Deletions of the ponA and ponB genes of Escherichia coli have been constructed in vitro and recombined into the chromosome to produce strains that completely lack penicillin-binding protein 1A or penicillin-binding protein 1B. In each case a DNA fragment internal to the gene was replaced by a fragment encoding an antibiotic resistance. The ponA and ponB deletions can therefore be readily introduced into other E. coli strains by P1 transduction of the antibiotic resistance. Although the complete absence of penicillin-binding protein 1A or penicillin-binding protein 1B was tolerated, the absence of both of these proteins was shown to result in bacterial lysis.

Penicillin-binding proteins 1A and 1 B of Escherichia coli are the major peptidoglycan transglycosylasetranspeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1 A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1 B, was also determined. Penicillin-binding protein 1 B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1 A and 1 B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identitiy. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C p-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1 A and 1 B and the other penicillin-binding proteins and fi-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1 A, 1B and 3, within the conserved sequence Gly-SerXaa-Xaa-Lys-Pro. The other region was 19 -40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.fi-Lactam antibiotics kill Escherichia coli by inactivating a series of penicillin-binding proteins (PBPs) that are located in the cytoplasmic membrane and which catalyse the final stages of peptidoglycan synthesis [l, 21. In E. coli there are seven well characterised PBPs with relative molecular masses (Mr) between 40000 and 91 000 [3]. Genetic analysis of the role of each of the E. coli PBPs in the lethal effects of p-lactam antibiotics has shown that the lower-Mr PBPs 4, 5 and 6 are non-essential for bacterial growth and are therefore not of primary importance in the killing action of p-lactams [l, 4-71. The higher-M, PBPs 1A/lB, 2 and 3 have been shown to be essential enzymes and p-lactams exert their lethal effects by the inactivation of one or more of these killing targets [l, 81. Inactivation of PBP 3 results in the inhibition of cell division and the formation of long filamentous cells (EC 3.1.23.10), EcoRI (EC 3.1.23.13), EcoRV (EC 3.1.23.-)), H@I Subsequently PB...

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Abstract:This study was purposed to sequence analysis of ESBLs genotype of K pneumoniae using partial sequence and Multilocus sequence typing (MLST). A total of 275 K. pneumoniae isolatesinvolved three general hospitals in Duhok, Erbil, and Sulymania, from September 2010 to June 2011. The Minimum Inhibitory Concentration (MIC) was measured by Phoenix system that confirmed 187 ESBL producing isolates followed by the Double Disk Synergy Test (DDST). Then, 12 isolates were selectedaccording to sample diversity, high resistancy to β-lactam and cephalosporins and harboring combination of three genes (TEM, SHV, and CTX-M). Partial sequence analysis of TEM; showed two different genotypes regarding blaTEM as 9 isolates (75%) from different samples (wound, sputum and blood) from three provinces harbor TEM-1 gene and 3 isolates (25%) only from urine in three provinces harbor TEM-198 gene. SHV analysis revealed characterization of selected isolates into six different genotypes. The common genotype was blaSHV-11 involved five isolates from sputum and blood in Erbil and Sulymania provinces, and wound in Duhok province. Only one genotype as all 12 isolates (100%) from different samples and different provinces was found harbored CTX-M-15 gene. Multilocus sequence typing (MLST) study performed using seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB). A total of 8 different sequence types (STs) were identified;ST11 was dominant sequence type, accounting 41.6 %( 5 isolates) and was harboring combination of TEM-1, SHV-11 and CTX-15 genes.

Abstract:Background: