Use of biotinylated beta-lactams and chemiluminescence for study and purification of penicillin-binding proteins in bacteria

Dargis, Michèle; Malouin, François

doi:10.1128/aac.38.5.973

Cited by 42 publications

(38 citation statements)

References 21 publications

(23 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The tolerance to lysis at low pH was also comparable for both the lysis induced by penicillin and the lysis induced by overproduction of inactive PBP1B. In the case of ␤-lactam-induced lysis, this tolerance effect was speculated to be the consequence of a suppressed murein hydrolase (autolytic) activity (6). Thus, we concluded that the observed lysis upon overproduction of inactive PBP1B is due to the unbalanced activity of murein hydrolases as in the case of penicillin-induced lysis.…”

Section: Discussionmentioning

confidence: 85%

See 1 more Smart Citation

Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

2003

View full text Add to dashboard Cite

Penicillin-binding protein 1B (PBP1B) of Escherichia coli is a bifunctional murein synthase containing both a transpeptidase domain and a transglycosylase domain. The protein is present in three forms (␣, ␤, and ␥) which differ in the length of their N-terminal cytoplasmic region. Expression plasmids allowing the production of native PBP1B or of PBP1B variants with an inactive transpeptidase or transglycosylase domain or both were constructed. The inactive domains contained a single amino acid exchange in an essential active-site residue. Overproduction of the inactive PBP1B variants, but not of the active proteins, caused lysis of wild-type cells. The cells became tolerant to lysis by inactive PBP1B at a pH of 5.0, which is similar to the known tolerance for penicillin-induced lysis under acid pH conditions. Lysis was also reduced in mutant strains lacking several murein hydrolases. In particular, a strain devoid of activity of all known lytic transglycosylases was virtually tolerant, indicating that mainly the lytic transglycosylases are responsible for the observed lysis effect. A possible structural interaction between PBP1B and murein hydrolases in vivo by the formation of a multienzyme complex is discussed.

show abstract

Section: Discussionmentioning

confidence: 85%

“…The PBPs present in the membranes were labeled with biotinylated ampicillin and detected as described previously (6). Following SDS-PAGE and transfer to nitrocellulose, the PBPs were detected with streptavidinhorseradish peroxidase by using chloronaphthol as the substrate (see above).…”

Section: Methodsmentioning

confidence: 99%

Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

2003

View full text Add to dashboard Cite

show abstract

“…The fractions containing PBP1B were dialyzed against 10 mM sodium acetate, 500 mM NaCl, 10 mM MgCl 2 , 10% glycerol, 0.02% NaN 3 , pH 5.0, and stored at Ϫ20°C. The purified PBP1B was active and bound biotinylated ampicillin (24).…”

Section: Methodsmentioning

confidence: 99%

In Vitro Murein (Peptidoglycan) Synthesis by Dimers of the Bifunctional Transglycosylase-Transpeptidase PBP1B from Escherichia coli

Bertsche

Breukink

Kast

et al. 2005

Journal of Biological Chemistry

141

195

View full text Add to dashboard Cite

PBP1B is a major bifunctional murein (peptidoglycan) synthase catalyzing transglycosylation and transpeptidation reactions in Escherichia coli. PBP1B has been shown to form dimers in vivo. The K D value for PBP1B dimerization was determined by surface plasmon resonance. The effect of the dimerization of PBP1B on its activities was studied with a newly developed in vitro murein synthesis assay with radioactively labeled lipid II precursor as substrate. Under conditions at which PBP1B dimerizes, the enzyme synthesized murein with long glycan strands (>25 disaccharide units) and with almost 50% of the peptides being part of cross-links. PBP1B was also capable of synthesizing trimeric muropeptide structures. Tri-, tetra-, and pentapeptide compounds could serve as acceptors in the PBP1B-catalyzed transpeptidation reaction.Most bacteria contain a murein (peptidoglycan) sacculus, an exoskeleton that is essential for the osmotic stability of the cell (1). Murein has a net-like structure and is composed of glycan strands of alternating ␤1,4-linked N-acetylmuramic acid and N-acetylglucosamine residues that are cross-linked by short peptides (2). Escherichia coli is rod-shaped and has a simple cell cycle; a newborn cell elongates with a constant diameter, and when the length of the cell has doubled, the cell divides at mid-cell, forming two new polar caps. Cell growth requires a well coordinated incorporation of the murein precursor, lipid II, into the existing sacculus (3).Two reactions are required for murein synthesis. First, disaccharide units of lipid II are oligomerized to murein glycan strands by transglycosylation. Second, peptide cross-links are formed by transpeptidation, the latter reaction being the target of ␤-lactam antibiotics. Murein synthases have a modular domain structure (4). E. coli has three bifunctional transglycosylases-transpeptidases, the class A penicillin-binding proteins (PBPs) 3 1A, 1B, and 1C (4 -6), two monofunctional transpeptidases, the class B PBPs 2 and 3, which contain a non-catalytic domain of unknown function (7), and one monofunctional transglycosylase, MtgA (8). Different molecular interactions between certain murein synthases and between murein synthases and hydrolases have been identified, indicating that the enlargement of the sacculus might be achieved by murein synthesis holoenzymes (9).PBP1B exists in three isoforms (␣, ␤, and ␥) that are encoded by the same gene (ponB) and is a major murein synthase in E. coli (10 -12). The protein has a short cytoplasmic part and is anchored in the cytoplasmic membrane via the amino acids 64 -88. Most of the enzyme is located in the periplasm (amino acids 89 -844), and the periplasmic part contains the transglycosylase (amino acids 198 -435) and transpeptidase (amino acids 447-780) domains (13). PBP1B was shown to form dimers in vivo (14, 15), and dimerization does not involve disulfide bridges (16). Moreover, PBP1B interacts with PBP3 and with the MltA-interacting protein A (MipA), that itself interacts with the murein hydrolase MltA (17)...

show abstract

“…PBPs were labeled using a biotinylated ampicillin (BIO-AMP) according to the method described by Dargis and Malouin (7). The reporter molecule, BIO-AMP, was also prepared as previously described (7). To selectively label PBP 2a from staphylococcal membrane preparations, samples were first incubated with 100 g of clavulanic acid/ml for 10 min at 35°C to saturate high-affinity PBPs (PBPs 1, 2, and 3).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

RWJ-54428 (MC-02,479), a New Cephalosporin with High Affinity for Penicillin-Binding Proteins, Including PBP 2a, and Stability to Staphylococcal Beta-Lactamases

Malouin

Blais

Chamberland

et al. 2003

Antimicrob Agents Chemother

View full text Add to dashboard Cite

RWJ-54428 (MC-02,479) is a new cephalosporin active against gram-positive bacteria, including methicillinresistant Staphylococcus aureus (MRSA). The potency of this new cephalosporin against MRSA is related to a high affinity for penicillin-binding protein 2a (PBP 2a), as assessed in a competition assay using biotinylated ampicillin as the reporter molecule. RWJ-54428 had high activity against MRSA strains COL and 67-0 (MIC of 1 g/ml) and also showed affinity for PBP 2a, with a 50% inhibitory concentration (IC 50 ) of 0.7 g/ml. RWJ-54428 also displayed excellent affinity for PBP 5 from Enterococcus hirae R40, with an IC 50 of 0.8 g/ml and a MIC of 0.5 g/ml. The affinity of RWJ-54428 for PBPs of ␤-lactam-susceptible S. aureus (MSSA), enterococci (E. hirae), and Streptococcus pneumoniae showed that the good affinity of RWJ-54428 for MRSA PBP 2a and E. hirae PBP 5 does not compromise its binding to susceptible PBPs. RWJ-54428 showed stability to hydrolysis by purified type A ␤-lactamase isolated from S. aureus PC1. In addition, RWJ-54428 displayed low MICs against strains of S. aureus bearing the four classes of staphylococcal ␤-lactamases, including ␤-lactamase hyperproducers. The frequency of isolation of resistant mutants to RWJ-54428 from MRSA strains was very low. In summary, RWJ-54428 has high affinity to multiple PBPs and is stable to ␤-lactamase, properties that may explain our inability to find resistance by standard methods. These data are consistent with its excellent activity against ␤-lactam-resistant gram-positive bacteria.

show abstract

Use of biotinylated beta-lactams and chemiluminescence for study and purification of penicillin-binding proteins in bacteria

Cited by 42 publications

References 21 publications

Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

Overproduction of Inactive Variants of the Murein Synthase PBP1B Causes Lysis in Escherichia coli

In Vitro Murein (Peptidoglycan) Synthesis by Dimers of the Bifunctional Transglycosylase-Transpeptidase PBP1B from Escherichia coli

RWJ-54428 (MC-02,479), a New Cephalosporin with High Affinity for Penicillin-Binding Proteins, Including PBP 2a, and Stability to Staphylococcal Beta-Lactamases

Contact Info

Product

Resources

About