Halocin H4 is activated through cleavage by halolysin HlyR4.

Halocins are antimicrobial peptides secreted by haloarchaea capable of inhibiting the growth of other haloarchaea or bacteria. Halocin H4 (HalH4) is secreted by the model halophilic archaeon Haloferax mediterranei ATCC 33500. Despite attempts to express halH4 heterologously in Escherichia coli and subsequent careful renaturation procedures commonly employed for haloarchaeal proteins, no active halocin was obtained. However, it was discovered that the antihaloarchaeal activity of this halocin could be activated through cleavage by halolysin R4 (HlyR4), a serine protease also secreted by Hfx. mediterranei ATCC 33500. Replacement of the cysteine at the number 115 amino acid with glycine and deletion of the internal trans-membrane region (15 aa) markedly abolished HalH4's antihaloarchaeal activity. Compared to the N-terminus, the C-terminal amino acid sequence was found to be more crucial for HalH4 to exert its antihaloarchaeal activity. Mass spectrometry analysis revealed that the biologically active antihaloarchaeal peptide produced after hydrolytic cleavage by HlyR4 was the C-terminus of HalH4, suggesting a potential mechanism of action involving pore formation within competitor species' cell membranes. Taken together, this study offers novel insights into the interplay between halocins and secreted proteases, as well as their contribution to antagonistic interaction within haloarchaea. IMPORTANCE: The antihaloarchaeal function of halocin H4 (HalH4) can be activated by extracellular proteases from haloarchaea, as demonstrated in this study. Notably, we report the first instance of halocin activation through proteolytic cleavage, highlighting its significance in the field. The C-terminus of HalH4 (CTH4) has been identified as the antihaloarchaeal peptide present in hydrolysates generated by HlyR4. The CTH4 exhibited inhibitory activity against a range of haloarchaeal species (Haloarchaeobius spp., Haloarcula spp., Haloferax spp., Halorubellus spp., and Halorubrum spp.), as well as selected bacterial species (Aliifodinibius spp. and Salicola spp.), indicating its broad-spectrum inhibitory potential across domains. The encoding gene of halocin HalH4, halH4, from the model halophilic archaeon Haloferax mediterranei ATCC 33500 can be expressed in Escherichia coli without codon optimization.

Genomic, Physiological, Biochemical, and Phenotypic Evidences Reveal a New Species, Halomicroarcula salaria sp. nov.

An extremely halophilic archaeon strain named FL173T was isolated from a salt mine (Anhui Province, China). Colonies on agar plate are orange-red, moist, and opaque. Cells are motile, Gram-stain-negative, polymorphic, and lyse in distilled water. Cells are able to grow at temperatures, NaCl concentrations, and pH ranging from 20 to 50 °C (optimum 42 °C), 2.6 to 5.1 M NaCl concentration (optimum 3.4 M), and 5.5 to 9.5 pH (optimum 7.0), respectively. Mg2+ is not necessary for growth. The major polar lipids of strain FL173T were phosphatidylglycerol (PG), phosphatidylglycerol phosphate methyl ester (PGP-Me), phosphatidylglycerol sulfonate (PGS), sulfonated mannosyl glycolipid (S-DGD-1). It has two copies of the 16S rRNA gene, which share the highest sequence similarity (93.04-99.02% sequence similarity) to the 16S rRNA genes of Halomicroarcula salinisoli F24AT, respectively. The rpoB' gene of strain FL173T showed the highest sequence similarity (93.76%) to that of H. salinisoli F24AT. The genome-based analysis showed that the average amino-acid identity (AAI), orthologous average nucleotide identity (ANI) and in silico DNA-DNA hybridization values between strains FL173T and H. salinisoli F24AT were 84.80%, 85.29%, and 29.70%, respectively, which are far below the threshold for the delineation of a prokaryotic new species. The DNA G+C content of strain FL173T is 64.9%. Genomic, physiological, biochemical, and phenotypic evidences showed that strain FL173T (CGMCC 1.18851=NBRC 114260) represents a new species of the genus Halomicroarcula, for which the name Halomicroarcula salaria sp. nov. is proposed.

Identification and biochemical characterization of a novel halolysin from Halorubellus sp. PRR65 with a relatively high temperature activity.

Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp154-His193-Ser348. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The Km, Vmax and Kcat for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg-1 and 1538.09 S-1, respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.

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.

How Disability Income Benefits Affect Employment for Persons with Disabilities in China: An Impairment-Based Work Disability Assessment Perspective.

Reconciling the potentially conflicting goals between income benefits and employment has become one of the key issues in international disability policy for working-age persons with disabilities. Inspired by the often-criticized experience of disability income benefits having an exclusory effect on employment of persons with disabilities in welfare states by two mechanisms-the disincentive effect of the generous benefits based on the work incapacity of persons with disabilities and the impairment-based work disability assessment for attaining these benefits-this paper aims to examine how disability income benefits affect employment for persons with disabilities in China. Using the life-story interviews method, this paper found that the disability income benefits for persons with disabilities based on their work incapability create "quasi-employment" perceptions among recipients with disabilities. The impairment-based work disability assessment for attaining these benefits excludes persons with disabilities from formal employment but does open them to more informal employment. Implications for policy are discussed.

Employment Legal Framework for Persons with Disabilities in China: Effectiveness and Reasons.

In order to promote the employment of persons with disabilities, two dominant legal approaches-anti-discrimination legislation based on the social model of disability and an employment quota scheme based on the medical model-are usually employed on a nation-state basis in disability policies. This article systematically examines the reasons why both the anti-discrimination and employment quota scheme legal frameworks have limited effectiveness in promoting employment of persons with disabilities in China. We found that the lack of a definition of disability, the lack of a definition of discrimination, and the absence of effective enforcement mechanisms are the reasons for poor outcomes of the anti-discrimination legal framework. For the employment quota scheme, conflicts between the mainstream labor market legal framework and the quota scheme legal framework have prompted employers to pay penalties rather than hire persons with disabilities. China should address these issues in the current legal system in the short term. Meanwhile, the CRPD should be more strongly emphasized in China. This article argues for the human rights model espoused by the CRPD, instead of the medical model, to develop a coherent and sustainable disability legal framework for promoting participation of persons with disabilities, rather than focusing on viewing them as recipients of care.

MMP-9-BDNF pathway is implicated in cognitive impairment of male individuals with methamphetamine addiction during early withdrawal.

Cognitive impairment is often concomitant with current and abstinent methamphetamine (METH) misuse. However, the mechanism underlying the pathogenesis of cognitive impairment induced by METH remains unclear. As evidence indicates that brain-derived neurotrophic factor (BDNF) is associated with METH addiction, the present study aimed to investigate whether BDNF and the proteins regulating the BDNF signaling pathway might be implicated in the cognitive impairment of the METH abusers during early withdrawal. A total of 171 male subjects were recruited, including 85 METH abstainers and 86 healthy controls. Cognitive function was evaluated with the Montreal Cognitive Assessment (MoCA) screening tool. The levels of serum proteins that regulate the BDNF signaling pathway were measured using enzyme-linked immunosorbent assay kits. 61.18% METH abstainers were determined to have cognitive impairment (MoCA＜26). The serum levels of mBDNF, proBDNF, and MMP-9, as well as the ratio of the mBDNF/proBDNF (M/P) were significantly decreased in the cognition-impaired METH abstainers than in the cognition-unimpaired METH abstainers. mBDNF, proBDNF, TrkB, MMP-9, MMP-9 activity, and M/P were significantly correlated with the MoCA score in the METH abstainers. The combination of mBDNF, TrkB, MMP-9, and MMP-9 activity demonstrated excellent diagnostic potential for cognitive impairment of METH abusers during early withdrawal (AUC = 0.978). The results provide the prospective evidence that the MMP-9-BDNF pathway may underlie the pathogenesis of cognitive impairment in METH abusers during early withdrawal.