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

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.

AbstractIn 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)

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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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
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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
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

Figures (4)
Citations
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Journal ArticleDOI
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.

8 citations


Journal ArticleDOI
TL;DR: The cell wall of Bacillus subtilis is a rigid structure on the outside of the cell that forms the first barrier between the bacterium and the environment, and at the same time maintains cell shape and withstands the pressure generated by the cell's turgor.
Abstract: The cell wall of Bacillus subtilis is a rigid structure on the outside of the cell that forms the first barrier between the bacterium and the environment, and at the same time maintains cell shape and withstands the pressure generated by the cell's turgor. In this review, the chemical composition of peptidoglycan, teichoic and teichuronic acids, the polymers that comprise the cell wall, and the biosynthetic pathways involved in their synthesis will be discussed, as well as the architecture of the cell wall. B. subtilis has been the first bacterium for which the role of an actin-like cytoskeleton in cell shape determination and peptidoglycan synthesis was identified and for which the entire set of peptidoglycan synthesizing enzymes has been localised. The role of the cytoskeleton in shape generation and maintenance will be discussed and results from other model organisms will be compared to what is known for B. subtilis. Finally, outstanding questions in the field of cell wall synthesis will be discussed.

7 citations


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

  • ...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)....

    [...]

  • ..., 2016), as well as modifying the PG to mature PG that is less sensitive to the activity of specific hydrolysases (Straume et al., 2020)....

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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.
Abstract: Three hundred and sixty different samples were collected from different sources, including wound, burn, nasal, and oral swabs from several hospitals in Baghdad. A number of 150 (53%) Staphylococcus aureus samples were isolated and identified among a total of 283 samples. Then, the spread of the Toxic Shock Syndrome Toxin-1 gene (tsst-1) was investigated in β-lactamase resistant S. aureus. According to the source of samples, the distribution of S. aureus isolates was found to be significantly higher (p < 0.01) in wound samples as compared to other sources. According to the age, a highly significant distribution (p < 0.01) was recorded in the age group of 15-30 years, whereas gender comparison showed no statistically significant differences. All the isolates were subjected to susceptibility test against eight β-Lactam antibiotics by using the disc diffusion method. The antimicrobial susceptibility test showed that S. aureus had maximum resistance percentage to Carbenicillin (97.3%), while the lowest resistance rate was against Meropenem, with a sensitivity rate of up to 82%. In addition, 144 (96%) out of the 150 S. aureus isolates have multiple drug resistance (MDR). All the isolates were subjected to polymerase chain reaction to amplify tsst-1 toxin gene. A number of 70 isolates (46.7%) were found to be positive for tsst-1 gene. The results showed no significant correlation between tsst-1 gene with the individual antibiotic resistance and the multi-drug resistance patterns of the isolates (p = 0.226).

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  • ...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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Journal ArticleDOI
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.

4 citations


Posted ContentDOI
12 Jun 2020-bioRxiv
TL;DR: Structural analysis reveals that FtsE contains all the conserved structural motifs associated with ATPase activity, and allowed interpretation of the in vivo dimeric arrangement in both ADP and ATP states, and provides evidences on how difference between ATP and ADP states in FTSE would dramatically alter FtsEX interaction with PG hydrolase PcsB in pneumococcal division.
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, where FtsX is an integral membrane protein and FtsE is an ATPase, required for mechanotransmission of the 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 interact with the modular peptidoglycan hydrolase PcsB at the septum. Here, we report the high-resolution structures of pneumococcal FtsE in complex with different nucleotides. Structural analysis reveals that FtsE contains all the conserved structural motifs associated with ATPase activity, and allowed interpretation of the in vivo dimeric arrangement in both ADP and ATP states. Interestingly, three specific FtsE regions were identified with high structural plasticity that shape the cavity in which the cytosolic region of FtsX would be inserted. The residues corresponding to the FtsX coupling helix, responsible for FtsE contact, 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, play 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 transporter, couple ATP hydrolysis by FtsE to mechanically transduce a conformational signal to activate 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 key for FtsX binding. The complementary region in FtsX has been also identified and validated in vivo. Our results provide evidences on how difference between ATP and ADP states in FtsE would dramatically alter FtsEX interaction with PG hydrolase PcsB in pneumococcal division.

3 citations


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

  • ...Furthermore, transmission electron microscopy analysis of these cells revealed that their septal cross-wall has not been split (58)....

    [...]


References
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Abstract: The peptidoglycan (murein) sacculus is a unique and essential structural element in the cell wall of most bacteria. Made of glycan strands cross-linked by short peptides, the sacculus forms a closed, bag-shaped structure surrounding the cytoplasmic membrane. There is a high diversity in the composition and sequence of the peptides in the peptidoglycan from different species. Furthermore, in several species examined, the fine structure of the peptidoglycan significantly varies with the growth conditions. Limited number of biophysical data on the thickness, elasticity and porosity of peptidoglycan are available. The different models for the architecture of peptidoglycan are discussed with respect to structural and physical parameters.

1,585 citations


Journal ArticleDOI
TL;DR: An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.
Abstract: Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.

950 citations


Journal ArticleDOI
TL;DR: It is shown that strain CP1200 produces a 17-residue peptide that induces cells of the Streptococcus pneumoniae species to develop competence and the hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.
Abstract: Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.

712 citations


Journal ArticleDOI
TL;DR: It is concluded that genetic properties of markers, such as size, linkage, and discreteness, are reflections of the actual distribution of determinants within DNA molecules.
Abstract: An enzyme of Pneumococcus responsible for the conversion of maltose to glucose and oligosaccharides was investigated. The genetic analysis of a group of mutants lacking this enzyme was accomplished by studying DNA-mediated transformations of the different strains. Transformation of the different mutants to maltose utilization by wild-type DNA occurred with different frequencies. Recombination of genetic material of the maltose-negative strains also gave rise to positive cells. Analysis of the recombinations indicated that the regions corresponding to the different markers are linked and arranged in a linear fashion. Some larger regions overlapped several smaller ones indicating that single mutations altered regions in the genetic material of different size. Heating the DNA used in transforming the mutant strains destroyed most rapidly the ability to transform the factors corresponding to the larger genetic regions. Factors corresponding to smaller regions were inactivated less rapidly. This is taken to indicate that a DNA molecule partly denatured by heat still retains part of its genetic activity. It is concluded, furthermore, that genetic properties of markers, such as size, linkage, and discreteness, are reflections of the actual distribution of determinants within DNA molecules.

523 citations


Journal ArticleDOI
Abstract: The regulation of competence for genetic transformation in Streptococcus pneumoniae depends on a quorum-sensing system, but the only molecular elements of the system whose specific role have been identified are an extracellular peptide signal and an ABC-transporter required for its export. Here we show that transcription of comC, the gene encoding a predicted 41-residue precursor peptide that is thought to be processed and secreted as the 17-residue mature competence activator, increased approximately 40-fold above its basal level of expression in response to exogenous synthetic activator, consistent with earlier experiments indicating that the activator acts autocatalytically. We also describe two new genes, comD and comE, that encode members of histidine protein kinase and response-regulator families and are linked to comC. Disruption of comE abolished both response to synthetic activator peptide and endogenous competence induction.

422 citations


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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.