Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems.

A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector. The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa. In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E. coli. These results indicated that PBP 1B of E. coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams.     

Analysis of the different molecular forms of penicillin-binding protein 1B in Escherichia coli ponB mutants lysogenized with specialized transducing lambda (ponB+) bacteriophages

Penicillin-binding protein (pbp) 1b, the main DD-transpeptidase/transglycosylase of Escherichia coli, is normally present in the cell in three molecular forms alpha, beta and gamma, differentiated by their mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The three molecular forms are enzymatically active in vitro and their relative amounts are kept fairly constant in most labelling experiments with radioactive beta-lactam antibiotics. In this paper, we have analyzed the expression of ponB (mrcB), the structural gene for pbp 1b, and the relation among the three forms of pbp 1b in ponB strains lysogenyzed by lambda 540 (ponB+) recombinant bacteriophages. Our data indicate that ponB is transcribed anti-clockwise on the E. coli chromosome and suggest that pbp 1b alpha is the first membrane-bound form of pbp 1b able to bind labelled beta-lactams, and is the precursor of pbp 1b beta which is, in turn, the precursor of pbp 1 beta gamma.     

Properties of the penicillin-binding proteins of Escherichia coli K12,.

Benzyl[14C]penicillin binds to six proteins with molecular weights between 40000 and 91000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to benzyl[14C]penicillin in whole cells. Proteins 5 and 6 released bound benzyl[14C]penicillin with half times of 5 and 19 min at 30 degrees C but the other binding proteins showed less than 50% release during a 60-min period at 30 degrees C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2-mercaptoethanol and neutral hydroxylamine. Release of benzyl[14C]penicillin did not occur if the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two [14C]cephalosporins. These beta-lactams bound to either all or some of those proteins to which benzyl[14C]penicillin bound. No binding proteins have been detected in the outer membrane of E coli with any beta-[14C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of benzyl[14C]penicillin to the six penicillin-binding proteins. These results, together with those obtained by direct binding experiments with beta-[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the target at which beta-lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3, and 4 correlate reasonably well with those expected for the above killing targets.     

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.     

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.     

Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12

5'-Phosphoribosylglycinamide transformylase (EC 2.1.2.2), encoded by the purN gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosylformylglycinamide from 5'-phosphoribosylglycinamide (GAR). The mature protein, as deduced from the purN structural gene sequence, contains 212 amino acid residues and has a calculated Mr of 23,241. The purN gene is located adjacent to and immediately downstream from the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase where the initiation codon for GAR transformylase overlaps the termination codon of AIR synthetase. Based on polarity studies, the expression of the purN gene originates from the purM control region and thus forms a purMN operon. The E. coli GAR transformylase shows greater homology to the GAR transformylase domain of the trifunctional Gart polypeptide of Drosophila than to the single GAR transformylase of Saccharomyces. Immediately downstream from the purN gene of the purMN operon is a region of dyad symmetry capable of forming a hairpin stem and loop structure characteristic of a rho-independent terminator.     

Comparison of the nucleotide sequences of the genes encoding the KS71A and F7(1) fimbrial antigens of uropathogenic Escherichia coli

DNA fragments encompassing the genes for the KS71A and F7(1) fimbrial subunits of Escherichia coli strains KS71 (O4:K12) and AD110 (O6:K2), respectively, have been subjected to DNA sequencing. The nucleotide sequences of the two fimbrillin genes were identical and they encode a polypeptide of 187 amino acids of which 21 amino acids probably will constitute the signal sequence. The primary structure of these fimbrillins showed significant homology with the primary structure of other E. coli fimbrillins.     

Isolation, characterization and nucleotide sequences of the aroC genes encoding chorismate synthase from Salmonella typhi and Escherichia coli

The aroC genes from Salmonella typhi and Escherichia coli, encoding 5-enolpyruvylshikimate-3-phosphate phospholyase (chorismate synthase) were cloned in E. coli and their DNA sequences were determined. The aroC gene from S. typhi was isolated from a cosmid gene bank by complementation of an E. coli aroC mutant. The corresponding E. coli gene was isolated from a pBR322 gene bank by colony hybridization using DNA encoding the aroC gene from S. typhi as a hybridization probe. Analysis of the nucleotide sequence revealed that both genes have an open reading frame capable of encoding proteins comprising 361 amino acids. The calculated molecular mass of the protein from S. typhi is 39,108 Da while that of the protein from E. coli is 39,138 Da. Homology is particularly strong between the coding regions of the genes: 95% when protein sequences are compared, and 83% when DNA sequences are examined. Use of a deletion variant of the E. coli aroC gene demonstrates that the C-terminal 36 amino acids are not essential for the correct folding or functional activity of the chorismate synthase enzyme.     

The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Escherichia coli

Abstract The nucleotide sequence of the gene encoding the K99 fimbrial subunit of enterotoxigenic Escherichia coli was determined. It appeared that the subunit is composed of 159 amino acid residues preceded by a N-terminal signal sequence of 22 amino acid residues. The secondary structure of the mature K99 polypeptide and the location of potential antigenic determinants were predicted. A comparison was made between the amino acid sequence of the K99 subunit and the subunits of other fimbrial adhesins.     

Expression and regulation of protein K, an Escherichia coli K1 porin, in Escherichia coli K-12

Using a modified lambda phage as a vector and a procedure developed in Dr. C. Schnaitman's laboratory, we have cloned the structural gene for protein K from an Escherichia coli K1 strain to an E coli K-12 strain. The cloned inserts consist of two HindIII fragments, 4 kb and 6.5 kb in size. The protein produced by the insert is nearly identical to "authentic" protein K when chymotryptic peptides of 125I-labeled proteins are compared. Protein K was found to respond to changes in the osmolarity of the medium, being favored in trypticase soy broth (high osmolarity). This fluctuation was not dependent on a functional ompR gene. However, protein K was not expressed in strains carrying the envZ-473 mutation. Thus, protein K appears to be within a class of exported proteins whose expression is regulated by the envZ gene independent of the ompR gene.     

Overexpression, solubilization and refolding of a genetically engineered derivative of the penicillin-binding protein 3 of Escherichia coli K12

Replacement of the amino-terminal 40-amino-acid region of the 588-amino-acid precursor of the membrane-bound penicillin-binding protein 3 (PBP3) by the decapeptide MKGKEFQAWI was carried out by altering the amino-coding end of the ftsI gene. Insertion of the modified gene into a runaway-replication plasmid under the control of a fused lpp promoter and lac promoter/operator, resulted in the overexpression by Escherichia coli of the modified PBP3 (designated PBP3**) in the cytoplasm. About 80% of the accumulated PBP3** underwent sequestration in the form of insoluble protein granules that were isolated by cell breakage or cell lysis. After selective removal of contaminants by an EDTA-lysozyme/DNase (deoxyribonuclease)/Nonidet extraction, treatment of the granules with guanidinium chloride followed by dialysis against buffer containing 0.5 M NaCl yielded a refolded, water-soluble PBP3**, which, upon chromatography on Superose 12, exhibited the expected 60,000 molecular mass. The refolded PBP3** bound benzylpenicillin in a 1 to 1 molar ratio, was highly sensitive to aztreonam and showed the same degree of thermostability, in terms of penicillin-binding capacity, as the parent, membrane-bound PBP3, suggesting that protein refolding occurred with formation of the correct intramolecular interactions. Two to three mg of refolded PBP3** can be obtained from 1 litre of culture of the overproducing strain.     

Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.     

The complete nucleotide sequence of the glnALG operon of Escherichia coli K12.

The nucleotide sequence of the E. coli glnALG operon has been determined. The glnL (ntrB) and glnG (ntrC) genes present a high homology, at the nucleotide and aminoacid levels, with the corresponding genes of Klebsiella pneumoniae. The predicted aminoacid sequence for glutamine synthetase allowed us to locate some of the enzyme domains. The structure of this operon is discussed.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.