A TrmBL2-like transcription factor mediates the growth phase-dependent expression of halolysin SptA in a concentration-dependent manner in Natrinema gari J7-2.

To cope with a high-salinity environment, haloarchaea generally employ the twin-arginine translocation (Tat) pathway to transport secretory proteins across the cytoplasm membrane in a folded state, including Tat-dependent extracellular subtilases (halolysins) capable of autocatalytic activation. Some halolysins, such as SptA of Natrinema gari J7-2, are produced at late-log phase to prevent premature enzyme activation and proteolytic damage of cellular proteins in haloarchaea; however, the regulation mechanism for growth phase-dependent expression of halolysins remains largely unknown. In this study, a DNA-protein pull-down assay was performed to identify the proteins binding to the 5'-flanking sequence of sptA encoding halolysin SptA in strain J7-2, revealing a TrmBL2-like transcription factor (NgTrmBL2). The ΔtrmBL2 mutant of strain J7-2 showed a sharp decrease in the production of SptA, suggesting that NgTrmBL2 positively regulates sptA expression. The purified recombinant NgTrmBL2 mainly existed as a dimer although monomeric and higher-order oligomeric forms were detected by native-PAGE analysis. The results of electrophoretic mobility shift assays (EMSAs) showed that NgTrmBL2 binds to the 5'-flanking sequence of sptA in a non-specific and concentration-dependent manner and exhibits an increased DNA-binding affinity with the increase in KCl concentration. Moreover, we found that a distal cis-regulatory element embedded in the neighboring upstream gene negatively regulates trmBL2 expression and thus participates in the growth phase-dependent biosynthesis of halolysin SptA. IMPORTANCE: Extracellular proteases play important roles in nutrient metabolism, processing of functional proteins, and antagonism of haloarchaea, but no transcription factor involved in regulating the expression of haloaechaeal extracellular protease has been reported yet. Here we report that a TrmBL2-like transcription factor (NgTrmBL2) mediates the growth phase-dependent expression of an extracellular protease, halolysin SptA, of haloarchaeon Natrinema gari J7-2. In contrast to its hyperthermophilic archaeal homologs, which are generally considered to be global transcription repressors, NgTrmBL2 functions as a positive regulator for sptA expression. This study provides new clues about the transcriptional regulation mechanism of extracellular protease in haloarchaea and the functional diversity of archaeal TrmBL2.

Hly176B, a low-salt tolerant halolysin from the haloarchaeon Haloarchaeobius sp. FL176.

Extracellular proteases of haloarchaea can adapt to high concentrations of NaCl and can find useful applications in industrial or biotechnology processes where hypersaline conditions are desired. The diversity of extracellular proteases produced by haloarchaea is largely unknown though the genomes of many species have been sequenced and are publicly available. In this study, a gene encoding the extracellular protease Hly176B from the haloarchaeon Haloarchaeobius sp. FL176 was cloned and expressed in Escherichia coli. A related gene homolog to hly176B, hly176A, from the same strain was also expressed in E.coli, but did not show any proteinase activity after the same renaturation process. Therefore, we focus on the enzymatic properties of the Hly176B. The catalytic triad Asp-His-Ser was confirmed via site-directed mutagenesis, indicating that Hly176B belongs to the class of serine proteases (halolysin). Unlike previously reported extracellular proteases from haloarchaea, the Hly176B remained active for a relatively long time in an almost salt-free solution. In addition, the Hly176B displayed prominent tolerance to some metal ions, surfactants and organic solvents, and exerts its highest enzyme activity at 40 °C, pH 8.0 and 0.5 M NaCl. Therefore, this study enriches our knowledge of extracellular proteases and expands their applications for various industrial uses.

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.