The Helix-Loop-Helix Motif at the N Terminus of HalI Is Essential for Its Immunity Function against Halocin C8

Abstract

Halocin C8 (HalC8) is a stable microhalocin exhibiting strong antimicrobial activity against a wide range of haloarchaea. HalI, a 207-amino-acid peptide derived from the N terminus of the HalC8 preproprotein, is the immunity protein of HalC8. In this study, the molecular mechanism of the immunity function of HalI was investigated. Both pull-down and surface plasmon resonance assays revealed that HalI directly interacted with HalC8, and a mixture of purified HalI and HalC8 readily formed a heterocomplex, which was verified by gel filtration. Interestingly, HalC8 tended to form a self-associated complex, and one immunity protein likely sequestered multiple halocins. Significantly, the helix-loop-helix (HLH) motif containing a 4-amino-acid repeat (RELA) at the N terminus of HalI played a key role in its immunity activity. Disruption of the HLH motif or mutagenesis of the key residues of the RELA repeat resulted in loss of both the immunity function and the ability of HalI to bind to HalC8. These results demonstrated that HalI sequestered the activity of HalC8 through specific and direct binding.Halocins are proteinaceous antibiotics that are ribosomally synthesized by extremely halophilic archaea and excreted into the environment to kill or inhibit other haloarchaeal cells (15). Halocins were first discovered in 1982 (23) and later were found to be produced by most rod-shaped haloarchaea (12, 32). So far, several halocins have been purified and characterized (7,11,15,17,31), and three halocin genes coding for H4 (halH4), S8 (halS8), and C8 (proC8) have been cloned (2,19,29). Although the amino acid sequences of these halocins are quite diverse, the halocins share many features. For example, halocins are usually produced upon transition from the exponential to stationary phase and are processed from larger precursor proteins (2,19,29). In addition, halocins appear to be exported by a twin-arginine translocation (Tat) pathway (24, 29), as their precursors usually contain a Tat signal at their N termini (2,19,29). Accordingly, halocin has been considered a good model for examining gene expression regulation and protein processing and transportation in haloarchaea.HalC8 is an extremely stable microhalocin with a wide inhibition spectrum (7). The HalC8 gene encodes a 283-aminoacid preproprotein (ProC8), which is processed into two functional peptides, the C-terminal peptide antibiotic HalC8 (76 amino acids) and the N-terminal immunity protein HalI (29). As far as we know, HalI is the only halocin immunity protein that has been characterized. It is likely oriented toward the outside of the cellular membrane and hence inhibits HalC8 by sequestration (29). However, the molecular details of the interaction between HalI and HalC8 have not been elucidated yet. In this study, we demonstrated that HalI blocked HalC8 activity by direct interaction with HalC8. The motif and residues of HalI required for this functional interaction were also determined. MATERIALS AND METHODSStrains and growth conditions. Escherichia coli DH5␣ (Clo...

Antimicrobial peptides and proteins in the face of extremes: Lessons from archaeocins

“…3A, Table 1) would result in release of the C-terminal acidic, hydrophobic and cysteine-rich 76 amino acid HalC8, with a molecular mass of 7.4 kDa, and a N-terminal protein HalI [46,48], which was hypothesized to be an immunity protein aiming to protect the producer strain against its antimicrobial peptide, similar to what is observed for bacteriocins. Evidence that HalI protein indeed assumed this function was obtained both in vitro and in vivo: (i) in vitro the strain AS7092 culture supernatant mixed with recombinant HalI failed to inhibit the growth of sensitive strains; (ii) in vivo, overexpression of HalI in Haloarcula hispanica conferred resistance to HalC8 [46].…”

“…Expression of an antimicrobial peptide and its immunity protein from a common gene encoding a mixed precursor is a unique feature of HalC8, as in the case of bacteriocins, when immunity of the producer is afforded by a specific immunity protein, it is generally encoded by a distinct and independent gene of the operon [7,11]. HalI would exert its immunity function by direct binding of a N-terminal helix-loophelix (HLH) motif to multiple HalC8 monomers, thus forming large complexes and sequestering HalC8 [48]. This is reminiscent of the mechanism by which the immunity protein protects the producing Lactococcus lactis from the action of nisin, a bacteriocin secreted by lactic acid bacteria [49].…”

“…Cysteines are in bold type and underlined. The helix-loop-helix region essential to the immunity function of HalI is underlined (adapted from Refs [46,48,50]…”

Halophilic archaea and their potential to generate renewable fuels and chemicals

“…strain AS7092 (formerly Halobacterium sp. strain AS7092; Y. Li et al, 2003; Mei et al, 2008). Halocin C8 exhibits strong antimicrobial activity and inhibits the growth of at least 16 haloarchaeal strains (Y. Li et al, 2003).…”

“…Halocin C8 exhibits strong antimicrobial activity and inhibits the growth of at least 16 haloarchaeal strains (Y. Li et al, 2003). The halC8 gene encodes a 283‐amino acid preproprotein (ProC8) that is processed into two functional peptides: the C‐terminal peptide antibiotic HalC8 (76 amino acids) and the N‐terminal immunity protein HalI (Mei et al, 2008; C. Sun et al, 2005). Having a single gene encode the peptide antibiotic and its immunity protein differs from other systems like bacteriocins and their associated immunity protein that are encoded by separate genes (Siegers & Entian, 1995).…”