Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin-binding protein 2 of Escherichia coli K12

We have determined the nucleotide sequence of the pbpA gene encoding penicillin-binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2-terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH2-terminal hydrophobic region (a stretch of 25 non-ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active-site serine residue of PBP 2 was predicted to be Ser-330. Around this putative active-site serine residue was found the conserved sequence of Ser-Xaa-Xaa-Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs 1A, 1B, 3, 5 and 6) and class A and class C beta-lactamases. In the higher-molecular-mass PBPs 1A, 1B, 2 and 3, Ser-Xaa-Xaa-Lys-Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.     

Identification of the penicillin-binding active site of penicillin-binding protein 2 of Escherichia coli

We determined the active site of penicillin-binding protein (PBP) 2 of Escherichia coli. A water-soluble form of PBP 2, which was constructed by site-directed mutagenesis, was purified by affinity chromatography, labeled with dansyl-penicillin, and then digested with a combination of proteases. The amino acid composition of the labeled chymotryptic peptide purified by HPLC was identical with that of the amino acid sequence, Ala-Thr-Gln-Gly-Val-Tyr-Pro-Pro-Ala-Ser330-Thr-Val-Lys-Pro (residues 321-334) of PBP 2, which was deduced from the nucleotide sequence of the pbpA gene encoding PBP 2. This amino acid sequence was verified by sequencing the labeled tryptic peptide containing the labeled chymotryptic peptide region. A mutant PBP 2 (thiol-PBP 2), constructed by site-directed mutagenesis to replace Ser330 with Cys, lacked the penicillin-binding activity. These findings provided evidence that Ser330 near the middle of the primary structure of PBP 2 is the penicillin-binding active-site residue, as predicted previously on the basis of the sequence homology. Around this active site, the sequence Ser-Xaa-Xaa-Lys was observed, which is conserved in the active-site regions of all E. coli PBPs so far studied, class A and class C beta-lactamases, and D-Ala carboxypeptidases. The COOH-terminal amino acid of PBP 2 was identified as His633.     

Determination of the cleavage site involved in C-terminal processing of penicillin-binding protein 3 of Escherichia coli.

Chromatographic peptide mapping of lysyl endopeptidase digests of penicillin-binding protein 3 (PBP 3) of Escherichia coli revealed peptides that differed in retention time between the precursor and mature forms. The peptides were purified from a processing-defective (prc) mutant and a wild-type (prc+) strain. These peptides were identified as the C-terminal region of the precursor form and mature PBP 3 by amino acid sequencing. Each of the C-terminal peptides was cleaved into two fragments by trypsin digestion. By sequencing the resultant carboxyl-side fragment derived from the mature form, it was concluded that the C-terminal residue of mature PBP 3 was Val-577, and thus the Val-577-Ile-578 bond is the cleavage site for processing. This conclusion was consistent with the amino acid compositions of the relevant peptides, which suggested that the peptide from the cleavage site to the end of the deduced sequence (Ile-578-Ser-588) was present in the precursor but absent in the mature form. One lysyl peptide bond resisted both lysyl endopeptidase and trypsin and remained uncleaved in the peptide analyzed above.     

Identification of a penicillin-binding protein 3 homolog, PBP3x, in Pseudomonas aeruginosa: gene cloning and growth phase-dependent expression.

A homolog of Pseudomonas aeruginosa penicillin-binding protein 3 (PBP3), named PBP3x in this study, was identified by using degenerate primers based on conserved amino acid motifs in the high-molecular-weight PBPs. Analysis of the translated sequence of the pbpC gene encoding this PBP3x revealed that 41 and 48% of its amino acids were identical to those of Escherichia coli and P. aeruginosa PBP3s, respectively. The downstream sequence of pbpC encoded convergently transcribed homologs of the E. coli soxR gene and the Mycobacterium bovis adh gene. The pbpC gene product was expressed from the T7 promoter in E. coli and was exported to the cytoplasmic membrane of E. coli cells and could bind [3H] penicillin. By using a broad-host-range vector, pUCP27, the pbpC gene was expressed in P. aeruginosa PAO4089. [3H]penicillin-binding competition assays indicated that the pbpC gene product had lower affinities for several PBP3-targeted beta-lactam antibiotics than P. aeruginosa PBP3 did, and overexpression of the pbpC gene product had no effect on the susceptibility to the PBP3-targeted antibiotics tested. By gene replacement, a PBP3x-defective interposon mutant (strain HC132) was obtained and confirmed by Southern blot analysis. Inactivation of PBP3x caused no changes in the cell morphology or growth rate of exponentially growing cells, suggesting that pbpC was not required for cell viability under normal laboratory growth conditions. However, the upstream sequence of pbpC contained a potential sigma(s) recognition site, and pbpC gene expression appeared to be growth rate regulated. [3H]penicillin-binding assays indicated that PBP3 was mainly produced during exponential growth whereas PBP3x was produced in the stationary phase of growth.     

Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion

A new beta-lactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other beta-lactam antibiotics has been widely found in highly beta-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a beta-lactam-inducible MRSA PBP.     

Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli

The pbp3 gene encoding PBP3 of Bacillus cereus was cloned and sequenced. For this purpose, PBP3 was first purified from B. cereus ts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereus ts-4 pbp3 gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3 structural gene was ligated into the pET17 x b expression vector and pET-pbp3 was constructed. A protein was produced by the cells of E. coli carrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

Cloning and analysis of the HaeIII and HaeII methyltransferase genes

The HaeIII methyltransferase (MTase) gene from Haemophilus aegyptius (recognition sequence: 5'-GGCC-3') was cloned into Escherichia coli in the plasmid vector pBR322. The gene was isolated on a single EcoRI fragment and on a single HindIII fragment. Clones carrying additional adjacent fragments were found to code also for the HaeII restriction endonuclease and HaeII modification MTase (recognition sequence: 5'-PuGCGCPy-3'). The sequence of the HaeIII modification gene was determined. The inferred amino acid sequence of the protein was found to share extensive similarity with other sequenced m5C-MTases. The central 'non-conserved' region of the M.HaeIII MTase, thought to form the nucleotide sequence-specificity domain, is almost identical to that of the M.BsuRI, M.BspRI and M.NgoPII MTases, which also recognize the sequence 5'-GGCC-3'.     

Unusual septum formation in Streptococcus pneumoniae mutants with an alteration in the D,D-carboxypeptidase penicillin-binding protein 3.

An internal 630-bp DNA fragment of the gene encoding penicillin-binding protein 3 (PBP 3) (dacA) of Streptococcus pneumoniae was identified in a lambda gt11 gene bank screened with anti-PBP 3 antiserum. The deduced 210-amino-acid sequence showed a high degree of homology to the low-molecular-weight PBPs 5 and 6 of Escherichia coli and Bacillus subtilis PBP 5. Viable mutants lacking a C-terminal part of PBP 3 were obtained after a plasmid containing the dacA fragment was integrated into the PBP 3 gene by homologous recombination. The truncated PBP 3* was still active in terms of beta-lactam binding. Most PBP 3 was found in the growth medium, indicating that membrane anchoring of PBP 3 is provided by the C terminus, as has been shown for other D,D-carboxypeptidases. The mutant cells grew with a slower generation time than the wild type in the shape of irregular enlarged spheres. In addition, as revealed by electron microscopy, cell separation was severely affected, septa were found unevenly distributed at multiple sites within the cells, and the murein layer appeared variable in thickness.     

Molecular analysis of the Rhodococcus sp. strain H1 her gene and characterization of its product, a heroin esterase, expressed in Escherichia coli.

The structural gene for heroin esterase was cloned from Rhodococcus sp. strain H1 and expressed in Escherichia coli BL21(DE3). The purified enzyme was found to be a tetramer with an M(r) of 137,000 and an apparent K(m) of 0.88 mM for 6-acetylmorphine. The G-x-S-x-G motif was observed in the deduced amino acid sequence, suggesting that the enzyme is a serin esterase.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Identification of the CTAG-recognizing restriction-modification systems MthZI and MthFI from Methanobacterium thermoformicicum and characterization of the plasmid-encoded mthZIM gene.

Two CTAG-recognizing restriction and modification (R/M) systems, designated MthZI and MthFI, were identified in the thermophilic archaeon Methanobacterium thermoformicicum strains Z-245 and FTF, respectively. Further analysis revealed that the methyltransferase (MTase) genes are plasmid-located in both strains. The plasmid pFZ1-encoded mthZIM gene of strain Z-245 was further characterized by subcloning and expression studies in Escherichia coli followed by nucleotide sequence analysis. The mthZIM gene is 1065 bp in size and may code for a protein of 355 amino acids (M(r) 42,476 Da). The deduced amino acid sequence of the M.MthZI enzyme shares substantial similarity with four distinct regions from several m4C- and m6A-MTases, and contains the TSPPY motif that is so far only found in m4C-MTases. Partially overlapping with the mthZIM gene and in reverse orientation, an additional ORF was identified with a size of 606 bp potentially coding for a protein of 202 amino acids (M(r) 23.710 Da). This ORF is suggested to encode the corresponding endonuclease R.MthZI.     

The Staphylococcus aureus mec determinant comprises an unusual cluster of direct repeats and codes for a gene product similar to the Escherichia coli sn-glycerophosphoryl diester phosphodiesterase.

The DNA sequence located between mecA, the gene that codes for penicillin-binding protein PBP2', and insertion sequence-like element IS431mec has been termed hypervariable because of its length polymorphism among different staphylococcal isolates. We sequenced and characterized the hypervariable region of the methicillin resistance determinant (mec) isolated from Staphylococcus aureus BB270. Within the 2,040-bp hypervariable region, we identified an unusual accumulation of long direct repeats. Analysis of the DNA sequence revealed a minimal direct repeat unit (dru) of 40 bp which was repeated 10 times within 500 bp. The dru sequences are responsible for the length polymorphism of mec. Moreover, we identified an open reading frame that codes for 145 amino acids (ORF145), whose deduced amino acid sequence showed 57% amino acid sequence similarity to the N terminus of the glycerophosphoryl diester phosphodiesterase (UgpQ) of Escherichia coli.     

Nucleotide sequences of genes encoding penicillin-binding proteins from Streptococcus pneumoniae and Streptococcus oralis with high homology to Escherichia coli penicillin-binding proteins 1a and 1b.

The nucleotide sequence of a 3,378-bp DNA fragment of Streptococcus pneumoniae that included the structural gene for penicillin-binding protein (PBP) 1a (ponA), which encodes 719 amino acids, was determined. Homologous DNA fragments from an S. oralis strain were amplified with ponA-specific oligonucleotides. The 2,524-bp S. oralis sequence contained the coding region for the first 636 amino acids of a PBP. The coding sequence differed by 437 nucleotides (27%) and one additional triplet, resulting in 87 amino acid substitutions (14%), from S. pneumoniae PBP 1a. Both PBPs are highly homologous to bifunctional high-M(r) Escherichia coli PBPs 1a and 1b.     

Cloning and expression of the aspartate carbamoyltransferase gene from Treponema denticola.

Treponema denticola seems to play a central role in the etiology of human periodontal disease. We have cloned an antigenic protein-coding sequence from T. denticola ATCC 33520. The protein-coding region was found to be a 3-kbp HindIII-HindIII fragment. The open reading frame consists of 1,426 bp and codes for a protein with an M(r) of 54,919. The deduced amino acid sequence showed 33.8% homology with that of the aspartate carbamoyltransferase of Escherichia coli. The gene products showed aspartate carbamoyltransferase activity.     

Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli

Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membrane-bound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltA-interacting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10(-6) M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.     

Cloning and expression of the aspartate carbamoyltransferase gene from Treponema denticola.

Treponema denticola seems to play a central role in the etiology of human periodontal disease. We have cloned an antigenic protein-coding sequence from T. denticola ATCC 33520. The protein-coding region was found to be a 3-kbp HindIII-HindIII fragment. The open reading frame consists of 1,426 bp and codes for a protein with an M(r) of 54,919. The deduced amino acid sequence showed 33.8% homology with that of the aspartate carbamoyltransferase of Escherichia coli. The gene products showed aspartate carbamoyltransferase activity.     

Expression and purification of a soluble form of penicillin-binding protein 2 from both penicillin-susceptible and penicillin-resistant Neisseria gonorrhoeae.

Resistance to penicillin in non-beta-lactamase-producing strains of Neisseria gonorrhoeae (CMRNG strains) is mediated in part by the production of altered forms of penicillin-binding protein 2 (PBP 2) that have a decreased affinity for penicillin. The reduction in the affinity of PBP 2 is largely due to the insertion of an aspartic acid residue (Asp-345a) into the amino acid sequence of PBP 2. Truncated forms of N. gonorrhoeae PBP 2, which differed only by the insertion of Asp-345a, were constructed by placing the region of the penA genes encoding the periplasmic domain of PBP 2 (amino acids 42-581) into an ATG expression vector. When the recombinant PBP 2 molecules were overexpressed in Escherichia coli, insoluble PBP 2 inclusion bodies, which could be isolated by low-speed centrifugation of cell lysates, were formed. These insoluble aggregates were solubilized and the truncated PBP 2 polypeptides were partially purified by cation-exchange chromatography and gel filtration in the presence of denaturant prior to the refolding of the enzyme in vitro. After renaturation, gel filtration was used to separate monomeric soluble PBP 2 from improperly folded protein aggregates and other protein contaminants. A 4-liter culture of induced E. coli cells yielded 1.4 mg of soluble PBP 2 or PBP 2' (PBP 2 containing the Asp-345a insertion), both of which were estimated to be 99% pure. The affinity of soluble PBP 2' for [3H]penicillin G was decreased fourfold relative to that of soluble PBP 2, and their affinities were found to be identical to the affinities of the full-length PBP 2 enzymes that were previously determined in N. gonorrhoeae membranes. Furthermore, soluble PBP 2 displayed a rank order of affinity for several other beta-lactam antibiotics that was consistent with the rank order of affinities previously reported for the native molecules. On the basis of these results, both of these soluble PBPs should be suitable for crystallization and X-ray crystallographic analysis.