Supporting Information Appendix Anaerobic biosynthesis of the lower ligand of vitamin B12   

1  

Supporting Information Appendix

Methods:

Cultivating Geobacter sulfurreducens PCA: G. sulfurreducens was cultivated anaerobically

with fumarate as the electron acceptor and acetate as the electron donor in the absence of added

vitamins as previously described (1). Briefly, the medium contained basal salts, 40 mM fumarate

and 20 mM acetate. The medium was flushed with 20% CO2:80% N2, inoculated inside an

anaerobic chamber, and cultures were incubated at 30 °C for 72 h.

Cultivating Eubacterium limosum ATCC 10825: E. limosum ATCC 10825 was grown in

modified Actinomyces broth composed of 17 g tryptone, 10 g yeast extract, 5 g dextrose, 5 g

NaCl, 13 g K2HPO4, 2 g KH2PO4, 1 g (NH4)2SO4, 1 g starch, 0.2 g MgSO4 x 7 H2O, and 10 mg

CaCl2 x 2 H2O per liter. The medium was additionally supplemented with 1 ml of trace elements

solution (SL-10) (2), 1 ml of Se/WO solution (4 mg Na2SeO3 x 5 H2O, 8 mg Na2WO4 x 2 H2O,

and 0.5 g NaOH per liter), and 1 ml of Wolin’s vitamin solution (with vitamin B12 omitted) per

liter (3). The medium was prepared under an atmosphere of N2 gas and reduced with a 0.01%

(w/v) cysteine-sulfide solution.

Cultivating Moorella thermoacetica ATCC 39073: M. thermoacetica ATCC 39073 was grown

in reinforced Clostridial medium supplemented with Wolin’s vitamin solution (with vitamin B12

omitted) (3, 4).

Growth curve experiments with Salmonella enterica serovar Typhimurium strain LT2:

Growth curves of S. enterica serovar Typhimurium strain LT2 were performed by inoculating

triplicate 5 ml cultures of LB medium with single colonies and growing to saturation at 37 ºC.

Supporting Information Appendix Anaerobic biosynthesis of the lower ligand of vitamin B12   

2  

Cells were harvested by centrifugation at 12,800 x g and washed three times with a minimal

medium containing glycerol as the carbon source and ethanolamine as the nitrogen source (5).

Each cell suspension was diluted to an optical density at 600 nm (O.D.600) of 0.001 in 5 mL of

the same medium containing the indicated supplements. The cultures were grown at 30 ºC with

aeration, and the O.D.600 was measured at the indicated time points. Supplements included 10

nM cobamide product purified by HPLC from E. coli , HPLC-purified buffer salts, 10 nM

HPLC-purified cyanocobalamin, or 10 nM cyanocobalamin.

Corrinoid extraction and HPLC analysis: Corrinoids were extracted with methanol from cell

pellets, then cyanated and desalted as described (6). An Agilent 1200 series HPLC system

equipped with a UV-diode array detector was used to analyze the extracted corrinoids. An

Agilent Zorbax SB-Aq reverse phase column (5m, 4.6 x 150 mm) was used at a flow rate of 1

ml min-1 at 30 C. Chromatograms were recorded at 365, 525, and 550 nm. Mobile phases used

were 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B).

Samples were analyzed by the following method: 25% solvent B over 2 min, followed by a

linear gradient of 25 to 34% solvent B over 11 min, 34 to 100% solvent B over 3 min, and 100 to

25% solvent B over 0.5 min.

Biochemical characterization of G. sulfurreducens BzaF: His-tagged G. sulfurreducens PCA

BzaF was expressed in E. coli BL21(DE3) containing a plasmid encoding the suf operon for in

vivo [4Fe-4S] reconstitution (7). A 15 ml culture of this strain was grown overnight in LB

medium contining kanamycin (40 mg/L) and chloramphenicol (25 mg/L). 1.9 L of M9 minimal

media containing 0.7% glucose, 40 mg/L kanamycin and 25 mg/L chloramphenicol was

Supporting Information Appendix Anaerobic biosynthesis of the lower ligand of vitamin B12   

3  

inoculated with this culture. The culture was incubated at 37 °C with shaking (180 rpm) until the

O.D.600 reached 0.50 to 0.55. The culture was then incubated at 4 °C without shaking for 2 h.

Subsequently, 50 mg of ferrous ammonium sulfate and 50 mg of cysteine were added, followed

by induction of the culture with 70 µM isopropyl β-D-1-thiogalactopyranoside (IPTG). The

culture was incubated at 15 °C with shaking (50 rpm) for 18-20 h. The cultures were then

incubated at 4 °C for 3 h without shaking. The cells were harvested and stored in liquid nitrogen

overnight before enzyme purification. All subsequent steps were performed in an anaerobic

chamber. The cell pellets were thawed at room temperature and suspended in lysis buffer (100

mM Tris-HCl, pH 7.5) in the presence of 2 mM DTT, lysozyme (0.2 mg/ml) and benzonase (100

units). This mixture was then cooled in an ice bath for 2 h. The cell suspension was sonicated

and centrifuged to obtain the cell-free extract. The enzyme was purified using standard Ni-NTA

chromatography. The column was first equilibrated with the lysis buffer. The cell-free extract

was then passed over the column, followed by 8-9 column volumes of wash buffer (100 mM

Tris-HCl, 300 mM NaCl, 20 mM imidazole, 2 mM DTT, pH 7.5). The enzyme was eluted with

100 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole, 2 mM DTT, pH 7.5. The purified enzyme

was buffer exchanged into 100 mM potassium phosphate, 30% glycerol, 2 mM DTT, pH 7.5

using an Econo-Pac 10DG desalting column (Bio-Rad) and the purified enzyme was stored in

liquid nitrogen. The purified protein contains a [4Fe-4S] cluster as verified by UV-Vis

spectroscopy (8).

Supporting  

 

g Information AAppendix Anaerobic

4 

biosynthesis oof the lower liggand of vitammin B12

Supporting  

 

Figure S1in the E. linvolved iindicate oannotatedpredicted

Figure S2from geneas thiC froThiC tran

g Information A

1. E. limosumlimosum KISTin cobalamin

open reading fd in the Joint G

to regulate th

2. An expandes annotated aom genomes

nslated gene se

Appendix

m genes adjacT612 genomebiosynthesis,

frames (ORFsGenomic Insthe expression

ded view of thas thiC in the that had multequences app

cent to cobale. Most of the, transport or s); ORF numbitute (JGI) daof any ORFs

he phylogeneentire Pfam s

tiple thiC hitspear in distinc

Anaerobic

5 

amin riboswe cobalamin rregulation, asbers are showatabase. Riboss.

etic tree showseed alignmen (200 sequenct clades. Exp

biosynthesis o

witches. Additriboswitches as indicated by

wn below and switch #7 is l

wn in Fig. 2Bnt (113 sequeces). The puterimentally v

of the lower lig

tional cobalamare associatedy the color schgene names aocated in an o

B. The tree waences) and all ative BzaA/B

verified functi

gand of vitam

min riboswitcd with genes heme. Arroware above, as orientation no

as constructedgenes annota

BzaB, BzaF anions for BzaA

min B12

ches

ws

ot

d ated nd

A,

Supporting  

 

BzaB andto boldfac

Figure S3A. 1-D 1Ha 1-D 1H Npeaks of t

B. UV-Vicyanocob

g Information A

d BzaF homolce in Fig. 2B)

3. Chemical aH NMR analyNMR of standthe lower liga

is spectrum ofalamin (red).

Appendix

ogs and for T.

analysis of thsis of the HPLdard cyanoco

and DMB are

f the cobamid

ThiC homolog

he cobamideLC-purified c

obalamin purishown in the

de product of

Anaerobic

6 

gs are indicate

produced bycobamide profied under the expanded vie

the bza opero

biosynthesis o

ed by * and #

y E. coli exprduct of the bze same HPLCew.

on (black) is i

of the lower lig

#, respectively

ressing the bzza operon (blaC conditions (

identical to th

gand of vitam

y (correspond

za operon. ack) compare(red). The pro

hat of standard

min B12

ding

ed to oton

d

Supporting  

 

Figure S4A. LC-MSexpressingcorrinoid different rB. Oxygeindicated traces) in shown. Incobamidecontainedstep in thegrown culwith 5-OHsynthesizebiosynthefrom the aculture cocould be f[5-OMe-6

g Information A

4. ExpressionS analysis of g the individuextracts. Theretention timen sensitivity ogenes from thminimal med

n the strain coes [5-OHBza]d predominante pathway is olture containiHBza; in the eed by the cell

esis can be peradded 5-OHBontaining the bformed at low6-MeBza]Cba

Appendix

n of E. limosuthe corrinoid

ual bzaA and e asterisk (*) e. of the E. limohe bza operondia containingntaining the cCba, [5-OMetly B12, indicaoxygen-sensitng the bzaA-bextract from tls under both rformed in th

Bza). A small bzaA-bzaB-co

w levels. The ca only under a

um bza geness extracted frbzaB genes. Nindicates an u

osum DMB bin were cultureg Cbi. LC-MScomplete bzaeBza]Cba andating that somtive, as evidenbzaB constructhis culture, [5anaerobic ande presence ofamount of [5

obT-bzaC conculture contaianaerobic con

Anaerobic

7 

s under aerobom E. coli ∆mNo benzimidaunknown corr

iosynthesis paed anaerobicaS analysis of c

operon, the ad B12 at low leme steps in the

nced by the act. The strain 5-OMeBza]Cd aerobic conf oxygen (in th-OMeBza]Cb

nstruct, suggeining the bzaA

nditions. An E

biosynthesis o

bic and anaemetE strains cazolyl cobamrinoid with th

athway. E. coally (black tracorrinoid extraerobic culturevels, whereae pathway areabsence of [5-expressing on

Cba was produnditions, indichis culture, [5ba was formeesting that theA-bzaB-cobT-E. coli ∆metE

of the lower lig

erobic growthcontaining the

mides are deteche same mass

oli strains expaces) or aerobracts from there contained ts the anaerobe sensitive to -OHBza]Cba nly bzaC wasuced from 5-Ocating that 5-O5-OHBza]Cbaed in the aerobe prior interm

T-bzaC-bzaD cE background

gand of vitam

h conditionse empty vectoctable in theseas FA but wi

ressing the bically (gray ese cultures isthe benzimidabic culture oxygen. The in the aerobic

s supplementeOMeBza OMeBza a was also forbically grownediate, 5-OHBconstruct prodwas used for

min B12

or or e ith a

s azolyl

first cally ed

rmed n Bza, duced the

Supporting  

 

plasmids cbackgrounC, D, E. Trespectivethe spectr

Figure S5abbreviati(9-13).

g Information A

containing bznd. The peaksThe UV-Vis sely, producedra of the corre

5. Benzimidaions of the low

Appendix

zaA-bzaB ands labeled withspectra of [5-O

by E. coli exesponding stan

azole lower liwer ligands a

d bzaC, whereh an asterisk (OHBza]Cba, xpressing bzandards.

igands in anaand the names

Anaerobic

8 

eas the remain(*) are unknow[5-OMeBza]Cgene combin

aerobic bactes of the organ

biosynthesis o

ning constructwn corrinoidsCba and [5-O

nations, are in

eria and archnisms reported

of the lower lig

ts were in a ∆s.

OMe-6-MeBzndistinguishab

haea. The strud to produce t

gand of vitam

∆metE ∆cobT

a]Cba ble from those

uctures and them are show

min B12

T

e of

wn

Supporting  

 

Figure S6Genetic loidentified benzimidaorganismsprevious biochemic

g Information A

6. Expanded oci containing

in the JGI azole lower s for which estudies (*),

cal characteri

Appendix

list of the pug predicted bz

database. Bligand that xperimental dor by heter

zation of the

utative bza geza genes adja

Based on thethese organi

data for cobamrologous expenzymes (@)

Anaerobic

9 

enes in bacteacent to cobam subset of bisms synthesmide productpression stud) in this study

biosynthesis o

eria and archmide biosynthbza genes prsize. The pretion are availdies performey.

of the lower lig

haea. hesis or metaresent, we hedictions areable (bold faced on the b

gand of vitam

abolism geneshave predictee validated ice) from this bza genes (&

min B12

s were ed the in the (#) or

&) or

Supporting  

 

Figure S7A: LC-Mstandard o

B: The Ublack) is o

Figure S8biosyntheLC‐MS/Mbza genesformationproductiostrain expbzaC wereΔmetE Δc

g Information A

7. Geobacter S/MS analysiof [5-OHBza]

UV-Vis spectroverlaid with

8. Expressionesis.

MS analysis ofs from M. thern of [5‐OMeBn in this strain

pressing only e expressed incobT backgro

Appendix

sulfurreduceis of corrinoi]Cba.

rum of the mthat of a stan

n of the Moor

f corrinoid exrmoacetica is

Bza]Cba, the pn is likely dubzaC was culn a ΔmetE baund.

ens producesds extracted f

major cobamidndard of [5-OH

rella thermoa

xtracts from as shown. The previously repe to the attachltured with 5‐ackground, wh

Anaerobic

10 

[5-OHBza]Cfrom G. sulfu

de produced bHBza]Cba (b

acetica bza op

anaerobically complete ope

ported cobamhment of the i‐OHBza. The hereas the rem

biosynthesis o

Cba. urreducens. T

by G. sulfurrlue).

peron in E. c

grown E. coleron, bzaA‐bz

mide of M. therintermediate plasmids con

maining const

of the lower lig

The major pea

reducens (10.

coli directs 5-

li expressing hzaB‐cobT‐bzarmoacetica. [5‐OHBza to a

ntaining only tructs were ex

gand of vitam

ak co-elutes w

.2 min in pan

-OMeBza

homologs of taC, directs the[5‐OHBza]Cbadded Cbi. ThbzaA‐bzaB o

xpressed in a

min B12

with a

nel A,

the e ba he r

Supporting  

 

Suppleme

Table S1.BzaF and (M. t.), G.using the

g Information A

ental Tables

. Percent ideThiC sequen

. sulfurreducepairwise align

Appendix

ntity matrix nces are from ens (G. s.), P.nment tool in

table of Bzathe organism propionicus

n BioEdit.

Anaerobic

11 

aA, BzaB, Bzams E. limosum

(P. p.) and M

biosynthesis o

aF and ThiC (E. l.), A. wo

M. barkeri (M.

of the lower lig

C proteins. Thoodii (A. w.), M

M. b.). The data

gand of vitam

he BzaA, BzaM. thermoacea were genera

min B12

aB, etica ated

Supporting  

 

Table S2.

g Information A

. Primers use

Appendix

ed in this stu

udy

Anaerobic

12 

biosynthesis oof the lower liggand of vitammin B12

Supporting  

 

Table S3.

Referenc

1. Mvb

2. Wb

3. WB

4. HanD

g Information A

. Strains and

es

Marsili E, Rooltammetry:iofilms. App

Widdel F, Koacteria that d

Wolin EA, WBiol Chem 23Hirsch A & Gnaerobic spo

Dairy Resear

Appendix

d plasmids u

ollefson JB, B direct electr

pl Environ Mohring G-W,decompose f

Wolin MJ, & 38:2882-288Grinsted E (1ore-formers frch 21(01):10

sed in this st

Baron DB, Hrochemical c

Microbiol 74( & Mayer F fatty acids. AWolfe RS (16.

1954) 543. Mfrom cheese01-110.

Anaerobic

13 

tudy

Hozalski RMcharacterizat(23):7329-73(1983) Stud

Arch of Micr1963) Forma

Methods for t, with observ

biosynthesis o

M, & Bond Dtion of catal337. dies on dissimrobiol. 134(4ation of Met

the growth avations on th

of the lower lig

DR (2008) Mlytic electrod

milatory sulf4):286-294. thane by Bac

and enumerahe effect of n

gand of vitam

Microbial biode-attached

fate-reducin

cterial Extrac

ation of nisin. Journa

min B12

film

ng

cts. J

al of

Supporting Information Appendix Anaerobic biosynthesis of the lower ligand of vitamin B12   

14  

5. Scarlett FA & Turner JM (1976) Microbial metabolism of amino alcohols. Ethanolamine catabolism mediated by coenzyme B12-dependent ethanolamine ammonia-lyase in Escherichia coli and Klebsiella aerogenes. J Gen Microbiol 95(1):173-176.

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8. Chatterjee A, et al. (2008) Reconstitution of ThiC in thiamine pyrimidine biosynthesis expands the radical SAM superfamily. Nat Chem Biol 4(12):758-765.

9. Irion E & Ljungdahl L (1965) Isolation of factor 3m coenzyme and cobyric acid coenzyme plus other B12 factors from Clostridium thermoaceticum. Biochemistry 4(12):2780-2790.

10. Krӓutler B, Kohler HP, & Stupperich E (1988) 5'-Methylbenzimidazolyl-cobamides are the corrinoids from some sulfate-reducing and sulfur-metabolizing bacteria. Eur J Biochem 176(2):461-469.

11. Pol A, van der Drift C, & Vogels GD (1982) Corrinoids from Methanosarcina barkeri: structure of the alpha-ligand. Biochem Biophys Res Commun 108(2):731-737.

12. Stupperich E & Eisinger H (1989) Function and the biosynthesis of unusual corrinoids by a novel activation mechanism of aromatic compounds in anaerobic bacteria. Advances in Space Research 9(6):117-125.

13. Stupperich E, Eisinger HJ, & Krӓutler B (1988) Diversity of corrinoids in acetogenic bacteria. P-cresolylcobamide from Sporomusa ovata, 5-methoxy-6-methylbenzimidazolylcobamide from Clostridium formicoaceticum and vitamin B12 from Acetobacterium woodii. Eur J Biochem 172(2):459-464.

14. Cheng J, Sibley CD, Zaheer R, & Finan TM (2007) A Sinorhizobium meliloti minE mutant has an altered morphology and exhibits defects in legume symbiosis. Microbiology 153 (Pt 2), 375-387.