Supporting Information
Site-specific activity of the acyltransferases HtrB1 and HtrB2 in
Pseudomonas aeruginosa lipid A biosynthesis
Lauren E. Hittle 1, Daniel A. Powell^ 1, Jace W. Jones^2, Majid Tofigh 1, David R. Goodlett2, Samuel M
Moskowitz3,4, and Robert K Ernst 1*
1
Department of Microbial Pathogenesis, University of Maryland School of Dentistry,
Baltimore, MD 21201
2
Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA**
3
Department of Pediatrics, Massachusetts General Hospital, Boston,
Massachusetts 20114
4
Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 20114
^
authors contributed equally
*
To whom correspondence should be addressed
Dr. Robert K. Ernst
Department of Microbial Pathogenesis
University of Maryland-Baltimore
School of Dentistry, Rm9205
650 W. Baltimore St -8 South
Baltimore, MD, 21201, U.S.A
Tel: 1 410 706 3622
E-mail: rkernst@umaryland.edu
**Current location for JWJ and DRG is Department of Pharmaceutical Sciences, School of Pharmacy,
University of Maryland, Baltimore, MD 21201
Supplemental Table 1. Strains and Plasmids Used
Strain or plasmid
Strains
E. coli
DH5a
P. aeruginosa
PAK
Plasmids
pDONR 201
pDONR 201 - PA11
pDONR 201 - PA3242
pUCP19-USER
pUCP19 - PA11
pUCP19 - PA3242
Genotype or description
Source or reference
F ∆(lacZYA-algF)U169 thi-l hsdR17 gyrA96 recAI end AI
supE44 relAI phoA F80 dlacZDM15
Invitrogen
Wild-type laboratory adapted strain
S. Lory, Harvard
Gateway cloning vector KanR
Gateway cloning vector KanR PA11 deletion
Gateway cloning vector KanR PA3242 deletion
Escherichia-Pseudomonas shuttle vectors CarbR
PA11 expression plasmid
PA3242 expression plasmid
Invitrogen
This study
This study
H. Schweizer, U Florida
This study
This study
SupplementalTable
Table 2.
2. Primers
Supplemental
Primers Used
Used
Primer name
For deletion PA 11
PA11 - 1
PA11 - 2
PA11 - 3
PA11 - 4
Sequence (5' - 3')
TGTACAAAAAAGCAGGCTTGCTCTGCCCAGTAGTTC (attB1 sequence underlined)
CCTTTCCTCAATACCATTTTTTCTCGGATCCACCTTTGAATTTCTCCACGAAACAC
GTGTTTCGTGGAGAAATTCAAAGGTGGATCCGAGAAAAAATGGTATTGAGGAAAGG
TGTACAAGAAAGCTGGGTCCATGAACGGATTGTCGC (attB2 sequence underlined)
For deletion PA3242
PA3242 - 1
PA3242 - 2
PA3242 - 3
PA3242 - 4
TGTACAAAAAAGCAGGCTGACACCAGCAATACCTCC(attB1 sequence underlined)
GGAACCGTCCATGGATCGCCCCCGTGGATCCCGGCGCAAGCGCTGAGGACGGGCAT
ATGCCCGTCCTCAGCGCTTGCGCCGGGATCCACGGGGGCGATCCATGGACGGTTCC
TGTACAAGAAAGCTGGGTGGTGATGTTCAACACGTC (attB2 sequence underlined)
For deletion - PA 11
USER0011F
USER0011R
GGAGACAUGGATGCGTTCGGATGTTC
GGGAAAGUCGGCAGAGCATAGTA
For deletion PA3242
USER3242F
USER3242R
GGAGACAUTATAACGAGAACGCCGGATG
GGGAAAGUCAAGGGGATCGGACACAG
Supplemental Table 3. Structural Analysis of wild type P. aeruginosa Lipid A Structures by ESI LTQ-FT
Mass Spectrometry.
Acyl Chain
Configuration
Experimental
m/z
Theoretical
m/z
1167.7293
1167.7289
1183.7250
Mass
Accuracy
(ppm)
C-3'
C-2'
C-3
C-2
Phosphate Configuration
0.34
C10(3-OH)
C12(3-OH):C12
H
C12(3-OH)
Monophosphate
1183.7239
0.93
C10(3-OH)
C12(3-OH)
H
C12(3-OH):C12(2OH)
Monophosphate
1211.7559
1211.7552
0.58
H
C12(3-OH):C12(2OH)
H
C12(3-OH):C12(2OH)
Monophosphate
1247.6966
1247.6953
1.04
C10(3-OH)
C12(3-OH):C12
H
C12(3-OH)
Diphosphate
1263.6930
1263.6902
2.22
C10(3-OH)
C12(3-OH)
H
C12(3-OH):C12(2OH)
Diphosphate
1275.7306
1275.7266
3.14
H
C12(3-OH):C12
H
C12(3-OH):C12(2OH)
Diphosphate
1365.8931
1365.8909
1.61
C10(3-OH)
C12(3-OH):C12
H
C12(3-OH):C12(2OH)
Monophosphate
1381.8882
1381.8858
1.74
C10(3-OH)
C12(3-OH):C12(2OH)
H
C12(3-OH):C12(2OH)
Monophosphate
1445.8596
1445.8572
1.66
C10(3-OH)
C12(3-OH):C12
H
C12(3-OH):C12(2OH)
Diphosphate
1461.8544
1461.8522
1.50
C10(3-OH)
C12(3-OH):C12(2OH)
H
C12(3-OH):C12(2OH)
Diphosphate
1536.0246
1536.0216
1.95
C10(3-OH)
C12(3-OH):C12
C10(3-OH)
C12(3-OH):C12(2OH)
Monophosphate
1552.0188
1552.0165
1.48
C10(3-OH)
C12(3-OH):C12(2OH)
C10(3-OH)
C12(3-OH):C12(2OH)
Monophosphate
1615.9903
1615.9879
1.49
C10(3-OH)
C12(3-OH):C12
C10(3-OH)
C12(3-OH):C12(2OH)
Diphosphate
1631.9853
1631.9828
1.53
C10(3-OH)
C12(3-OH):C12(2OH)
C10(3-OH)
C12(3-OH):C12(2OH)
Diphosphate
1684.0911
1684.0869
2.49
C10(3-OH):C16
C12(3-OH):C12
H
C12(3-OH):C12(2OH)
Diphosphate
Acyl chain and phosphate configuration were putatively assigned based on accurate mass (< 5 ppm) and tandem
mass spectrometry (MSn). The monophosphate label indicated the lipid A structure had one phosphate group
and the configuration was a heterogeneous mixture of C-1 monophosphate and/or C-4 monophosphate. The
diphosphate label indicated the lipid A structure had two phosphate groups and the configuration was a
heterogeneous mixture of C-1 and C-4 bisphosphate and C-4 pyrophosphate. Example tandem mass
spectrometry spectra (MS2 and MS3) for m/z 1446 was detailed in Figure S2.
Supplemental Figure 1. ESI LTQ-FT mass spectrum for WT P. aeruginosa
Supplemental Figure 1. The mass accuracy of the reported m/z values were less than 5 ppm. Refer to Table S1
for putative structures for all designated m/z values. The inset lipid A structure corresponded to the base peak
(m/z 1446). Acyl chain and phosphate configuration were putatively assigned based on accurate mass (< 5 ppm)
and tandem mass spectrometry (MSn). The ESI mass spectrum displayed slightly different abundances for some
m/z values when compared to the MALDI TOF mass spectrum (Figure 2). For example, m/z 1366 was of low
relative abundance in the MALDI TOF spectrum but was of high abundance in the ESI LTQ-FT mass spectrum.
The differences in abundance were attributed to the different ionization processes.
Supplemental Figure 2. ESI LTQ-FT tandem mass spectra for m/z value 1446 from WT P. aeruginosa
Supplemental Figure 2. A.) The MS2 mass spectrum of the precursor ion at m/z 1446 with m/z isolation and
fragmentation in the LTQ and mass analysis in the FT. The mass accuracy of the reported m/z values were less
than 5 ppm. The inset lipid A structure corresponded to the following: 1° C12(3-OH) acyl chains at the C-2 and
C-2, 1° C10(3-OH) acyl chain at the C-3, and 2° C12(2-OH) and C12 acyl chains at the C-2 and C-2
positions, respectively. A series of diagnostic cross-ring product ions (0,2A2 and 0,4A2) localized the 1° and 2°
acyl chains to their specific positions. For example, the abundant 0,2A2 product ions indicated the C-3 position
did not contain a 1° acyl chain (MacArthur, Jones, Goodlett, Ernst and Preston, 2011; Hittle, Jones, Hajjar,
Ernst and Preston, 2015). Conversely, when the C-3 position contains a 1° acyl chain the 0,4A2 product ion series
are the primary cross-ring dissociation pathway (Jones, Shaffer, Ernst, Goodlett and Turecek, 2008; Jones,
Cohen, Tureĉek, Goodlett and Ernst, 2010; Pelletier, Casella, Jones, Adams, Zurawski, Hazlett, Doi and Ernst,
2013). The 0,2A2 and 0,4A2 product ions confidently assigned the 1° C10(3-OH) to the C-3 position and the 2°
C12 acyl chain to the C-2 position (m/z values 990.5, 892.5, 740.4, 722.4, 722.4, and 680.4). Likewise, absence
of the C12(2-OH) acyl chain on the distal disaccharide confidently assigned this acyl chain to the C-2 position.
Corroborating evidence via the observed abundances of the neutral losses of the acyl chains confirmed the lipid
A structure (Jones, Shaffer, Ernst, Goodlett and Turecek, 2008). B.) The MS3 mass spectrum of the second
generation precursor ion at m/z 1348 (from m/z 1446) with m/z isolation, fragmentation, and mass analysis in
the LTQ. The m/z value at 1348 corresponded to the neutral loss of H3PO4 from m/z 1446. The neutral loss of
H3PO4 from m/z 1446 was a major dissociation channel and left the fatty acyls intact which provided a means
for further evaluating the acyl chain configuration.. The lipid A structure described in A.) was confirmed via
MS3 of m/z 1348 as evident by a series of cross-ring (0,2A2 and 0,4A2) and glycosidic cleavages (B1) product
ions. In addition, the presence and abundance of product ions corresponding to the neutral loss of specific acyl
chains further solidified the location of the 1° and 2° fatty acyls.
Supplemental Figure 3. ESI LTQ-FT tandem mass spectra for m/z value 1248 from the ∆htrB1 mutant
Supplemental Figure 3 A.) The MS2 mass spectrum of the precursor ion at m/z 1248 with m/z isolation and
fragmentation in the LTQ and mass analysis in the FT. The mass accuracy of the reported m/z values were less
than 5 ppm. The inset lipid A structure corresponded to the following: 1° C12(3-OH) acyl chains at the C-2 and
C-2, 1° C10(3-OH) acyl chain at the C-3, and 2° C12 acyl chain at the C-2 position. Analogous to Figure S2
for m/z 1446 a series of diagnostic cross-ring and acyl chain neutral loss product ions were identified that
allowed for confident assignment of the fatty acyls. B.) The MS3 mass spectrum of the second generation
precursor ion at m/z 1150 (from m/z 1248) with m/z isolation, fragmentation, and mass analysis in the LTQ. The
m/z value at 1150 corresponded to the neutral loss of H3PO4 from m/z 1248. The neutral loss of H3PO4 from m/z
1248 was a major dissociation channel and left the fatty acyls intact which provided a means for further
evaluating the acyl chain configuration. The lipid A structure described in A.) was confirmed via MS 3 of m/z
1150 as evident by a series of cross-ring (0,2A2 and 0,4A2) and glycosidic cleavages (B1) product ions. In
addition, the presence and abundance of product ions corresponding to the neutral loss of specific acyl chains
further solidified the location of the 1° and 2° fatty acyls.
Supplemental Figure 4. ESI LTQ-FT tandem mass spectra for m/z value 1264 from the ∆htrB2 mutant
Supplemental Figure 4. ESI LTQ-FT tandem mass spectra for m/z value 1264 from the ∆htrB2 mutant. A.) The
MS2 mass spectrum of the precursor ion at m/z 1264 with m/z isolation and fragmentation in the LTQ and mass
analysis in the FT. The mass accuracy of the reported m/z values were less than 5 ppm. The inset lipid A
structure corresponded to the following: 1° C12(3-OH) acyl chains at the C-2 and C-2, 1° C10(3-OH) acyl
chain at the C-3, and 2° C12(2-OH) acyl chain at the C-2 position. Analogous to Figure S2 and S3 a series of
diagnostic cross-ring and acyl chain neutral loss product ions were identified that allowed for confident
assignment of the fatty acyls. B.) The MS3 mass spectrum of the second generation precursor ion at m/z 1166
(from m/z 1264) with m/z isolation, fragmentation, and mass analysis in the LTQ. The m/z value at 1166
corresponded to the neutral loss of H3PO4 from m/z 1264. The neutral loss of H3PO4 from m/z 1264 was a major
dissociation channel and left the fatty acyls intact which provided a means for further evaluating the acyl chain
configuration. The lipid A structure described in A.) was confirmed via MS3 of m/z 1166 as evident by a series
of cross-ring (0,2A2 and 0,4A2) and glycosidic cleavages (B1) product ions. In addition, the presence and
abundance of product ions corresponding to the neutral loss of specific acyl chains further solidified the location
of the 1° and 2° fatty acyls.
Supplemental Figure 5. MALDI-TOF mass spectra for ∆htrB complemented mutants
A
∆htrB1 complemented
Relative Intensity
100
1446
1462
1616
1643
50
1430
1600
0
1400
1600
1800
B
∆htrB2 complemented
Relative Intensity
100
1462
1446
1643
50
1616
0
1400
1600
1800
Supplemental Figure 5. MALDI-TOF mass spectra for ∆htrB complemented mutants. A) ∆htrB1 mutant
complemented with a WT copy of htrB1 gene in trans; B) ∆htrB2 mutant complemented with a WT copy of
htrB2 gene in trans. Interestingly, the htrB2 complemented strain shows increased levels of hydroxylation
further suggesting the role for this enzyme in the addition of 2-OH C12 to Pseudomonas aeruginosa lipid A.
Supplemental Information -References
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