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Identification and characterization of a periplasmic trilactone esterase, Cee, revealed
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unique features of ferric enterobactin acquisition in Campylobacter
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Supporting Information
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Ximin Zenga, Yiming Moa, Fuzhou Xua,b, and Jun Lina*
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a
Department of Animal Science, The University of Tennessee, 2640 Morgan Circle Drive,
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Knoxville, TN 37996, USA; and bInstitute of Animal Science and Veterinary Medicine, Beijing
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Academy of Agriculture and Forestry Sciences, Beijing 100097, China
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Running title: Campylobacter ferric enterobactin esterase
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*Corresponding author:
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Dr. Jun Lin
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Department of Animal Science
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The University of Tennessee
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2640 Morgan Circle Drive
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Knoxville, TN 37996-4574, USA
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Phone: (865)974-5598; Fax: (865)974-7297
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Email: jlin6@utk.edu
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1
1
Results
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Genome sequencing of C. jejuni ATCC 33560 (JL11). The genomic DNA of C. jejuni
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ATCC 33560 was sequenced using a 454 GS FLX sequencer with Titanium series reagents. The
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sequencing statistics were summarized in Table S1. Total 46 contigs were aligned and ordered
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against the completed reference genomes of three C. jejuni strains NCTC 11168 (Parkhill et al.,
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2000), 81-176 (Hofreuter et al., 2006) and RM1221 (Fouts et al., 2005) using Mauve (Rissman
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et al., 2009). The ATCC 33560 contigs were best ordered against the C. jejuni RM1221
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reference (Fig. S1). Totally 46 pairs of PCR primers were designed for gap closure based on the
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C. jejuni RM1221 genome as well as the contig sequences. Approximately 1,100 PCR reactions
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were performed and 150 purified PCR products were subjected to Sanger sequencing. The three
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copies of ribosomal contigs (Parkhill et al., 2000) were assembled into the scaffold, which has
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been confirmed by PCR. Following gap closure, the high quality draft genome sequence was
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subjected to automatic annotation using the RAST server (Aziz et al., 2008), which revealed
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1,954 coding sequences and 48 RNAs in C. jejuni ATCC 33560 genome.
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Genomic evidence showing Cee is the sole Ent trilactone esterase in Campylobacter.
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Amino acid sequences of the three characterized Ent trilactone esterases, Fes (C0671), IroD
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(C1252) and IroE (C1251) from uropathogenic Escherichia coli CFT073 (Lin et al., 2005), were
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used to search for Ent esterase homologs in sequenced Campylobacter genomes using tblastn
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program with the default setting (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi).
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genome sequences examined including those of 20 C. jejuni strains and 2 C. coli strains. Each
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genome either contains only one Ent esterase homolog (Cee) or does not have any genes
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The
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homologous to the three query esterases. Fig. S3 shows the representative search for C. jejuni
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NCTC11168 and 81-176, indicating no Ent esterase homologs other than Cee (Cj1376) in the
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genome.
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Table S1. 454 sequencing statistics of C. jejuni ATCC 33560.
Parameter
Corresponding Value
Total number of reads
172,427
Total number of bases
66,731,820
Total number of reads used in Newbler
171,669
Total number of bases used in Newbler
66,433,940
Average read length
387.0
No. of total bases in all contigs
1,732,440
No. of total bases in large contigs
1,730,042
No. of all contigs
45
No. of large contigs
38
Size (bp) of large contigs
601 to 312,882
N50 of large contig size
192,284
Average size (bp) of large contigs
44,373
Size (bp) of small contigs
101 to 489
Average coverage depth fold
38.3
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Table S2. Genes implicated in FeEnt acquisition among four C. jejuni strains based on genome sequence
analysis.
C. jejuni Strains
NCTC11168
81-176
ATCC 33560
RM1221
FeEnt utilization
Yes
No
Yes
No
cfrA
+
–
–
+
cfrB
pseudogene
+
+
pseudogene
tonB1
+
–
–
+
exbB1/exbD1
+
–
–
+
tonB2
+
+
+
pseudogene
exbB2/exbD2
+
+
+
+
tonB3
+
–
–
+
exbB3/exbD3
+
+
+
+
ceuBCD
+
+
+
+
ceuE
+
+
+
pseudogene
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+, presence of the gene.
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-, absence of the gene.
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Table S3. Genes present in C. jejuni NCTC 11168 and ATCC 33560, but absent in 81-176
Coding Length in
IronJL11(aa)
Regulateda
NCTC 11168 Locus
Gene Annotation
Cj0030
hypothetical protein
465
-
Cj0422c
putative helix-turn-helix containing protein
69
-
Cj0501
pseudogene (putative ammonium transporter)
372
-
Cj0668
putative ATP /GTP-binding protein
135
-
Cj1301
hypothetical protein
138
-
Cj1376b
putative periplasmic protein
269
-
Cj1521c
putative CRISPR-associated protein
143
-
Cj1522c
putative CRISPR-associated protein
296
-
Cj1523c
putative CRISPR-associated protein
984
-
Cj1585c
Lactate oxidase
923
-
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3
a
4
5
b
Based on available transcriptome profiling of C. jejuni NCTC11168 in response to iron (Palyada et al.,
2004).
The adjacent genes (Cj1375 and Cj1377c) are induced under iron-restricted conditions (Palyada et al.,
2004).
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Table S4. Key bacterial plasmids and strains used in this study
Plasmids or strains
Description
Source or Reference
Plasmids
pRY107
E. coli-Campylobacter shuttle vector, kanamycin resistant (Kmr)
(Cmr)
(Yao et al., 1993)
pRY111
E. coli-Campylobacter shuttle vector, chloramphenicol resistant
pCfrB
pRY107 derivative containing cfrB plus its promoter region
(Xu et al., 2010)
pCj1375-76
pRY107 derivative containing cj1375-76 operon plus its promoter region
This study
pCj1376
pCj1375-76 derivative with in-frame deletion of Cj1375 but still containing the
promoter of Cj1375
This study
pCj1376B
The BamHI/PstI fragment from pCj1376, containing Cj1376 gene, was sub-cloned intoThis study
pRY111. The function of this plasmid and pCj1376 is deemed same except resistance
marker difference.
pQE30
Expression vector for N-His6 fusion recombinant protein, Ampr
Qiagen
pCj1376N30
pQE30 expression vector containing Cj1376 with the removal of first 29 aa
This study
Ampr
(Yao et al., 1993)
pGEMT-Easy
PCR cloning vector,
pCee
T-easy vector derivative containing Cj1376
Promega
This study
pCee(Cm)
pCee derivative with cat cassette inserted at SwaI site, used as a suicide vector
This study
pCc1376
pRY107 derivative containing Cj1376 homolog and two upstream genes from C. coli
JL170
This study
pC1376
T-easy vector derivative containing Cj1376 homolog from C. coli JL170
This study
pC1376(Cm)
pC1376 derivative with cat cassette inserted at HindIII site, used as a suicide vector
This study
pCeuBCDE
pRY107 derivative containing ceuBCDE operon plus its promoter region
This study
pABC-cj1375-76
pRY111 derivative containing cj1375-76 operon plus the promoter of cmeABC
(Jeon et al., 2011)
pTonB3
pRY107 derivative containing tonB3
This study
pCfrA
pRY107 derivative containing intact cfrA plus its promoter
(Zeng et al., 2009)
JL11
ATCC 33560, bovine isolate
(Zeng et al., 2009,
Xu et al., 2010)
JL849
JL11 derivative, cee-
This study
JL241
NCTC 11168, human isolate
(Parkhill et al.,
2000)
JL491
11168 derivative, cfrA-
Dr. Richard Haigha
JL531
JL491/pCfrB
This study
JL818
11168 derivative, cee-
This study
JL673
JL531 derivative,
cee-
This study
JL593
JL531 derivative, ceuE-
JL583
11168 derivative,
ceuE-
JL590
11168 derivative, ceuE- cee-
(Palyada et al.,
2004)
JL242
81-176, human isolate
(Black et al., 1988)
JL229
81-176/pRY111
This study
JL689
81-176/pRY107
This study
JL535
81-176/pCj1375-76
This study
JL536
81-176/pCj1376
This study
Strains
C. jejuni
This study
(Palyada et al.,
2004)
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JL726
81-176 derivative containing plasmid pCc1376
This study
JL648
81-176/pCj1376(S157A)
This study
JL649
81-176/pCj1376(H251A)
This study
JL870
81-176 derivative containing plasmid pABC-cj1375-76
This study
JL595
81-176 derivative, ceuE-/pCj1376
This study
JL1050
81-176 derivative,
feoB-/pCj1376B
This study
(cfrA+
tonB3+
tonB2-
JL538
RM1221, a chicken isolate with FeEnt utilization defect
ceuBCD+ ceuE- cfrB- cee- )
(Wang et al., 2009)
JL547
RM1221/pCeuBCDE
This study
JL674
RM1221/pCj1376
This study
JL170
W13A, turkey isolate
(Xu et al., 2010)
JL922
QZ6, swine isolate
(Xu et al., 2010)
C. coli
cfrA-
JL923
QZ6 derivative,
JL925
QZ6 derivative, cfrA- cee-
This study
This study
DH5α
F- Φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17 (rk-, mk+) phoA
supE44 thi-1 gyrA96 relA1 λ-
Invitrogen
JM109
endA1 recA1 gyrA96 thi hsdR17 (rk-, mk+) relA1 supE44 Δ(lac-proAB) [F’ traD36
lacIqZΔM15]
Promega
JL48
DH5α/pRK2013
(Zeng et al., 2009,
Xu et al., 2010)
JL633
BL21/pET22b, produce His-tagged recombinant IroE
(Lin et al., 2005)
JL634
BL21/pET28b, produce His-tagged recombinant IroD
(Lin et al., 2005)
JL637
BL21/pET28b, produce His-tagged recombinant Fes
(Lin et al., 2005)
JL640
DH5α/pCee(S157A)
This study
JL641
DH5α/pCee(H251A)
This study
JL542
DH5α/pCeuBCDE
This study
JL751
JM109/pCj1376N30
This study
E. coli
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a
Dr. Richard Haigh is at University of Leicester.
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Table S5. Major primers used in this study
Product size Target gene/ operon or
(bp)
purpose
Primer
DNA Sequence (5’-3’)a
Cj1376F
GCATTAAAAACCGCTCTTGC
Cj1376R
TTGCAAGGCAAAAGTGATGA
Cj1376F1
GGAAATTGATAATGAAAAAAATATTATTTTTATTCTGT
TCTTTTTTTCTAGC
Cj1376R1
GAATAAAAATAATATTTTTTTCATTATCAATTTCCTATT
AAATTTGAAATTTAAATTAGG
rCj1376F2
AAAGGATCCAAACAAAACTTTATTTTAGAA (BamHI)
rCj1376R
CGGGGTACCTTAATTAAGATCAAGAAGCTTTTTCATCC
(KpnI)
C1376F
GCGCAAAAAGAAGCGTAAGT
C1376R
TTTCTTGCGTAGGAGCCTGT
TonB3F
TGGCAACACTTTACATAG
TonB3R
CATTGATAGTAGCAGGAG
Cj1376_F
TTTATCGCTATGGGCTTTGC
Cj1376_R
TTGCAAAATGTTTTAAAAGAGCA
CHFL1
TTTGCTAGCTGCTCGGCGGTGTTCCTTT
CHFR1
TTTGCTAGCGCGCCCTTTAGTTCCTAAAG
C1376_F
TAGCCCTTTGGCGTTTGATA
C1376_R
AATTCACTACCACCCCACCA
C1376_1F
CATACGCCTTTTGGCAAGAT
C1376_1R
TTTCTTGCGTAGGAGCCTGT
J1376F
CCCAAGCTCTCAAAACAAGC
J1376R
ATGGCAAAAAGTCCTCCAAA
Ceu_F
TGGCTATGGAGAAAGCCTTA
Ceu_R
CTCTCTGTCCGCCACTAAAACT
S157A-F
CTTTGGACATgCTTTTGGAGGACTTTTTGCCATTGATAC
S157A-R
CCTCCAAAAGcATGTCCAAAGAGAATTTGATGGCTAAA
ATC
H251A-F
CTTTTTAAAAATGAAACTgcTGGAAGTGTCATATCTAAA
GCTATGC
H251A-R
GATATGACACTTCCAgcAGTTTCATTTTTAAAAAGTTTA
AAATGAG
2,474
Complete Cj1375-76 operon
from C. jejuni NCTC11168
7,930
Inverse PCR to generate inframe deletion of Cj1375
from pCj1375-76
738
Recombinant Cee production
2,634
Complete cee operon from C.
coli JL170
1,343
tonB3
1,917
C. jejuni cee for construction
of isogenic cee mutant
802
Chloramphenicol resistance
cassette (Turcatti et al.)
285
Confirm the insertional
mutation in C. coli cee
2,212
C. coli cee for construction of
isogenic mutant
425
Confirm the insertional
mutation in C. jejuni cee
3904
ceuBCDE operon from C.
jejuni NCTC11168.
7,930
Create S157A mutation in
Cee
7,930
Create H251A mutation in
Cee
2
3
4
a
Restriction sites are underlined in the primer sequence and the names are identified in parentheses. The
nucleotides in lower case are designed for desired aa substitution mutagenesis.
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Figure S1. Ordering of C. jejuni JL11 (ATCC 33560) contigs against three finished genomes (C.
jejuni NCTC11168, 81-176 and RM1221). Red vertical lines delineate boundaries of contigs. The
unordered contigs are put on the right side of each ordering diagram.
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A
fldA
C. jejuni 81-176
fldA
C. jejuni NCTC 11168
CCO1491 fldA
C. coli RM2228
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3
B
C
4
5
6
7
8
9
10
11
12
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14
15
16
17
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Figure S2. Sequence analysis of Cj1376 (Cee). (A) Genetic organization of cee in C. jejuni and C.
coli. Genes are colored according their GC contents. The Cj1376 homolog in C. coli RM2228 is
named as CCO1491. Dashed double arrows indicate the inverse organization of cee and its
downstream genes in C. jejuni and C. coli. Solid arrows denote key primers to amplify cee operon
from C. jejuni NCTC11168 (in red) and C. coli JL170 (in blue), respectively. (B) Structural
modeling of Cee using IroE as the template (backbone of IroE in green and Cee in red). The
catalytic site was indicated as the side chains within the yellow box area. (C) Cross-species
sequence alignment of Cee in Campylobacter species with IroE from different organisms. The
specific amino acids in E. coli CFT073 IroE that have been demonstrated to contribute to in vitro
catalytic activity (Larsen et al., 2006) were shown above the alignment. The corresponding
conserved amino acids in Campylobacter Cee were below the alignment. These conserved amino
acids were encompassed with black boxes. Asterisks (*) above the alignment denote the conserved
residues across all tested organisms.
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2
Query Sequence
Blast Result
Fes
IroD
Cj1376 in C. jejuni
NCTC11168
IroE
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Figure S3. No Ent esterase homolog other than Cee was observed in Campylobacter. This is a
representative searching result by using the known Ent esterases (Fes, IroD and IroE) from
Escherichia coli CFT073 (NC_004431) to blast against genomes of C. jejuni NCTC11168 and 81-176.
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