**4. Materials and methods**

Here, we evaluated the evolutionary relationship between bacteriocin- like-producing haloarchaea members based on comparisons of their amino acid sequences retrieved from annotated genomes sequences deposited in the IMG database [41].

#### **4.1 Database search of halocin gene clusters**

Schematic workflow of the methodology employed of amino acid sequences retrieving and phylogenetic assessment is illustrated in **Figure 2**. The methodology consisted of: first, complete and nearly complete genome sequences of *Halobacteriales* members were retrieved from IMG database. Then, in *silico* screening for gene sequences encoding halocins was done based on the BLASTP algorithm with default parameters [42]. All redundant and low-quality sequences were eliminated from datasets.

#### **4.2 Phylogenetic reconstruction**

Multiple sequences alignment of retrieved amino acid sequences were performed using ClustalW [43]. The evolutionary history was inferred using the Unweighted pair group method with arithmetic mean (UPGMA) method [44] implemented in MEGA X [45, 46]. The optimal tree with the sum of branch length = 18.99 is shown. Percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [47] and are in the

**89**

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

units of the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). In the final

*Schematic workflow of the methodology employed for amino acids sequences retrieving and phylogenetic* 

A total of 1858 of complete and nearly complete genome sequences of

ing to *Halobaceria* class including *Natrinema*, *Haloferax*, *Haloterrigena*,

*Halobacteriales class* members were retrieved from the IMG database and creened for halocin encoding gene based on the BLASTP algorithm with default parameters [42]. A total of 61 amino acid sequences were retrieved from 15 genera belong-

*Natronorubrum*, *Halobacterium*, *Haladaptatus, Halorubrum, Halococcus, Halopiger*, *Natrialba, Halolamina*, *Natronococcus, Haloarcula, Halapricum and Halorussus*. Furthermore, some other unclassified halohilic archaea were present as well, including uncultured halophilic archaeon, halophilic archaeon sp. DL31 and

Results showed that some species present more than one copy for halocin encoding genes. In fact, three (n = 3) classes of halocins were identified in this study (**Table 1**). The first class is halocin C8-like bacteriocin domain (HalC8), the best known bacteriocin like sequences in archaea, it has been demonstrated to be produced from a ProC8 precursor, targeted to the membrane by the Tat pathway, and cleaved by an unknown mechanism to yield the active mature peptide HalC8 and an immunity protein HalI, protecting the producing strain against its own AMP [22]. HalC8 was identified in all species except *Natrialba aegyptia* DSM 13077. Indeed, *Natrinema* genus members appears to be more represented in terms of C8-like bacteriocin production. It's worth noting that among the six officially described species within the *Natrinema* genus, five are described in the current analysis. Several studies report the production of HalC8 and/or the presence of the *halC8* gene among *Natrinema* species isolated from different geographical origins like Chaidan Salt Lake in Qinghai province, China [33], Ichekaben salterns Chotts and sebkhas in algeria [48, 49]. The second class is halocin H4 (HalH4) identified in *Haloferax mediterranei* strain ATCC 33500, *Natrialba aegyptia and Natrinema gari* JCM 14663 species.

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

dataset, a total of 405 positions was obtained.

**5.1 Amino acid sequence of halocins**

Haloarchaeon S8 (**Table 1**).

**5. Results**

**Figure 2.**

*assessment.*

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

#### **Figure 2.**

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

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.

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

Here, we evaluated the evolutionary relationship between bacteriocin- like-producing haloarchaea members based on comparisons of their amino acid sequences retrieved from annotated genomes sequences deposited in the IMG database [41].

Schematic workflow of the methodology employed of amino acid sequences retrieving and phylogenetic assessment is illustrated in **Figure 2**. The methodology consisted of: first, complete and nearly complete genome sequences of *Halobacteriales* members were retrieved from IMG database. Then, in *silico* screening for gene sequences encoding halocins was done based on the BLASTP algorithm with default parameters [42]. All

Multiple sequences alignment of retrieved amino acid sequences were performed using ClustalW [43]. The evolutionary history was inferred using the Unweighted pair group method with arithmetic mean (UPGMA) method [44] implemented in MEGA X [45, 46]. The optimal tree with the sum of branch length = 18.99 is shown. Percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method [47] and are in the

controlling the growth of haloarchaea in salted food products [40].

redundant and low-quality sequences were eliminated from datasets.

*3.1.2.2 Halocin H1*

**3.2 Applications of halocins**

**4. Materials and methods**

**4.2 Phylogenetic reconstruction**

**4.1 Database search of halocin gene clusters**

**88**

*Schematic workflow of the methodology employed for amino acids sequences retrieving and phylogenetic assessment.*

units of the number of amino acid substitutions per site. All ambiguous positions were removed for each sequence pair (pairwise deletion option). In the final dataset, a total of 405 positions was obtained.

#### **5. Results**

#### **5.1 Amino acid sequence of halocins**

A total of 1858 of complete and nearly complete genome sequences of *Halobacteriales class* members were retrieved from the IMG database and creened for halocin encoding gene based on the BLASTP algorithm with default parameters [42].

A total of 61 amino acid sequences were retrieved from 15 genera belonging to *Halobaceria* class including *Natrinema*, *Haloferax*, *Haloterrigena*, *Natronorubrum*, *Halobacterium*, *Haladaptatus, Halorubrum, Halococcus, Halopiger*, *Natrialba, Halolamina*, *Natronococcus, Haloarcula, Halapricum and Halorussus*. Furthermore, some other unclassified halohilic archaea were present as well, including uncultured halophilic archaeon, halophilic archaeon sp. DL31 and Haloarchaeon S8 (**Table 1**).

Results showed that some species present more than one copy for halocin encoding genes. In fact, three (n = 3) classes of halocins were identified in this study (**Table 1**).

The first class is halocin C8-like bacteriocin domain (HalC8), the best known bacteriocin like sequences in archaea, it has been demonstrated to be produced from a ProC8 precursor, targeted to the membrane by the Tat pathway, and cleaved by an unknown mechanism to yield the active mature peptide HalC8 and an immunity protein HalI, protecting the producing strain against its own AMP [22]. HalC8 was identified in all species except *Natrialba aegyptia* DSM 13077. Indeed, *Natrinema* genus members appears to be more represented in terms of C8-like bacteriocin production. It's worth noting that among the six officially described species within the *Natrinema* genus, five are described in the current analysis. Several studies report the production of HalC8 and/or the presence of the *halC8* gene among *Natrinema* species isolated from different geographical origins like Chaidan Salt Lake in Qinghai province, China [33], Ichekaben salterns Chotts and sebkhas in algeria [48, 49].

The second class is halocin H4 (HalH4) identified in *Haloferax mediterranei* strain ATCC 33500, *Natrialba aegyptia and Natrinema gari* JCM 14663 species.


**91**

**Table 1.**

*IMG database.*

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

**Taxonomy Genus Species level Class of Halocins**

*Halobacterium Halobacterium* sp. DL1 Halocin C8-like

*Halobacterium salinarum* 

*Halobacterium salinarum* 

*Halobacterium salinarum* 

*Halobacterium salinarum* 

*paucihalophilus* DX253 *Haladaptatus* sp. R4 *Haladaptatus paucihalophilus* DSM

*Halorubrum trapanicum*

*Halococcus salifodinae* DSM

*DSM 670*

*DSM 671*

*DSM 6692*

*DSM 3754*

*DSM 668*

18195 *Halorubrum Halorubrum lacusprofundi* R1S1

CBA1232

8989

CGMCC

H5-DGR

CBA1105

uncultured halophilic archaon J07HX5

J07ABHX67 Uncultured

SG1\_71\_5 *Haloarchaeon S8a Haloarchaeon S8a* Halocin S8

uncultured haloarchaeon

Halobacteriaceae archaea

*Halococcus Halococcus* sp. 197A

*Halopiger Halopiger* sp. IIH3 *Natrialba Natrialba aegyptia* DSM 13077

*Halolamina Halolamina pelagica*

*Halapricum Halapricum salinum*

*Halorussus Halorussus amylolyticus YC93*

**Halophilic archaeon** halophilic archaeon sp. DL31

*Classes of Halocins identified by in silico analysis of all genomes of halophilic archaea domain available in* 

**uncultured halophilic** 

**archaeon**

*Natronococcus Natronococcus occultus* SP4 *Haloarcula Haloarcula salaria*

*Haladaptatus Haladaptatus* 

bacteriocin *Halobacterium salinarum* 

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

Domain: *Archaea* Kingdom: *Euryarchaeota* Phylum: *Euryarchaeota* Class: *Halobacteria* Order: *Halobacteriales*

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


#### **Table 1.**

*Classes of Halocins identified by in silico analysis of all genomes of halophilic archaea domain available in IMG database.*

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

Domain: *Archaea* Kingdom: *Euryarchaeota* Phylum: *Euryarchaeota* Class: *Halobacteria* Order: *Halobacteriales*

Family: *Halobacteriaceae*

**Taxonomy Genus Species level Class of Halocins**

*Natrinema Natrinema pellirubrum* 157

JCM 10476

DSM 15624 *Natrinema altunense* 1A4-DGR

3751

12890

*Haloferax Haloferax mediterranei*

*Natrinema* sp. J7-2 *Natrinema pallidum* DSM

*Natrinema pellirubrum*

*Natrinema altunense* JCM

*Natrinema gari* JCM 14663 Halocin C8-like

*Natrinema* sp. J7-1 *Natrinema altunense* AJ2 *Natrinema atunense* 4.1

ATCC 33500

*14919*

JCM 10717

13917

*ZY19*

*13891*

*WANU15*

DSM 13204

GA33

*Natronorubrum Natronorubrum tibetense*

*thermotolerans*

*Haloterrigena Haloterrigena* 

*Haloferax mediterranei* R-4 *Haloferax lucentense DSM* 

*Haloferax* sp. ATCC BAA-646

*Haloferax volcanii* DS2 *Haloferax alexandrinus*

*Haloferax sp.* ATCC BAA-645

*Haloferax larsenii* CDM 5 *Haloferax larsenii* JCM

*Haloterrigena salifodinae* 

*Haloterrigena jeotgali A29 Haloterrigena mahii H13 Haloterrigena salina JCM* 

*Haloterrigena sp. P1A Haloterrigena turkmenica* 

*Natronorubrum tibetense*

*Natronorubrum sediminis* CGMCC 1.8981

Halocin C8-like bacteriocin

bacteriocin and Halocin H4

Halocin C8-like bacteriocin

Halocin H4

Halocin C8-like bacteriocin

Halocin C8-like bacteriocin

Halocin H4

**90**

HalH4 was first characterized from *H. mediterranei* isolated from solar saltern lakes of Spain [50]. HalH4 is a 40 kDa protein with an N-terminal 46 aa leader peptide which is cleaved off leaving a 313 aa mature halocin [51].

The third class is halocin S8, a microhalocin of 36 amino acids (3580 Da) initially purified from an unidentified haloarchaeal strain S8a, isolated from the Great Salt Lake (Utah, 109 United States) [52].

#### **Figure 3.**

*Phylogenetic tree of halocin amino acid sequences of halophilic archaeal species. The evolutionary history was inferred using the UPGMA algorithm implemented in MEGA X software. Numbers at the nodes indicate the percentage of occurrence in 100 bootstrapped trees (bootstrap values > 50% are shown).*

**93**

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

Phylogenetic analysis of retrieved halocin peptide sequences was conducted and the result is illustrated in **Figure 3**. Results showed a clear segregation of the three halocins classes (C8, H4 and S8), where halocin S8 is phylogenetically more close to HalH4. Furthemore, no clear separation of species was observed based on HalC8 amino acids sequences analyses. HalC8 was detected in 12 genera belonging to three orders of *Halobacteria* class [53]: *Natrialbales* (*Natrinema*, *Haloterrigena*,

*Halobacteriales* (*Halorussus*, *Halobacterium*, *Haladaptatus*, *Halococcus*, *Haloarcula*, *Halapricum*) and uncultured halophilic archaeon (J07ABHX67) phylogenetically related to species *Halorussus amylolyticus* YC93. The halocin S8 was detected only in the strain Haloarchaeon S8a (Q9HHA8). HalH4 is identified in *Natrialbales* (*Natrinema gari* JCM 14663 (ELY77239.1), *Haloterrigena thermotolerans*, *Natrialba* 

It's worth noting that HalH4/HalC8 halocins were identified in *Haloferax, Haloterrigena* and *Natrinema* genera with only the species *Natrinema gari* JCM 14663 (ELY77239.1) being able to produce the two classes in the same time. Earlier studies reported that several described halocins, with broad inhibitory properties, are derived from *Haloferax* and *Natrinema* strains [24, 28, 45, 51, 54] and it has been suggested that halocin production may explain their dominance in some saline

On the basis of our *in silico* analyses, we can conclude that halocin production is considered as a general feature of some members of halophilic archaea, particularly members of *Natrialbales* and *Haloferacales* orders with the occurrence of Halocin C8-like production. This group can thrive in saline ecosystems in which several other microorganisms are not able to live. Thus, the dominance of certain species isolated in some saline ecosystems could be attributed to halocin production as a mechanism of competition between microrganisms. This chapter will open new research lines that will shed light on halocins as marker for haloarchaeal phylogentic delineation.

*Natronorubrum*, *Halopiger*); *Haloferacales* (*Haloferax*, *Halolamina*) and

*aegyptia* DSM 13077) and *Haloferacales* (*Haloferax*).

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

**5.2 Phylogenetic analysis**

ecosystems [54, 55].

**Conflict of interest**

We have *no conflict of interest* to declare.

**6. Conclusion**

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

## **5.2 Phylogenetic analysis**

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

which is cleaved off leaving a 313 aa mature halocin [51].

Lake (Utah, 109 United States) [52].

HalH4 was first characterized from *H. mediterranei* isolated from solar saltern lakes of Spain [50]. HalH4 is a 40 kDa protein with an N-terminal 46 aa leader peptide

*Phylogenetic tree of halocin amino acid sequences of halophilic archaeal species. The evolutionary history was inferred using the UPGMA algorithm implemented in MEGA X software. Numbers at the nodes indicate the* 

*percentage of occurrence in 100 bootstrapped trees (bootstrap values > 50% are shown).*

The third class is halocin S8, a microhalocin of 36 amino acids (3580 Da) initially purified from an unidentified haloarchaeal strain S8a, isolated from the Great Salt

**92**

**Figure 3.**

Phylogenetic analysis of retrieved halocin peptide sequences was conducted and the result is illustrated in **Figure 3**. Results showed a clear segregation of the three halocins classes (C8, H4 and S8), where halocin S8 is phylogenetically more close to HalH4. Furthemore, no clear separation of species was observed based on HalC8 amino acids sequences analyses. HalC8 was detected in 12 genera belonging to three orders of *Halobacteria* class [53]: *Natrialbales* (*Natrinema*, *Haloterrigena*, *Natronorubrum*, *Halopiger*); *Haloferacales* (*Haloferax*, *Halolamina*) and *Halobacteriales* (*Halorussus*, *Halobacterium*, *Haladaptatus*, *Halococcus*, *Haloarcula*, *Halapricum*) and uncultured halophilic archaeon (J07ABHX67) phylogenetically related to species *Halorussus amylolyticus* YC93. The halocin S8 was detected only in the strain Haloarchaeon S8a (Q9HHA8). HalH4 is identified in *Natrialbales* (*Natrinema gari* JCM 14663 (ELY77239.1), *Haloterrigena thermotolerans*, *Natrialba aegyptia* DSM 13077) and *Haloferacales* (*Haloferax*).

It's worth noting that HalH4/HalC8 halocins were identified in *Haloferax, Haloterrigena* and *Natrinema* genera with only the species *Natrinema gari* JCM 14663 (ELY77239.1) being able to produce the two classes in the same time. Earlier studies reported that several described halocins, with broad inhibitory properties, are derived from *Haloferax* and *Natrinema* strains [24, 28, 45, 51, 54] and it has been suggested that halocin production may explain their dominance in some saline ecosystems [54, 55].
