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

Daniel Straume1, Katarzyna Wiaroslawa Piechowiak1, Silje Olsen1, Gro Anita Stamsås1, Kari Helene Berg1, Morten Kjos1, Maria Victoria Heggenhougen1, Martín Alcorlo2, Juan A. Hermoso2, Leiv Sigve Håvarstein1 •
Norwegian University of Life Sciences1, Spanish National Research Council2
10 Jun 2019-bioRxiv (Cold Spring Harbor Laboratory)-pp 665463
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 (2 min read)

Jump to: [Introduction] – [Purification and properties of CbpD-B6] – [The S2 phase] – [Discussion] – [Methods] – [DNA cloning] – [CbpD-B6 resistance assay] and [Acknowledgments]

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.

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Figures (4)

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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
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
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
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
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
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
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
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
Citations
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Journal Article•DOI•
Organization of peptidoglycan synthesis in nodes and separate rings at different stages of cell division of Streptococcus pneumoniae.

[...]

Amilcar J. Perez1, Michael J. Boersma1, Kevin E. Bruce1, Melissa M. Lamanna1, Sidney L. Shaw1, Ho-Ching Tiffany Tsui1, Atsushi Taguchi2, Erin E. Carlson3, Michael S. VanNieuwenhze1, Malcolm E. Winkler1 •
Indiana University1, Harvard University2, University of Minnesota3
01 Jun 2021-Molecular Microbiology
TL;DR: Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.
Abstract: Bacterial peptidoglycan (PG) synthesis requires strict spatiotemporal organization to reproduce specific cell shapes. In ovoid-shaped Streptococcus pneumoniae (Spn), septal and peripheral (elongation) PG synthesis occur simultaneously at midcell. To uncover the organization of proteins and activities that carry out these two modes of PG synthesis, we examined Spn cells vertically oriented onto their poles to image the division plane at the high lateral resolution of 3D-SIM (structured-illumination microscopy). Labeling with fluorescent D-amino acids (FDAA) showed that areas of new transpeptidase (TP) activity catalyzed by penicillin-binding proteins (PBPs) separate into a pair of concentric rings early in division, representing peripheral PG (pPG) synthesis (outer ring) and the leading-edge (inner ring) of septal PG (sPG) synthesis. Fluorescently tagged PBP2x or FtsZ locate primarily to the inner FDAA-marked ring, whereas PBP2b and FtsX remain in the outer ring, suggesting roles in sPG or pPG synthesis, respectively. Pulses of FDAA labeling revealed an arrangement of separate regularly spaced "nodes" of TP activity around the division site of predivisional cells. Tagged PBP2x, PBP2b, and FtsX proteins also exhibited nodal patterns with spacing comparable to that of FDAA labeling. Together, these results reveal new aspects of spatially ordered PG synthesis in ovococcal bacteria during cell division.

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The Cell Wall of Bacillus subtilis

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Danae Morales Angeles1, Dirk-Jan Scheffers2•
Norwegian University of Life Sciences1, University of Groningen2
01 Feb 2021-Current Issues in Molecular Biology
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  • ...Nevertheless, it is evident that Class A PBPs play an important role in overall PG synthesis, being responsible for the major portion of PG in normal cells, crucial for width regulation, and modification of PG to a more mature form (Cho et al., 2016; Dion et al., 2019; Straume et al., 2020)....

    [...]

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Structural Characterization of the Essential Cell Division Protein FtsE and Its Interaction with FtsX in Streptococcus pneumoniae.

[...]

Martín Alcorlo1, Daniel Straume2, Joe Lutkenhaus3, Leiv Sigve Håvarstein2, Juan A. Hermoso1 •
Spanish National Research Council1, Norwegian University of Life Sciences2, University of Kansas3
01 Sep 2020-Mbio
TL;DR: High-resolution structures of pneumococcal FtsE bound to different nucleotides are reported, revealing regions with high structural plasticity which are key for FtsS binding to FtsX and all the conserved structural motifs associated with ATPase activity.
Abstract: FtsEX is a membrane complex widely conserved across diverse bacterial genera and involved in critical processes such as recruitment of division proteins and in spatial and temporal regulation of muralytic activity during cell division or sporulation. FtsEX is a member of the ABC transporter superfamily. The component FtsX is an integral membrane protein, whereas FtsE is an ATPase and is required for the transmission of a conformational signal from the cytosol through the membrane to regulate the activity of cell wall hydrolases in the periplasm. Both proteins are essential in the major human respiratory pathogenic bacterium Streptococcus pneumoniae, and FtsX interacts with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report high-resolution structures of pneumococcal FtsE bound to different nucleotides. Structural analysis revealed that FtsE contains all the conserved structural motifs associated with ATPase activity and afforded interpretation of the in vivo dimeric arrangement in both the ADP and ATP states. Interestingly, three specific FtsE regions with high structural plasticity were identified that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for contacting FtsE, were identified and validated by in vivo mutagenesis studies showing that this interaction is essential for cell growth and proper morphology.IMPORTANCE Bacterial cell division is a central process that requires exquisite orchestration of both the cell wall biosynthetic and lytic machineries. The essential membrane complex FtsEX, widely conserved across bacteria, plays a central role by recruiting proteins to the divisome apparatus and by regulating periplasmic muralytic activity from the cytosol. FtsEX is a member of the type VII family of the ABC-superfamily, but instead of being a transporter, it couples the ATP hydrolysis catalyzed by FtsE to mechanically transduce a conformational signal that provokes the activation of peptidoglycan (PG) hydrolases. So far, no structural information is available for FtsE. Here, we provide the structural characterization of FtsE, confirming its ATPase nature and revealing regions with high structural plasticity which are key for FtsE binding to FtsX. The complementary binding region in FtsX has also been identified and validated in vivo Our results provide evidence on how the difference between the ATP/ADP-bound states in FtsE would dramatically alter the interaction of FtsEX with the PG hydrolase PcsB in pneumococcal division.

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10 Nov 2020-bioRxiv
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Abstract: Streptococcus pneumoniae is an opportunistic pathogen that is a common cause of serious invasive diseases such as pneumonia, bacteremia, meningitis, and otitis media. Transmission of this bacterium has classically been thought to occur through inhalation of respiratory droplets and direct contact with nasal secretions. However, the demonstration that S. pneumoniae is desiccation tolerant, and therefore environmentally stable for extended periods of time, opens up the possibility that this pathogen is also transmitted via contaminated surfaces (fomites). To better understand the molecular mechanisms that enable S. pneumoniae to survive periods of desiccation, we performed a high throughput transposon sequencing (Tn-seq) screen in search of genetic determinants of desiccation tolerance. We identified 42 genes whose disruption reduced desiccation tolerance, and 45 genes that enhanced desiccation tolerance. The nucleotide excision repair pathway was the most enriched category in our Tn-seq results, and we found that additional DNA repair pathways are required for desiccation tolerance, demonstrating the importance of maintaining genome integrity after desiccation. Deletion of the nucleotide excision repair gene uvrA resulted in decreased transmission efficiency between infant mice, indicating a correlation between desiccation tolerance and pneumococcal transmission. Understanding the molecular mechanisms that enable pneumococcal persistence in the environment may enable targeting of these pathways to prevent fomite transmission, thereby preventing the establishment of new colonization and any resulting invasive disease.

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Cites background from "Class A PBPs have a distinct and un..."

  • ...The function of these two proteins is still not fully understood, however they are known to 294 be required for maturation of the cell wall as opposed to the construction of nascent peptidoglycan 295 (45)....

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A Molecular Study of Toxic Shock Syndrome Toxin gene (tsst-1) in β-lactam Resistant Staphylococcus aureus Clinical Isolates

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TL;DR: The spread of the Toxic Shock Syndrome Toxin-1 gene (tsst-1) was investigated in β-lactamase resistant S. aureus and showed no significant correlation with the individual antibiotic resistance and the multi-drug resistance patterns of the isolates.
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5 citations

Cites background from "Class A PBPs have a distinct and un..."

  • ...Earlier studies reported a high prevalence of tsst-1 gene among MSSA and β-lactam sensitive strains, but MRSA and β-lactam resistant strains typically do not and/ or less produce these superantigens (i.e. TSST-1, SEB, and SEC exotoxins)[19, 54]....

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  • ...Both MRSA and MSSA can harbor one or more superantigenic toxin genes....

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  • ...Skin and soft tissue infections (SSTIs) are commonly caused by S. aureus, specifically MRSA....

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  • ...The pathogenic mechanism and virulence factors are assumed to be different between MRSA and MSSA [15]....

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  • ...This allows for resistance to all βlactam antibiotics and obviates their clinical use during MRSA infections....

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References
More filters
Journal Article•DOI•
A predatory mechanism dramatically increases the efficiency of lateral gene transfer in Streptococcus pneumoniae and related commensal species.

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Ola Johnsborg1, Vegard Eldholm1, Martha Langedok Bjørnstad1, Leiv Sigve Håvarstein1•
Norwegian University of Life Sciences1
01 Jul 2008-Molecular Microbiology
TL;DR: This work describes a mechanism for active acquisition of homologous DNA that dramatically increases the efficiency of gene exchange between and within the streptococcal species mentioned above.
Abstract: Bacteria that are competent for natural genetic transformation, such as pneumococci and their commensal relatives Streptococcus mitis and Streptococcus oralis, take up exogenous DNA and incorporate it into their genomes by homologous recombination. Traditionally, it has been assumed that genetic material leaking from dead bacteria constitutes the sole source of external DNA for competent streptococci. Here we describe a mechanism for active acquisition of homologous DNA that dramatically increases the efficiency of gene exchange between and within the streptococcal species mentioned above. This mechanism gives competent streptococci access to a common gene pool that is significantly larger than their own genomes, a property representing a considerable advantage when these bacteria are subjected to external selection pressures, such as vaccination and treatment with antibiotics.

126 citations

"Class A PBPs have a distinct and un..." refers methods in this paper

  • ...pneumoniae were constructed by overlap extension PCR as previously described by Johnsborg et al.(22)....

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  • ...Amplicons used to transform S. pneumoniae were constructed by overlap extension PCR as previously described by Johnsborg et al.22....

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Journal Article•DOI•
Attenuation of penicillin resistance in a peptidoglycan O-acetyl transferase mutant of Streptococcus pneumoniae.

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M. Inês Crisóstomo1, Waldemar Vollmer2, Arun S. Kharat1, Silja Inhülsen2, Florian Gehre2, Florian Gehre1, Stephan Buckenmaier2, Alexander Tomasz1 •
Rockefeller University1, University of Tübingen2
01 Sep 2006-Molecular Microbiology
TL;DR: DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O‐acetylase, and complementation experiments showed that both phenotypes – reduced resistance and lysozyme sensitivity – were linked to the defective adr gene.
Abstract: Summary The level of penicillin resistance in clinical isolates of Streptococcus pneumoniae depends not only on the reduced affinity of penicillin binding proteins (PBPs) but also on the functioning of enzymes that modify the stem peptide structure of cell wall precursors. We used mariner mutagenesis in search of additional genetic determinants that may further attenuate the level of penicillin resistance in the bacteria. A mariner mutant of the highly penicillin-resistant S. pneumoniae strain Pen6 showed reduction of the penicillin minimum inhibitory concentration (MIC) from 6 to 0.75 μg ml−1. Decrease in penicillin MIC was also observed upon introduction of the mutation (named provisionally adr, for attenuator of drug resistance) into representatives of major epidemic clones of penicillin-resistant pneumococci. Attenuation of resistance levels was specific for β-lactams. The adr mutant has retained unchanged (low affinity) PBPs, unaltered murM gene and unchanged cell wall stem peptide composition, but the mutant became hypersensitive to exogenous lysozyme and complementation experiments showed that both phenotypes – reduced resistance and lysozyme sensitivity – were linked to the defective adr gene. DNA sequence comparison and chemical analysis of the cell wall identified adr as the structural gene of the pneumococcal peptidoglycan O-acetylase.

107 citations

Journal Article•DOI•
Requirement of essential Pbp2x and GpsB for septal ring closure in Streptococcus pneumoniae D39.

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Adrian D. Land1, Ho-Ching Tiffany Tsui1, Ozden Kocaoglu1, Stephen A. Vella1, Sidney L. Shaw1, Susan K. Keen1, Lok-To Sham1, Erin E. Carlson1, Malcolm E. Winkler1 •
Indiana University1
01 Dec 2013-Molecular Microbiology
TL;DR: It is reported here that GpsB is essential in ellipsoid‐shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GPSB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent‐vancomycin staining, and eventual lysis.
Abstract: Bacterial cell shapes are manifestations of programs carried out by multi-protein machines that synthesize and remodel the resilient peptidoglycan (PG) mesh and other polymers surrounding cells. GpsB protein is conserved in low-GC Gram-positive bacteria and is not essential in rod-shaped Bacillus subtilis, where it plays a role in shuttling penicillin-binding proteins (PBPs) between septal and side-wall sites of PG synthesis. In contrast, we report here that GpsB is essential in ellipsoid-shaped, ovococcal Streptococcus pneumoniae (pneumococcus), and depletion of GpsB leads to formation of elongated, enlarged cells containing unsegregated nucleoids and multiple, unconstricted rings of fluorescent-vancomycin staining, and eventual lysis. These phenotypes are similar to those caused by selective inhibition of Pbp2x by methicillin that prevents septal PG synthesis. Dual-protein 2D and 3D-SIM (structured illumination) immunofluorescence microscopy (IFM) showed that GpsB and FtsZ have overlapping, but not identical, patterns of localization during cell division and that multiple, unconstricted rings of division proteins FtsZ, Pbp2x, Pbp1a and MreC are in elongated cells depleted of GpsB. These patterns suggest that GpsB, like Pbp2x, mediates septal ring closure. This first dual-protein 3D-SIM IFM analysis also revealed separate positioning of Pbp2x and Pbp1a in constricting septa, consistent with two separable PG synthesis machines.

102 citations

Journal Article•DOI•
Interplay between Penicillin-binding proteins and SEDS proteins promotes bacterial cell wall synthesis

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Sophie Leclercq1, Adeline Derouaux1, Samir Olatunji1, Claudine Fraipont1, Alexander J. F. Egan2, Waldemar Vollmer2, Eefjan Breukink3, Mohammed Terrak1 •
University of Liège1, Newcastle University2, Utrecht University3
24 Feb 2017-Scientific Reports
TL;DR: It is found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects, suggesting a tight regulatory mechanism consistent with the cell’s need to ensure appropriate use of the limited pool of lipid II.
Abstract: Bacteria utilize specialized multi-protein machineries to synthesize the essential peptidoglycan (PG) cell wall during growth and division. The divisome controls septal PG synthesis and separation of daughter cells. In E. coli, the lipid II transporter candidate FtsW is thought to work in concert with the PG synthases penicillin-binding proteins PBP3 and PBP1b. Yet, the exact molecular mechanisms of their function in complexes are largely unknown. We show that FtsW interacts with PBP1b and lipid II and that PBP1b, FtsW and PBP3 co-purify suggesting that they form a trimeric complex. We also show that the large loop between transmembrane helices 7 and 8 of FtsW is important for the interaction with PBP3. Moreover, we found that FtsW, but not the other flippase candidate MurJ, impairs lipid II polymerization and peptide cross-linking activities of PBP1b, and that PBP3 relieves these inhibitory effects. All together the results suggest that FtsW interacts with lipid II preventing its polymerization by PBP1b unless PBP3 is also present, indicating that PBP3 facilitates lipid II release and/or its transfer to PBP1b after transport across the cytoplasmic membrane. This tight regulatory mechanism is consistent with the cell's need to ensure appropriate use of the limited pool of lipid II.

100 citations

Journal Article•DOI•
Immobilization and single-step purification of fusion proteins using DEAE-cellulose.

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José María Sánchez-Puelles1, Jesús Sanz1, José Luis García1, Ernesto García1•
Spanish National Research Council1
01 Jan 1992-FEBS Journal
TL;DR: A choline-binding-domain--beta-galactosidase chimera is constructed, which can be purified by this procedure and shows a high beta-galactsidase activity when immobilized in the column.
Abstract: We have developed a new single-step system, using a DEAE matrix, to immobilize and/or purify fusion proteins containing the choline-binding domain of the Streptococcus pneumoniae murein hydrolases. We have constructed a choline-binding-domain--beta-galactosidase chimera, which can be purified by this procedure and shows a high beta-galactosidase activity when immobilized in the column. A vector plasmid, pCUZ1, containing the lppp-5/lac promoter as well as 13 restriction sites, was constructed to facilitate the cloning and expression of gene fusions. This plasmid also allows the selection of recombinants by the well-known blue/white 5-bromo-4-chloro-3-indolyl-beta-D-galactoside procedure. A chimera between the choline-binding domain and the pneumococcal hemolysin was also constructed and purified using pCUZ1.

97 citations

"Class A PBPs have a distinct and un..." refers methods in this paper

  • ...The cells were lysed using the Fast Prep method with ≤106 µm glass beads at 6.5 m s−1 and insoluble material were removed by centrifugation at 20 000 × g. CbpD-B6 was purified from the soluble protein fraction by performing DEAE cellulose chromatography as described by Sanchez-Puelles et al.30, but using TBS (pH 7.4) instead of a phosphate buffer (pH 7.0)....

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  • ...CbpD-B6 was purified from the soluble protein fraction by performing DEAE cellulose chromatography as described by Sanchez-Puelles et al.(30), but using TBS (pH 7....

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Related Papers (5)
Class A PBPs have a distinct and unique role in the construction of the pneumococcal cell wall.

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Identification of a Functionally Unique Family of Penicillin-Binding Proteins

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30 Nov 2017-Journal of the American Chemical Society
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Active site restructuring regulates ligand recognition in class A penicillin-binding proteins

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18 Jan 2005-Proceedings of the National Academy of Sciences of the United States of America
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Regulation and function of class A Penicillin-binding proteins

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18 Feb 2021-Current Opinion in Microbiology
Manuel Pazos, Waldemar Vollmer
PBP1B Glycosyltransferase and Transpeptidase Activities Play Different Essential Roles during the De Novo Regeneration of Rod Morphology in Escherichia coli.

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14 Mar 2017-Journal of Bacteriology
Dev K. Ranjit, Matthew A. Jorgenson, Kevin D. Young
Frequently Asked Questions (2)
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. 

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