-10.0), as well as good tolerance to some metal ions. However, at 60 °C, the stability is reduced. It has a good tolerance to some metal ions. The proteolytic activity was completely inhibited by phenylmethanesulfonyl fluoride, suggesting that the Hly34 is a serine protease. This study further deepens our understanding of haloarchaeal extracellular protease, most of which found in halophilic archaea are classified as serine proteases. These proteases exhibit a certain level of alkaline resistance and moderate heat resistance, and they may emerge with higher activity under low-salt conditions than high-salt conditions. The protease Hly34 is capable of degrading a number of proteins, including substrate proteins, such as azocasein, whey protein and casein. It has promising applications in industrial production.

Microbial community structure and shift pattern of industry brine after a long-term static storage in closed tank.

Brine from Dingyuan Salt Mine (Anhui, China), an athalassohaline hypersaline environment formed in the early tertiary Oligocene, is used to produce table salt for hundreds of millions of people. However, halophiles preserved in this niche during deposition are still unknown. Here, we employed cultivation and high-throughput sequencing strategies to uncover the microbial community and its shift after a long-term storage in the brine collected from Dingyuan Salt Mine. High-throughput sequencing showed (1) in the fresh brine (2021), Cyanobium_stocktickerPCC-6307 spp. (8.46%), Aeromonas spp. (6.91%) and Pseudomonas spp. (4.71%) are the dominant species in bacteria while Natronomonas spp. (18.89%), Halapricum spp. (13.73%), and Halomicrobium spp. (12.35%) in archaea; (2) after a 3-year-storage, Salinibacter spp. (30.01%) and Alcanivorax spp. (14.96%) surpassed Cyanobium_stocktickerPCC-6307 spp. (8.46%) becoming the dominant species in bacteria; Natronomonas spp. are still the dominant species, while Halorientalis spp. (14.80%) outnumbered Halapricum spp. becoming the dominant species in archaea; (3) Alcanivorax spp. and Halorientalis spp. two hydrocarbons degrading microorganisms were enriched in the brine containing hydrocarbons. Cultivation using hypersaline nutrient medium (20% NaCl) combined with high-throughput 16S rRNA gene sequencing showed that (1) the biomass significantly increased while the species diversity sharply declined after a 3-year-storage; (2) Halorubrum spp. scarcely detected from the environment total stocktickerDNA were flourishing after cultivation using AS-168 or NOM medium; (3) twelve possible new species were revealed based on almost full-length 16S rRNA gene sequence similarity search. This study generally uncovered the microbial community and the dominant halophiles in this inland athalassohaline salt mine, and provided a new insight on the shift pattern of dominant halophiles during a long-term storage, which illustrated the shaping of microorganisms in the unique environment, and the adaptation of microbe to the specific environment.

Genomic features of a new head-tail halovirus VOLN27B infecting a Halorubrum strain.

Relatively few viruses infecting haloarchaea (haloviruses) have been reported. In this study, the genome sequence of VOLN27B, a recently described archaeal tailed virus (arTV) with a myovirus morphotype was described, along with the sequence of its host, Halorubrum spp. LN27. Halovirus VOLN27B contains a linear, dsDNA genome of 76,891 bp which is predicted to encode 109 proteins and four tRNAs (tRNAThr, tRNAArg, tRNAGly and tRNAAsn). The DNA G + C content of VOLN27B genome is 56.1 mol%, nearly 10% lower than that of its host strain. A 315 bp LTR (long terminal repeat) was detected in the genome. The genome of its host strain LN27 was 3,301,211 bp (chromosome and 1 plasmid) with a DNA G + C content of 68.3 mol% and 3142 annotated protein coding genes. At least two hypothetical proviruses were detected in the genome. It lacked a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus. Sequence similarity and phylogenetic tree reconstructions placed it within the genus Halorubrum as a potential new species. VOLN27B exhibits a distinct difference in the frequency of codon usage against its host strain Halorubrum sp. LN27. The organization of VOLN27B genome shows remarkable synteny and amino acid sequence similarity to the genomes and predicted proteins of HF1-like haloviruses (genus Haloferacalesvirus) and a provirus in the genome of Halorubrum depositum Y78. VOLN27B and its host Halorubrum sp. LN27 comprise a new virus-host system from a hypersaline ecosystem and can be used to further understand the novel biology at extreme salt concentration.

Impact of long-term industrial contamination on the bacterial communities in urban river sediments.

BACKGROUND: The contamination of the aquatic environment of urban rivers with industrial wastewater has affected the abiotic conditions and biological activities of the trophic levels of the ecosystem, particularly sediments. However, most current research about microorganism in urban aquatic environments has focused on indicator bacteria related to feces and organic pollution. Meanwhile, they ignored the interactions among microorganisms. To deeply understand the impact of industrial contamination on microbial community, we study the bacterial community structure and diversity in river sediments under the influence of different types of industrial pollution by Illumina MiSeq high-throughput sequencing technology and conduct a more detailed analysis of microbial community structure through co-occurrence networks. RESULTS: The overall community composition and abundance of individual bacterial groups differed between samples. In addition, redundancy analysis indicated that the structure of the bacterial community in river sediments was influenced by a variety of environmental factors. TN, TP, TOC and metals (Cu, Zn and Cd) were the most important driving factors that determined the bacterial community in urban river sediments (P < 0.01). According to PICRUSt analysis, the bacterial communities in different locations had similar overall functional profiles. It is worth noting that the 15 functional genes related to xenobiotics biodegradation and metabolism were the most abundant in the same location. The non-random assembly patterns of bacterial composition in different types of industrially polluted sediments were determined by a co-occurrence network. Environmental conditions resulting from different industrial pollutants may play an important role in determining their co-occurrence patterns of these bacterial taxa. Among them, the bacterial taxa involved in carbon and nitrogen cycles in module I were relatively abundant, and the bacterial taxa in module II were involved in the repair of metal pollution. CONCLUSIONS: Our data indicate that long-term potential interactions between different types of industrial pollution and taxa collectively affect the structure of the bacterial community in urban river sediments.