scholarly journals Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

2000 ◽  
Vol 350 (1) ◽  
pp. 75 ◽  
Author(s):  
Sebabrata MAHAPATRA ◽  
Sanjib BHAKTA ◽  
Jasimuddin AHAMED ◽  
Joyoti BASU
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Mycobacterium Leprae ◽  
High Molecular Mass ◽  
Penicillin Binding Protein ◽  
Derivatives Of

scholarly journals Characterization of derivatives of the high-molecular-mass penicillin-binding protein (PBP) 1 of Mycobacterium leprae

2000 ◽  
Vol 350 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Sebabrata MAHAPATRA ◽  
Sanjib BHAKTA ◽  
Jasimuddin AHAMED ◽  
Joyoti BASU
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Mycobacterium Leprae ◽  
Binding Activity ◽  
High Molecular Mass ◽  
Class A ◽  
C Terminus ◽  
Fusion Junction ◽  
Derivatives Of

Mycobacterium leprae has two high-molecular-mass multimodular penicillin-binding proteins (PBPs) of class A, termed PBP1 and PBP1* [Lepage, Dubois, Ghosh, Joris, Mahapatra, Kundu, Basu, Chakrabarti, Cole, Nguyen-Disteche and Ghuysen (1997) J. Bacteriol. 179, 4627–4630]. PBP1-Xaa–β-lactamase fusions generated periplasmic β-lactamase activity when Xaa (the amino acid of PBP1 at the fusion junction) was residue 314, 363, 407, 450 or 480. Truncation of the N-terminal part of the protein up to residue Leu-147 generated a penicillin-binding polypeptide which could still associate with the plasma membrane, whereas [∆M1–R314]PBP1 (PBP1 lacking residues Met-1 to Arg-314) failed to associate with the membrane, suggesting that the region between residues Leu-147 and Arg-314 harbours an additional plasma membrane association site for PBP1. Truncation of the C-terminus up to 42 residues downstream of the KTG (Lys-Thr-Gly) motif also generated a polypeptide that retained penicillin-binding activity. [∆M1–R314]PBP1 could be extracted from inclusion bodies and refolded under appropriate conditions to give a form capable of binding penicillin with the same efficiency as full-length PBP1. This is, to the best of our knowledge, the first report of a soluble derivative of a penicillin-resistant high-molecular-mass PBP of class A that is capable of binding penicillin. A chimaeric PBP in which the penicillin-binding (PB) module of PBP1 was fused at its N-terminal end with the non-penicillin-binding (n-PB) module of PBP1* retained pencillin-binding activity similar to that of PBP1, corroborating the finding that the n-PB module of PBP1 is dispensable for its penicillin-binding activity.

Antigenic and genetic characterization of a putative hybrid transferrin-binding protein B fromNeisseria meningitidis

1999 ◽  
Vol 45 (12) ◽  
pp. 1050-1054
Author(s):  
Tamara Menéndez ◽  
Mairet Pérez ◽  
Anabel Alvarez
Keyword(s):  
Neisseria Meningitidis ◽  
Molecular Mass ◽  
Reference Strain ◽  
Binding Protein ◽  
High Molecular Mass ◽  
Putative Hybrid ◽  
Family Strain ◽  
Transferrin Binding Proteins ◽  
Meningococcal Strain

The transferrin-binding protein Bs (TbpBs) from the bacterium Neisseria meningitidis have been divided into two families according to genetic and antigenic features. TbpB from meningococcal strain B385 showed a molecular mass similar to that exhibited by TbpBs belonging to the high molecular mass family of TbpBs. TbpB was recognized by immunoassay using a specific serum directed against the TbpB of the reference strain for this family (strain M982). It was also recognized by a serum elicited against the TbpB of the reference strain for the low molecular mass family (strain B16B6). The tbpB gene from strain B385 was cloned and sequenced. The highest degree of sequence homology was found to be with the TbpBs belonging to the high molecular mass family, although a region of 14 amino acids that is only present in the TbpB from strain B16B6 was also found. This report illustrates a TbpB that shows hybrid antigenic and genetic behaviour.Key words: Neisseria meningitidis, transferrin-binding proteins, TbpB families.

scholarly journals Cloning and Characterization of a Gene,pbpF, Encoding a New Penicillin-Binding Protein, PBP2B, inStaphylococcus aureus

1999 ◽  
Vol 43 (7) ◽  
pp. 1578-1583 ◽  
Author(s):  
Hitoshi Komatsuzawa ◽  
Gil H. Choi ◽  
Kouji Ohta ◽  
Motoyuki Sugai ◽  
Monique T. Tran ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Protein Band ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Putative Signal Peptide ◽  
C Terminus ◽  
Histidine Tag

ABSTRACT A previously unrecognized penicillin binding protein (PBP) gene,pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and β-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed inEscherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region ofpbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

scholarly journals Characterization of a Mycobacterium smegmatis Mutant Lacking Penicillin Binding Protein 1

1999 ◽  
Vol 43 (12) ◽  
pp. 3011-3013 ◽  
Author(s):  
Helen Billman-Jacobe ◽  
Ruth E. Haites ◽  
Ross L. Coppel
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Liquid Culture ◽  
Binding Protein ◽  
Mycobacterium Leprae ◽  
Deficient Mutant ◽  
Penicillin Binding Protein

ABSTRACT The ponA gene of Mycobacterium smegmatisencodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of Mycobacterium tuberculosis and Mycobacterium leprae. Transposon disruption of ponA in M. smegmatis resulted in a PBP1-deficient mutant that was sensitive to β-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.

Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1∗ and its soluble derivative from Mycobacterium tuberculosis

2002 ◽  
Vol 361 (3) ◽  
pp. 635-639
Author(s):  
Sanjib BHAKTA ◽  
Joyoti BASU
Keyword(s):  
Molecular Mass ◽  
Inclusion Bodies ◽  
Biochemical Characterization ◽  
Binding Protein ◽  
High Molecular Mass ◽  
Proteinase K ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
E Coli ◽  
Class A

The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacteriumtuberculosis, has been named penicillin-binding protein 1∗ (PBP1∗), by analogy to the previously characterized PBP1∗ of M. leprae. This gene has been overexpressed in Escherichia coli. His6-tagged PBP1∗ localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1∗ binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272–663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly95—Gln143 results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1∗ binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.

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