TABLE 1 | List of Lactobacillus genomes examined in this in silico screen.

No. Spesies Strain Accession No. Source Country

1 Lactobacillus crispatus ST1 NC_014106.1 - -
2 Lactobacillus helveticus CAUH18 NZ_CP012381.1 koumiss China
3 Lactobacillus acidophilus NCFM NC_006814.3 - -
4 Lactobacillus gasseri ATCC 33323 NC_008530.1 - -
5 Lactobacillus johnsonii Byun-jo-01 NZ_CP029614.1 jejunum South Korea
6 Lactobacillus amylovorus 30SC NC_015214.1 - -
7 Lactobacillus kefiranofaciens ZW3 NC_015602.1 - -
8 Lactobacillus sakei FAM18311 NZ_CP020459.1 food Switzerland
9 Lactobacillus reuteri DSM 20016 NC_009513.1 - -
10 Lactobacillus paracasei JCM 8130 NZ_AP012541.1
11 Lactobacillus salivarius UCC118 NC_007929.1
12 Lactobacillus plantarum WCFS1 NC_004567.2
13 Lactobacillus rhamnosus ATCC 53103 NC_013198.1 -
14 Lactobacillus casei ATCC 393 NZ_AP012544.1 - -
15 Lactobacillus gallinarum HFD4 NZ_CP012890.1 faecal India
sample
16 Lactobacillus paragasseri strain JCM NZ_AP018549.1
5343
17 Lactobacillus paraplantarum CK401 NZ_CP053337.1 fermented South Korea
vegetable

MATERIALS AND METHODS

3.1 Bacteriocin Identification and Visualization

Using the in silico bacteriocin prediction tool BAGEL4, complete genome sequences belonging
to the genus Lactobacillus (Table 1) were aquired and analyzed. Amino acid sequences of all
bacteriocins belongs to lactobacillus were aquired from BAGEL4 and aligned against the
genomes as driver sequences using blastP. Bacteriocin peptides sekuens were aligned using the
Multiple Sequence Alignment (MSA) tool MUSCLE and then visualized using Jalview. The
previously generated MUSCLE peptide alignments were then input into the MEGA-X software
package for phylogenetic analysis. Using a neighbor-joining method, an unrooted phylogenetic
tree was generated and bootstrap replication values of 1,000. In alignments where specific
sequences contained no common sites, these were deleted.

3.2 Phylogenetic Analysis of Lactobacillus Species

The 16S rRNA sequences from all Lactobacillus species were acquired from
https://www.ncbi.nlm.nih.gov.
All 16S rRNA sequences were then collated and aligned as before using
the MSA tool MUSCLE. The resulting alignment output was then input into MEGA-X. A
neighbor-joining tree was generated using bootstrap values based on 1,000 replications. Where
no common sites were found for specific peptides in the generation of the phylogenetic tree they
were not included in the phylogenetic arrangement. The bacteriocin predictions by BAGEL4
were subsequently overlaid onto the phylogenetic tree using microsoft Powerpoint.
RESULTS

BAGEL4 Predicted Bacteriocin Class 1

Species Predicted Bacteriocin Amino Acid E-value

Lactobacillus Gassericin E MKQFNYLSHKDLAVVVGGRNNWQTNVGGAVGSAM Evalue=1e-25
johnsonii IGATVGGTICGPACAVAGAHYLPILWTGVTAATGGFG match=61.33%
KIRK
Lactobacillus Gassericin MKNFNTLSFETLANIVGGRNNWAANIGGVGGATVAG Evalue=4e-48
paragasseri WALGNAVCGPACGFVGAHYVPIAWAGVTAATGGFG match=98.67%
KIRK

BAGEL4 Predicted Bacteriocin Class II

Species Predicted Bacteriocin Amino Acid E-value

Lactobacillus Amylovorin MKQLNSEQLQNIIGGNRWTNAYSAALGCAVPGVKYG Evalue=7e-42
amylovorus KKLGGVWGAVIGGVGGAAVCGLAGYVRKG match=100.00%;

Lactobacillus Lactacin_F,subunit_lafX MKLNDKELSKIVGGNRWGDTVLSAASGAGTGIKACK Evalue=9e-42

johnsonii SFGPWGMAICGVGGAAIGGYFGYTHN match=100.00%

Lactobacillus putative_bacteriocin MMIFKKLSEKELQKISGGVGIQKCSLGFSSREYLNKIT Evalue=3e-28

sakei KWIKHH match=100.00%;

Lactobacillus Plantaricin_A MKIQIKGMKQLSNKEMQKIVGGKSSAYSLQMGATAI Evalue=3e-29

plantarum KQVKKLFKKWGW match=100.00%;
 Lactobacillus Acidocin_LF221B(Gasseri MALKTLEKHELRNVMGGNKWGNAVIGAATGATRGV Evalue=6e-43
paragasseri cinK7B) SWCRGFGPWGMTACGLGGAAIGGYLGYKSN match=98.46%;

Lactobacillus Plantaricin A VIIMKIQIKGMKQLSNKEMQKIVGGKSSAYSLQMGAT Evalue=3e-29

paraplantarum AIKQVKKLFKKWGW match= 100%

BAGEL4 Predicted Bacteriocin Class III

Species Predicted Bacteriocin Amino Acid E-value

L. acidophilus Bacteriocin_helveticin_J MVGSITPKLVYRLNGMHHVVAQVGAVNGDHVFALQ Evalue=0.0
LLHSAHDVLVYRKHKGLTKDINYTNPHLVMTGFGHT match=100.00%;
QTWVPANDNDEYFVGAKPNSGNWTTQIARVKYPRLL
SENYTSNTQLPRLSHLNRVTDVPYDGHNHLHRVEAS
VSPNGKYFMIASIWNNGSGHFGLFDLDEVNQKLDEN
GTTNTPITDLHCLSAFHIDNFDNPSVAPDEEEPTMIDS
VQGYAIDNDKNIYISNQLSPKINHETGEVTTWARKIV
KFPWGETDSNNWQVAMIDGIDLPDRYSEVESIHVNAP
DDIYLTVAYHQKIVKGDEYALRTLENQIFHIDNL
Lactobacillus Bacteriocin_helveticin_J MHHVVAQVGVVNGDHVFALQLLHSAHDVLVYRKH Evalue=7e-167
amylovorus EGLTKNIDYTDPHLVMMGFGHTQTWVPANDKDEYF match=91.25%;
VGAKPNSGNWTTQIARVKYPRLLPERYTSNTQLPRLS
HLNHVTDVPYDGHDHLHRVEASVSPNGKYFMIASIW
DDGSGHFGLFDLNEVNQKLDENGTKNTPITDLHCLN
AFHIDNFDNPSVAPNEETPQMIDSVQGYAIDDDKNIYI
SNQLSPKINHETGEVTTWSRKIVKFPWGETNSDNWQ
VAMVDGIDLPDRYSEMESIHVNAANDIYLTVAYHQK
YIKGGEYKLRTLENQIFHITDL
Lactobacillus Bacteriocin_helveticin_J MVKSITPHLVYRLNGMHHVVAQVGVVNDDHVFALQ Evalue=8e-172
crispatus LLHSAHDVLVYRKHEGLTKNIDYTDPHLVMMGFGHT match=88.63%
QTWVAANDKDEYFVGAKPNSGNWTTQIARVKYPRL
LPERYTSNTQLPRLSHLNRATDVPYDGHNHLHRVEAS
 VSPNGKYFMIAAIWDDDSGHFALYDLNEVNQKLDEN
GTTNTPITDLHCLSAFHIDNFDHPSVAPSEEAPQMIDS
VQGYAIDDDKNIYISNQLSPKIDHATGEVTTWSRKIVK
FPWGETNPENWQVAMIDGIDLPDRYSEVESIHVQVPD
DIYLTVAYHQKYVKDGEFKLRTLENQIFHISDLG
Lactobacillus Bacteriocin_helveticin_J MIGKETQIRLVNKLENIHHVVVQASAIDGNNVFALQL Evalue=1e-61
gasseri LHKQSDVIVYQTPNDSKTVTFNEDHPILYLKGPNSAG match=44.96%;
TAGGHTQTWVQSGENNKWFVGTKPKRHGNTYWTT
QIARVAVSGYQTQTFTNNTELPRLSYLNRAGSGYGDG
SVAYPGKDLVRVEAAVSPNRQYFLIASIDINHTGHFA
VYNLDEVNKKLDEAEEKTEDINIQNLNCLGAFNVPHF
NDQKIISIQGYGIDDNKNIYISSQPSPYTTFLGFPKQGK
PREIVKIPWGISDPSKWSVVNLDNSLKLDALNFCTEFE
GIQVTGDCLYLTVAYHQRNSDLTTLMNRIYQVEKF
Lactobacillus Bacteriocin_helveticin_J MVKSITPHLVYRLNGMHHVVAQVGVVNGDHVFALQ Evalue=5e-177
helveticus LLHSAHDVLVYRKHEGLTKNIDYTDPHLVMMGFGHT match=91.37%
QTWVPANDKDEYFVGAKPNSGNWTTQIARVKYPRLL
PERYTSNTQLPRLSHLNHVTDVPYDGHDHLHRVEAS
VSPNGKYFMIASIWDDGSGHFGLFDLNEVNQKLNEN
GTKNTPITDLHCLSAFHIDNFDNPSVAPDEEMPQMIDS
VQGYAIDDDKNIYISNQLSPKINHETGEVTTWSRKIVK
FPWGETNSDNWQVAMVDGIDLPDRYSEMESIHVNAA
NDIYLTVAYHQKYIKGGEYKLRTLENQIFHITDL
Lactobacillus Bacteriocin_helveticin_J MVKNITPDLVYRLNGMHNVVAQVGAVMDKHVFAL Evalue=9e-169
kefiranofacien QLLHSAHDVLVYRKHEGLTKNIDYTEPHLVMTGFGH match=87.06%;
s TQTWIPANDKDEYFVGAKPNSGNWTTQIARVKYPKL
LSENYTSNTQLPRLSHLNHATDVPYDGHNHLHRVEA
CVSPNGKYFMIAAIWNDDSGHFALYDLNEVNQKLDE
NGTKNTPITDLHCLSAFHIDNFDHPSVAPNQEEDQVID
SVQGYAIDNDKNIYISNQLSPEINHTTGEVTTWKRKIV
KFPWGETNSNNWDVSMIDGIDLPDRYSEVESIQVNAP
DDIYLTVAYHQKYIKNGEFSLRTLENQVFHISDL
Lactobacillus Bacteriocin_helveticin_J LPENYTSNTQLPRLSHLNHATDVPYDGHNHLHRVEA Evalue=5e-49
gallinarum AVSPNGKYFMIAAIWNDDSSHFALYDLNEVNQKLDE match=85.54%;
NGTTNTPITRL
 2. MSA dan Pohon Filogenetik.

2.1 Class II Bacteriocin

Figure 2. Multiple Sequence Alignment (MSA) of class III bacteriocin predicted by BAGEL4.

Figure 3. Phylogenetic arrangement of predicted class II bacteriocin (left) and 16SrRNA (right) within Lactobacillus genomes
investigated in this study.
 2.1 Class III Bacteriocin

Figure 4. Multiple Sequence Alignment (MSA) of class III bacteriocin predicted by BAGEL4.

Figure 5. Phylogenetic arrangement of predicted class III bacteriocin (left) and 16SrRNA (right) within Lactobacillus genomes
investigated in this study.
Result

This study uses an in silico approach to determine both the prevalence and diversity of bacteriocin gene clusters within the
genus Lactobacillus. Utilizing the genome sequences available in https://www.ncbi.nlm.nih.gov databases, 17 genomes of
Lactobacillus species (Table 1) were analyzed. This study showed the prediction of bacteriocin gene class, of which 2
species matched Class I, 6 spesies matched Class II and 7 spesies matched Class III bacteriocin. No homologies were
found in Lactobacillus reuteri, paracasei, salivarius, rhamnosus and herbinensis when predicted by BAGEL4.

In order to reveal associations between bacteriocin gene presence within genomes and their phylogenetic position within
the overall Lactobacillus genus, we superimposed the BAGEL4 bacteriocin predictions onto a Lactobacillus neighbor-
joining phylogenetic tree constructed from 16S rRNA sequences. Here we can see that bacteriocin clusters are both
diverse and common across those genomes examined in this study (Figure 1). Class I, II and II bacteriocin are spread
across the whole genus, but Class II and III bacteriocin were the most frequent to be found.

Figure 1. Phylogenetic arrangement of Lactobacillus genomes investigated in this study. The

BAGEL4 bacteriocin predictions are overlaid in order to examine associations between
bacteriocin gene presence and position within the Lactobacillus phylogenetic arrangement.