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.

Adsorption, Gas-Sensing, and Optical Properties of Molecules on a Diazine Monolayer: A First-Principles Study.

Using first-principles calculations, the structural, electronic, and optical properties of CO2, CO, N2O, CH4, H2, N2, O2, NH3, acetone, and ethanol molecules adsorbed on a diazine monolayer were studied to develop the application potential of the diazine monolayer as a room-temperature gas sensor for detecting acetone, ethanol, and NH3. We found that these molecules are all physically adsorbed on the diazine monolayer with weak adsorption strength and charge transfer between the molecules and the monolayer, but the physisorption of only NH3, acetone, and ethanol remarkably modified the electronic properties of the diazine monolayer, especially for the obvious change in electric conductivity, showing that the diazine monolayer is highly sensitive to acetone, NH3, and ethanol. Further, the adsorption of NH3, acetone, and ethanol molecules remarkably modifies, in varying degrees, the optical properties of the diazine monolayer, such as work function, absorption coefficient, and the reflectivity, whereas adsorption of other molecules has infinitesimal influence. The different adsorption behaviors and influences of the electronic and optical properties of molecules on the monolayer show that the diazine monolayer has high selectivity to NH3, acetone, and ethanol. The recovery time of NH3, acetone, and ethanol molecules is, respectively, 1.2 µs, 7.7 µs, and 0.11 ms at 300 K. Thus, the diazine monolayer has a high application potential as a room-temperature acetone, ethanol, and NH3 sensor with high performance (high selectivity and sensitivity, and rapid recovery time).