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Phosphatidylcholine biosynthesis in Xanthomonas campestris via a
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yeast-like acylation pathway
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Roman Moser, Meriyem Aktas and Franz Narberhaus*
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Microbial Biology, Ruhr University Bochum, Germany
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This file contains:
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Supplementary table S1: Bacterial strains and plasmids used in this study
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Supplementary table S2: Oligonucleotides used in this study
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Figure S1: S. meliloti and X. campestris PmtA sequence alignment (A) and
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phylogenetic tree of sinorhizobial and rhodobacterial Pmt enzymes (B)
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Figure S2: Verification of chromosomal pmtA deletion via Southern blot hybridisation
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Figure S3: Motility of Xanthomonas WT and pmtA mutant strains and corresponding
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lipid profiles (B) and growth curves (C)
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Figure S4: Constructed Xanthomonas mutants and respective phenotypes
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Supplementary references
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* Corresponding author. Mailing address: Lehrstuhl für Biologie der
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Mikroorganismen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität
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Bochum, Universitätsstrasse 150, NDEF 06/783, D-44780 Bochum, Germany.
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Phone: 49 (234) 322 3100. Fax: 49 (234) 321 4620.
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E-mail: franz.narberhaus@rub.de
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Table S1: Bacterial strains and plasmids used in this study
Relevant characteristicsa
Source or reference
JM83
Host for plasmid amplification
(Vieira and Messing, 1982)
S17-1
Donor for biparental mating
(Simon et al., 1983)
BL21(DE3)
Host for expression
(Studier and Moffatt, 1986)
Wild-type; RifR
U. Bonas, Halle-Wittenberg,
Strain or plasmid
E. coli strains
X. campestris pv. campestris strains
8004
Germany
R
BO2612
Wild-type derivative, deletion of pmtA; Rif ,
BO2669
xc_3949 plasmid-integration mutant; RifR, KmR
R
This study
This study
BO2676
xc_0084 plasmid-integration mutant; Rif , Km
R
This study
BO2677
xc_0174 plasmid-integration mutant; RifR, KmR
This study
R
BO2678
xc_0762 plasmid-integration mutant; Rif , Km
R
This study
BO2683
xc_0313 plasmid-integration mutant; RifR, KmR
This study
BO2684
xc_0952 plasmid-integration mutant; RifR, KmR
This study
R
BO2685
xc_4211 plasmid-integration mutant; Rif , Km
R
This study
BO2699
xc_0238 plasmid-integration mutant; RifR, KmR
This study
R
BO3200
xc_0188 plasmid-integration mutant; Rif , Km
R
This study
BO3201
xc_0517 plasmid-integration mutant; RifR, KmR
This study
R
BO3202
xc_4099 plasmid-integration mutant; Rif , Km
R
This study
BO3206
xc_0505 plasmid-integration mutant; RifR, KmR
This study
R
BO3247
xc_0471 plasmid-integration mutant; Rif , Km
R
This study
BO3249
xc_1129 plasmid-integration mutant; RifR, KmR
This study
R
BO3250
xc_2748 plasmid-integration mutant; Rif , Km
R
This study
BO3251
xc_3542 plasmid-integration mutant; RifR, KmR
This study
BO3252
xc_0231 plasmid-integration mutant; RifR, KmR
This study
R
BO3253
xc_2692 plasmid-integration mutant; Rif , Km
R
This study
BO3255
xc_0998 plasmid-integration mutant; RifR, KmR
This study
R
BO3256
xc_1343 plasmid-integration mutant; Rif , Km
R
This study
BO2668
xc_4097 plasmid-integration mutant; RifR, KmR
This study
R
BO3294
xc_4230 plasmid-integration mutant; Rif , Km
R
This study
BO3295
xc_4294 plasmid-integration mutant; RifR, KmR
This study
A. tumefaciens C58
Wild-type
C. Baron, Montreal, Canada
C58 ∆pmtA
Wild-type derivative, deletion of pmtA
(Wessel et al., 2006)
C58 ∆pcs
Wild-type derivative, deletion of pcs
(Wessel et al., 2006)
High-copy His tag expression vector; KmR
Novagen, Darmstadt,
A. tumefaciens strains
Plasmids
pET28b
Germany
pK19mobsacB
mobilisable E. coli vector for construction of the
(Schäfer et al., 1994)
Xc_pmtA deletion mutant; KmR
pBBR1MCS-5
Broad-host-range vector; GmR
(Kovach et al., 1995)
Plasmids for recombinant protein expression
pBO801
pET24b derivative with At_pmtA; KmR
(Klüsener et al., 2009)
pBO803
pET24b derivative with At_pcsA; KmR
(Klüsener et al., 2009)
pBO2604
pET28b derivative with Xc_pmtA; Km
R
This study
pBO3222
pET28b derivative with xc_0188; KmR
This study
pBO3272
pET28b derivative with xc_1417; Km
R
This study
pBO3273
pET28b derivative with xc_0238; KmR
This study
pBO3274
pET28b derivative with xc_3542; Km
R
This study
pBO3275
pET28b derivative with xc_1129; KmR
This study
pBO3276
pET28b derivative with xc_2692; Km
R
This study
pBO3277
pET28b derivative with xc_0505; KmR
This study
pBO3279
pET28b derivative with xc_0517; KmR
This study
pBO3280
pET28b derivative with xc_0231; Km
R
This study
pBO3281
pET28b derivative with xc_3949; KmR
This study
pBO3283
pET28b derivative with xc_4220; Km
R
This study
pBO3284
pET28b derivative with xc_4294; KmR
This study
pK19mobsacB derivative carrying the up- and
This study
Xc_pmtA deletion
pBO2612
downstream fragments of Xc_pmtA for pmtA
deletion; KmR
∆pmtA mutant complementation
pBO2634
pBBR1MCS-5 derivative carrying Xc_pmtA and
This study
the upstream region for complementation; Gm R
Plasmid-integration mutagenesis
pBO2668
pK19mobsacB derivative carrying a xc_4097
This study
fragment from 126 bp to 815 bp; KmR
pBO2669
pK19mobsacB derivative carrying a xc_3949
This study
fragment from 136 bp to 955 bp; KmR
pBO2676
pK19mobsacB derivative carrying a xc_0084
fragment from 298 bp to 830 bp; Km
pBO2677
pK19mobsacB derivative carrying a xc_0174
fragment from 561 bp to 1130 bp; Km
pBO2678
This study
R
pK19mobsacB derivative carrying a xc_0238
fragment from 41 bp to 420 bp; Km
pBO2683
This study
R
pK19mobsacB derivative carrying a xc_0762
fragment from 563 bp to 1060 bp; Km
pBO2699
This study
R
This study
R
pK19mobsacB derivative carrying a xc_0313
This study
fragment from 271 bp to 940 bp; KmR
pBO2684
pK19mobsacB derivative carrying a xc_0952
This study
fragment from 406 bp to 985 bp; KmR
pBO2685
pK19mobsacB derivative carrying a xc_4211
This study
fragment from 387 bp to 1016 bp; KmR
pBO3200
pK19mobsacB derivative carrying a xc_0188
This study
fragment from 41 bp to 480 bp; KmR
pBO3201
pK19mobsacB derivative carrying a xc_0517
This study
fragment from 24 bp to 423 bp; KmR
pBO3202
pK19mobsacB derivative carrying a xc_4099
This study
fragment from 131 bp to 551 bp; KmR
pBO3206
pK19mobsacB derivative carrying a xc_0505
fragment from 137 bp to 676 bp; Km
pBO3247
pK19mobsacB derivative carrying a xc_0471
fragment from 8 bp to 287 bp; Km
R
This study
R
This study
pBO3249
pK19mobsacB derivative carrying a xc_1129
fragment from 10bp to 287bp; Km
pBO3250
pK19mobsacB derivative carrying a xc_2748
fragment from 13bp to 410bp; Km
pBO3251
This study
R
pK19mobsacB derivative carrying a xc_3542
fragment from 5 bp to 272 bp; Km
pBO3252
This study
R
This study
R
pK19mobsacB derivative carrying a xc_0231
This study
fragment from 4 bp to 273 bp; KmR
pBO3253
pK19mobsacB derivative carrying a xc_2692
This study
fragment from 8 bp to 317 bp; KmR
pBO3255
pK19mobsacB derivative carrying a xc_0998
This study
fragment from 11 bp to 299 bp; KmR
pBO3256
pK19mobsacB derivative carrying a xc_1343
This study
fragment from 9 bp to 299 bp; KmR
pBO3294
pK19mobsacB derivative carrying a xc_4230
This study
fragment from 12 bp to 298 bp; KmR
pBO3295
pK19mobsacB derivative carrying a xc_4294
fragment from 29 bp to 310 bp; KmR
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a
At, Agrobacterium tumefaciens; Km, kanamycin; Rif, rifampicin; Xc, Xanthomonas campestris.
This study
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Table S2: Oligonucleotides used in this study
Oligonucleotide
Sequence (5‘→3‘)a
Xc_pmtA deletion
Xcc0035-UR_up
AAACCATGGGCTGTCGTCTAGTTGGC
Xcc0035-UR_rv
AAAGCTAGCCAATCCTCACACCGGTT
Xcc0035-DR_up
AAAGGATCCTGCCGCAAAGCCTACAT
Xcc0035-DR_rv
AAAGCTAGCTTCGCGCAATCCGGT
Plasmid-integration mutagenesis
Xcc_0188-AT-fw
AAAGCGCGCTGCGTTGGTTCCGT
Xcc_0188-AT-rv
AAACTTGCCGCTGCGCAGGCGCA
Xcc AT-0238_fw
CCAACATCCTGTTGCAACCC
Xcc_0238-AT-rvII
GCCTTCCGGGGTGATCACGT
Xcc_0505-AT-fw
AAATCGACGCGGCGCAACGCACG
Xcc_0505-AT-rv
AAAGGGTCAGGCGCATGATCGCG
Xcc AT-0517_fw
GCGTGCCTGCAGTGGCGCCA
Xcc_0517-AT-rvII
CCGCGCCAGGTGGTACAGCC
Xcc AT-3949_fw
TTCTTCGTGCTGAGCGGGTT
Xcc AT-3949_rv
AGGTCTGGGTAACGTGGAAC
Xcc AT-4097_fw
CTTCCTGCTGGTGCGCGGGC
Xcc AT-4097_rv
CGCATGAGCACGGCCAGCAC
Xcc AT-4099_fw
CGGACCGGCTTCTGCGCATG
Xcc_4099-AT-rvII
CACCGGCACCACCTGCACAC
xc0231-fw
ACCGCGGCACAGTTGCGTGC
xc0231-rv
GTGCGCGGCAACCACGATGC
xc0998-fw
AGCGTTGCGGCGCTGCTGCA
xc0998-rv
CCCGCACGACGCAGCGCGAT
xc1129-fw
ATCCGCCGCGCCACGCCCGA
xc1129-rv
TAGAGCCGCTTGAGCTCCCC
xc1343-fw
CACCACCTTCCGCACCGCCA
xc1343-rv
ATACCGCCGCCCTGCAGGGT
xc2692-fw
TGCTGATCCGCGAGGCGAGC
xc2692-rv
AAGGTGGCCAGGGCCGCCTG
xc2748-fw
GGCCCGGCGCAGATCGTCAC
xc2748-rv
CCTGCTGGCACCTCCTCTGC
xc3542-fw
GCATCGCGTGCGTTGCCGAC
xc3542-rv
GCCTGCGGCTGCACGTACAG
xc4230-fw
GCCGGAGCACACCGTGACCG
xc4230-rv
CCGGCAAGGTGTCGCGGCAC
xc4294-fw
CGATTGACGCCATGCAGGGC
xc4294-rv
CGATCAGAAAGTCGGCGTCGC
xc0084-fw
GGTGATCGCTATGGCCGGCA
xc0084-rv
AACACCAGCACCAGGCCCAC
xc0174-fw
GGCCATCGAGGTCACCGATC
xc0174-rv
CCGGCATGCGTGTTACGCAC
Xcc_0313-fw
AAATTGCCGTTGTCGCTGGCGAC
Xcc_0313-rv
AAACCGCACCGGCCACTGATGAA
xc0471-fw
AGGCGCCGCTGCAGATCCGC
xc0471-rv
CGCGCTTGCGGCCGCACGAA
xc0762-fw
ACGGGCCAATCGGCTATACC
xc0762-rv
CCAGCACGAACTCGCGGATG
Xcc_0952-fw
AAAGCATGGTTTCCCACGGCCGA
Xcc_0952-rv
AAATGCCAACCACGATCGGCGTC
Xcc_4211-fw
AAACGCGTATGCGCTTGGCTTGC
Xcc_4211-rv
AAAATCAACGGCGCGCCCAGTTG
Construction of expression plasmids
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XccPmt_N-fw
AAAACATATGGCGCGTTCGTACAG
XccPmt_N-rv
AAACTCGAGTCAGCAGGCGTACGA
xc_0188 ohne His-fw
AAACATATGAGCGAGTCATCCCTGCC
xc_0188 ohne His-rv
TTTAAGCTTTCAGGACTCCATCGCG
xc0231-fw
AAACATATGACCGCGGCACAGTTGCGT
xc0231-rv
AAAAAGCTTTTACAGCGATCTTTCCAGGTATTG
xc0238-fw
AAACATATGAGTGACACTGACCCGTCGTTG
xc0238-rv
AAAAAGCTTTCAAAGCGTATCGCGGTGCTTG
xc0505-fw
AAACATATGTCCGGTCACAGTCAGTTCGC
xc0505-rv
AAAAAGCTTTCAGGCGTGCTCCTGGCGT
xc0517-fw
AAACATATGATTGCCCGTTTGATCGCGCG
xc0517-rv
AAAAAGCTTTCATGCGCGCGCCTCCA
xc1129-fw
AAACATATGACCCGCATCCGCCGCG
xc1129-rv
AAAAAGCTTCTACGGTTCCGCGACCACC
xc1417-fw
AAACATATGGCCATCCGCAATCGCATGC
xc1417-rv
AAAAAGCTTTCAAGCATCCAACTCCAGCAC
xc2692-fw
AAACATATGAGTGTGCTGATCCGCGAGG
xc2692-rv
AAAAAGCTTTCAGCCCAGTCCGAAGGC
xc3542-fw
AAACATATGCGCATCGCGTGCGTTGCC
xc3542-rv
AAAAAGCTTTCACCGCACTACTGCCGGTT
xc3949-fw
AAACATATGCGCTATCCGGCTCTCGATCT
xc3949-rv
AAAAAGCTTCTACGCCGGCTCGGCCA
xc4220-fw
AAACATATGCCTGGTGTGTGTATGTGCTC
xc4220-rv
AAAAAGCTTTTACTCCGCCGGCGGCGAC
xc4294-fw
AAACATATGCGTTGTCTGGCACCTATCCAC
xc4294-rv
AAACTCGAGTCAACTGAGCGGCGTGG
a
Enzyme restriction sites are underlined.
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Fig. S1. A. Comparison of PmtA sequences of S. meliloti (Sm) and X. campestris
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(Xc). Identical amino acids are highlighted in black, similar residues in grey.
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B. Phylogenetic tree of rhodobacterial- and sinorhizobial-type Pmt enzymes. The tree
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was constructed using the MABL phylogeny program (http://www.phylogeny.fr).
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Distances between sequences are expressed as 0.5 changes per amino acid
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residue. Gene ID numbers (NCBI) are indicated in braces. The Xanthomonas PmtA
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(Xc_PmtA) is marked by an asterisk.
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Fig. S2. Verification of the chromosomal pmtA deletion. Chromosomal DNA from
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Xanthomonas wild-type (WT) and pmtA mutant was isolated and 5 µg of DNA were
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separated on a 2% agarose gel. DNA was transferred on a Hybond-C membrane.
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The pmtA upstream region of Xanthomonas WT (3778 bp) or pmtA mutant (3149 bp)
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was detected using a specific DNA-probe. For probe construction primers Xcc0035-
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UR_up and Xcc0035-UR_rv were used. M, marker.
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Fig. S3. A. Motility of Xanthomonas wild-type (WT) and pmtA mutant. Strains
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carrying the empty vector (EV) were used as control. For complementation, plasmid
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pBO2634 carrying the pmtA gene with its own promoter was transferred into WT and
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∆pmtA strains. Cells were cultured on NYG agar plates for 24 h at 30°C. With a
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sterile tip, cell material was inoculated onto XOLN plates (Fu and Tseng, 1990)
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containing 0.3% (w/v) agar. Plates were incubated in darkness for 96 h at 30°C.
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Motility was measured in 3 independent experiments and 4 replicates per strain.
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Diameters of swim rings were measured (right panel). Swimming diameter of the WT
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strains were defined as 100%. Standard deviations are indicated.
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B. Lipid profiles of the analysed strains grown in NYG medium for 24 h at 30°C were
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analysed via TLC using solvent 2. MMPE of Xanthomonas WT is marked by a solid
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arrow, lack of MMPE by a dashed arrow.
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C. Growth of Xanthomonas wild-type (WT) and pmtA mutant. Xanthomonas WT and
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pmtA mutant and the strains carrying the empty vector (EV) or the complementation
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plasmid pBO2634 were grown in NYG complex medium for 24 h at 30°C. Cells were
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transferred to fresh medium and the OD600 was adjusted to 0.1. The OD600 was
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measured every 1.5 h-2.0 h.
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Fig. S4. Mutagenesis of genes putatively involved in GPC-dependent PC production
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in Xanthomonas.
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A. Overview of mutated genes and respective phenotypes of the mutants.
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B. Phospholipid analysis of selected mutants. Cells were grown in the presence of 1
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mM GPC. Total phospholipids were analysed via one-dimensional TLC using solvent
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2. Presence of PC is indicated by solid arrows, lack of lipids by a dashed arrow. Lack
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of the yellow pigment xanthomonadin in the xc_4097 mutant is indicated by a
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triangle.
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References
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Klüsener, S., Aktas, M., Thormann, K.M., Wessel, M., and Narberhaus, F. (2009) Expression
and physiological relevance of Agrobacterium tumefaciens phosphatidylcholine
biosynthesis genes. J Bacteriol 191: 365-374.
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Peterson, K.M. (1995) Four new derivatives of the broad-host-range cloning vector
pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166: 175-176.
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mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids
pK18 and pK19: selection of defined deletions in the chromosome of
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Studier, F.W., and Moffatt, B.A. (1986) Use of bacteriophage-T7 RNA-polymerase to direct
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113-130.
Vieira, J., and Messing, J. (1982) The pUC plasmids, an M13mp7-derived system for
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Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial
membrane. Mol Microbiol 62: 906-915.