molecular genetic studies on pediocin-like...

Molecular genetic studies on pediocin-like bacteriocin Appendix

193 S. Manjulata Devi

SEQUENCES DEPOSITED AT THE GENBANK

NCBI DATABASE

>gi|330003952|gb|GU222444.2| Streptococcus equinus strain AC1 16S

ribosomal RNA gene, partial sequence

TGCTAAAGTTGGAAGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTGCCTACTAGCGGGGGATAAC

TATTGGAAACGATAGCTAATACCGCATAACAGCATTTAACCCATGTTAGATGCTTGAAAGGAGCAATTG

CTTCACTAGTAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATA

CATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA

GCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTC

GGATCGTAAAGCTCTGTTGTAAGAGAAGAACGTGTGTGAGAGTGGAAAGTTCACACAGTGACGGTAACT

TACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTGTCCGGA

TTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTAATAAGTCTGAAGTTAAAGGCAGTGGCTTAACCATTG

TTCGCTTTGGAAACTGTTAGACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATG

CGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAA

AGCGTGGGGAGCAAA

>gi|270266325|gb|GU222445.1| Pediococcus acidilactici strain Cb1 16S ribosomal

RNA gene, partial sequence

TTTTAACACGAAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGCAGGGGATAAC

ACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCCTGGTTTTCTTTTAAAAGATGGCTCTG

CTATCACTTCTGGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGATGAT

GCGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC

AGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTT

CGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTGAGAGTAACTGTTCACCCAGTGACGGTATTT

AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGA

TTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAG

AAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAAT

GCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGA

AAGCATGGGTAGCGAA

>gi|270266326|gb|GU222446.1| Pediococcus pentosaceus strain Cb4 16S


TTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCAGAAGTAGGGGATAACACC

TGGAAACAGGTGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTGAAAGATGGCTCTGCTATC



ACTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGC

CGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGG

GAATCTTCCACAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAA

GCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCAGTGACGGTATTTAACCAGAAAGCCAC

GGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAG

CGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGG

GAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAAC

ACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAA

>gi|270266327|gb|GU222447.1| Lactobacillus plantarum strain Acr2 16S


TTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAAC

ACTTGGAAACAGGTGCTAATACCGTATAATAGAGAAAACCGCATGGTTTTCTTTTGAAAGATGGCTCTG

CTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGAT

ACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGC

AGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTT

CGGCTCGTAAAACTCTGTTGTTAGAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTT

AACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGA

TTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAG

AAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAAT

GCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGCCGCTGAGGCTCGA

AAGTATGGGTAGCAAA

>gi|270266328|gb|GU222448.1| Enterococcus faecium strain V3 16S ribosomal


CACCGGAAAAAGAGGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAAC

ACTTGGAAACAGGTGCTAATACCGTATAACAATCAAAACCGCATGGTTTTGATTTGAAAGGCGCTTTCG

GGTGTCGCTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGA

TGCATAGCCGACCTGAGAGGGTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGG

CAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTT

TCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAACTGTTCATCCCTTGACGGTATC

TAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGG

ATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGG

GAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAA

TGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCG

AAAGCGTGGGGAGCAAA



>gi|270266329|gb|GU222449.1| Enterococcus faecium strain BL1 16S ribosomal












AAAGCGTGGGGAGCAAA

>gi|270266330|gb|GU222450.1| Enterococcus faecium strain Acr4 16S ribosomal












AAAGCGTGGGGAGCAAA

DEFINITION Enterococcus faecium strain Acr4 plasmid pEnt4 pediocin PA-1

operon, partial sequence.

LOCUS HQ876214 6742 bp DNA linear BCT 18-JUN-2011

ACCESSION HQ876214

VERSION HQ876214.1 GI:335906199

KEYWORDS .

SOURCE Enterococcus faecium



ORGANISM Enterococcus faecium

Bacteria; Firmicutes; Lactobacillales; Enterococcaceae;

Enterococcus.

REFERENCE 1 (bases 1 to 6742)

AUTHORS Devi,S.M.

TITLE Evidence for pediocin PA-1 like bacteriocin production by

Enterococcus faecium Acr4 and studies on organization of

operon

JOURNAL Unpublished


AUTHORS Devi,S.M.

TITLE Direct Submission

JOURNAL Submitted (11-JAN-2011) Food Microbiology, Central Food

Technological Research Institute, Mysore, Karnataka 570020,

India

FEATURES Location/Qualifiers

source 1..6742

/organism="Enterococcus faecium"

/mol_type="genomic DNA"

/strain="Acr4"

/db_xref="taxon:1352"

/plasmid="pEnt4"

operon <1..6742

/operon="pediocin PA-1 operon"

gene <1..758

/gene="repB"


CDS <1..758

/gene="repB"


/note="involved in plasmid replication and

maintenance;frequently found in theta-replicating

plasmids from lactic acid bacteria"

/codon_start=3

/transl_table=11

/product="replication protein"

/protein_id="AEH68221.1"

/db_xref="GI:335906200"

/translation="LQNNRFEDDIQRTYEKMMGLHFGRRSKSGLNREFFVMFTEFEIK GEAEIPYVDIRVYPKALHLLNDLESWVRYALAEFRDLKSSYAKTMFRLLKQFRTTGYAYFSKA

DFDELLDIPKTYRQGDINKKVIKPIKEELTPLFRGLTVRKKYGKGRGKPVIGYSFTWKPEKKD

ANDFSQGQFQDERQKLFNIQHNGELTEQEKWRAIDKVKGLTLGSTEKQALAVKQAEHDKKIRD

QARKEALAELRKGFGNHA

http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=1352


http://www.ncbi.nlm.nih.gov/nuccore/335906199?from=1&to=6742



http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi?mode=c#SG11

http://www.ncbi.nlm.nih.gov/protein/335906200



CDS 751..1329

/note="orf912; involved in plasmid replication and

maintenance; frequently found in theta-replicating

plasmids from lactic acid bacteria"

/codon_start=1

/transl_table=11

/product="hypothetical protein"


/db_xref="GI:335906201" /translation="MPKTIRELADELKVSKQTIQYHYQRLPTKNRQKDSQGTNMISLT AERIIRDKVAKPSVANTQQTGSKKVTKTSKENNELIATLRREIEDLKSQRDKQLATKDRQIDHLTKLVD

QQQQLQLATVADNRRLKDHVQKLSGQLTQKTNDNLSTGNDLFNIQDK

ESKIAKQKIVKSGSNKDGIHTNRAIKRWWKFW"

gene 2270..2458

/gene="pedA"


CDS 2270..2458

/gene="pedA"


/codon_start=1

/transl_table=11

/product="pre-pediocin"


/db_xref="GI:335906202" /translation="MKKNEKLTEKEMANIIGGKYYGNGVTCGKHSCSVDWGKATTCII

NNGAMAWATGGHQGNHKC"

gene 2496..2834

/gene="pedB"


CDS 2496..2834

/gene="pedB"


/codon_start=1

/transl_table=11

/product="immunity portein"


/db_xref="GI:335906203" /translation="MNKTKSEHIKQQALDLFTRLQFLLQKHDTIEPYQYVLDILETGI SKTKHNQQTPERQARVVYNKIASQALVDKLHFTAEENKVLAAINELAHSQKGWGEFNMLDTTNTWPSQ"

gene 2858..3382

/gene="pedC"


CDS 2858..3382

/gene="pedC"

















/codon_start=1

/transl_table=11

/product="accessory protein"


/db_xref="GI:335906204" /translation="MSKKFWSNIFLALGVFLAFAGVATISVSADSSATIESNTSSKII

DGATYEENIRGVIPITLTQYLHKAQTGEKFIVFVGFKECVHCRKFSPVMKQYLQQSQHPIYYLDYGNNG

SFSMASQKQITDFYSTFATPMSFMGTPTVALLDNGKVVSMTAGDDTTLSDLQQITADYNNQ"

gene 3481..5655

/gene="pedD"


CDS 3481..5655

/gene="pedD"


/codon_start=1

/transl_table=11

/product="ABC transporter"


/db_xref="GI:335906205"

/translation="MWTQKWHKYYTAQVDENDCGLAALNMILKYYGSDYMLAHLRQLAKTTADGTTVLGLVKAAKHLNLNAEAVRADMDALTASQLPLPVIVHVFKKNKLPHYYVVYQVTENDLIIGDPDPTVKTTKI

SKSQFAKEWTQIAIIIAPTVKYKPIKESRHTLIDLVPLLIKQKRLIGLIITAAAITTLISIAGAYFFQL

IIDTYLPHLMTNRLSLVAIGLIVAYAFQAIINYIQSFFTIVLGQRLMIDIVLKYVHHLFDLPMNFFTTR

HVGEMTSRFSDASKIIDALGSTTLTLFLDMWILLAVGLFLAYQNINLFLCSLVVVPIYISIVWLFKKTF

NRLNQDTMESNAVLNSAIIESLSGIETIKSLTGEATTKKKIDTLFSDLLHKNLAYQKADQGQQAIKAAT

KLILTIVILWWGTFFVMRHQLSLGQLLTYNALLAYFLTPLENIINLQPKLQAARVANNRLNEVYLVESE

FSKSREITALEQLNGDIEVNHVSFNYGYCSNILEDVSLTIPHHQKITIVGMSGSGKTTLAKLLVGFFEP

QEQHGEIQINHHNISDISRTILRQYINYVPQEPFIFSGSVLENLLLGSRPGVTQQMIDQACSFAEIKTD

IENLPQGYHTRLSESGFNLSGGQKQRLSIARALLSPAQCFIFDESTSNLDTITEHKIVSKLLFMKDKTI

IFVAHRLNIASQTDKVVVLDHGKIVEQGSHRQLLNYNGYYARLIHNQE"

ORIGIN

1 TACTGCAAAA TAACCGTTTT GAAGATGACA TTCAGAGAAC TTATGAAAAA ATGATGGGAT

61 TACATTTTGG TAGACGAAGT AAAAGTGGCT TAAATCGAGA ATTTTTTGTT ATGTTTACCG

121 AATTTGAAAT TAAAGGCGAA GCTGAAATAC CTTACGTTGA TATCCGAGTT TATCCTAAAG

181 CCTTACACTT ACTAAACGAT TTAGAAAGTT GGGTTCGTTA TGCGTTGGCA GAGTTTAGAG

241 ATTTAAAAAG TAGTTACGCA AAAACAATGT TTCGGTTACT AAAACAATTT AGAACTACTG

301 GGTACGCTTA CTTTTCCAAA GCAGATTTTG ATGAGTTACT TGATATTCCA AAAACTTATC

361 GGCAAGGCGA CATTAACAAA AAAGTGATAA AACCAATCAA AGAAGAACTT ACCCCCCTAT

421 TTCGTGGGCT AACTGTCCGA AAGAAATACG GTAAAGGGCG AGGAAAGCCT GTTATTGGCT

481 ATTCGTTTAC CTGGAAACCC GAAAAGAAAG ACGCTAACGA CTTCTCACAA GGTCAATTTC

541 AAGATGAACG TCAAAAACTC TTTAATATTC AGCATAATGG CGAATTAACA GAACAGGAAA

601 AATGGCGTGC CATTGATAAA GTTAAGGGGT TAACTTTAGG CTCTACTGAA AAGCAAGCAT

661 TGGCTGTCAA ACAAGCCGAA CATGATAAAA AAATAAGAGA TCAAGCAAGA AAAGAAGCAC

721 TTGCTGAACT CCGAAAGGGG TTTGGAAATC ATGCCTAAAA CAATTAGAGA ACTTGCTGAC

781 GAATTGAAGG TCTCTAAACA AACTATTCAA TACCACTACC AAAGACTACC AACAAAGAAC









841 CGACAAAAAG ATAGTCAAGG TACAAACATG ATCAGCCTTA CAGCTGAAAG GATTATTAGG

901 GACAAGGTAG CAAAGCCTTC GGTAGCAAAT ACCCAACAAA CAGGTAGCAA AAAAGTGACA

961 AAGACTAGCA AAGAAAATAA TGAGCTAATT GCCACTCTAA GAAGAGAAAT AGAAGATTTA

1021 AAGTCTCAAC GTGACAAACA GCTTGCTACC AAAGACCGAC AAATAGATCA TCTAACAAAA

1081 TTGGTGGATC AGCAGCAACA ATTACAATTA GCAACAGTAG CAGATAACCG TCGATTAAAA

1141 GATCATGTAC AAAAGCTAAG TGGGCAACTA ACTCAAAAAA CTAACGACAA CTTGTCGACC

1201 GGAAATGATC TTTTTAACAT CCAAGATAAA GAAAGCAAAA TAGCTAAACA GAAGATTGTT

1261 AAATCTGGTA GTAATAAAGA TGGCATACAC ACAAATAGAG CTATTAAACG CTGGTGGAAA

1321 TTCTGGTAAA AGTTAATGTA AGCCTTAAGG TTTCAACTAA AGCAATTACA GTCAACCATA

1381 ACCATAGTAT TGGATTGTCA TTTTATTGGC TATAAAATAG TAAATCAGTG AATTTCATTA

1441 CAAAAGGGCT CACAAAAAAT TGTTTTCTTC CTCCAACAAT AGCGAGACGC TTTTCTAATT

1501 GCTTGACCCA AAGAGCAATA GAATATTTTG AAGGTCCAAA TTATTCTGTT AATGATTTAA

1561 GTGAACGGCC TTCTTGGTGA AATTTAACCA ATGAATCTTT GAAATCTTGT GAATAACGAA

1621 TTGACATAAA AATGCTCCTA TATTTTCATT TTACGGACTG AATAAAAATA GTCCATTTTT

1681 TTAGTATAAG AGCAGTAAAA CCAGACGTGG AAACCACGTA GTCTTTTAGT TGATTCAGTA

1741 AAAGAAGCCG AAACCAACGT TTTCACGTTG GTTTCGGCTT CTTTGGCTTT TAATTGCGGG

1801 AACGCACACA AAGAGCCAAA AAAGATTTGA TAAAATCAAA GCTAGAAACT AGCTCCGGTC

1861 ATGCTTGTTG CGATCATTAT CGCGTAAGTC TTCTACGTGG GCATCACCAC TCGTATCGAT

1921 ATCTAGTTCT TCGCGGCCGA CGTTTTCACT TACTTGTTTC ATATCTTCAT GTTATTGTAG

1981 TATAGTGTTA AATTTTTCAT TTACGACCGG GCGTTTGTTG ACATCGGTAG ATGCAGCCGC

2041 ACCATCTCCG GGCTTTCTTT CGATCACGAT TTCTTCTCGT TTAAAATGAA TATATAAACT

2101 GTGTCATAAC TTAAAAGATA CTGCGTTGAT AGGCCAGGTT TCAAAAATTG ACCAAGATCG

2161 TTAACCAGTT TTGGTGCGAA AATATCTAAC TAATACTTGA CATTTAAATT GAGTGGGAAC

2221 TAGAATAAGC GCGTATTAAG GATAATTTAA GAAGAAGGAG ATTTTTGTGA TGAAAAAAAA

2281 TGAAAAATTA ACTGAAAAAG AAATGGCCAA TATCATTGGT GGTAAATACT ACGGTAATGG

2341 GGTTACTTGT GGCAAACATT CCTGCTCTGT TGACTGGGGT AAGGCTACCA CTTGCATAAT

2401 CAATAATGGA GCTATGGCAT GGGCTACTGG TGGACATCAA GGTAATCATA AATGCTAGCA

2461 TTATGCTGAG CTGGCATCAA TAAAGGGGTG ATTTTATGAA TAAGACTAAG TCGGAACATA

2521 TTAAACAACA AGCTTTGGAC TTATTTACTA GGCTACAGTT TTTACTACAG AAGCACGATA

2581 CTATCGAACC TTACCAGTAC GTTTTAGATA TTCTGGAGAC TGGTATCAGT AAAACTAAAC

2641 ATAACCAGCA AACGCCTGAA CGACAAGCTC GTGTAGTCTA CAACAAGATT GCCAGCCAAG

2701 CGTTAGTAGA TAAGTTACAT TTTACTGCCG AAGAAAACAA AGTTCTAGCA GCCATCAATG

2761 AATTGGCGCA TTCTCAAAAA GGGTGGGGCG AGTTTAACAT GCTAGATACT ACCAATACGT

2821 GGCCTAGCCA ATAGTACTGA TAAAGGGGAT ATTGTAGTTG TCTAAGAAAT TTTGGTCAAA

2881 TATCTTTTTA GCATTAGGCG TCTTTCTTGC TTTTGCAGGA GTTGCTACCA TATCGGTGAG

2941 TGCTGACAGT TCCGCTACTA TAGAATCAAA TACTAGCTCG AAAATCATCG ATGGTGCAAC

3001 TTATGAAGAA AACATCAGGG GCGTTATTCC TATTACGCTA ACTCAATATT TGCATAAAGC

3061 TCAAACTGGA GAAAAATTTA TTGTCTTTGT CGGGTTCAAG GAGTGTGTGC ATTGTCGTAA

3121 ATTTTCTCCA GTCATGAAAC AGTACTTACA ACAAAGTCAG CATCCCATTT ATTACTTAGA

3181 CTATGGGAAC AACGGGTCTT TCAGCATGGC TTCTCAAAAA CAAATAACTG ATTTCTATTC

3241 AACTTTTGCA ACCCCCATGA GTTTTATGGG AACGCCAACT GTTGCCTTGC TCGATAATGG

3301 TAAGGTGGTA TCAATGACCG CTGGTGATGA TACCACTTTA TCTGATTTAC AACAGATTAC

3361 TGCTGATTAC AATAATCAGT AGTCACCTGG TTAATATGGT TTTGTAACCA ATGTAAAAGG

3421 CGATGGATCT TTGAAATCGT CTTTTTTTAT GCACAAATTT TAAAGATCGG TGGTTTGCTT

3481 ATGTGGACTC AAAAATGGCA CAAATATTAT ACAGCACAAG TTGATGAAAA TGACTGTGGT



3541 TTAGCTGCAC TAAATATGAT CCTAAAATAC TATGGCTCCG ATTACATGTT GGCCCATCTT

3601 CGACAGCTTG CCAAAACAAC TGCTGACGGT ACAACTGTTT TGGGGCTTGT TAAAGCAGCA

3661 AAACACTTAA ATTTAAATGC CGAAGCTGTG CGTGCTGATA TGGATGCTTT GACAGCCTCA

3721 CAATTGCCAT TACCAGTCAT TGTTCATGTA TTCAAGAAAA ATAAGTTACC ACACTACTAT

3781 GTTGTCTATC AGGTAACTGA AAACGATTTA ATTATTGGTG ATCCTGATCC AACCGTTAAA

3841 ACCACTAAAA TATCGAAATC ACAATTTGCT AAAGAATGGA CCCAGATTGC AATTATCATA

3901 GCCCCAACAG TTAAATATAA ACCCATAAAA GAATCACGGC ACACATTAAT TGATCTAGTG

3961 CCTTTATTGA TTAAACAAAA AAGATTAATT GGACTAATTA TTACCGCAGC AGCTATAACA

4021 ACATTAATCA GTATTGCTGG TGCATATTTC TTTCAGTTAA TTATCGATAC TTATTTGCCG

4081 CACTTGATGA CTAATAGGCT TTCACTAGTT GCCATTGGTC TGATTGTAGC TTATGCTTTC

4141 CAAGCAATTA TCAACTATAT ACAAAGTTTT TTTACGATTG TATTAGGACA ACGTCTCATG

4201 ATCGACATCG TTTTAAAATA CGTTCACCAT CTTTTTGATT TACCAATGAA TTTTTTTACT

4261 ACCCGTCATG TCGGTGAAAT GACCTCACGC TTTTCTGATG CAAGCAAAAT TATTGATGCA

4321 CTTGGAAGTA CAACGCTCAC CCTTTTTTTA GACATGTGGA TTTTATTAGC AGTAGGGTTA

4381 TTTTTGGCCT ATCAAAACAT CAATTTATTT TTATGCTCGT TAGTTGTGGT TCCAATTTAC

4441 ATCTCGATTG TTTGGCTATT TAAAAAAACT TTTAATCGTT TAAATCAAGA TACAATGGAA

4501 AGCAATGCAG TTCTTAATTC TGCTATTATT GAAAGTCTCA GTGGCATAGA AACCATTAAA

4561 TCACTAACTG GTGAAGCAAC TACAAAAAAA AAGATTGACA CACTATTTTC TGACTTATTG

4621 CATAAAAACT TGGCTTATCA AAAAGCTGAT CAAGGACAAC AAGCTATCAA AGCAGCTACT

4681 AAATTAATCC TAACTATTGT TATCCTTTGG TGGGGTACTT TTTTTGTTAT GCGACACCAA

4741 CTGTCTTTAG GTCAGCTGTT AACTTATAAT GCTTTGCTCG CTTACTTCTT GACCCCATTA

4801 GAAAATATTA TTAATTTACA GCCTAAACTA CAAGCTGCCA GAGTGGCTAA TAATCGATTA

4861 AATGAGGTTT ATCTAGTAGA GTCTGAATTT TCTAAATCTA GGGAAATAAC TGCTCTAGAG

4921 CAACTAAATG GTGATATTGA GGTTAATCAT GTTAGTTTTA ACTATGGCTA TTGTTCTAAT

4981 ATACTTGAGG ATGTTTCTCT AACAATTCCA CATCATCAGA AGATTACTAT TGTAGGCATG

5041 AGTGGTTCGG GGAAAACGAC CCTAGCCAAG TTGCTAGTTG GTTTTTTTGA GCCTCAAGAA

5101 CAGCACGGTG AAATTCAGAT TAATCATCAC AATATATCTG ATATTAGTCG CACAATTTTA

5161 CGCCAATATA TTAATTATGT TCCTCAAGAA CCTTTCATTT TTTCGGGCTC TGTATTAGAA

5221 AATTTATTGT TAGGTAGCCG TCCTGGAGTA ACTCAACAAA TGATTGATCA AGCTTGTTCC

5281 TTTGCTGAAA TCAAAACTGA TATAGAAAAT TTGCCTCAAG GTTATCATAC TAGATTAAGT

5341 GAAAGTGGAT TCAACTTATC TGGTGGGCAA AAACAGCGGT TATCAATAGC TAGAGCATTA

5401 TTGTCTCCGG CACAATGTTT CATTTTTGAC GAATCAACCA GTAATTTAGA CACCATTACT

5461 GAACATAAAA TAGTCTCTAA GCTATTATTC ATGAAAGACA AAACGATAAT TTTTGTAGCA

5521 CATCGTCTCA ATATTGCGTC TCAAACCGAT AAAGTTGTCG TTCTTGATCA TGGAAAGATT

5581 GTTGAACAGG GATCACATCG ACAATTGTTA AATTATAATG GGTATTATGC ACGGTTAATT

5641 CATAATCAAG AATAGCCTGA CAAGAACCAG TCTGCTATTG ATAGACTATT CTTGTCCGTG

5701 AAATCCTCGC GTATTTCCGT GAGGAGCATA GTATATTTAG CGATCTTCAA ATTTTAAGTA

5761 TATTGATTCA TATGTTTATC CTCCTAAGTT TGAGGACAAA CCGGTACATG TTATAATACT

5821 AATAAATTCT ATGTGTAATT GCGCATTGAC CTTGTTTAAC TCGGTCTTAT GATAACCATA

5881 AAAACTCAAT TTTTGCCGTT GTTAAATGGA ATGTATTCTT AATTTAATTT ATGCTTACAC

5941 TTAACTGATT TTGTTATGCT TTAAATACTA AAAATTGTTG CATATCCCGC TGCTTTCAAC

6001 CAAATTTTTT TAATTGCTTT GTGCAGAGAA TCTTCATAAA CCTGGCAACA AAATTTGAAT

6061 AACTAACTTT AAGAACTGTA AATCTAAATT GAAATTATTA TTTTAATGTT AGGAGTAATT

6121 AAGCTGGACT AAAAGAAAGT ATTAAAACCA ATTGATGAAA TGCTTGTTGA TCCTTGGCAA

6181 GTTGATATTC AAGAATTGTT TGAAGCTTCT TTCAATGAAC CTGATGAGAT CAAAAGGAAC



6241 TTGTATGATT CCTTATACAC TTATGTTTTG CAAAAAAGAC AAGAAGATAT TATTAATCGT

6301 CCTGGCTTCG TTATTTAGAC CTCTAAAAGC CTGCTGAGGG CTTTTTGTTT TGCTTTGATA

6361 TAAATGTATA TGAATGGTCT TAAAATCGCT AGAAACGAAA AATAAGACCC TTAAAAACGA

6421 ACATAGCAGC TAAAATCTTT TTGAGATTCA AAAAACTAAC TGTTTGCTGT CAATGGTAGC

6481 GGACGAGCAA AGCGTGGGAG CATAAGGAAT TGACAGCTCT AAACCAGTCT TAACACTGAA

6541 TTGGCGAAAG CCAAAGTTTC TATAAAACTT TGCTTTCCTG CCTAACGGCG AGTGAAAAAG

6601 CGGTCAAGCT GGCTCAGCTT GGACGGGGTT CGGGGCGTTA GCGCCCGAAT TAATGTGGCT

6661 TGCCACACCT TTTAGGCAAC GAACAGAGTG AGGCGCAAGG AGCATAGCGA CTGGAGTTTA

6721 ATGTGAGCCC TGTTTTTTTG GG

>gi|386656287|gb|JQ434263.1| Enterococcus faecium strain Acr4 plasmid

insertion sequence ISLpl1 putative transposase TraISLpl1-like gene, partial

sequence

TTTAACCGGTATGCCCAAAGAAACCGTGATTTTCAATCGATTAAAGTTAATAAAACTGCCTTACTATGC

CCACGAGGACCAACGACTGTATCTAGTTCAAAATCGCCGATGCGATTACGTTGATTAATCATCATGGGA

CGCTGTTCAATTGATCGCCCCAAAGATTGATTATATTTGGATCGTTGGTCAACGTTACGCCGTTGGCGT

ACGCCATGTTCAGGTAGATCATTCAAGGAGAAACCAATTCTCCCCTGATTTAGCCAATTATAAATAGAT

T

> gi|386656284|gb|JQ434262.1| Enterococcus faecium strain Acr4 plasmid MobC

(mobC) and MobA (mobA) genes, complete cds.

ACCESSION JQ434262

VERSION JQ434262.1 GI:386656284

KEYWORDS

SOURCE Enterococcus faecium

ORGANISM Enterococcus faecium

Bacteria; Firmicutes; Lactobacillales; Enterococcaceae;

Enterococcus.


AUTHORS Devi,S.M. and Halami,P.M.

TITLE Detection of mobile genetic elements in the intergeneric pediocin

PA-1 bacteriocin in Enterococcus faecium NCIM 5423

JOURNAL Unpublished


AUTHORS Devi,S.M. and Halami,P.M.

TITLE Direct Submission

JOURNAL Submitted (22-JAN-2012) Food Microbiology Department, Central

Food

Technological Research Institute, Chavalamba Mansion, Mysore,

Karnataka 500020, India

FEATURES Location/Qualifiers

source 1..1290




/organism="Enterococcus faecium"

/mol_type="genomic DNA"

/strain="Acr4"

/isolation_source="fermented carrot"

/culture_collection="NCIM:5423"

/db_xref="taxon:1352"

/plasmid="unnamed"

gene 14..394

/gene="mobC"

CDS 14..394

/gene="mobC"

/note="putative mobilization protein"

/codon_start=1

/transl_table=11

/product="MobC"

/protein_id="AFJ19237.1"

/db_xref="GI:386656286" /translation="MSEQNQNLASDPSKKYTYRSEPKQISFRVSESEFAKLKQSAEAL QMSVPAFVKAKAQNARLVTPKVAPDIAQAIARDLAKAGGNINQIAKWCNTHQHDVAPGDAQRLSENLKI

MQKELQKIWQQLK"

gene 376..1290

/gene="mobA"

CDS 376..1290

/gene="mobA"

/note="relaxase/mobilization nuclease domain protein"

/codon_start=1

/transl_table=11

/product="MobA"

/protein_id="AFJ19236.1"

/db_xref="GI:386656285" /translation="MATVKVSRTTSCSRAINYAEPRATVKTGINCDIDYAKSEMKQIR MLYGKDDHVQAHLLIQSFRPGEITAEKANQLGKEYAEKIAPEHQIAIYTHTDKDHIHNHIVINSVNLET

GKKFQAHGQAFLDKCYDINDEICLTHGLSITERGKKEEKRTMSEIKLKEKNEPVWKDEIRFAIDQTMKN

PKTRTYDQFCDSLKIFGIHCFNRGKNFTYELIKKKKKVRSNKLGKDYEKETILREPDRREQTYNQQRTN

ELRRARASIRETAEEVRREQRTLTPEQPEIRPARRNHEQHVERGTDHQQELSL"

ORIGIN

1 AAGGGTGGGA CTTATGAGCG AACAAAATCA AAATTTGGCT AGCGATCCTT CTAAAAAATA

61 TACCTATCGA TCCGAGCCAA AACAAATCAG TTTTCGAGTG AGCGAATCCG AATTTGCAAA

121 GCTGAAGCAG TCAGCTGAAG CTTTGCAAAT GAGTGTGCCG GCTTTCGTGA AAGCCAAGGC

181 ACAAAACGCG CGTCTGGTGA CACCGAAAGT GGCGCCAGAC ATTGCCCAGG CAATCGCGCG

241 CGATTTAGCC AAAGCTGGAG GGAATATCAA TCAAATTGCT AAGTGGTGCA ACACACATCA

301 ACATGACGTT GCTCCTGGAG ACGCCCAACG CTTATCCGAA AACCTAAAAA TCATGCAAAA

361 GGAGCTACAA AAGATATGGC AACAGTTAAA GTAAGTCGAA CCACTTCATG TAGCCGAGCA












421 ATCAATTACG CAGAACCACG AGCAACCGTC AAAACAGGAA TAAACTGTGA CATTGATTAT

481 GCCAAAAGTG AAATGAAACA AATACGTATG CTTTACGGAA AAGACGATCA TGTACAAGCA

541 CACTTATTGA TTCAGTCCTT TCGACCAGGA GAAATTACCG CCGAAAAAGC CAACCAGCTA

601 GGAAAAGAAT ACGCTGAAAA AATCGCACCA GAACACCAAA TCGCGATTTA TACGCACACG

661 GACAAAGACC ATATCCACAA TCACATTGTC ATTAATTCGG TCAATTTAGA AACAGGAAAA

721 AAGTTTCAAG CACATGGTCA AGCATTCCTA GATAAGTGTT ACGATATCAA CGACGAAATT

781 TGTTTGACTC ATGGTTTAAG TATCACTGAA CGAGGAAAAA AAGAAGAAAA ACGAACTATG

841 TCTGAAATCA AACTAAAAGA AAAAAATGAA CCTGTTTGGA AAGACGAAAT TCGCTTTGCC

901 ATTGATCAAA CCATGAAAAA TCCAAAAACC AGAACCTATG ACCAATTTTG TGATTCCCTC

961 AAAATATTTG GGATTCATTG CTTCAATCGT GGGAAAAATT TTACTTACGA ATTAATCAAA

1021 AAAAAGAAAA AAGTACGATC CAACAAACTA GGAAAGGACT ATGAAAAGGA GACAATTTTA

1081 CGTGAGCCGG ACAGACGAGA ACAAACCTAT AACCAACAGC GAACCAACGA GCTACGAAGA

1141 GCTCGAGCTA GCATTCGCGA AACTGCAGAA GAAGTACGAA GAGAACAACG AACTCTTACG

1201 CCTGAACAAC CGGAAATTAG ACCGGCTCGA AGAAATCATG AACAACACGT TGAAAGAGGA

1261 ACAGACCACC AACAAGAATT ATCGCTCTAA

Detection and Characterization of Pediocin PA-1/AcH likeBacteriocin Producing Lactic Acid Bacteria

S. Manjulata Devi • Prakash M. Halami

Received: 19 March 2011 / Accepted: 30 May 2011

� Springer Science+Business Media, LLC 2011

Abstract Fifty-five bacteriocinogenic lactic acid bacteria

(LAB) isolated from seven different sources. Eight isolates

were found to produce pediocin PA-1 like bacteriocin as

detected by pedB gene PCR and dot-blot hybridization.

The culture filtrate (CF) activity of these isolates exhibited

strong antilisterial, antibacterial activity against tested

food-borne pathogens and LAB. The identification and

genetic diversity among the selected LAB was performed

by conventional morphological and molecular tools like

RFLP, RAPD, and 16S rDNA gene sequencing. The iso-

lates were identified as, 1 each of Pediococcus acidilactici

Cb1, Lactobacillus plantarum Acr2, and Streptococcus

equinus AC1, 2 were of P. pentosaceus Cb4 and R38, and

other 3 were Enterococcus faecium Acr4, BL1, V3. Partial

characterization of the bacteriocins revealed that the pep-

tide was heat-stable, active at acidic to alkaline pH, inac-

tivated by proteolytic enzymes, and had molecular weight

around 4.6 kDa and shared the properties of class IIa

pediocin-family. The bacteriocin production at different

temperatures, pH, and salt concentrations was studied to

investigate the optimal condition for application of these

isolates as a starter culture or as a biopreservative in either

acidic or non-acidic foods.

Introduction

The pediocin PA-1/AcH (pediocin PA-1) represent a class

IIa bacteriocin of low molecular weight, unmodified anti-

listerial peptides with a consensus motif of YGNGVXC at

their N-terminal end [12]. Among all the class IIa

bacteriocins, pediocin PA-1 is widely distributed and is more

potent in inhibiting the growth of several pathogens associ-

ated with food spoilage and food related health hazards and

hence can be a potential food bio-preservative agent [14].

Pediocin PA-1 is a plasmid encoded bacteriocin initially

characterized from the strains of P. acidilactici PAC 1.0

[8]. Subsequently, other species viz P. pentosaceus,

P. parvulus and other genera viz. Lactobacillus plantarum

and B. coagulans were reported for the production of same

bacteriocin where in different environmental conditions are

known to influence bacteriocin production [3, 5, 7, 10]. The

gene organization and sequences of pediocin PA-1 operon

were found to be highly conserved and resides on plasmid

size that ranges from 9 to 14 kb [10]. These reports are in

concurrent observation that distribution of pediocin PA-1

operon among different bacteria took place by integration

into the native plasmids [7]. In order to study such transfer,

there is a need for detection and characterization of large

number of pediocin PA-1 producers from different sources.

Although pediocin PA-1 producers are reported from

different LAB, specific isolation of intergeneric and inter-

specific pediocin PA-1 like bacteriocin producers are not

reported. Hence, in this study an attempt was made for the

rapid detection of pediocin PA-1 like bacteriocin producers

in different genera and species of LAB by using molecular

tools. Influence of cultural conditions for the production of

pediocin PA-1 like bacteriocin was also investigated.

Materials and Methods

Bacterial Strains and Maintenance

Standard pediocin PA-1 producers viz. Pediococcus acid-

ilactici PAC1.0 [8], P. acidilactici K7 [6] and enterocin A

S. M. Devi � P. M. Halami (&)

Department of Food Microbiology, CFTRI, Mysore, India

e-mail: [email protected]

123

Curr Microbiol

DOI 10.1007/s00284-011-9963-8

producer Enterococcus faecium MTCC 5153 (MTCC,

Chandigarh, India) were used in this study. All the above

LAB cultures as well as P. acidilactici DK7, P. acidilactici

DPAC1.0 (plasmid cured strains, obtained by novobiocin

treatment), Leuconostoc mesenteroides NRRL B640

(NRRL, Peoria, USA) and the LAB isolates of the study were

grown in de Man, Rogosa and Sharpe (MRS) broth or on

MRS agar (Hi Media, Mumbai, India) at 37�C in static

condition. The food-borne pathogenic indicator strains viz.

Listeria monocytogenes ScottA, L. innocua FB 21, and

L. murrayi FB 69 (obtained from Dr. AK Bhunia, Purdue

University, USA); Aeromonas hydrophila NRRL B445;

Yersinia entericolitica MTCC859 and Escherichia coli

MTCC118, Staphylococcus aureus FR1722, Salmonella

typhi FB231, and S. paratyphi FB254 (from Dr. E. Noterman,

National Institute of Public health, Netherlands), were grown

in Nutrient broth or BHI broth (Hi Media, Mumbai) at 37�C

under shaking (200 rpm). The above mentioned strains were

maintained at -40�C in lactobacilli MRS media and BHI or

Nutrient media with 40% glycerol (v/v). Before being used,

strains were propagated twice in their respective broth.

Isolation of Bacteriocinogenic LAB

The isolation of antilisterial bacteriocin producing LAB from

fermented vegetable sources like carrot, cucumber, beans, and

betel leaves was performed using ScottA as indicator descri-

bed previously [6]. The other sources like fermented milk

(curd) and chicken intestine sample were diluted and pour

plated and observed for zone of inhibition against ScottA, and

further characterized as described previously [6].

PCR Amplification of Pediocin PA-1 Genes

Total DNA from LAB was isolated as described by Mora

et al. [11]. All the oligonucleotide primers were obtained

from Sigma-Aldrich (Bangalore, India) and the PCR

components were from Bangalore GeNei (Bangalore). The

pedB gene was amplified by using primers, pedBF (50GG

TGATTTTATGAATAAGACTAAGTCG30) and pedBR

(50CCCCTTTATCAGTACTATTGGCTAGGC30) posi-

tioned at 3488–3514 and 3823–3849 as per the sequence of

pSMB74 of P. acidilactici H (Accession number-U02482).

The standard procedure for PCR amplification was fol-

lowed as described by Sambrook and Russell [15] with

annealing at 60�C. Similarly, pedAB gene was amplified as

described earlier [6]. All the PCR amplicons were analyzed

by 1.5% agarose (SRL, Mumbai, India) gel electrophoresis.

DNA Dot-Blot Hybridization

The PCR product of pedB gene obtained from P. acidilactici

PAC1.0 was labelled with digoxigenin-dUTP using random

primed DNA labeling kit (Roche Chemicals, Germany) and

used as a probe for dot-blot analysis. Ten microlitre of total

DNA (approximately 25–50 ng ll-1) of test culture was

heat denatured, spotted on a Hybond Nylon membrane

(Amersham International, UK) according to the method

described earlier [15] and hybridized using probe. Hybrid-

ization and stringency washes were carried out at 42�C

according to manufacturer instructions (Roche chemicals,

Germany).

Phenotypic and Biochemical tests

The Gram-staining, catalase, fermentation of carbohydrate

viz. 1% each of glucose, lactose, maltose, sucrose, man-

nitol, sorbitol, etc., gas production from glucose was per-

formed as per standard microbiological methods. Growth

of test cultures in MRS broth at different temperatures (10,

37, and 45�C) was evaluated upon incubation for 16 h.

Similarly, growth in MRS broth containing 5 and 8% NaCl

and at pH (4, 8, and 10) was also tested.

RAPD, RFLP, and 16S rDNA Gene Sequencing

Random Amplified Polymorphic DNA (RAPD) PCR of

total DNA was carried out by primer M13 (50GAG-

GGTGGCGGTTCT30) in a 25 ll reaction volume as

described earlier [17]. Digestion of 16S rDNA gene PCR

product with HaeIII and AluI enzymes (Bangalore GeNei,

Bangalore) for Restriction fragment length polymorphism

(RFLP) analysis was performed. The primers and the PCR

conditions used for amplification were followed as

described earlier [13]. DNA sequences of 16S rDNA PCR

product was sequenced at the sequencing facility of Vimta

Labs (Hyderabad, India). The gene sequences obtained

were analyzed using the BLAST search programme [1].

Antibacterial Activity Assay

The test cultures were grown in MRS broth at 37�C for

16 h under static condition. The cultures were centrifuged

at 9000 rpm in 4�C for 15 min and the culture filtrate (CF)

was collected, filtered through 0.4 l filter (Millipore) and

stored at 4�C until further use. The inhibitory effect of the

CF was tested against food-borne pathogens and LAB

cultures by spot-on-lawn assay [4].

Characteristics of Antimicrobial Compound

The CF of the test culture was subjected to treatment

with different proteolytic enzymes such as proteinase K,

papain, trypsin, pepsin (SRL) at a final concentration of

1 mg ml-1, reducing agents (conc. 10%) like b-mercap-

toethanol (SRL) and Dithiothreitol (DTT) (SRL) were also

S. M. Devi, P. M. Halami: Pediocin PA-1/AcH like Bacteriocin Producing Lactic Acid Bacteria

123

used. Reaction mixture was incubated at 37�C for 2 h and

residual activity was determined using ScottA, as described

previously [13]. Similarly, stability of CF at different

temperature and varying pH was also tested as above. The

chloroform extracted bacteriocin from CF was subjected

for bioassay by Tricine–SDS-PAGE [16] and overlaid with

ScottA.

Bacteriocin Production at Different Temperatures, pH,

and NaCl Concentration

MRS broth with pH of 4, 8, and 10 (adjusted by HCl or

NaOH), as well as MRS with 4 and 8% sodium chloride

(w/v) (SRL) was prepared and inoculated with 1% freshly

grown test cultures and allowed growth for 16 h at static

conditions at 37�C. Growth (OD 600 nm) and bacteriocin

production of test cultures in MRS broth at different tem-

peratures (15, 37, and 50�C) was also studied as described

above. Antilisterial activity expressed as arbitrary unit per

ml (AU ml-1) and defined as the highest dilution of test

sample exhibiting the zone of inhibition against indicator

ScottA.

Results and Discussion

Detection of Putative Pediocin PA-1 like Bacteriocin

Producing LAB

In order to study intergeneric and interspecific pediocin

PA-1 producers, we have screened large number of antilis-

terial bacteriocin producing LAB. Among the screened

sources, the LAB isolated from vegetables displayed strong

antilisterial activity. From each representative source, the

cultures with high activity were selected and subsequently

tested for the presence of immunity protein of pediocin PA-1

(pedB) gene by PCR. Among 55, 8 cultures gave expected

amplicons of 362 bp for pedB and 600 bp for pedAB genes.

The results of CF activity against ScottA and pedB PCR

analysis of the selected native isolates is shown in Fig. 1a, b.

PCR results were additionally confirmed by dot-blot

hybridization using pedB gene probe. As expected, cultures

K7, BL1, Acr2, Acr4, Cb1, Cb4, V3, AC1, and R38 gave

positive signal suggesting a conserved pediocin PA-1 gene

in native isolates, whereas, E. faecium MTCC 5153 and

DPAC1.0 did not react with the probe (data not shown). The

detection of pediocin PA-1 by molecular tools was earlier

reported in P. parvulus [3] and P. acidilactici [9].

Characteristics of Native LAB

All the selected isolates were Gram-positive, catalase

negative and cocci in shape except the isolate Acr2, which

was rod shaped. The gas production was observed only for

Acr2 and V3 isolate. The isolate Acr2, was unable to grow

at 45�C, 8% NaCl and at pH 10. Similarly AC1 isolate was

unable to grow in 8% NaCl concentration, 10 and 45�C

temperatures and also at pH 4 and 10. The isolates were

able to ferment different carbohydrates tested, except AC1

which could not utilize lactose. The other isolates were

able to grow at all the parameters used. These results

suggested that, the isolates had distinct characteristic

features.

Molecular Typing of Putative Pediocin PA-1 Producers

In order to differentiate the isolates among each other and

also from native P. acidilactici K7, RAPD PCR was per-

formed. RAPD showed five different banding profiles

indicating, the selected isolates were different from each

other. Similarly, RFLP also showed variability at their 16S

rDNA gene (Fig. 2a, b). For species level identification,

16S rDNA gene sequencing followed by BLAST analysis

was performed. DNA sequence homology in combination

with the results of physiological and biochemical tests, the

putative pediocin producers were identified as follows—

Streptococcus equinus AC1, Pediococcus acidilactici Cb1,

Pediococcus pentosaceus Cb4 and R38, Lactobacillus

plantarum Acr2, Enterococcus faecium Acr4, BL1, and

V3. The 16S rDNA gene sequences (*700 bp) have been

deposited in the GenBank database under the Accession

numbers GU222444–GU222450 for the LAB strains AC1,

Cb1, Cb4, Acr2, V3, BL1, and Acr4, respectively. The

bacteriocin producing LAB isolates reported in this study

are deposited in the National Collection of Industrial

Microorganisms (NCIM) at the National Chemical Labo-

ratory, Pune, India.

Antibacterial Spectrum and Properties of Bacteriocin

All test isolates were studied for their ability to inhibit

various food-borne pathogens as well as closely related

LAB species like E. faecium 5153 and Leuconostoc mes-

enteroides NRRL B640. The tested isolates were able to

inhibit all the Listeria spp., mutants of K7 and PAC 1.0 as

well as Gram-negative Aeromonas and Yersinia sp, with an

inhibition zone size of around 10–18 mm. Isolates St.

equinus AC1, Lb. plantarum Acr2, E. faecium Acr4, and

P. pentosaceus R38 were also able to inhibit Gram-positive

Staphylococcus aureus. None of the isolates inhibited

P. acidilactici K7, PAC 1.0, as well as Escherichia coli and

Salmonella typhi. Inhibitory spectra of pediocin PA-1 to

selected Gram-positive bacteria were earlier reported

[12, 14]. The Gram-negative Aeromonas hydrophila B445

was similarly inhibited by pediocin SA1 [2]. In general, an

antibacterial spectrum of Cb4, Acr2, Acr4, BL1, and V3


123

was found to be different than the pediocin PA-1 producing

in P. acidilactici K7.

The protease sensitivity and inactivation by reducing

agents suggested the proteinaceous nature and involvement

of disulfide bridge, respectively, of the AMC. Optimum

activity of CF for most of the isolate was found to be at pH

7–8 and temperature between 40 and 80�C. However, at pH

2–4 and temperature at 100 and 121�C, the activity was

reduced to *50%. Antilisterial activity was retained at

even pH 10 and at 90�C suggesting heat stable and wide pH

range AMC. Upon Tricine SDS-PAGE analysis, all isolates

had the active peptide of 4.6 kDa (data not shown). The

above reported observations for the selected isolates are

similar to that described for pediocin PA-1 [14].

Effect of Cultural Conditions on Production

of Bacteriocin

The bacteriocin production for all the cultures was more at

37�C when compared to 15 and 50�C. All the strains of

E. faecium V3, Acr4, BL1 were able to grow and produce

bacteriocin at all the temperatures, pH, and NaCl concen-

tration, this could be due to the fact that E. faecium has wider

adaptability to environment. The isolate P. acidilactici K7,

Cb1, and Lb. plantarum Acr2 were able to grow at 15 and

37�C. The isolate St. equinus AC1 was unable to grow at 15

and 50�C, pH 4 and 10, and at 8% NaCl concentration.

Similarly, the isolate Lb. plantarum Acr2 was unable to

grow at pH 4 and 10 and did not produce bacteriocin

(Fig. 3). The bacteriocin production is greatly influenced by

the nutrients, temperature, pH, NaCl concentration [4]. The

optimum condition for pediocin AcH, SA-1, and other class

IIa bacteriocins was found to be at temperature 30–35�C, pH

5–7, and NaCl 1.5–3% [12]. Results obtained suggested that

these native isolates could be used as a protective culture in

acidic foods like pickles and yogurt, as they exhibited good

growth and bacteriocin production at different cultural

conditions.

In conclusion, we detected the presence of pediocin

PA-1 gene cluster and PA-1 like bacteriocin properties in

E. faecium and St. equinus besides P. pentosaceus and

Lb. plantarum of vegetable and dairy origin. The molecular

typing tools have proven to be useful in differentiation,

characterization, and identification of LAB in spite of their

high heterogeneity and phylogenetic inter-mixing. These

cultures can be further used to study the pediocin PA-1

operon integration, revealing the possible mechanism of

horizontal operon transfer as reported for B. coagulans I4

and Lb. plantarum 423 [7, 18]. We are presently investi-

gating the flanking regions of the operon to discover the

Fig. 1 Detection of antilisterial

activity of native LAB isolates

by spot-on-lawn assay (a), pedB

gene specific PCR (b). a, b 1K7, 2 Cb1, 3 Cb4, 4 R38, 5AC1, 6 Acr4, 7 Acr4, 8 BL1, 9V3. b 10 MTCC 5153 (Negative

control), M 10 Kb molecular

marker

Fig. 2 Differentiation of the native pediocin PA-1 like bacteriocin

producers by RAPD PCR (a) and RFLP of 16S rDNA gene digested

with HaeIII and AluI (b). a Lane 1 AC1, 2 K7, 3 Cb1, 4 Cb4, 5 Acr2, 6

BL1, 7 V3, 8 Acr4, 9 R38, 10 PAC1.0 b Lane 1 K7, 2 PAC1.0, 3 Cb1, 4Cb4, 5 BL1, 6 Acr2, 7 Acr4, 8 V3, 9 R38, 10 AC1, 11 MTCC 5153. M is

a 10 Kb molecular marker (GeNei, Bangalore) in both the gels


123

novel mobile genetic elements involved in such recombi-

nation events. Hence, Pediocin PA-1 produced by LAB

other than P. acidilactici can be of industrial significance in

different food systems because of their wider environ-

mental adaptability.

References

1. Altschul SF, Maddan TL, Schaffer AA, Zhang J, Zhang Z, Miller

W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new

generation of protein database search programs. Nuc Acids Res

25:3389–3402

2. Anastasiadou S, Papagianni M, Filiousis G, Ambrosiadis I,

Koidis P (2008) Pediocin SA-1, an antimicrobial peptide from

Pediococcus acidilactici NRRL B5627: production conditions,

purification and characterization. Biores Technol 99:5384–5390

3. Bennik MHJ, Smid EJ, Gorris LGM (1997) Vegetable-associated

Pediococcus parvulus produces pediocin PA-1. Appl Environ

Microbiol 63:2074–2076

4. Biswas SR, Ray P, Johnson MC, Ray B (1991) Influence of

growth conditions on the production of a bacteriocin, pediocin

AcH, by Pediococcus acidilactici H. Appl Environ Microbiol

57:1265–1267

5. Ennahar S, Aoude-Werner D, Sorokine O, van Dorsselaer A,

Bringel F, Hubert JC, Hasselmann C (1996) Production of ped-

iocin AcH by Lactobacillus plantarum WHE92 isolated from

cheese. Appl Environ Microbiol 62:4381–4387

6. Halami PM, Ramesh A, Chandrashekar A (2005) Fermenting

cucumber, a potential source for the isolation of pediocin-like

bacteriocin producers. World J Microbiol Biotech 21:1351–1358

7. Le Marrec C, Hyronimus B, Bressollier P, Verneuil B, Urdaci

MC (2000) Biochemical and genetic characterization of coagulin,

a new antilisterial bacteriocin in the pediocin family of bacteri-

ocins, produced by Bacillus coagulans I4. Appl Environ Micro-

biol 66:5213–5220

8. Marrug JD, Gonzalez CF, Kunka BS, Ledeboer AM, Pucci MJ,

Tooner MY, Walker SA, Zoetmulder LCM, Vandenbergh PA

(1992) Cloning, expression, and nucleotide sequence of genes

involved in production of pediocin PA-I, a bacteriocin from

Pediococcus acidilactici PAC1.0. Appl Environ Microbiol 58:

2360–2367

9. Martinez JM, Martinez MI, Herranz C, Suarez AM, Cintas LM,

Fernandez MF, Rodriguez JM, Hernandez PE (2000) Use of

genetic and immunological probes for pediocin PA-1 gene

detection and quantification of bacteriocin production in Pedio-coccus acidilactici strains of Meat origin. Food Agric Immunol

12:299–310

10. Miller KW, Ray P, Steinmetz T, Hanekamp T, Ray B (2005)

Gene organization and sequences of pediocin AcH/PA-1 pro-

duction operons in Pediococcus and Lactobacillus plasmids. Lett

Appl Microbiol 40:56–62

11. Mora D, Fortina MG, Parini C, Daffonchio D, Manachini PL

(2000) Genomic sub-populations within the species Pediococcusacidilactici detected multilocus typing analysis: relationships

between pediocin AcH/PA-1 producing and non-producing

strains. Microbiol 146:2027–2038

12. Papagianni M, Anastasiadou S (2009) Pediocins: The bacterio-

cins of pediococci. Sources, production, properties and applica-

tions. Microb Cell Fact 8:1–16

13. Rai AK, Bhaskar N, Halami PM, Indirani K, Suresh PV,

Mahendrakar NS (2009) Characterization and application of a

native lactic acid bacterium isolated from tannery fleshings for

fermentative bioconversion of tannery fleshings. Appl Microbiol

Biotechnol 83:757–766

14. Rodriguez JM, Martinez MI, Kok J (2002) Pediocin PA-1, a

wide-spectrum bacteriocin from lactic acid bacteria. Cr Rev Food

Sci Nutr 42:91–121

15. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory

manual, 3rd edn. Cold Spring Harbor, New York

16. Schagger H, Von Jagow W (1987) Tricine-sodium dodecyl sul-

fate-polyacrylamide gel electrophoresis for separation of proteins

in the range from 1 to 100 kDa. Anal Biochem 166:368–379

17. Schillinger U, Yousif NMK, Sesar L, Franz CMAP (2003) Use of

group-specific and RAPD-PCR analyses for rapid differentiation

of Lactobacillus strains from probiotic yogurts. Curr Microbiol

47:453–456

18. Van Reenen CA, Van Zyl WH, Dicks LMT (2006) Expression of

the immunity protein of plantaricin 423, produced by Lactobacillusplantarum 423, and analysis of the plasmid encoding the bacte-

riocin. Appl Environ Microbiol 72:7644–7651

Fig. 3 Production of bacteriocin by native isolates at various

temperatures (a), NaCl concentration (b), and pH (c). Bacteriocin

production indicated in bars and was determined by CFS activity

(KAU ml-1) against ScottA. Results are the mean of ±SD (n = 2

trails)


123

Journal of Basic Microbiology 2012, 52, 1–7 1

© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jbm-journal.com

Research Paper

Detection of mobile genetic elements in pediocin PA-1 like producing lactic acid bacteria

Sundru Manjulata Devi and Prakash M. Halami

Food Microbiology Department, CSIR-Central Food Technological Research Institute, Mysore 570020, India

To evaluate the presence of mobile genetic elements in intergeneric and interspecific pediocin producing lactic acid bacteria (LAB) the flanking regions of the pediocin PA-1/AcH (pediocin PA-1) operon was characterized. In Enterococcus faecium Acr4 and Lactobacillus plantarum Acr2 a variation in the amplicon size in the downstream region of the operon was identified, suggesting a deletion in this region. Beyond that, in pediocin PA-1 encoding plasmids mobile genetic elements such as ISLpl1 and mobilization regions were detected by Southern hybri-dization analysis. Phylogenetic analyses of the E. faecium Acr4 ISLpl1 gene sequence suggested the gene transfer from lactobacilli in the environment. The tyrosine recombinase detected in pediocin plasmids of P. acidilactici H and K7 indicate a possible transfer of the entire operon among LAB. Since these elements are known to be associated with transfer of genes linked to the bacteriocin production, antibiotic resistance and sugar utilization, we suggest similar mechanism for natural spread of pediocin PA-1 operon among different bacterial species.

Keywords: Lactic acid bacteria / Pediocin PA-1 / Mobile genetic elements / ISlpl1 / Plasmids

Received: February 08, 2012; accepted: April 21, 2012

DOI 10.1002/jobm.201200079

Introduction*

Lactic acid bacteria (LAB) is a heterogenous group of bacteria with complex nutritional requirements. The majority of LAB species have multiple requirements for amino acids and vitamins. They have been found asso-ciated with animal oral cavities and intestines, plant leaves as well as decaying plant or animal matter such as rotting vegetables, fecal matter, and compost. LAB are masters in environmental adaptation indicated by their association with loss and gain of a gene or a set of genes [1]. Horizontal gene transfer (HGT) or lateral gene transfer is very common in LAB due to the presence of mobile genetic elements (MGEs) like transposons, inser-tion elements, conjugative and mobilizable plasmids [1, 2]. The mobilization (Mob) proteins and the insertion elements like ISLpl1 of Lactobacillus plantarum, ISL2 in L. helveticus, ISL3 in L. delbrueckii etc. are usually linked to the bacteriocin production, sugar utilization, antibi-

Correspondence: Prakash M. Halami, Food Microbiology Department, Central Food Technological Research Institute, Mysore 570020, India E-mail: [email protected] Phone: +91-821-2517539 Fax: +91-821-2517233

otic resistance, cold shock protein etc. and are found to be transferred among LAB by a means of HGT [1, 3–5]. Pediocin PA-1/AcH (pediocin PA-1), represents a class IIa bacteriocin produced by Pediococcus acidilactici. The pediocin PA-1 operon of size 3.5 Kb is localized on the plasmids of 9 to 14 Kb and found to be highly con-served [6]. The natural spread of pediocin PA-1 like bac-teriocin encoding plasmid into other bacterial strains of LAB and non-LAB was reported earlier [6–8]. Mlalazi et al. [9] detected the pediocin PA-1/AcH structural genes in L. casei, L. paracasei and L. rhamnosus, but the flanking regions of the pediocin operon was not analyzed. How-ever, reports indicating possible mechanism of plasmid integration from the host bacterium to the recipient with reference to pediocin PA-1 operon are lacking. Several of the gene clusters of lantibiotics like nisin, lacticin 3147 and 481 are located on transposons or composite transposons and are found to transfer among LAB [10]. The possibility in transfer of natural pediocin plasmids in other LAB can be interesting to develop industrially important starter cultures, because of their broad spectrum activity after nisin. The distribution of the mob gene and ISLpl1in the pe-diocin plasmids among LAB has been reported in pedio-

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2 S. M. Devi and P. M. Halami Journal of Basic Microbiology 2012, 52, 1–7


cin AcH producer in Bacillus coagulans I4, L. plantarum 423 and L. plantarum WHE 92 [7, 8, 11] and also in bac-teriocin-negative mutant of Ruminococcus gnavus E1 [10]. In the present work, we evaluated the MGEs in the intergeneric as well as interspecific LAB by molecular methods like PCR and Southern hybridization. The possible role in natural distribution of pediocin PA-1 production among LAB is also discussed.

Materials and methods

Bacterial strains and maintenance Standard pediocin PA-1 producers P. acidilactici K7 [12], P. acidilactici Cb1, E. faecium Acr4, P. pentosaceus Cb4, and L. plantarum Acr2 producing pediocin PA-1 like bacterio-cin [13] were used. All strains were grown at 37 °C un-der static condition in deMan, Rogosa and Sharpe (MRS) broth (HiMedia, Mumbai, India). Listeria monocytogenes ScottA (obtained from Dr. AK Bhunia, Purdue Univer-sity, USA) used as indicator for bacteriocin activity was grown in brain hearth infusion (BHI) broth (HiMedia) at 37 °C under shaking conditions The culture mainte-nance and growth conditions were followed accordingly as described earlier [13].

Profiling and curing of plasmid DNA For plasmid extraction, one ml of 16 h culture grown in MRS broth was harvested by centrifugation and the cell pellet was resuspended in 379 μl lysis buffer (8% su-crose, 50 mM Tris-HCl, 1 mM EDTA [pH 8.0], 97 µl of 20 mg ml–1 lysozyme [Sigma, Bangalore, India] and 10 U of mutanolysin (Sigma) and incubated for 1 h at 37 °C. Subsequent isolation of plasmid DNA was performed according to the protocol of Anderson and McKay [14]. Plasmid curing by 10 µg ml–1 of novobiocin (SRL, Mum-bai) was done as described by Miller et al. [6]. The colo-nies that were unable to produce zone of inhibition on the lawn of Listeria monocytogenes ScottA were purified and considered as plasmid cured strains. Concomitantly loss of plasmid was confirmed by plasmid DNA profil-ing as described above and was compared with the parental culture.

Southern transfer and hybridization After gel electrophoresis on 0.7% agarose, plasmid DNAs were transferred to Hybond-N+ nylon membrane with 10X SSC solution as described by Sambrook and Russell [15]. Probe labeling and hybridization of the pediocin immunity gene (pedB), ISLpl1 element and mob gene was done with the DIG high prime DNA labeling and detection kit II (Roche Inc., Germany).

PCR amplification of bacteriocin encoding genes, ISLpl1 mobile insertion element and mob gene The bacterial cultures P. acidilactici K7, P. acidilactici Cb1, E. faecium Acr4, P. pentosaceus Cb4, and L. plantarum Acr2 were subjected to the amplification of pediocin PA-1 gene cluster and its flanking regions. Primers were designed based on DNA sequence of pSMB74 (Accession number U02482), a plasmid encoding the production of pediocin AcH in Pediococcus acidilactici. For amplification of flanking region of pediocin PA-1 operon inverse PCR was performed by using the XT 20 PCR system kit (GeNeiTM, Bangalore). The standard procedure for PCR amplification was followed as described by Sambrook and Russell [15]. Similarly, the amplification for the ISLpl1 and mobilization genes was performed. PCR pri-mers and required annealing temperatures are enlisted in Table 1.

Nucleotide sequences analysis, phylogenetic tree construction and accession numbers Amplified PCR products were purified with the Qiagen gel purification kit (Qiagen, Germany) and were sent to Vimta Labs (Hyderabad, India) for sequencing. The chromatograms were examined with the Chromas Lite version 2.01 (Technelysium Pty Ltd, Australia) and the processed sequences were aligned with other sequences available in NCBI database by using GeneDoc software (http://www.psc.edu/biomed/genedoc). The sequence ho-mology search was done with the BLAST programme [16]. Neighbour joining tree were constructed in MEGA 5.0 using Kimura-2 parameter model [17]. The partial sequence of pediocin harboring plasmid (around 6.7 Kb), mob region and ISLpl1 element of E. faecium Acr4 was deposited in GenBank under the accession nos. HQ876214, JQ434262 and JQ434263, respectively.

Results

Plasmid profiling of LAB and bacteriocin production In plasmid profiling, PA-1 like producing isolates K7, Acr4, Acr2, Cb1, Cb4 showed the presence of low and high molecular sized plasmids in the range of 50 to 3.0 Kb (Fig. 1A). The pediocin PA-1 immunity gene (pedB) of K7 showed 100% homology to pSMB74. A strong signal around 8 and 3 Kb plasmids was observed for K7, Cb1 and Cb4, whereas in Acr4 12, 7 and 3 Kb plasmids were detected to encode the immunity gene. Similarly, the isolate Acr2 showed a signal at around 5 Kb. Plasmid curing experiments with novobiocin resulted in the loss of anti-listerial activity. PCR ampli-fication analysis with pediocin specific primers gave no

Journal of Basic Microbiology 2012, 52, 1–7 Mobile genetic elements in lactic acid bacteria 3


Table 1. Primers used for PCR amplification and sequencing of pediocin harboring plasmids.

Positions as per pSMB74

Primer sequences (5′- 3′) Expected product size (bp)

Annealing temperature (°C)

Reference

3488–3514 PedBF- GGTGATTTTATGAATAAGACTAAGTCG 362 60 3828–3849 PedBR- CCCCTTTATCAGTACTATTGGC

13

3126–3144 Ped1F- GTTGATAGGCCAGGTTTCA 5072–5091 Ped2R- TCATCAAGTGCGGCAAATAA

1967 62 This study

3813–3833 Ped2F- CAATACGTGGCCTAGCCAAT 5072–5091 Ped2R- TCATCAAGTGCGGCAAATAA

1278 62 This study

5005–5024 Ped3F- CCGCAGCAGCTATAACAACA 6228–6247 Ped3R- CCAGGACGGCTACCTAACAA

1243 60 This study

6094–6113 Ped4F-CTCAAGAACAGCACGGTGAA 8948–8929 Ped4R-GCCAAGGATCAACAATCATT

2800a 62 This study

Primers used for Inverse PCR

3328–3307 PedinAR- ATTTACCACCAATGATATTGGC 3488–3514 PedBF- GGTGATTTTATGAATAAGACTAAGTCG

8000 * 53 This study

Primers used for ISLpl1 and mobilization gene

ISLpl1 F- CAGGAATGATTGCTCACGAA ISLpl1 primers ISLpl1 R- TTTAACCGGTATGCCCAAAG

313 61 This study

Mob F-AAGGGTGGGACTTATGAGCG Mobilization gene Mob R-TTGTTGGTAGTCTGCTCCTC

1274 56 3

* variation in the amplicon sizes was observed

result. The plasmid profiling resulted in loss of low molecular sized plasmids encoding the pediocin and no signal with the pedB gene probe was detected by South-ern (Fig. 1 B, C).

PCR analysis of the flanking regions of pediocin operon By using inverse PCR different sizes of amplicons (6 to 12 Kb) were obtained for the isolates (Fig. 2A) Similarly, a variation in the amplification of the downstream region of the operon was observed by using the primer pair ped4F and ped4R, giving a 1.1 Kb size in Acr4, 2.8 Kb in the case of K7, Cb1 and Cb4 and a 300 bp product in Acr2 (Fig. 2B). Sequencing of this region in K7, Cb1 and Cb4 showed 100% homology to the

pSMB74 of P. acidilactici H, that encodes a part of pedD, pemK, pemI and orf264 (tyrosine recombinase). While in Acr4 it was 3′ of pedD and orf 264 and in Acr2 it was only 3′ of pedD.

Detection of ISLpl1 element and mob gene The sequence analysis of the 313 bp amplicon of ISLpl1 insertion element in E. faecium Acr4 and L. plantarum Acr2 showed 99% homology to the IS-30 related ISLpl1 of several Lactobacillus sp. like, L. casei BL23, L. brevis ATCC 367, L. plantarum 256 etc. The plasmid profiling of pediocin PA-1 like bacteriocin producing LAB is shown in Fig. 3A. The probe for ISLpl1 from Acr4 showed a strong signal in the region of chromosome as well as plasmid in Acr4 and Acr2, whereas in K7 and Cb1 the

Figure 1. Plasmid profiling of pediocin PA-1 like producing LAB (A), plasmid profiling (B) and southern detection of the immunity gene (C) in plasmid cured strains. A, B, C: lane 1, λ HindIII molecular marker (GeNei, Bangalore), lane 2, P. acidilactici K7; lane 3, E. faecium Acr4; lane 4, P. pentosaceus Cb4; lane 5, L. plantarum Acr2; lane 6, P. acidilactici Cb1; lane 7, 10Kb molecular marker (GeNei).



Figure 2. PCR characterization of flanking regions of pediocin PA-1 operon in bacteriocin producing LAB. A: inverse PCR ampli-fication using PedinAR and PedBF; B: PCR amplification using Ped4F and Ped4R. A: 1, P. acidilactici K7; 2, P. acidilactici Cb1; 3, E. faecium Acr4; 4, L. plantarum Acr2 and 5, P. pentosaceus Cb4; M, 10 Kb molecular marker (GeNei). B: 1, P. acidilactici K7; 2, P. acidilactici Cb1; 3, P. pentosaceus Cb4; 4, E. faecium Acr4; 5, L. plantarum Acr2; M, 3 Kb molecular marker (GeNei). signal was detected at band corresponding to chromo-some. The probe did not react for Cb4 (Fig. 3B). It was observed that, both high and low molecular sized plasmids probably harbor the ISLpl1 in Acr4 and Acr2. But the plasmids around 12 Kb in Acr4 and 6 Kb in Acr2 which reacted for pedB probe also reacted with the insertion element ISLpl1. Subsequently, the mob gene was characterized in the pediocin PA-1 producing organisms. Upon PCR amplifi-cation with mobF and mobR primers, a product size of 1274 bp in Acr4 was observed. Sequencing of the mob gene from Acr4 showed 98–99% homology to the E. faecium L50, E. durans 41D, E. hirae JM79 which are

Figure 3. Plasmid profiling (A) Southern detection of the ISLpl1 (B) and mob region (C) in LAB producing pediocin PA-1 like. A and B: lane 1, λ HindIII molecular marker; lane 2, P. acidilactici K7; lane 3, E. faecium Acr4; lane 4, P. pentosaceus Cb4; lane 5, L. plantarum Acr2; lane 6, P. acidilactici Cb1. Arrows indicate the bands corres-ponding to the ISLpl1 and mob gene.

linked to the production of bacteriocin such as entero-cin L50, duracin and hiracin, respectively [18, 19]. The mob gene probe reacted with the 12 Kb plasmid harbor-ing the pediocin operon in Acr4 whereas, the other isolates has not reacted to the probe (Fig. 3C).

Phylogeny of ISLpl1 and mobA gene in E. faecium Acr4 To find the close clustering of the ISLpl1 and mobA gene (relaxases) to other LAB species, a Neighbor Joining (NJ) phylogenetic tree was constructed. Based on the avail-able sequences in the NCBI database, a close clustering of the Acr4 to several of the bacteriocin and horA asso-ciated plasmids of Lactobacillus sp. was observed for ISLpl1 (Fig. 4A). Similarly, NJ tree of the mobA gene showed a close clustering of the Acr4 to the different bacteriocins of Enterococcus sp. revealed the presence of a conjugative mobilization plasmid (Fig 4B).

Discussion

The Pediocin PA-1/AcH (pediocin PA-1), represents a class IIa bacteriocin produced by Pediococcus acidilactici. However, the natural spread of pediocin PA-1 like bac-teriocin encoding plasmid into other strains of LAB and non-LAB was described in Lactobacillus plantarum WHE92, L. plantarum 423 and Bacillus coagulans I4 [6–8]. Earlier reports of Le Marrec et al. [7] and Van Reenen et al. [8] showed the presence of a mobilization protein and replication genes in the flanking region of pediocin PA-1 like operon suggesting the conjugative mobiliza-tion and/or inter-plasmid recombination events. These evidences supported the intergeneric transfer of pedio-cin operon in B. coagulans and L. plantarum 423. The present study focused on the detection of MGEs in the plasmids of different LAB species encoding the pediocin PA-1 like operon. Earlier reports revealed the association of pediocin PA-1 in plasmids of P. acidilactici PAC1.0 (pSRQ11; 9.4 Kb), P. acidilactici H (pSMB74; 8.9 Kb), P. pentosaceus S34 (pS34; 8.9), L. plantarum WHE92 (pWHE92; 11 Kb), B. coagulans I4 (pI4; 14 Kb) [6, 7, 20]. Hence, several of the LAB are found to harbor both high and low molecular sized plasmids encoding for e.g. bacteriocin production (like pediocin PA-1), antibi-otic resistance, carbohydrate utilization, cold shock proteins [3, 4, 6, 7, 20, 21]. Suggesting that pediocin PA-1 is indeed a plasmid linked phenotype in the inves-tigated LAB selected isolates as described for other pediocin PA-1 like bacteriocin producers [6]. The PCR characterization of flanking regions of the pediocin PA-1 operon was useful in determination of any recombi-



Figure 4. Phylogenetic tree of the ISLpl1 element (A) and the mob region (B).

nation events in the intergeneric and interspecific pediocin PA-1 producing LAB isolates. A variation in the downstream region of the operon in E. faecium Acr4 and L. plantarum Acr2 suggests the involvement of recombination events after pedD. The tyrosine recombinases belonging to an integrase or recombinase family are usually associated with site specific recombination events. Such MGEs contribute to the stabilization of the integrated element like antibi-otic resistance or other gene cassettes in the host or excision of bacteriocin like genes from one bacterium to the other [22]. The analysis of the complete sequence data of pSMB74 reveals the association of replicative (rep) genes in the upstream region of the operon, which are considered as putative ori regions. Such genes have the ability to cleave and join the plasmid DNA by strand transferase enzymatic activity [23, 24]. Hence, the pres-ence of rep and the tyrosine recombinase in the up-stream and downstream of the operon respectively, suggests the possible excision of the operon from the pediocin harboring Pediococcus species. Similar report was observed in the horA encoding plasmids which are also associated with rep genes and integrases [24]. These elements are found to help in the horizontal transfer between closely related populations by plasmid inde-pendent manner and subsequently integrate into plas-mids encoding bacteriocin and antibiotic resistance gene [24, 25]. To investigate the means of HGT in pediocin PA-1 like bacteriocin production among LAB species, the TraISLpl1 and mob regions were studied by molecular tools. These elements are known to be involved in the transfer of a gene or a set of genes among different genera. The TraISLpl1 was found to be linked to the genetic determinants that encode bacteriocin biosyn-

thetic operon, antibiotic resistance, Hop resistance, cold shock protein etc. [4, 25]. Similarly, the Mob proteins are also reported in pediocin PA-1 producing bacterio-cin in B. coagulans I4 and L. plantarum 423, suggesting the possible role of pediocin operon transfer in these bacte-ria [7, 8]. The ISLpl1 was also reported in the L. planta-rum WHE 92 plasmid encoding the pediocin PA-1 bacte-riocin [21]. Till date, the presence of TraISLpl1 insertion element in E. faecium was not reported. Suzuki et al. described that the TraISLpl1 in plasmid of horA gene encoding plasmids played a major role in the intracellu-lar transfer [24]. The high homology of the ISLpl1 to other Lactobacillus sp. helps in understanding the trans-fer of pediocin PA-1 like bacteriocin by horizontal means. The ISLpl1 is reported as a strong MGE, encod-ing a transposase (TraISLpl1) are found in several spe-cies of Lactobacillus, Pediococcus and Oenococcus [3]. The distribution of such IS elements in different genera not only helps in identification of the flanking regions of bacteriocin encoding or antibiotic resistance genes but also to characterize the species at strain level [11]. The presence of additional copies of ISLpl1 in the plasmids of Acr4 and Acr2, suggests the active insertion of this element by a replicative mechanism of transposition, as described for ISRgn1, ISLpl1 etc. [10, 11]. The plasmid localization of this insertion element helps in gene exchange between LAB species [4]. Such IS elements which are usually associated in the chromosomal or in the extrachromosomal elements contribute to the ge-netic rearrangements by gene loss, decay, acquisition and/or duplication processes [26, 27]. Similarly, Todokoro et al. [3] reported the presence of the mobilization proteins with bacteriocin associated plasmids in several of the vancomycin resistant E. fae-cium strains. The mobA gene of the mobilization regions



encodes a relaxase enzyme and binds to the oriT site to form a complex and cleaves the DNA due to the nicking activity. The mobB and mobC gene product acts as acces-sory proteins and further help in strand separation, thereby leading to the transfer of the genes by these mobilizable plasmids [2]. The presence of such relax-ase/mobilization nuclease domain was reported in a multiple bacteriocin producer E. faecium L50 and hiracin from E. hirae DCH5 suggesting the integration of the bacteriocin encoding gene cluster into the genome [18, 19]. The MGEs like rep, integrases, ISLpl1 and mob genes are responsible for the integration or excision of the pediocin PA-1 operon and is transferred among closely related LAB species by HGT phenomenon. The phylogeny of the ISLpl1 reveals the transfer of pediocin PA-1 from the pediocin associated plasmids of Lactobacillus strains to the E. faecium Acr4. Gomez et al. [10] reported the association of the genes encoding the ruminococcin A (a trypsin-dependent lantibiotic) to the ISRgn1 element belonging to IS3 family. The ISRgn1 element of R. gnavus was found to have a close phyloge-netic grouping to Clostridium coccoides suggesting the same ecological niche by these bacteria and strongly supports the transfer of the bacteriocin by this MGE by HGT. Devi and Halami [13] reported the isolation of L. plantarum Acr2 and E. faecium Acr4 from the same source, supporting the HGT phenomenon in the same ecological niche for the transfer of this bacteriocin from pediocin producing Lactobacillus strains. The shar-ing of conserved motifs of such Tra and Mob proteins helps in understanding the plasmid transfer among these bacteria [28]. Therefore the flanking region of the plasmid harboring pediocin PA-1 operon showed plas-ticity and such MGEs act as mediators in the integra-tion of a gene or a set of genes from one organism to other. In conclusion, the MGEs may have crucial role in the intergeneric and interspecific transfer of pediocin PA-1 operon. They play a major role in the transfer of pedio-cin from pediococcal strains to other LAB. Our observa-tion on the variation in the PCR amplicons suggested that the flanking regions of the operon underwent some recombination events. The presence of integrases, relaxases and TraISLpl1 in the flanking regions of the pediocin operon detected in Acr4 and Acr2 could be associated with the acquiring of this bacteriocin from other pediocin PA-1 producing LAB into their native plasmids. The phylogeny of the ISLpl1 suggested the same ecological niche for the HGT of pediocin PA-1 between Pediococcus, Enterococcus and Lactobacillus species in the evolutionary time span and has given a scope in understanding the HGT theory. These plasmids with

intergeneric transferring ability play an incredible role in the development of potential starter culture with bio-preservative properties, carbohydrate fermentation etc. Further efforts will be put forward to elucidate the arrangement and function of the DNA modules adja-cent to the bacteriocin operon that contributes towards the transfer and mobilization among different LAB species by conjugation experiments.

Acknowledgement

This work was carried out within the FAST TRACK pro-ject of DST, Government of India, New Delhi. SMD ac-knowledges CSIR, New Delhi, for the fellowship.

References

[1] Van Reenen, C.A., Dicks, L.M.T., 2010. Horizontal gene transfer amongst probiotic lactic acid bacteria and other intestinal microbiota: what are the possibilities? A Re-view. Arch. Microbiol., 193, 157–168.

[2] Francia, M.V., Varsaki, A., Garcillan-Barcia, M.P., Latorre, A. et al., 2004. A classification scheme for mobilization re-gions of bacterial plasmids. FEMS Microbiol. Rev., 28, 79–100.

[3] Todokoro, D., Tomita, H., Inoue, T., Ike, Y., 2006. Genetic analysis of bacteriocin 43 of vancomycin-resistant Entero-coccus faecium. Appl. Environ. Microbiol., 72, 6955–6964.

[4] Nicoloff, H., Bringel, F., 2003. ISLpl1 is a functional IS30-related insertion element in Lactobacillus plantarum that is also found in other lactic acid bacteria. Appl. Environ. Microbiol., 69, 6032–6040.

[5] Sørvig, E., Skaugen, M., Naterstad, K., Eijsink, V.G.H., Axelsson, L., 2005. Plasmid p256 from Lactobacillus plan-tarum represents a new type of replicon in lactic acid bac-teria, and contains a toxin – antitoxin-like plasmid main-tenance system. Microbiol., 151, 421–431.

[6] Miller, K.W., Ray, P., Steinmetz, T., Hanekamp, T., Ray, B., 2005. Gene organization and sequences of pediocin AcH/ PA-1 production operons in Pediococcus and Lactobacillus plasmids. Lett. Appl. Microbiol., 40, 56–62.

[7] Le Marrec, C., Hyronimus, B., Bressollier, P., Verneuil, B., Urdaci, M.C., 2000. Biochemical and genetic characteriza-tion of coagulin, a new antilisterial bacteriocin in the pe-diocin family of bacteriocins, produced by Bacillus coagu-lans I4. Appl. Environ. Microbiol., 66, 5213–5220.

[8] Van Reenen, C.A., Van Zyl, W.H., Dicks, L.M.T., 2006. Expression of the immunity protein of plantaricin 423, produced by Lactobacillus plantarum 423, and analysis of the plasmid encoding the bacteriocin. Appl. Environ. Mic-robiol., 72, 7644–7651.

[9] Mlalazi, M., Winslow, A.R., Beanubrun, J.J., Erido, B.E, 2011. Occurrence of pediocin PA-1/AcH-like bacteriocin in native non-starter Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus from retail cheddar cheese. In-ternet J. Food Safety, 13, 325–331.



[10] Petrovic, T., Niksic, M., Bringel, F., 2006. Strain typing with ISLpl1 in lactobacilli. FEMS Microbiol. Lett., 255, 1–10.

[11] Gomez, A., Ladire, M., Marcille, F., Nardi, M., Fons, M., 2002. Characterization of ISRgn1. a novel Insertion se-quence of the IS3 family isolated from a bacteriocin-negative mutant of Rumnicoccus gnavus E1. Appl. Environ. Microbiol., 8, 4136–4139.

[12] Halami, P.M., Ramesh, A., Chandrashekar, A., 2005. Fer-menting cucumber, a potential source for the isolation of pediocin-like bacteriocin producers. World J. Microbiol. Biotechnol., 21, 1351–1358.

[13] Devi, S.M., Halami, P.M., 2011. Detection and characteri-zation of pediocin PA-1/AcH like bacteriocin producing lactic acid bacteria. Curr. Microbiol., 63, 181–185.

[14] Anderson, D.G., McKay, L.L., 1983. Simple and rapid me-thod for isolating large plasmid DNA from lactic Strepto-cocci. Appl. Environ. Microbiol., 46, 549–552.

[15] Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, third ed. Cold Spring Harbor Labora-tory Press.

[16] Altschul, S.F., Maddan, T.L., Schaffer, A.A., Zhang, J. et al., 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res., 25, 3389–3402.

[17] Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) soft-ware version 4.0. Mol. Biol. Evol., 24, 1596–1599.

[18] Sanchez, J., Diep, D.B., Herranz, C., Nes, I.F. et al., 2007. Amino acid nucleotide sequence, adjacent genes, and het-erologous expression of hiracin JM79, a sec-dependent bacteriocin produced by Enterococcus hirae (Anas platyrhhyn-chos). FEMS Microbiol. Lett., 270, 227–236.

[19] Criado, R., Diep, D.B., Aakra, A., Gutierrez, J. et al., 2006. The complete sequence of the enterocin encoding plasmid pCIZ2 from the multiple bacteriocin producer Enterococcus faecium L50 and characterization of enterocin Q produc-

tion and immunity. Appl. Environ. Microbiol., 72, 6653–666.

[20] Papagianni, M., Anastasiadou, S., 2009. Pediocins: The bacteriocins of pediococci. sources, production, properties and applications. Microbial Cell Fact., 8, 1–16.

[21] Ennahar, S., Aoude-Werner, D., Sorokine, O., van Dorsse-laer, A. et al., 1996. Production of pediocin AcH by Lacto-bacillus plantarum WHE92 isolated from cheese. Appl. En-viron. Microbiol., 62, 4381–4387.

[22] Toussaint, A., Merlin, C., 2002. Mobile elements as a com-bination of functional modules. Plasmid, 47, 26–35.

[23] del Solar, G., Giraldo, R., Ruiz-Echevarria, M.J., Espinosa, M., Diaz-Orejas, R., 1998. Replication and control of circu-lar bacterial plasmids. Microbiol. Mol. Biol. Rev., 62, 434–464.

[24] Suzuki, K., Sami, M., Iijima, K., Ozaki, K., Yamashita, H., 2006. Characterization of horA and its flanking regions of Pediococcus damnosus ABBC478 and development of more specific and sensitive horA PCR method. Lett. Appl. Mic-robiol., 42, 392–399.

[25] Ehrmann, M.A., Remiger, A., Eijsink, V.G., Vogel, R.F., 2000. A gene cluster encoding plantaricin 1.25β and other bacteriocin-like peptides in Lactobacillus plantarum TWM1.25. Biochimica. Biophysica. Acta, 1490, 335–361.

[26] Bačun-Družina, V., Mrvčić, J., Butorac, A., Stehlik-Tomas, V., Gjuračić, K., 2009. The influence of gene transfer on the lactic acid bacteria evolution. Mljekarstvo, 59, 181–192.

[27] Callanan, M., Kaleta, P., O’Callaghan, J., O’Sullivan, O. et al., 2008. Genome sequence of Lactobacillus helveticus, an organism distinguished by selective gene loss and inser-tion sequence element expansion. J. Bacteriol., 190, 727–735.

[28] Mills, S., McAuliffe, O.E., Coffey, A., Fitzgerald, G.F., Ross, R.P., 2006. Plasmids of lactococci-genetic accessories or genetic necessities? FEMS Microbiol. Rev., 30, 243–273.

((Funded by

● FAST TRACK project of DST, Government of India, New Delhi))

molecular genetic studies on pediocin-like...

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