Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

TL;DR: The first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis in S. pneumoniae, which constitute an autonomous functional entity which processes or repairs nascent peptidoglycan synthesized by FtsW/PBP2x.

Abstract: In oval shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is performed by independent functional complexes; the divisome and the elongasome. Penicillin binding proteins (PBPs) were long considered as the key peptidoglycan synthesizing enzymes in these complexes. Among these were the bifunctional class A PBPs, which are both glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only transpeptidase activity. Recently, however, it was established that the monofunctional class B PBPs work together with non-PBP glycosyltransferases (FtsW and RodA) to make up the core peptidoglycan synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question. Here we utilize the peptidoglycan hydrolase CbpD to show that class A PBPs have an autonomous role during cell wall synthesis in S. pneumoniae. Purified CbpD was shown to target the septum of S. pneumoniae cells. Using assays to specifically inhibit PBP2x, we demonstrate that CbpD specifically target nascent peptidoglycan synthesized by the divisome. Notably, class A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant form. The class A PBP mediated processing was independent of divisome and elongasome activities. Class A PBPs thus constitute an autonomous functional entity which processes or repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our knowledge this is the first time a specific function has been identified for class A PBPs in bacterial cell wall synthesis.

Summary

Introduction

• The peptidoglycan layer covering the pneumococcal cell provides shape and rigidity, and is essential for growth and survival.
• Peptidoglycan is synthesized from lipid II precursors at the outside the cytoplasmic membrane by glycosyltransferases that polymerize the glycan chains and transpeptidases that interconnect the chains through peptide cross-links.
• So far the exact bond cleaved has not been identified.
• The SH3b domain is essential for the function of CbpD, and experimental evidence indicates that it binds to the peptidoglycan portion of the cell wall27.
• Hence, the authors have used the unique specificity of CbpD to study the functional relationships between different peptidoglycan synthesizing enzymes in S. pneumoniae.

Purification and properties of CbpD-B6

• The gene encoding cbpD from S. mitis B6 was amplified by PCR, ligated into the pRSET-A vector, and expressed using E. coli BL21 cells as a host.
• The recombinant CbpD-B6 protein was further purified by size-exclusion chromatography (SEC) on a SuperdexTM 75 10/300 GL column (see Methods section for details).
• CbpD-B6 attacks the septal area of the pneumococcal cell wall.
• The SEM microscopy analysis clearly showed that CbpD-B6 attacks only the septal region of the peptidoglycan sacculus, resulting in cells that are split in half along their equators (Fig. 2A and B).
• At the highest oxacillin concentrations used (50 and 100 µg ml-1), the pneumococci became as sensitive as untreated control cells (Fig. 3A).

The S2 phase

• During the S1-phases the oxacillin concentration increases gradually resulting in progressively stronger inhibition of PBP2x.
• The authors observed that the R-phase disappears if oxacillin (0.8 µg ml-1) and CbpD-B6 are added simultaneously to pneumococcal cultures.
• This finding suggested a plausible explanation for the S2-phase.
• The fact that pneumococcal cells need either PBP1a or PBP2a to survive, indicates that these PBPs can, at least to a certain extent, substitute for each other.
• In both cases the authors observed the usual S1, R and S2 phases (Fig. 4 and Fig. S5C and D), demonstrating that PBP2a can substitute for PBP1a in the process that transforms CbpD-B6-sensitive peptidoglycan into a resistant structure.

Discussion

• Recently it has become clear that FtsW/PBP2x and RodA/PBP2b constitute cognate pairs of interacting proteins that make up the core peptidoglycan synthesizing machineries within the pneumococcal divisome and elongasome, respectively9,10,11.
• This discovery has important implications for their understanding of pneumococcal cell wall synthesis, and the role played by class A PBPs in this process.
• The authors results show that class A PBPs act downstream of the FtsW/PBP2x machinery to produce alterations in the cell wall.
• This demonstrates that the elongasome is active even in the absence of a functional divisome.
• The authors clearly show that class A PBPs together with their associated auxiliary proteins somehow remodels the primary peptidoglycan synthesized by the PBP2x/FtsW machinery.

Methods

• All strains used in the present study are listed in Table S1.
• Liquid cultures were grown aerobically with shaking.
• Chemically competent E. coli cells were transformed by heat-shocking at 42ºC.
• Following incubation at 37°C for two hours, transformants were selected by plating 30 µl cell culture on TH-agar plates containing the appropriate antibiotic; kanamycin (400 µg ml-1), streptomycin (200 µg ml-1) or spectinomycin (200 µg ml-1).

DNA cloning

• All primers used in this study are listed in Table S2.
• The sf-gfp gene was amplified using the kp116 and kp119 primers and SPH370 genomic DNA as template, and the cbpD-B6-Δchap gene was amplified from SO7 genomic DNA using the primer pair kp117/kp118.
• The resulting sf-gfp-cbpD-B6 amplicon was cleaved with NdeI and HindIII and ligated into pRSET A giving the pRSET-sfGFP-cbpD-B6 plasmid.
• Amplicons used to transform S. pneumoniae were constructed by overlap extension PCR as previously described by Johnsborg et al.22.
• The spectinomycin resistant marker aad9 was employed to knock out lytA in strain ds789.

CbpD-B6 resistance assay

• Pneumococcal cells were grown in 96-wells microtiter plates and OD550 was measured every five minutes.
• The cells were grown for 10 minutes in the presence of antibiotics before purified CbpD-B6 was added to a final concentration of five µg ml-1. CbpD-sensitive cells were observed as a drop in OD550.
• Both cultures were incubated further for 10 minutes at 37°C before formaldehyde was added to a final concentration of 2.5%.
• Cells were then incubated in 100 µl PBS containing 0.05% Non-bound sfGFP-CbpD-B6 was washed off the cells by rinsing the glass slide by submerging the glass slide in five tubes each containing 40 ml PBS.
• Phase contrast pictures and GFP fluorescence pictures were captured using a Zeiss AxioObserver with an ORCA Flash4.0 V2 Digital CMOS camera (Hamamatsu Photonics) through a 100x PC objective.

Acknowledgments

• This work was supported by grants from the Research Council of Norway (no. 240058 and 250976) and the Norwegian University of Life Sciences.
• The percent reduction in cell density caused by cell lysis was determined.
• The experiments were repeated three times with the same results.
• Δpbp2a/Δpbp1b mutant (strain khb225) and Δpbp1a/Δpbp1b mutant (strain khb224) as well as a Δpbp2b mutant (strain ds789) all developed the typical S1, R, S2 phases upon increasing concentrations of oxacillin.

Figures

Fig. 3. Inhibition of PBP2x results in CbpD-resistant peptidoglycan. A. Increasing concentrations of oxacillin was added to exponentially growing wild type cells (RH425) at OD550 0.25 (black arrow). After ten minutes, CbpD-B6 was added (red arrow) to a final concentration

Fig. 2. Scanning (A and B) and transmission (C and D) electron micrographs of pneumococci subjected to 0.5 µg ml-1 CbpD-B6 for 60 seconds before they were fixed and prepared for electron microscopy. Arrows indicate areas in the cell wall attacked by the muralytic enzyme.

Fig. 4. Class A PBPs are essential for transforming new peptidoglycan into a CbpD-B6-resistant form. The figure shows a schematic presentation of the sensitivity to CbpD-B6 in different genetic backgrounds based on CbpD-B6-resistance assays (see Fig. 3 and supplementary Fig. S5 for relevant growth curves). Development of the S1, R and S2 phases is indicated with the relevant concentration of oxacillin shown on top. Strain khb321 expressing a low-affinity PBP2x (LA-PBP2x) did not develop the R-phase. The same pattern was seen for WT cells (RH425) treated with moenomycin for 10 minutes (WT + M) before addition of CbpD-B6. When the class A PBP1a was changed to a low-affinity version (LA-PBP1a, strain khb332), the cells did not develop the S2-phase when treated with supra-MIC concentrations of oxacillin. The double

Fig. 1. Functionality of CbpD-B6 in S. pneumoniae. A. Exponential growing pneumococci (strain RH425) was added purified CbpD-B6 (arrow) to a final concentration of 0.3 µg ml-1 (blue circles). Growth of non-treated cells are represented by green circle. B and C. Wild type S. pneumoniae (strain A), pneumococci expressing CbpD-B6 from the native cbpD-locus (strain C)

Full text

1
Class A PBPs have a distinct and unique role in the construction of the
pneumococcal cell wall.
Daniel Straume
¶1
, Katarzyna Wiaroslawa Piechowiak
¶1
, Silje Olsen
1
, Gro Anita Stamsås
1
, Kari
Helene Berg
1
, Morten Kjos
1
, Maria Victoria Heggenhougen
1
Martin Alcorlo
2
, Juan A. Hermoso
2
and Leiv Sigve Håvarstein
1
.
¶
These authors contributed equally to this work.
1
Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences,
NO-1432 Ås, Norway.
2
Department of Crystallography and Structural Biology, Instituto Química-Física `Rocasolano'
CSIC (Spanish National Research Council), Serrano 119, 28006 Madrid, Spain.
Running title: Class A PBPs remodel the cell wall
Key words: Class A PBPs, CbpD, peptidoglycan, Streptococcus pneumoniae
* Corresponding author:
Leiv Sigve Håvarstein
Faculty of Chemistry, Biotechnology, and Food Science,
Norwegian University of Life Sciences, P.O. Box 5003, NO-1432 As, Norway.
Tlf: 47-67232493
E-mail: sigve.havarstein@nmbu.no
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2
Abstract
In oval shaped Streptococcus pneumoniae, septal and longitudinal peptidoglycan synthesis is
performed by independent functional complexes; the divisome and the elongasome. Penicillin
binding proteins (PBPs) were long considered as the key peptidoglycan synthesizing enzymes in
these complexes. Among these were the bifunctional class A PBPs, which are both
glycosyltransferases and transpeptidases, and monofunctional class B PBPs with only
transpeptidase activity. Recently, however, it was established that the monofunctional class B
PBPs work together with non-PBP glycosyltransferases (FtsW and RodA) to make up the core
peptidoglycan synthesizing machineries within the pneumococcal divisome (FtsW/PBP2x) and
elongasome (RodA/PBP2b). The function of class A PBPs is therefore now an open question.
Here we utilize the peptidoglycan hydrolase CbpD to show that class A PBPs have an
autonomous role during cell wall synthesis in S. pneumoniae. Purified CbpD was shown to target
the septum of S. pneumoniae cells. Using assays to specifically inhibit PBP2x, we demonstrate
that CbpD specifically target nascent peptidoglycan synthesized by the divisome. Notably, class
A PBPs could process this nascent peptidoglycan from a CbpD-sensitive to a CbpD-resistant
form. The class A PBP mediated processing was independent of divisome and elongasome
activities. Class A PBPs thus constitute an autonomous functional entity which processes or
repairs nascent peptidoglycan synthesized by FtsW/PBP2x. Our results support a model in which
pneumococcal peptidoglycan is made by three functional entities, the divisome, the elongasome
and a peptidoglycan-repairing or -remodelling complex consisting of bifunctional PBPs. To our
knowledge this is the first time a specific function has been identified for class A PBPs in
bacterial cell wall synthesis.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted June 10, 2019. ; https://doi.org/10.1101/665463doi: bioRxiv preprint

3
Introduction
The peptidoglycan layer covering the pneumococcal cell provides shape and rigidity, and is
essential for growth and survival. It consists of linear chains of two alternating amino sugars, N-
acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), interlinked by peptide
bridges between MurNAcs on adjacent strands
1,2
. Peptidoglycan is synthesized from lipid II
precursors at the outside the cytoplasmic membrane by glycosyltransferases that polymerize the
glycan chains and transpeptidases that interconnect the chains through peptide cross-links. S.
pneumoniae produce five different p
enicillin-binding proteins (PBPs) with transpeptidase
activity, namely PBP1a, PBP1b, PBP2a, PBP2b and PBP2x
3
. The first three of these, designated
class A PBPs, are bifunctional enzymes that catalyse transglycosylation as well as
transpeptidation, while PBP2x and PBP2b are monofunctional transpeptidases (class B PBPs)
4
.
Monofunctional glycosyltransferases that have homology to the glycosyltransferase domains of
class A PBPs are present in some bacterial species, but are absent from S. pneumoniae. PBP2x is
an essential constituent of the divisome, a multiprotein division machine that synthesizes the
septal cross-wall
3,5,6,7
. The other monofunctional transpeptidase, PBP2b, is a key component of
another multiprotein complex, the elongasome, which is responsible for longitudinal
peptidoglycan synthesis
3,5,6,7,8
. Until recently, it was believed that only class A PBPs were able to
polymerize glycan chains in S. pneumoniae. Consequently, the divisome as well as the
elongasome would have to include at least one class A PBP in order to be functional. Recently,
however, it was discovered that FtsW and RodA, two proteins belonging to the SEDS (s
hape,
e
longation, division, and sporulation) family, function as peptidoglycan polymerases that
synthesize glycan strands from lipid II
9,10,11
. FtsW and RodA were originally reported to be lipid
II flippases, a function now assigned to MurJ
12
. However, it is still not entirely clear whether
these polytopic membrane proteins are monofunctional glycan polymerases or bifunctional
flippases and polymerases
13,14
. Previous research has shown that FtsW and RodA are essential,
and work in conjunction with PBP2x and PBP2b, respectively
9,11
.
Peptidoglycan synthesis requires the concerted action of enzymes that carry out
transglycosylation and transpeptidation reactions. Thus, in principle, peptidoglycan synthesis
might be performed by monofunctional transglycosylase working together with monofunctional
transpeptidase, by single bifunctional enzymes such as the class A PBPs, or by a combination of
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4
monofunctional and bifunctional enzymes. As mentioned above, class A PBPs have traditionally
been considered to be essential components of bacterial divisomes and elongasomes. However, it
has been known for a long time that Bacillus subtilis is viable without class A PBPs
15
. Thus,
considering the recent discovery of the SEDS partners of PBP2x and PBP2b, it is conceivable
that the pneumococcal divisome and elongasome perform the primary synthesis of septal and
peripheral peptidoglycan without the involvement of class A PBPs. If so, the function of class A
PBPs is an open question, and their role in peptidoglycan synthesis must be re-examined. Here,
we have addressed this question by exploiting the unique properties of the peptidoglycan
hydrolase CbpD (c
holine-binding protein D).
When developing competence for natural transformation, streptococci belonging to the
mitis phylogenetic group express a set of core competence genes controlled by the competence
stimulating peptide (CSP)
16
and the competence-specific two-component regulatory system
ComDE
17
. Most proteins whose expression are controlled by this quorum-sensing-like system
are involved in DNA-binding, DNA-uptake, DNA-processing or genomic integration of
internalized DNA
18,19
. However, among the CSP/ComDE-regulated genes is a peptidoglycan-
degrading enzyme, CbpD, which does not seem to have a role in any of the transformation steps
mentioned above
19,20
. Instead, evidence strongly suggests that CbpD, which is encoded by a late
competence gene, is part of a DNA-acquisition mechanism consisting of CbpD and the cognate
immunity protein ComM
21,22,23
. Previous research has shown that ComM, a polytopic membrane
protein encoded by an early competence gene, protects competent pneumococci from being lysed
by their own CbpD. The mechanism behind this protection is not understood
24,25
.
CbpD is composed of three different domain types: an N-terminal c
ysteine, histidine-
dependent a
midohydrolase/peptidase (CHAP) domain, one or two Src homology 3b (SH3b)
domains, and a C-terminal choline-binding domain (Cbd) consisting of four choline-binding
repeats. CHAP domains are present in many peptidoglycan hydrolases, and function as either N-
acetylmuramoyl-L-alanine amidases or endopeptidases
19,26
. Hence, the CHAP domain of CbpD
must cleave somewhere within the peptide bridges of streptococcal peptidoglycan. However, so
far the exact bond cleaved has not been identified. The SH3b domain is essential for the function
of CbpD, and experimental evidence indicates that it binds to the peptidoglycan portion of the
cell wall
27
. CbpDs from Streptococcus mitis and Streptococcus oralis contain only one SH3b
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5
domain, sandwiched between the CHAP and the Cbd domain, while many (but not all) strains of
S. pneumoniae contain two successive SH3b domains. The choline-binding repeats of the Cbd
domain anchor CbpD to cell wall teichoic acid, and possibly also lipoteichoic acid, through non-
covalent interactions with the choline residues decorating these polymers
28
. Mitis group
streptococci such as S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis and S. infantis
produce choline-decorated teichoic acids, while this type of teichoic acid is not present in mitis
group streptococci that are more distantly related to S. pneumoniae
29
. Similar to the CHAP and
SH3b domains, the Cbd domain is essential for the biological function of CbpD
27
.
Even though CbpD appears to be a key component of the pneumococcal gene transfer
machinery it is still poorly characterized. It has proved very difficult to express pneumococcal
CbpD in a soluble and active form in Escherichia coli and other hosts, and we therefore
investigated whether homologs of pneumococcal CbpD from various mitis group streptococci
might be more amenable to heterologous expression. This strategy turned out to be successful, as
we were able to purify milligram amounts of the CbpD protein produced by S. mitis B6 (CbpD-
B6) in soluble form. In the present study, we show that CbpD-B6 is active against S.
pneumoniae, and that its properties appear to be very similar to pneumococcal CbpD.
Interestingly, we found that CbpD-B6 cleaves only a distinct subset of the peptide bridges that
cross-link the carbohydrate chains in pneumococcal peptidoglycan. It is highly specific for
nascent peptidoglycan formed by PBP2x and FtsW. We realized that this property can be
exploited as a research tool. Hence, we have used the unique specificity of CbpD to study the
functional relationships between different peptidoglycan synthesizing enzymes in S.
pneumoniae. Our results strongly indicate that class A PBPs are not part of the core machinery of
the divisome and elongasome, but have an important autonomous role in construction of the fully
matured peptidoglycan layer.
Results
Purification and properties of CbpD-B6
The gene encoding cbpD from S. mitis B6 was amplified by PCR, ligated into the
pRSET-A vector, and expressed using E. coli BL21 cells as a host. Since choline-binding
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Frequently asked questions

Q1. What contributions have the authors mentioned in the paper "Class a pbps have a distinct and unique role in the construction of the pneumococcal cell wall" ?

These authors contributed equally to this work. 

Q2. What future works have the authors mentioned in the paper "Class a pbps have a distinct and unique role in the construction of the pneumococcal cell wall" ?

Further confirmation or rejection of the repairosome hypothesis will be an important topic for future studies.