*3.1.1.3 Halocin C8 (HalC8)*

Halocin C8 is produced by *Natrinema* sp. AS7092, a strain isolated from the large Chaidan Salt Lake, China (7.44 kDa, 76 amino acids) [28]. It is a unique polypeptide with an isoelectric point of 4.4 [33]. Its activity is retained after desalting, boiling and frozing [33]. Halocin C8 has a very broad spectrum of activity against several species and genera of *Halobacteriales* members including *Natronobacterium gregoryi*, *Nbt*. comb. nov and *Natronomonas pharaonis* [28]. The *halC8* gene encodes both halocin C8 and its immunity protein HalI.

**87**

*Halocins, Bacteriocin-Like Antimicrobials Produced by the Archaeal Domain: Occurrence…*

Halocin A4, also called also halocin U1, is produced by an uncharacterized haloarchaea strain isolated from a Tunisian saltern [34]. Its molecular weight is 7.435 Da, as determined by the spectrometric mass, and is both acidic (pH = 4.14) and hydrophobic (eluent at ~85% acetonitrile) [26]. Halocin A4 has been reported to inhibit the growth of crenarchaeal *Sulfolobus* sp. strains [26]. G*ene* encoding HalA4 is *located* on a 300 kpb megaplasmid, pHM300 (NC\_017943) [29].

Halocin H6 is produced by *Haloferax gibbonsii* Ma 2.39 species [27]. Its activity is resistant to trypsin. Stabilities of this peptide were studied and have shown that HalH6 can be desalted and it retained its activity after heat treatment up to 10 min at 100°C [27]. Halocin H6 is considered as a bactericidal substance which causes cell

Halocin Sech7a was excreted by the extremely halophilic haloarchaeon Sech7a, isolated from brine samples of Secovlje solar salterns crystallizers in Slovenia [36]. Sech7a is about 11 kDa. It is stable over a wide pH range and is heat labile at temperatures above 80°C. Its optimal activity was observed in the early exponential phase growth at 45°C. It loses activity under low salt conditions, but its activity can

Halocin SH10 is produced by *Natrinema* sp. BTSH10, a strain isolated from the Kanyakumari salt marsh, Tamil Nadu, India [37]. The optimal production of halocin SH10 is at 42°C, pH 8.0 and 3 M NaCl at the stationary phase. In this context, it was reported that the activity is lost under acidic conditions [37]. Production of SH10 is influenced by the carbon source composition of the medium — *Natrinema*

This class comprises halocins composed of proteins greater than 10 kDa in size. Currently, there are two characterized protein halocins, HalH1 and HalH4, in the

Halocin H4, produced by *Haloferax mediterranei* R4 (ATCC 33500) was isolated from a Spanish solar salt pond in Alicante. It is the first halocin that was studied [20]. The optimal activity was detected at the midpoint between exponential and stationary phases [20]. Halocin H4 is sensitive to proteases, high temperature and desalting. HalH4 has an antimicrobial activity against other haloarchaeons. It Interacts with the membrane of the target cells where it causes permeability changes that result in an ionic imbalance leading to cell lysis and death [21, 38]. The *halH4* gene, encoding halocin activity, is located on the pHM300 megaplasmid, a single

sp. BTSH10 could produce maximal halocin in the presence of beef [37].

lysis and the specific target of HalH6 is the Na+/H+ antiport [27, 35].

be restored after dialysis against initial saline conditions [36].

*DOI: http://dx.doi.org/10.5772/intechopen.94765*

*3.1.1.4 Halocin A4 (HalU1)*

*3.1.1.5 Halocin H6 (HalH6)*

*3.1.1.6 Halocin Sech7a*

*3.1.1.7 Halocin SH10*

*3.1.2 Protein halocins*

*3.1.2.1 Halocin H4*

range of 30 to 35 kDa [28].

polypeptide of 34.9 kDa.

*Halocins, Bacteriocin-Like Antimicrobials Produced by the Archaeal Domain: Occurrence… DOI: http://dx.doi.org/10.5772/intechopen.94765*

### *3.1.1.4 Halocin A4 (HalU1)*

*Extremophilic Microbes and Metabolites - Diversity, Bioprospecting and Biotechnological...*

of them have been characterized and purified.

Halocins, bacteriocins-like peptides and proteins produced by extremely halophilic archaea, were first discovered in 1982 by F. Rodriguez Valera [24, 25]. They are classified according to their size into two major classes: high molecular mass (protein, > 10 kDa) and low molecular mass (peptide, ≤10 kDa) called microhalocins [26, 27]. It has been shown that halocins are effective against *Haloarchaea* and *Crenarchaea* such as *Sulfolobus* spp. and *Methanosarcina thermopila*, and thus act across the main subdivision of the archaeal domain. These compounds represent a general class of antiarchaeal toxins and there is no confirmation about the inhibition of bacteria [26, 27]. Production of halocin is a practically universal feature of archaeal halophilic rods [27]. Although several halocins were identified, only some

These halocins are composed of a peptide with size below or in the range of 10 kDa. Seven halocins have been characterized including HalS8, HalR1, HalC8, HalU1, HalH6, Sech7a and Sech10. They are hydrophobic and retain their activity in the absence of salt and can be stored at 4°C. They are relatively insensitive to heat

HalS8 is the first characterized microhalocin with 36 amino acids (3580 Da), it is synthesized by the uncharacterized S8a haloarchaea [29]. Halocin S8 showed a narrow inhibitory spectrum and can only inhibit *Halobacterium salinarum* NRC817, *Halobacterium* GRB and *Haloferax gibbonsii* [29]. It can be desalted and it is heat resistant. Its activity is resistant to trypsin but sensitive to proteinase K and is undetectable in the transition to the stationary phase [29, 30]. The *halS8 gene* is *encoded*

Halocin R1, the second characterized microhalocin, is produced by

*Halobacterium salinarum* GN101, a strain isolated from solar salt marsh in Mexico [31]. Initially HalR1 was described with a molecular weight of 6.2 kDa [32] and later on, it was shown that the HalR1 peptide is composed of 38 amino acids [24, 29]. Like HalS8, the activity is not affected by desalting and is resistant to acids, bases, organic solvents DNase and RNase, and against some proteases such as papain, trypsin or thermolysin, but it is sensitive to proteinase K, pronase P and

Halocin C8 is produced by *Natrinema* sp. AS7092, a strain isolated from the large Chaidan Salt Lake, China (7.44 kDa, 76 amino acids) [28]. It is a unique polypeptide with an isoelectric point of 4.4 [33]. Its activity is retained after desalting, boiling and frozing [33]. Halocin C8 has a very broad spectrum of activity against several species and genera of *Halobacteriales* members including *Natronobacterium gregoryi*, *Nbt*. comb. nov and *Natronomonas pharaonis* [28]. The *halC8* gene encodes both

**3.1 Halocins**

*3.1.1 Microhalocins*

and organic solvents [28].

*3.1.1.1 Halocin S8 (HalS8)*

on a ∼200-kbp megaplasmid [29].

*3.1.1.2 Halocin HalR1 (HalR1)*

elastase [20, 32].

*3.1.1.3 Halocin C8 (HalC8)*

halocin C8 and its immunity protein HalI.

**86**

Halocin A4, also called also halocin U1, is produced by an uncharacterized haloarchaea strain isolated from a Tunisian saltern [34]. Its molecular weight is 7.435 Da, as determined by the spectrometric mass, and is both acidic (pH = 4.14) and hydrophobic (eluent at ~85% acetonitrile) [26]. Halocin A4 has been reported to inhibit the growth of crenarchaeal *Sulfolobus* sp. strains [26]. G*ene* encoding HalA4 is *located* on a 300 kpb megaplasmid, pHM300 (NC\_017943) [29].

### *3.1.1.5 Halocin H6 (HalH6)*

Halocin H6 is produced by *Haloferax gibbonsii* Ma 2.39 species [27]. Its activity is resistant to trypsin. Stabilities of this peptide were studied and have shown that HalH6 can be desalted and it retained its activity after heat treatment up to 10 min at 100°C [27]. Halocin H6 is considered as a bactericidal substance which causes cell lysis and the specific target of HalH6 is the Na+/H+ antiport [27, 35].

### *3.1.1.6 Halocin Sech7a*

Halocin Sech7a was excreted by the extremely halophilic haloarchaeon Sech7a, isolated from brine samples of Secovlje solar salterns crystallizers in Slovenia [36]. Sech7a is about 11 kDa. It is stable over a wide pH range and is heat labile at temperatures above 80°C. Its optimal activity was observed in the early exponential phase growth at 45°C. It loses activity under low salt conditions, but its activity can be restored after dialysis against initial saline conditions [36].

### *3.1.1.7 Halocin SH10*

Halocin SH10 is produced by *Natrinema* sp. BTSH10, a strain isolated from the Kanyakumari salt marsh, Tamil Nadu, India [37]. The optimal production of halocin SH10 is at 42°C, pH 8.0 and 3 M NaCl at the stationary phase. In this context, it was reported that the activity is lost under acidic conditions [37]. Production of SH10 is influenced by the carbon source composition of the medium — *Natrinema* sp. BTSH10 could produce maximal halocin in the presence of beef [37].

#### *3.1.2 Protein halocins*

This class comprises halocins composed of proteins greater than 10 kDa in size. Currently, there are two characterized protein halocins, HalH1 and HalH4, in the range of 30 to 35 kDa [28].

## *3.1.2.1 Halocin H4*

Halocin H4, produced by *Haloferax mediterranei* R4 (ATCC 33500) was isolated from a Spanish solar salt pond in Alicante. It is the first halocin that was studied [20]. The optimal activity was detected at the midpoint between exponential and stationary phases [20]. Halocin H4 is sensitive to proteases, high temperature and desalting. HalH4 has an antimicrobial activity against other haloarchaeons. It Interacts with the membrane of the target cells where it causes permeability changes that result in an ionic imbalance leading to cell lysis and death [21, 38]. The *halH4* gene, encoding halocin activity, is located on the pHM300 megaplasmid, a single polypeptide of 34.9 kDa.

#### *3.1.2.2 Halocin H1*

Halocin H1 is produced by *Haloferax mediterranei* M2a (previously known as *H*. *mediterranei* Xia3) isolated from salt ponds in Santa Pola (Alicante, Spain) [20]. Halocin H1 is a single 31 kDa polypeptide characterized by a broad inhibitory spectrum among *Halobacteriales* members. HalH1 activity is temperature and salt dependant. It is stable at 50°C only and requires a salt concentration of 1.5 M to maintain its activity [38, 39]. Optimum activity was observed at mid-exponential phase. The sensitivity to proteases and the gene encoding activity were not determined yet.

### **3.2 Applications of halocins**

Some studies reported the role of halocins in a variety of environmental, industrial and biotechnological applications \*\*\*(REFERENCES?). However, this topic is poorly documented and somewhat controversial. One of these applications is the use of halocin producing strains in the textile industry during the tanning process characterized by high salinity concentration, halocins could inhibit the growth of pathogenic microbes affecting the quality of products. [7, 10]. Moreover, some halocins have also been reported for biomedical and therapeutic uses, for example, Halocin H7 has been shown to inhibit the Na+/H+ antiport in *Haloarchaea*, can be used as a treatment to reduce the injuries caused when ischemic organ transplantation is re-infused [35]. The therapeutic potential of halocins needs more research on their physical structures and their modes of action. On the other hand, halocins are known also to have a potential application in food industry as preservative agents by controlling the growth of haloarchaea in salted food products [40].
