Ð. ÑепоÑеÑаÑÑа. B. cepacia. B. contaminas. B. diffusa. B. dolosa. B. lata. B. latens. B. metallica. B. multivorans. B. pseudomultivorans. B. pyrrocinia. B. seminalis.
Mar. Drugs 2016, 14, 83
S1 of S18
Supplementary Materials: Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from Ross Sea (Antarctica) Pietro Tedesco, Isabel Maida, Fortunato Palma Esposito, Emiliana Tortorella, Karolina Subko, Chidinma Christiana Ezeofor, Ying Zhang, Jioji Tabudravu, Marcel Jaspars, Renato Fani and Donatella de Pascale Table of contents: Figure S1. Phylogenetic trees (using the complete deletion option) of the Arthrobacter (A), Pseudomonas (B) and Psychrobater (C) genera. Figure S2. 1H-NMR spectrum of compound 1 in CD3OD at 600 MHz. Figure S3. Edited-HSQC NMR spectrum of compound 1 in CD3OD. Figure S4. COSY NMR spectrum of compound 1 in CD3OD. Figure S5. HMBC spectrum of compound 1 in CD3OD at 600 MHz. Figure S6. 1H-NMR spectrum of compound 1 in DMSO-d6 at 400 MHz. Figure S7. 1H-NMR spectrum of compound 2 in CD3OD at 600 MHz. Figure S8. Edited-HSQC NMR spectrum of compound 2 in CD3OD. Figure S9. COSY NMR spectrum of compound 2 in CD3OD. Figure S10. HMBC NMR spectrum of compound 2 in CD3OD. Figure S11. 1H-NMR spectrum of compound 2 in DMSO-d6 at 400 MHz. Figure S12. 2D ROESY spectrum of compound 2 in DMSO-d6 with Rhamnose protons labelled. Figure S13. Compound 2 showing selected ROE correlations of the rhamnose moiety. Figure S14. 1H-NMR spectrum of compound 3 in CD3OD at 600 MHz. Figure S15. Edited-HSQC spectrum of compound 3 in CD3OD at 600 MHz. Figure S16. COSY NMR spectrum of compound 3 in CD3OD at 600 MHz. Figure S17. HMBC NMR spectrum of compound 3 in CD3OD. Figure S18. HRESIMS spectrum (M + Na)+ of compound 1. Figure S19. MS/MS data of compound 1. Figure S20. HRESIMS spectrum (M + Na)+ of compound 2. Figure S21. MS/MS data of compound 2. Figure S22. HRESIMS spectrum (M + Na)+ of compound 3. Figure S23. MS/MS data for compound 3. Table S1. Cross- streaking experiments of BTN isolates against a wide panel of Bcc strains. The Bcc strains highlighted in red correspond to type strains. Symbols: +, growth; ±, reduced growth; -, no growth; C+, positive controls, i.e., Bcc strains grown in the absence of the tester strain(s). Abbreviations: Ps, Pseudomonas; Ar, Arthrobacter; CF, Cystic Fibrosis; Env; environmental.
Mar. Drugs 2016, 14, 83
S2 of S18
Arthrobacter _hist
s_X80 738 ns_X83406
cter Art X 807 _m hro 45 yso ba ren Ar cte s_A t hr r _ J61 so l ob Ar 7 i _E ac 482 th ter F6 ro 60 _b Ar ba 74 erg th cte 8 ro e rei r_ ba _A ar ct dle J6 er 09 ye _c 63 n re sis 0 at _A in J5 ol 51 yt ic 16 us 3 _D 88 21 1
hor m iae_
80739
9628
nae_X
itensis_AJ60
rob a
Arth
89
Arth
roba cter_ p
rotop
nicotia bacter_
0
10
81
84
15
96
40
2267
s (T); A-6; AF10
hlorophenolicu Arthrobacter_c
hro
Arthrobacter_phenanthrenivorans_AM176541
_p
ter
ac
Art
Ar t hr ob
98 19
AJ 31 50 ari 09 et i 69 ba s_ 75 cte A Arth 25 r _t J6 73 e rob 39 cti_ act 83 AJ er _ 49 0 63 flav 9 us_ 82 Arth 9 AB roba 537 6859 cter_ 168 agilis _X 8 Arthro 11 0 bacter_ 748 1 niigate 62 8 nsis_A B 2485 26 Arthrobacter 21 _defluvii_AM 409361
e_
_D Q
us
ne
_t um ba
te r
te rra
ac
ub
ro b
te r
_s
Ar th
ac ro b Ar th
Arthrobacte
(A)
Arthro
88 45 94
73
Arth
Arth
Art
roba
rob a
r_livingstonen sis
bacter_
cter_
cter
_alp
stacke b
p syc
_GQ406811
randtii
hrola
ctop
_AJ64
hilus
0198
82 _A F inus bac 1341 _GQ ter BT 79 Ar _ps 227 t N4 ych 413 Ar hrob A roc rth th a h c r ro itin ob ter ba i ph ac _r t er ilus cte am _A r_ os B5 pa us 886 _X sc 33 80 en 74 s_ 2 X8 07 40
71
hro
89 98 27 90 98 AB 27 e_ AB za 36 ry l a_ 07 _o ico X8 er m 091 is_ ct hu 235 rm F ba r_ ifo ro s_A cte lob th ran ba r _g Ar ivo 2 ro cte th lfon 069 72 r a A F 33 _su ob ter e_A t hr 58 ma Ar bac 0741 lero hro s_X8 _ sc e r Art n e acte 77 5 m og rob 5 lychro Arth r _po bacte 8 X8340 Arthro xydans_
te
11
er_o 94 hrobact 2 Art
ac
r_su bacte 1709 _AM93 Arthro tarcticus 82 ter_an rthrobac 60 A 50 ssicus_AB0719 Arthrobacter_ru
44
97 1
b ro
2
Arthrobacter_sanguinis_EU086805
54 44 afaciens_X807 Arthrobacter_ure 6 405 ens_X83 sc re 04 98 acter_au 5125 Arthrob J A 6 _ 5 licus 07 78 ajaco 834 rogu 7 r_nit is_X 852 _ilic bacte r 2 4 ro h te 2 B Art 21 1 90 bac A 0 o _ r 35 ilus Arth F2 liph A 0 a 7 s_ alk 88 hu 53 ter_ 27 r op 33 bac l ot AJ hro X9 s_ thy _ Art e u is se r _m ns _ro cte we ba ter ac ol u hro t b r w A r_ hro
th Ar
81
60
Arthrobacter_nicotinovorans_X80743 76
t Ar
06
5 63 Y1 67 LI 3 ); 61 bi _ (T AJ m _ s s o an rh licu ler r_ no 2 he ot o cte 606 rop cry ba h r o c J60 r sy _A cte th sis _p r n ba Ar e le o t 061 ac 606 t hr gue ob _A J Ar ker t hr nsis er _ t ie Ar c tr o ba ang hro 9 r _g 66 Art 8340 acte us_X rob 39 lfure Arth
30 29
poiete
idinolovora
51
ko
Q4 ;G
5 88
27
ter _
32
ac
hro
51
s_ um AJ mi 24 ns 34 i i ree _X 21 bac ns 93 ter is_ Arth 35 _lu A rob 4 Y1 t eo acte 1 lu 1 64 r _g s_A and 96 00 J24 ave 342 Arth nsis 92 roba 2 _A J cter_ 316 57 citre 140 us_X Arthro 87 bacter_ 8073 crysta 7 llo Art
0.02
31
_c
lbu
Arthro
_a
31
on _m
te r
Arthrobacter _arila
r te
ter
46 39
r te ac
ac ac
ac
_ ter ac
b ro
b ro ro b
4 236 54 725 J29 X 80 93 _A 7 oli_ cae 82 a_c 39 pho igell 6 J asi /Sh A 6 n ti_ ichia er _ 82 70 n her e act 39 b 50 Esc igm J6 hro p 31 Art _A i l l AJ te ti_ as en _c um ob t hr Ar
th Ar th Ar Ar t hr ob
Ar t hr ob
Ar th
Arthrobacter_uratoxydans_X83410
Supplementary material
Mar. Drugs 2016, 14, 83
Pseudo
67 Pseudomomonas_viridiflava_AY180972 nas_congela 33 ns_AJ49282 33 Pseudom 8 24 PPseudom onas_syringa se on e_DQ31 5 8866 P udom as_ficu
21 28 22 65
6621 domonas_lut ea_AY364 537 Pseudomonas_gramini s_Y11150
do
Ps
(B)
sere
on
AJ u s 1 a 0 s PsPseeu eud domona _ars ndel 3599 49282 0 7 eu uddomom ona s_k enic ii_AF 6 do om o ona s_ ilon oxy 058 m o nas s_ thiv ens dan 286 on na _ br er is_ s_ as s_ bra ass val AJ FN6 4 _f co ss ica ens 292 re rru ica ce is 4 5213 de g ce a _A 26 rik ata ar rum F10 sb _D um _A 03 er 84 _E F 1 23 ge 0 U 0 ns 12 39 032 13 1 is 88 _A J2 49 38 2 80
do m P s on eu as_ do m mo Ps on dom cre Ps nas_ eud as_ on mo eu x a r do caen omo ant s_p ico Ps mona i_E nas hom ara lora U 6 _m a f u t a eu s Ps dom _pse 206 end rina lva _A eu on u 7 _ _ B Pse dom as_o doal 9 ocin AB AB 060 a_ 17 04 1 c udo ona s_a leovo alige D8 69 69 37 mo 4 5 9 Pse nas_ lcali rans nes fus phi _D _Z 0164 9 udo mon cov la_ Q8 76 99 agin AB 42 66 Pseu Pse 1 a s _ 0 u 6 dom onas domon asplenae_FJ3058018 Pseu as_ ii _vra 3 4 domo _ 4 AB0 835 38 a 4 n g n o 3 a v a 8 s e 1 0 Pseud rici_ 213 1996 09 omona _rhizosph nsis_AY Z76 97 66 a s_abie 2 tanip erae_A 9170951652 54 Pseudom 5267 onas_jap hila_AJ01Y 3 15 47 onica_A B12 04 Pseu
se
a s_ ctae_A P ud 3 B0213 P seud omon trema 78 40 Ps seud omon as_sa e_AJ492 82 3 6528 PPseeud omo as_c vastan oi_A 6 3759 se ud omo nas arica B _ o p 31 n 0 u c P a m 2 an a pay s d 2 ae_ 1402 21587 P eu om ona s_lin nab D84 0 P Ps se domona s_m i_AY ina_ 0
60
eu
ynxa
38
eu
eu ud do o na as_ fluo _AJ alis_ A F4 do om mo nas s_lu sal resc 936 053 m on na _c rid omo ens 933 28 on a s_ os a_ _ n as s_ pa tan AJ ii_A D840 _a tol lle tin 581 Y09 13 zo aa ron ii_A 99 1 52 to sii_ ian F3 9 8 fo A a rm F2 _A 744 an 55 Y0 72 s_ 33 91 52 D8 6 7 40 09
602
do
AF 3 98 a_ 667 98 ne Z7 Y50 2655 8 rra is_ _A 9 ite ph is Q6 53 ed ora raph s_D 168 _m lor ro phi _FJ 4 as ch hlo ra his 665 27 7 on s_ _c oro ap 40 395 m na as chl ror li_Z D8 5 1 do o on s_ hlo gda ns_ B02 188 eu om om ona s_c my trole is_A AB04 Pseudeud om ona as_a _tae dens hila_ 3 p Ps Pseuddommon onas _lun chro 09473 2 4577 Psseuudo dom onas s_psy gi_AF Y97095 P se seu dom ona _fra 70 50 _A P P eu om nas 7 nsis 4684 Ps seud domo moravie ensis_AF u P _ 7 g 29 Psemonas_umson _AM29356 4370 o s ii 55P8s 9eudomona s_mohn 468452 ud omona koreensis_AF 8 P se 3565 4 6 Pseudomonas_ kei_AM29 Pseudom onas_reine sis_AJ011507 Pseuddo s_vancouveren Pseu mona 259 Pseudomonas_jessenii_AF068 Pseudomonas_moorei_AM293566
eu
as as on n s m mo ona o o 7 d d m s 45 eu eu do ona s 64 Ps Ps eu m na s 0 F0 1 6_ Ps udo mo ona lis_A 9283 N _ 7_ e do m ta J4 61 BT TNN_ Ps seu udo rien a_A 0644 B T N8 P se _o drin AF 3 8 B T 9 P n a s ce n a _ 3 B TN 10 o as_ dri AF074 68 B T N d o m o n _ ce e _ 6 689 s 2 la B m F a u u A ig e o n 84 93 Psseuddomo as_m enneri_ AF0743 03 P seu mon s_br ardii_ 6 J5376 P u d o o n a g e ss A _ a c ti 7 1 7397Pseeudom onas_ proteoly 0 4 8 P s dom s_ lens_D 70 Pseuudomona s_mucido 025 ntha_D84 Pse mona 9 Pseuudomonas_s 166 57645 Pse anensis_AF0 4084 Pseudomonas_lib 87208 AY7 acis_ _pan onas dom 60 Pseu 4388 Pseudomonas_cichorii_Z76658
54 34 34 40 63
Ps
a P e m 0 4 PPs se udo on s_e ialis_ 92829 588 P seueud udo mo as_s xtrem AJ49 PsPseseu do omo mon nas_ imiae orien 2831 m t
1
Ps
0.01
5
P eu seu do do Ps Ps mon mo eu eu as na do m P do _p s_ Ps ona seu mo oha ma eu s_ do na n rin c d m s Ps om uat on _ta gen col eu on roc as ea si a_ do as ie _ n s_ A Pse _ m n b e D B udo P on ang eg orb ns Q 3 Pse mona seud as_g uilli ase ori_ is_ 33 010 om ui se ns A FJ 91 7 s_ ud Pse udo omona delhie onasneae pticais_EM1 42444 1 mon s_c nsis _p _A _X U7 14 8 Pseu a i t s _D el i M 4 9 9 52 13 r _ o dom 28 onas knackm nellol Q33_AM1 91895401287 20 Pseu _pan 1 ussii is_Z 915 14 10 domo ip 9 aten _AF0 7665 3 99534 n a s_ 23 s 9 6 1 Pseud nitroreduc is_EF4239489 67 omona e 4 84 55 19 Pseudom s_jinju ns_AM08401 onas_pa 58 8 e n 4 sis 73 chastrella 35 e_A B _A F 468448 Pseudomon as_duriflava 125366 2 _EU046271 96 Pseudomonas_luteola_ D84002 2 27 Pseudomonas_psychrotolerans_AJ575816 1 44 018 _D84 orans oleov 99 Pseudomonas_ 06 genes_D840 ali alc s_ 9 na 52 4 mo Pseudo 1 U1433 1 ulinigri_E 63 onas_sab gia_EU88898105 Pseudom 6 46 la e 8 p 2 _ U s a 7 E 7 64 2 is_ mon 3 0 9 Pseudo xinjiangens Q0886640 1 1988 16 onas_ nensis_D B02138AY69116 m o d 9 u e 3 _A nsis_ U01 0236996 m P se a ia n x _ e 4 04 4 8 2 520 e s _ g a 8 s in o n t n o in D dom rtuc _argen vesce nea_ _AB8400268 29 2289 e p _ 6 i 4 a 8 Pseu s a as fla m lv D 7 3 5 mon domon nas_ _stra s_fu ans_ AF0 10 44 457 udo o a bit lii_ EU 06 9 20 u s e m n a s Pse o P on mo iha se is_ F 00 0 ud Pse udom eudo oryz mos ens ilii_AAB 84 e Ps s_ s_ n te a_ _D Ps na na iwa on id da mo mo ta m sic ti do udo as_ as_ los _pu u e n n g Ps Pse mo mo co nas o o le ud ud _p mo e o P s P se n a s u d o se m P do eu Ps
6454
7 314 35 8 3 Y 95 is_A 684 68 25 807 _otitid _X06 Z766945295 8 4896 96 7 1 li_X _ s a _co mona ginos rans B18302264 9471 ella do aeru ovo ns_A_AF _U F0 69 91 Shig A seu in P hia/ as_ res ifige dica rica ri_ 770 06 n heric mo nas_ azot s_in alea utze AY Y77 Esc o b st _ udo a s_ m Pse eudo ona mon as_ as_ etis is_A n Ps udom udo mo on seg get e se do om s_ se P Ps e u u d n a s_ Ps Pse mo na do mo eu do P s eu Ps
Ps
63 Pseudomonas_marginalis_Z76663 50 Pseudom 27 _rhodesiae_AF064459 Pseudomoonas nas_ 16 Pse omonas_vgrimontii_AF268029 Pseuud eronii_AF0 1236 P d omo 6446 882 Psseudomo nas_extrem eudo australis 0 n a s_ a 2 7 P se m n _ A J5 8 u ta o P 3501 46 P seuddomon nas_me rctica_AJ 78 53760 Psseeudoomon as_po ridiana 1 238 6 _ a a A J5 3 7 Ps udo mon s_triv e_AJ4 7
S3 of S18
Mar. Drugs 2016, 14, 83
1 Psy BTN1
95
23
ct e
te
r_o
hro
r
k
bac
b chro
sy 85 P
Psych
ts ho
ro
te
a
ke
ns
is
ti_A r_lu
9 B0 _A
J4 3
47
082
8
us_ ritim _ma cter A ticus_
r_arc bacte
94
AJ6
092
Y 4448
BTN24_
19
19
0
42259 r_AY8
us_
r_cele robacte Psych
sals ter_ bac chro Psy
Psychrobacter_
04 391 A J5
M1 65 e_F
oca tiph r_lu cte rob a
ych Ps
namhaensis_AY7 228
05
94 09 J3 s_ A
nu ma ri ub r_s
cte ba
59
29
35
41 99 J3 0
_A
co la
r_ m ar in
ac te
hro
BTN22_Psychrobacter
yc
Ps
yc
hro
Ps
ba
ct e
yc
yc
hr ob
82
c
94
J _A
3
88 16 28
21
chr oba
hr ob
25 34 31 AJ 5 68 is_ 60 ar Y6 im _A niv tis r_ en te l ol ac ha yo ob r hr _c r yc cte Ps ba ter bac hro yc hr o c Ps Psy N3 BT cter 52 r oba h c y 5 Ps B TN r robacte _Psych BTN12
2 31
27
c la _g er
45
hrob
Psy
16
Ps
36
a ol
95 Psyc
59
16 52
56
51
BTN20B_Psychrobacter
94
ac
te
r_ p
ac
te
ro t
eo
Psychro
acte
cte
r_p
r_ p
r_a q
isc
bacter
r_va ll
is_A
uat icu
at o
rii_
s_A
AB
ac
J5 8 4
45
37
ific
en
lyt ic
us
_A
J2
72
30
sis _A
B0 1
832
J58
483
3
00
60
72
22
chrobacter
580
95
s riu
Ps yc hr ob
o hr
yc Ps
21
44
36
0 J6 _A
Ps
66
y
Ps
ac
99
b ro ch
41
12
t er
in
t
yc
73
a r_
92
ob hr
te ac
96
yc
a
yc
yc
ba hro
30
48
71
Y _A
t en lim
8 N1
Ps
99
bac
la
60
76
ac
ico
58
46
72
cter roba
igid
28
b hro
_fr t er
15
1 B TN
s 7_P
BT
s _P
c ba
BTN20A_Psy
sy BTN14 P
ro ych
AJ
89
0.02
1
er chrobact
21 B TN
_ sus
3 J4 43
t er
3
BTN19_Psychrobacter
ch _Psy
42
AB
ac
Y6
AJ
_AF44120
no
ro b
s_
A is_
ne
725
are s_ al i
tivo
X 80
er_
s_A
on
ter_jeotgali
Ps
ec
sy
ch
riu
_ coli
a ct
m ul
_P
vig e
39 87 ran 1 s_A J 6 095 rii_EU 5 9397 5 18
Psychrobac
Ps
a r_f
r_ fu l
BTN15_P
lla_
rob
p r_
te
N2 3
cte r_c iba
ura
ct e
estua
ba
ter_a
bac ter_
te ac
robac
ba
hro
Psych
BT
ac
BTN13 Psychrobacter
e Shig ych yc
Psy chro
hr ob
sychroba cter
hia/ heric Esc Ps Ps
Ps ych ro
yc
ob hr yc Ps
Ps
chrob acter B TN 2P syc Ps hr o ych bac ter rob ac Ps t er yc _a hr qu ob im ac ari BT t er s_ N1 AY _fo 72 6_ zii 28 _A Ps 04 J4 yc 30 hr 82 ob 7 ac te r
S4 of S18
57
3
(C) Figure S1. Phylogenetic trees (using the complete deletion option) of the Arthrobacter (A); Pseudomonas (B) and Psychrobater (C) genera.
Mar. Drugs 2016, 14, 83
S5 of S18
MayV-00635.10.1.1r UserName Aberdeen
17000000
SampleName BTN1-C8
16000000 15000000 14000000 13000000 12000000 11000000 10000000 9000000 8000000 7000000 6000000 5000000 4000000 3000000 2000000 1000000
-1000000
6.00
42.47
7.15
5.38
2.54
2.69
1.38
1.22
1.36
0
6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 f1 (ppm)
Figure S2. 1H-NMR spectrum of compound 1 in CD3OD at 600 MHz.
10
MayV-00635.12.1.2rr UserName Aberdeen SampleName BTN1-C8 eHSQCedSolvent: MeOH
20
30
40
60
70
80
90
100
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2 3.0 f2 (ppm)
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
Figure S3. Edited-HSQC spectrum of compound 1 in CD3OD at 600 MHz.
1.0
0.8
f1 (ppm)
50
Mar. Drugs 2016, 14, 83
S6 of S18
MayV-00635.11.1.2rr UserName Aberdeen
1.0
SampleName BTN1-C8 Experiment: eCOSY Solvent: MeOH 1.5
2.0
3.0
3.5
4.0
4.5
5.0
5.5
5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 f2 (ppm)
Figure S4. COSY NMR spectrum of compound 1 in CD3OD at 600 MHz.
0
MayV-00635.13.1.2rr UserName Aberdeen
10
SampleName BTN1-C8 Experiment: eHMBC_NUS.MeOH
20 30 40 50 60
80 90 100 110 120 130 140 150 160 170 5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2 3.0 f2 (ppm)
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
Figure S5. HMBC spectrum of compound 1 in CD3OD at 600 MHz.
1.0
0.8
0.6
f1 (ppm)
70
f1 (ppm)
2.5
Mar. Drugs 2016, 14, 83
S7 of S18
BTN1-C8_DMSO.37.fid 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0
6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 f1 (ppm)
Figure S6. 1H-NMR spectrum of compound 1 in DMSO-d6 at 400 MHz. MayV-00633.10.fid UserName Aberdeen BTN1-C6
19000 18000 17000 16000 15000 14000 13000 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0 f1 (ppm)
3.5
3.0
2.5
2.0
1.5
-1000
6.00
21.72
2.31
1.80 2.18 1.13 1.15 2.18
1.11
1.03 2.29
1.18
1.16 1.25 1.27
0
1.0
Figure S7. 1H-NMR spectrum of compound 2 in CD3OD at 600 MHz.
0.5
0.0
Mar. Drugs 2016, 14, 83
S8 of S18
MayV-00633.12.ser UserName Aberdeen
10
SampleName BTN1-C6 Experiment: eHSQCed_NUS.
20 30 40 50
70 80
f1 (ppm)
60
90 100 110 120 130 140 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 f2 (ppm)
Figure S8. Edited-HSQC NMR spectrum of compound 2 in CD3OD.
MayV-00633.11.ser UserName Aberdeen SampleName BTN1-C6 COSY
1.0
1.5
2.0
2.5
3.5
4.0
4.5
5.0
5.5
6.0
6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 f2 (ppm)
Figure S9. COSY NMR spectrum of compound 2 in CD3OD.
f1 (ppm)
3.0
Mar. Drugs 2016, 14, 83
S9 of S18
0
MayV-00633.13.1.2rr _BTN1-C6_180515_1H-2DCOSY-2DHSQCed-2DHMBC_av600_MayV-00633
10 20 30 40 50 60 70
90 100 110 120 130 140 150 160 170 180 6.0
4.5
5.0
5.5
4.0
3.5 f2 (ppm)
2.0
2.5
3.0
1.0
1.5
0.5
Figure S10. HMBC NMR spectrum of compound 2 in CD3OD. BTN1-C6_DMSO.38.fid
12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0 f1 (ppm)
3.5
3.0
2.5
2.0
1.5
1.0
Figure S11. 1H-NMR spectrum of compound 2 in DMSO-d6 at 400 MHz.
0.5
0.0
f1 (ppm)
80
Mar. Drugs 2016, 14, 83
S10 of S18
Figure S12. 2D ROESY spectrum of compound 2 in DMSO-d6 with Rhamnose protons labelled.
C5H12
C7H15
O O
H
O OH
O
H H
O HO
HO H
H OH
COSY ROE
Figure S13. Compound 2 showing selected ROE correlations of the rhamnose moiety and COSY correlations showing position of unsaturation of the lipid chain.
Mar. Drugs 2016, 14, 83
S11 of S18
MayV-00637.14.1.1r SampleName BTN1-C10 Experiment: eSpresat Solvent: MeOH
3000000 2800000 2600000 2400000 2200000 2000000 1800000 1600000 1400000 1200000 1000000 800000 600000 400000 200000 0 -200000
5.8
5.4
4.6
5.0
4.2
3.8
2.6 3.0 f1 (ppm)
3.4
1.8
2.2
1.4
1.0
0.6
0.2
-0.2
Figure S14. 1H-NMR spectrum of compound 3 in CD3OD at 600 MHz.
10
MayV-00637.12.1.2rr SampleName BTN1-C10 Experiment: eHSQCed_NUS.highres Solvent: MeOH Holder: 11 Group
20 30 40 50
70 80 90 100 110 120 130 5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
f2 (ppm)
Figure S15. Edited-HSQC NMR spectrum of compound 3 in CD3OD.
1.0
f1 (ppm)
60
Mar. Drugs 2016, 14, 83
S12 of S18
MayV-00637.11.1.2rr SampleName BTN1-C10 Experiment: eCOSY Solvent: MeOH Holder:
1.0
1.5
2.0
3.0
3.5
f1 (ppm)
2.5
4.0
4.5
5.0
5.5
5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 f2 (ppm)
Figure S16. COSY NMR spectrum of compound 3 in CD3OD.
MayV-00637.13.1.2rr SampleName BTN1-C10 Experiment: eHMBC_NUS.highres Solvent: MeOH
20 30 40 50 60 70
90 100 110 120 130 140 150 160 170 180 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 f2 (ppm)
Figure S17. HMBC NMR spectrum of compound 3 in CD3OD.
f1 (ppm)
80
Mar. Drugs 2016, 14, 83
S13 of S18
Retention Time: 21.149
Ion Mode: ESI+ 555.351 555.351
22 20 18 16 14 12 10 556.356 556.356
8 6 4 2
554.783 554.783
ESI+
554.406 554.406 554.083 554.083
557.359 556.624 556.624
555.920 555.920
557.632 557.632 558.359 558.359
0 554.0
554.5
555.0
555.5
556.0
556.5
557.0
557.5
558.0
Figure S18. HRESIMS spectrum (M + Na)+ of compound 1.
Figure S19. ESI MS/MS data of compound 1.
558.5
m/z
Mar. Drugs 2016, 14, 83
S14 of S18
Retention Time: 20.240
Ion Mode: ESI+
70 65 60 55 553.337 553.337
50 45 40 35 30 25 20
554.339 554.339 15 10 5
ESI+
555.341 555.341 554.603 554.603
553.148 553.148
552.073 552.073 551.329 551.329
552.770 552.770
551.5
552.5
555.608 555.608 556.346 556.346
0 552.0
553.0
553.5
554.0
554.5
555.0
555.5
Figure S20. HRESIMS spectrum (M + Na)+ of compound 2.
Figure S21. ESI MS/MS data of compound 2.
556.0
556.5
m/z
Mar. Drugs 2016, 14, 83
S15 of S18 581.368 581.368
Retention Time: 21.698
Ion Mode: ESI+
96 88 80 72 64 56 48 40 582.370 582.370 32 24 16 8
ESI+ 580.352 580.352
583.374 583.374 581.977 581.977 581.571 581.571
580.757 580.757
582.983 582.983
583.742 583.742
584.376 584.376
0 580.5
581.0
581.5
582.0
582.5
583.0
583.5
Figure S22. HRESIMS spectrum (M + Na)+ of compound 3.
Figure S23. ESI MS/MS data for compound 3.
584.0
m/z
Mar. Drugs 2016, 14, 83
S16 of S18
Table S1. Cross- streaking experiments of BTN isolates against a wide panel of Bcc strains. The Bcc strains highlighted in red correspond to type strains.Symbols: +, growth; ±, reduced growth; -, no growth; C+, positive controls, i.e., Bcc strains grown in the absence of the tester strain(s). Abbreviations: Ps, Pseudomonas; Ar, Arthrobacter; CF, Cystic Fibrosis; Env; environmental. Bcc Species
B. cepacia
B. multivorans
B. cenocepacia
Source
Strain
C+
CF
FCF 1
CF
FCF 2
CF
Ps
Psychrobacter BTN21 BTN24 BTN23
BTN5
Ar
BTN1
BTN2
BTN15
BTN3
BTN19
BTN20B
BTN12 BTN14 BTN17 BTN16 BTN18 BTN11 BTN13
BTN22
BTN4
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
FCF3
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
LMG 2161
+
-
-
-
-
±
-
-
-
-
-
-
-
-
-
CF
FCF 5
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 6
+
-
-
-
-
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 7
+
-
-
-
-
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 8
+
-
-
-
-
±
-
-
-
-
-
-
CF
FCF 9
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 10
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 11
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 18822
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
LMG 17588
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 12
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 13
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 14
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 15
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 16
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 17
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 18
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 19
+
±
-
-
±
-
-
-
-
±
-
±
±
-
±
-
±
-
-
±
CF
FCF 20
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 21
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 22
+
-
-
±
-
-
±
±
-
-
±
-
-
-
-
-
-
-
-
-
CF
FCF 23
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 24
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 25
+
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
±
-
CF
FCF 26
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Mar. Drugs 2016, 14, 83
B. stabilis B. vietnamensis B. dolosa B. ambifaria B. anthina
B. pyrrocinia
S17 of S18
CF
FCF 27
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 28
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 29
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 30
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 31
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 16654
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
C 5424
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
CEP 511
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
MVPC 1/16
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
MVPC 1/73
+
-
-
-
-
-
-
-
-
±
-
-
-
-
-
-
-
-
-
-
CF
LMG 24506
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
LMG 19230
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
LMG 19240
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 32
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 33
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 34
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 36
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 37
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 38
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 39
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 21462
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 40
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 41
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 42
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 18941
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 18942
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
MCI 7
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 19467
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
LMG 16670
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
LMG 20983
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 43
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 44
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CF
FCF 45
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Mar. Drugs 2016, 14, 83
B. lata
S18 of S18
CF
FCF 46
+
-
-
-
-
-
-
-
-
Env
MVPC 1/26
+
-
-
-
-
-
-
-
Env
MVPC 2/77
+
-
-
-
-
-
-
-
CF
LMG 21824
+
-
-
-
-
-
±
CF
LSED 4
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
±
-
±
-
-
-
-
-
-
-
-
-
-
±
-
±
-
-
-
-
-
-
±
-
-
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Env
LMG 6991
+
-
-
-
-
±
±
±
-
±
-
-
-
-
±
-
-
-
-
±
B. ambifaria
Env
LMG 19182
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
±
-
-
-
B. anthina
Env
LMG 20980
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. cenocepacia
CF
LMG 16656
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. cepacia
Env
LMG 1222
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. contaminas
AI
LMG 23361
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. diffusa
CF
LMG 24065
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. dolosa
CF
LMG 18943
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. lata
Env
LMG 22485
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. latens
CF
LMG 24064
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. metallica
CF
LMG 24068
+
-
-
+
-
-
±
±
±
±
±
+
-
-
-
-
-
-
-
-
B. multivorans
CF
LMG 13010
+
-
-
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. pseudomultivorans
CF
LMG 26883
+
-
-
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. pyrrocinia
Env
LMG 14191
+
-
-
+
-
-
-
-
±
±
-
+
-
-
-
-
-
-
-
-
B. seminalis
CF
LMG 24067
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. stabilis
CF
LMG 14294
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. uborrensis
Env
LMG 20358
+
-
-
±
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
B. vietnamensis
Env
LMG10929
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
