Supplemental Figure 1a. SDS-PAGE analysis showing OMPs expressed by V. tubiashii strain 19105. From left to right, Lane 1, cells grown in the presence of 3% NaCl compared to those grown with 1% NaCl in Lane 2; Lane 3 cells grown in the presence of 0.3% bile salts compared to those grown without bile salts Lane 4; Lane 5 cells grown in the presence of 2% maltose compared to those grown without maltose Lane 6; Lane 7 represents the molecular markers, sizes are reported in kDa. Note the sequenced and uniquely expressed OMPs at each environmental condition are identified with an arrow.

50 kDa

70 kDa

45 kDa

40 kDa

90 kDa

25 kDa

20 kDa

15 kDa

65 kDa

35 kDa

14.4

20.1

30.0

43.0

67.0

94.0

Bile salts

0.3% 0%

NaCl

3% 1%

Maltose

2% 0% MW

1a.

14.4

20.1

30.0

43.0

67.0

94.0

Temperature

37° 30°

pH

8.5 7 6 MW

50 kDa

70 kDa

45 kDa

40 kDa

90 kDa

25 kDa

20 kDa

15 kDa

65 kDa

35 kDa

Supplemental Figure 1b. SDS-PAGE analysis showing OMPs expressed by V. tubiashii strain 19105. From left to right, Lane 1-2, cells grown at different temperatures; and Lane 3-5, cells grown at various pH conditions; Lane 6 represents the molecular markers, sizes are reported in kDa. Note the sequenced and uniquely expressed OMPs at each environmental condition are identified with an arrow.

1b.

Supplemental Figure 2a. SDS-PAGE analysis showing OMPs expressed by V. tubiashii strain 19109. From left to right, Lane 1, cells grown in the presence of 3% NaCl compared to those grown with 1% NaCl Lane 2; Lane 3, cells grown in the presence of 0.3% bile salts compared to those grown without bile salts Lane 4; Lane 5, cells grown in the presence of 2% maltose compared to those grown without maltose, Lane 6; Lane 7 represents the molecular markers, sizes are reported in kDa. Note the sequenced and uniquely expressed OMPs at each environmental condition are identified with an arrow.

50 kDa

70 kDa

45 kDa40 kDa

90 kDa

25 kDa

20 kDa

15 kDa

65 kDa

35 kDa

14.4

20.1

30.0

43.0

67.0

94.0

NaCl3% 1%

Bile salts0.3% 0%

Maltose2% 0% MW

2a.

14.4

20.1

30.0

43.0

67.0

94.0

Temperature37° 30°43°

pH8.5 7 6 MW

50 kDa

70 kDa

45 kDa40 kDa

90 kDa

25 kDa

20 kDa

15 kDa

65 kDa

35 kDa

Supplemental Figure 2b. SDS-PAGE analysis showing OMPs expressed by V. tubiashii strain 19109. From left to right, Lane 1-3, cells grown at various temperatures; and Lane 4-6, cells grown at various pH conditions; Lane 7 represents the molecular markers, sizes are reported in kDa. Note the sequenced and uniquely expressed OMPs at each environmental condition are identified with an arrow.

2b.

a.

Vv4965-T1

CTCTAGAAGTTGGTGGCCGTGCTGAAGCTCGCCTATCTCTGAAAGATGGTAAGGCACAAG

Vc395

CTCTAGAAGTTGGTGGCCGTGCTGAAGCTCGCCTATCTCTGAAAGATGGTAAGGCACAAG

Vt109

CTCTAGAGATCGGCGGTCGTGCTGAAGCTCGTATGTCTCTAAAAGATGGTAAAGCTGAAG

Vt105

CTATCGAAATGGGTGGTCGCGCGGAAGCACGTCTAGCTCTTAAAGATGGNAAGGCAGAAN

** * ** * ** ** ** ** ***** ** * **** ******** ** ** **

Vv4965-T1 ACAACTCTCGCGTACGTCTAAACTTCTTGGGTAAAGCAGAAATCAATGACAGCCTATACG

Vc0395 ACAACTCTCGCGTACGTCTAAACTTCTTGGGTAAAGCAGAAATCAATGACAGCCTATACG

Vt109 ACAAAACACGTGTACGTCTAAACTTCCTAGGTAAGACTCAAATCACTGATAGCCTATATG

Vt105 ACAAATCTCGCGTACGTCTAAACTTCCTAGGTACAGCTCAAATCACTGACAGCCTATACG

**** * ** *************** * **** * ****** *** ******** *

Vv4965-T1 GTGTTGGTTTCTACGAAGGCGAGTTTACTACTAATGATCA------------AGGTAAAA

Vc395 GTGTTGGTTTCTACGAAGGCGAGTTTACTACTAATGATCA------------AGGTAAAA

Vt109 GTCTAGGTTTCTACGAAGCTGAGTACACTACAGCAGACAACACTGTAACTGTAGACGGCA

Vt105 GTGTGGGTTTCTACGAAGGTGAGTTCACTACTAATGACAATGGCGGCGCTACAGATGGCA

** * ************* **** ***** ** * ** *

Vv4965-T1 ACG-CGTCTAACA---ACAGCCTANACAACCGTTATACCTACGCTGGTATCGGTGGCACT

Vc395 ACG-CGTCTAACA---ACAGCCTAGACAACCGTTATACCTACGCTGGTATCGGTGGCACT

Vt109 AAA-CAACTGACGGTGATAACATTGAGCACCGTTACACTTACGCTGGTATTGGTGGTGAC

Vt105 ACAACAGCTCATC----TGATCTAACTAACCGTTACCTATACGGTGGTATCGGCGGAGCT

* * ** * * ******* **** ****** ** **

Vv4965-T1 TACGGTGAAGTGACTTACGGTAAAAACGATGGCGCATTGGGCGTAATCACTGACTTCACC

Vc395 TACGGTGAAGTGACTTACGGTAAAAACGATGGCGCATTGGGCGTAATCACTGACTTCACC

Vt109 TTTGGTGAAGTAACTTACGGTAAAAACGATGGTGCACTANGCGTTATCACTGACTTTACT

Vt105 TTCGGTGAAGTAACTTACGGTAAGAACGACGGTGCGCTANGTGTTATCACTGATTTCACT

* ******** *********** ***** ** ** * * ** ******** ** **

Supplemental Figure 3. Clustal X alignment of nucleotide sequence of ompU gene from V. tubiashii strains 19105 and 19109, V. vulnificus 4965-T1 and V. cholerae 395, respectively. ompU gene sequences were obtained in this study. Indicated by underlying are the PCR primer sequences used in the study. a. Forward sequence, b. Reverse sequence. The symbol “*” represents conserved nucleotides among the sequences.

Vv4965-T1 NCNNTGGACTGACC-CAGTTCGTC-TTCTGATGCTACTTTACCAA----------CTTTG

Vc395 GCNNNGNANTTANG-CAATACNTNGTTCTGATGCTACTTTACCAAA---------CTTTG

Vt109 GCNNNGAACTGAANATAGCTCGTC-TTCTGCACCAGCTTTACCTACAGTACCAGTTGCTG

Vt105 NCCNTNGACNNAAA-AAGCTCGTC-TTCTGCATCTGCTTTAGAGATG----TAAGATTTG

* * * * * * ***** * ***** * **

Vv4965-T1 TCTGA--ATCT------AGCAGATTGAACTGGTAAGAGATGTAAGAGCGNNAGTTTGGCT

Vc395 GCTGA--ATCT------AGCAGATTGAACTGGTAAGAGATGTAAGAGCGNAAGTTTGGCT

Vt109 TCTTATCATCTTTACTAAGTAGGTTGAAGTTGTAAGAGATGTAAGTACGGAAGTTTGGCT

Vt105 TCGTC--GTCT------AGCAGGTTAAAGTTGTACGATACGTAGNCACGGAAGTTAGGCT

* *** ** ** ** ** * *** ** * *** ** **** ****

Vv4965-T1 TGAAGTAGTAAGTTGCGTCGATAGCAAAATTATCTGCTGAAGT--------T-TCTTTCG

Vc395 TGAAGTAGTAAGTTGCGTCGATAGCAAAATTATCTGCTGAAGT--------TCTCTTTCG

Vt109 TGAAGTAGTATGTTGCATCGATCGCTACATTGTCAGCTGCATCATACGTAGTACCTGAAG

Vt105 TAAAGTAGTATGTTGCGTCGATAGCGATGTTATCTGCAGCATCTTCTTTAGTTGAACCTG

* ******** ***** ***** ** * ** ** ** * * * *

Vv4965-T1 CTGTTTCTGCA---TTGTTGTATGTCGCAGTAAACGCAGCTTGACCTAGTTTGTAACCAG

Vc395 CTGTTTCTGCA---TTGTTGTATGTCGCAGTAAACGCAGCTTGACCTAGTTTGTANCCAG

Vt109 TTTTCTCTTCTAGCTTATTGTATGTTGTAGTGAATGCTACTTTTTCTAGAGTGTAAGCTG

Vt105 TTTTGTTTTCTAGGTAGTTGTAAGACGTTGTGAATACAGTCTGGCCCATAGTGTAAGCTG

* * * * * ***** * * ** ** * * * * ***** * *

Vv4965-T1 CAGCTAGCTCGTAGCCAGTGTAGTCAACGT-----CTTTCGC---TAGCTC-------AC

Vc395 CANCTANCTCGTAGCCAGTGTAGTCAACGT-----CTTNCGC---TAGCTC-------AC

Vt109 CTGCGAATTCGTAACCAGTTTTGTCTGTTTT---GCTTGAGC---CGTCTTTGTCTTTAC

Vt105 CTGCTAGTTCGTAACCTGTCTTATCAGTTGTTGAGCTAGAACCTTTCACTTGGTCTTCGC

* * * ***** ** ** * ** ** * ** *

b.

Supplemental Figure 3. Clustal X alignment of nucleotide sequence of ompU gene from V. tubiashii strains 19105 and 19109, V. vulnificus 4965-T1 and V. cholerae 395, respectively. ompU gene sequences were obtained in this study. Indicated by underlying are the PCR primer sequences used in the study. a. Forward sequence, b. Reverse sequence. The symbol “*” represents conserved nucleotides among the sequences.

Vt109 NCNNNNTCCNNNNCTTACCCAGCCTGACTCAAGCCATCTCAACCCTACGTAAAATGCTGA

Vt105 ----------NCNNNTTCAAAACCTGACTCAGGCCATCTCATCCCTACGTAAAATGCTGA

Vf33809 ---------------TGGTGGAATGCACACCGGCGGACTCGGCCCCAAAGAAGTCATCGC

* ** * ** * *** *** * ** *

Vt109 AAGACTCGACCAAGTCGCC--TCAGTTTGTGAAA-ACGGTTCCAAAACGTGGTTATCAGT

Vt105 AAGACTCGACTAAATCCCCCATCAATCCGTGGAAGACGGTTCCAAAACGTGGTTATCAGT

Vf33809 TACCGGCGGTCAAAAC--------------------------------------------

* ** **

Vt109 TGATCGCGACCGTCGAAAGCGTTCAGCTCGATGCAAGTAAAGATCCTGATGCCATCGAGC

Vt105 TGATCGCGACCGTCGAAACCCTGGAGCTCGATGCAAGTAAAGATCCTGATGCCATCGAGC

Vf33809 ------------------------------------------------------------

Vt109 AGTCAGATGAGTCTTACTCAAGTGCCGTATCCTCTTCAGAACTTGTCAGTCAAGCTGCTG

Vt105 AATCATATGAGTCTTACTCAAGTGCCGTATCCTCTTCACAACTTGTCAGTCAAGCTGCTG

Vf33809 ------------------------------------------------------------

Vt109 TTTCTGAAGCAGTTATTACTCAGACGTCGCCTTCTGTAGACTCGACACCTGCGGCACCAA

Vt105 TTTCTGAATCAGTTATTACTCAGACGTCGCCTTCTGTAGACTCGACACCTGCAGCACCAA

Vf33809 ------------------------------------------------------------

Vt109 AACCACGAATGGATCTGTTCACTAAATTGATTCTGTTGGTTGCCGTCATACTGCCAT---

Vt105 AACCACGAATGGATCTGTTCACTAAATTGATTCTGTTGGTTGCCGTCATACTGCCATCNN

Vf33809 ------------------------------------------------------------

a.

Supplemental Figure 4. Clustal X alignment of nucleotide sequence of toxR gene from V. tubiashii strains 19105 and 19109 compared to that of V. cholerae 395 and V. fluvialis strain ATCC 33809. toxR gene sequences were obtained in this study and gene sequence for V. fluvialis strain ATCC 33809 was obtained from the NCBI accession number AF170885. a. Forward sequence, b. Reverse sequence. The symbol “*” represents conserved nucleotides among the sequences.

Vt109 TGAACAGATCCATTCGTGGTTTTGGTGCCGCAGGTGTCGAGT-CTACAGAAGGCGACGTC

Vt105 TAAAGGATAACCGTAAGGATGTCGGAGTTCCAATTATTACGCACTCCAGCAGTCGATGTG

Vf33809 TGGACGACTCCAGCCTGACTCAAGCCATCTCAACC--------CTACGTAAAATGCTGAA

* * * * * ** ** * * * *

Vt109 TGAGTAATAACTGCTTCAGAAACA-------GCAGCTTG-ACTGACAAGTTCTGAAGAGG

Vt105 TGAGCCAAGAGGACTCCATGAACACTTAGNCGCGGTTTCTGCGGACCGATTCCGTGAAGG

Vf33809 AGACT--CGACCAAGTC---GCCT--------CAGTTTGTGAAAAC-GGTTCCAAAACGT

** * * * * ** * *** *

Vt109 AT------------ACGGTACTTGAGTAAGACTCATCTGACTGCTCGATG--GCATCAGG

Vt105 ATGGATAGGGCCGGACGATAATTGTGCAAAATTAACTTGACTGCTCGATTATGCATCAGG

Vf33809 GGTTATCAGTT--GATCGCGACCGTCGAAAGCGTTCAGCTCGATGCAAGTAAAGATCCTG

* * ** * * * ** *

Vt109 ATCT-TTACTT--GCATCGAGC-TGAACGCTT-TCGACGGTCGCGATC--AACTGATAAC

Vt105 ATCTATTACCTATGCATCGAGCCTGAACGCTTATTAACGCTCGCTACCCNGAGTGATAAC

Vf33809 ATGC--CATCGA-GCAGTCAGA-TGAGTCTTACTCAAGTGCCGTATCC--TCTTCAGAAC

** * *** ** *** * * * ** * * * ***

Vt109 CAC---GTTTTGGAACCG--TTT-----TCACAAACTGAGGCGACTTGGTCGAGT-----

Vt105 CACAGTGGTTGGTAACCGGATTTGNCGATCATAAATGGGTGCGACTTGGTGGTCTAGCTC

Vf33809 TTGTCAGTCAAGCTGCTG---TT-----TCTGAAGCAGTTATTACTCAGACGTCG-----

* * * * ** ** ** * *** * *

Vt109 -CTTTCAGCATTTTACGTA------GGGTTGAG-ATGGCTTGAGTCAGGCTGGA--GTCG

Vt105 GCTCTCAAACTTTTACAAACGTTAGGGGTTGAACATGGCTTGACTCAGGCTGCAACGTCT

Vf33809 -CCTTCTGTAGACTCGACACCTGCGGCACCAAA---ACCACGAATGGATCTGTT----CA

* ** * * * ** *** *

Vt109 TCCACCT-CAAACCCCTGAG---AATACGTCTCCCNCNCTCTTNCAATCTCACAGTTCCA

Vt105 TCCACCTTCAAACCCCTGAACCAAATACCTGTACTNCNCTCTTNCGTTTCCATAGTCACA

Vf33809 CTAAATT--GATTCTGTTGGT--TGCCGTCATACTGCCTATTTGTGTCATTTTGTTTACG

* * * * * * ** * *

b.

Supplemental Figure 4. Clustal X alignment of nucleotide sequence of toxR gene from V. tubiashii strains 19105 and 19109 compared to that of V. cholerae 395 and V. fluvialis strain ATCC 33809. toxR gene sequences were obtained in this study and gene sequence for V. fluvialis strain ATCC 33809 was obtained from the NCBI accession number AF170885. a. Forward sequence, b. Reverse sequence. The symbol “*” represents conserved nucleotides among the sequences.