Functional redundancy of division specific penicillin‐binding proteins in <i>Bacillus subtilis</i>

Sassine, Jad; Xu, Meizhu; Sidiq, Karzan R.; Emmins, Robyn; Errington, Jeff; Daniel, Richard A.

doi:10.1111/mmi.13765

Cited by 36 publications

(62 citation statements)

References 56 publications

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“…This suggests that PBP1 has a second role, as previously proposed 19 . Compatible results have been obtained in B. subtilis, where introduction of a point mutation in the TP active site of the essential protein PBP2B, did not lead to loss of cell viability 20 .…”

Section: Roda-pbp3 and Ftsw-pbp1 Form Cognate Pairs In S Aureusmentioning

confidence: 87%

SEDS-bPBP pairs direct Lateral and Septal Peptidoglycan Synthesis in Staphylococcus aureus

Reichmann

Tavares

Saraiva

et al. 2019

Preprint

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Peptidoglycan (PGN) is the major component of the bacterial cell wall, a structure essential for the physical integrity and shape of the cell. Bacteria maintain cell shape by directing PGN incorporation to distinct regions of the cell, namely through the localisation of the late stage PGN synthesis proteins. These include two key protein families, SEDS transglycosylases and the bPBP transpeptidases, proposed to function in cognate pairs. Rod-shaped bacteria have two SEDS-bPBP pairs, involved in cell elongation and cell division. Here, we elucidate why coccoid bacteria, such as Staphylococcus aureus, also possess two SEDS-bPBP pairs. We determined that S. aureus RodA-PBP3 and FtsW-PBP1 likely constitute cognate pairs of interacting proteins. Lack of RodA-PBP3 decreased cell eccentricity due to deficient pre-septal PGN synthesis, whereas the depletion of FtsW-PBP1 arrested normal septal PGN incorporation. Although PBP1 is an essential protein, a mutant lacking PBP1 transpeptidase activity is viable, showing that this protein has a second function. We propose that the FtsW-PBP1 pair has a role in stabilising the divisome at midcell. In the absence of these proteins, the divisome appears as multiple rings/arcs that drive lateral PGN incorporation, leading to cell elongation. We conclude that RodA-PBP3 and FtsW-PBP1 mediate lateral and septal PGN incorporation, respectively, and that the activity of these pairs must be balanced in order to maintain coccoid morphology.Peptidoglycan (PGN) synthesis is an essential process that is both spatially and temporally regulated to ensure that the bacterial cell shape is maintained 1 . Rod-shaped bacteria elongate by synthesising PGN along the length of the cell in a process directed by the cytoskeletal protein MreB 2 . In Escherichia coli and Bacillus subtilis, this protein polymerises into short filaments that move processively around the cell diameter, and organise a multi-protein machinery, including PGN synthesis proteins, referred to as the elongasome or the Rod system 3-5 . Cell division is dependent on another cytoskeletal protein, FtsZ, which polymerises to form the Z-ring and recruits a multi-protein complex responsible for septum synthesis, known as the divisome 6,7 . This complex directs PGN incorporation to the midcell, resulting in inward PGN synthesis, and eventually bisects the mother cell, leading to daughter cell separation.Ovococci such as Streptococcus pneumoniae and Lactococcus lactis lack MreB, and FtsZ is proposed to coordinate both elongation and septation 8,9 . In these organisms PGN is

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Section: Roda-pbp3 and Ftsw-pbp1 Form Cognate Pairs In S Aureusmentioning

confidence: 87%

SEDS-bPBP pairs direct Lateral and Septal Peptidoglycan Synthesis in Staphylococcus aureus

Reichmann

Tavares

Saraiva

et al. 2019

Preprint

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“…(9,40,41) PBP2b, encoded by pbpB, is the only PBP that is essential for growth in B. subtilis, which is attributed to its major role in septum formation. (10,41,42) More recently, it was revealed that the transpeptidation activity of PBP2b is dispensable and cells containing deactivated PBP2b maintain normal growth and morphology. (10, 11) Thus, the development of chemical tools that can selectively target the activity of these transpeptidases can complement the genetic studies and deepen our knowledge about these PBPs, which will be the subject of future work.…”

Section: Selectivity Profile Of β-Lactams For B Subtilis Pbpsmentioning

confidence: 99%

“…(7,8) Functional redundancy within the PBPs has compounded the difficulties in establishing the role of individual PBPs in peptidoglycan biosynthesis. (9)(10)(11) Single PBPs have been studied by conjugation to fluorescently labeled protein tags. (10,12) However, these data alone are insufficient to fully characterize their roles as protein localization does not provide information about the activity state of the PBPs.…”

Section: Introductionmentioning

confidence: 99%

“…(9)(10)(11) Single PBPs have been studied by conjugation to fluorescently labeled protein tags. (10,12) However, these data alone are insufficient to fully characterize their roles as protein localization does not provide information about the activity state of the PBPs. Moreover, protein tags are bulky and can perturb protein abundance, localization, or function.…”

Section: Introductionmentioning

confidence: 99%

“…(29)(30)(31)(32)(33) However, as described previously, the specific tasks of individual members have been difficult to assign due to functional redundancy of these enzymes. (9,10,12,32) In order to facilitate the development of chemical tools to study cell growth and division in B. subtilis, we assessed the PBP inhibition profile of a library of β-lactam antibiotics in this model microorganism using the fluorescent whole cell assay mentioned above. Briefly, B. subtilis cells were treated with increasing concentrations of different β-lactam antibiotics, followed by Boc-FL as the readout probe to determine which PBPs were blocked by the antibiotic.…”

Section: Introductionmentioning

confidence: 99%

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Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins inBacillus subtilis

Sharifzadeh

Dempwolff

Kearns

et al. 2019

Preprint

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Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 β-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b or PBP4. Almost all penicillin scaffolds were co-selective for all or combinations of PBP2a, 2b and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin), 2b (cefotaxime) or 2a, 2b and 4 (cephalothin).Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid) and various co-selective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for β-lactams and speculated to be involved in resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

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Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria

Kawai

Mickiewicz

Errington

2018

Cell

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101

115

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Summaryβ-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient “L-form” bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

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Functional redundancy of division specific penicillin‐binding proteins in Bacillus subtilis

Cited by 36 publications

References 56 publications

SEDS-bPBP pairs direct Lateral and Septal Peptidoglycan Synthesis in Staphylococcus aureus

SEDS-bPBP pairs direct Lateral and Septal Peptidoglycan Synthesis in Staphylococcus aureus

Harnessing β-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins inBacillus subtilis

Lysozyme Counteracts β-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria

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