of 10
1
Discovery of chlamydial peptidoglycan reveals
bacteria with murein sacculi but without FtsZ
Supplem
entary Data
2
3
Supplementary Figure S1.
Protochlamydia
HPLC fractions 1-
3 contain modified
muropeptides
.
MS/MS analysis of the three
Protochlamydia
muropeptides (Figure 2 E) show similar
fragmentation patterns. The masses of the fragment ions A
-G are consistent with modified
muropeptides, whereby two modifications add an extra
mass of 314 Da compared to the
canonical muropeptides with tri-
, tetra-
and pentapeptide, respectively.
Note: While MS/MS fragments are often referred to with y and b ion designations, according
to IUPAC, there is no general nomenclature for mixed compound
s like the reduced
muropeptides analyzed here. The situation is further complicated by the presence of unknown
modifications preventing the clear numbering of the y ions. Therefore we have referred to the
fragments as A, B, C, etc.
4
5
Supplementary Figure S
2.
Protochlamydia
sacculi are degraded by lysozyme.
Representative negative stain EM images of
Protochlamydia
sacculi. The undigested control
sample (A
-C) contained many coccoid sacculi. After digestion with lysozyme (D
-F), fewer
intact sacculi were present and degraded material was abundant. Bars, 5μm.
6
7
Supplementary Figure S3. Fluorescent D-
amino acids label intracellular but not purified
Protochlamydia
cells or uninfected amoebae.
Shown is an amoeba cell infected with
Protochlamydia
and stained by DAPI to detect
chlamydial cells and fluorescently labeled D
-alanine
35
(BADA) to detect sites of PG
synthesis (A). BADA results in multiple strong signals (some of them halo-
shaped). Because
FISH/DAPI never stains all chlamydial cells inside amoebae, not all FDAA signals have a
corresponding signal. Purified chlamydial cells (B, C) or uninfected amoebae (D, E) showed
no signal with HADA (C, E) and BADA (B, D). Note that chlamydiae cannot divide in host-
free media, and thus would not incorporate FDAA. Amoeba cells are stained (white) with
eukaryote-
specific FISH in A, D and E. Bars, 5 μm.
8
9
Supplementary Figure S
4. Fluorescent D-
amino acids label neither intracellular, nor
purified
Simkania
cells.
Shown are amoeba cells infected with
Simkania
(A, B) and stained
by FISH or DAPI to detect chlamydial cells and fluorescently labeled D
-alanine
35
(HADA in
A, BADA in B). BADA labeling showed no signals. When labeled with HADA, no signals
were visible with the image acquisition settings
used for
Protochlamydia
; in images recorded
at maximum sensitivity few
Simkania
cells showed very weak signals (just above the
detection limit) for HADA. Purified
Simkania
elementary bodies (C, D) are not stained by
FDAA labeling. Amoeba cells are stained (white) with eukaryote-
specific FISH in A, B. Bars,
5 μm.
10
Supplementary Table S1. PG synthesis genes in chlamydial genomes.
Protein
A
Function
Comment
MurA
UDP
-N-acetylglucosamine 1-
carboxyvinyltransferase
Chlamydiaceae
are resistant to fosfomycin due
to
replacement of Cys115
-> Asp in MurA
13
MurB
UDP
-N-acetylmuramate
dehydrogenase
MurC
B
UDP
-N-acetylmuramate alanine ligase
MurC of
C. trachomatis
uses L
-alanine, L-
serine and
glycine with comparable efficiency
in vitro
22
MurD
UDP
-N-acetylmuramoylalanine D
-
glutamate ligase
MurE
UDP
-N-acety
lmuramoyl
-L-alanyl
-D-
glutamate 2,6-
diaminopimelate ligase
MurE of chlamydiae has Arg at position 416, typical of
m-DAP specific enzymes
57
MurF
UDP
-N-acetylmuramoyl
-tripeptide D
-
alanyl
-D-alanine ligase
MraY
Phospho
-N-acetylmuramoyl
pentapeptide
transferase
MurG
N-acetylglucosaminyl transferase
Class A HMW
PBPs
Bifunctional transpeptidase/
transglycosylase
Class B HMW
PBPs
PBP2
(PBP1)
PBP3
(PBP2)
LMW PBP
PBP6
(PBP3)
Transpeptidase
Transpeptidase
D-alanyl
-D-alanine carboxypeptidase
In
contrast to environmental chlamydiae, members of the
Chlamydiaceae
are susceptible to beta-
lactams
Alr
Alanine racemase
Chlamydiae possess DagA, a D
-alanine glycine
permease
MurI
Glutamate racemase
Ddl
B
D-alanine-
D-alanine ligase
Although
Chlamydiaceae
lack Alr, Ddl of
C. trachomatis
uses D
-Ala as a substrate
20
UppP
Undecaprenyl
-diphosphate
phosphatase
Chlamydiaceae
lack UppP, but are
sensitive to
Bacitracin
58
m-DAP pathway
Synthesis of m
-DAP
Like plants and cyanobacteria chlamydiae use the
aminotransferase pathway for generation of m
-DAP
59
A
Genes for proteins
highlighted in dark blue are absent from the genomes of all chlamydiae
.
Proteins
highlighted in light blue are encoded in the genomes of
Protochlamydia
and
Parachlamydia
, but not in pathogenic chlamydiae (
Chlamydiaceae
) and
Simkania
.
Underlined
Proteins
were characterized previously
21,
22,
23,
24,
25
. GlcNac, N
-
acetlyglucosamine; MurNAc, N
-acetyl muramic acid; HMW PBP, high molecular weight
penicillin binding protein;
LMW PBP, low molecular weight penicillin binding protein; UPP,
Undecaprenyl
-diphosphate; m
-DAP, meso
-diamino pimelic acid
B
MurC and Ddl are encoded as fusion protein in
Chlamydiaceae