Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic b-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared b-lactam sensitivity of the mutants and the parent strain. b-Lactam resistance was complemented by wild-type, but not DD-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for b-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter b-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored b-lactam resistance of the dacA mutant. Therefore, PBP5DD-carboxypeptidase activity is essential for intrinsic b-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot. INTRODUCTIONEscherichia coli encodes 12 penicillin-binding proteins (PBPs), four of which (PBP4, -5, -6 and DacD) have been reported to have DD-carboxypeptidase (DD-CPase) activity (Höltje, 1998; Denome et al., 1999;Ghosh et al., 2008). All these proteins are low molecular mass (LMM) PBPs (Ghuysen, 1991) and are dispensable for survival in vitro (Denome et al., 1999). PBP5 and PBP6 are 62 % identical at the amino acid level and share 48 and 47 % identity with DacD, respectively (Baquero et al., 1996). It has been suggested that these proteins might have similar physiological functions based upon their homology but only PBP5 appears to play a prominent role in maintenance of cell shape (Nelson & Young, 2001;Nelson et al., 2002;Ghosh & Young, 2003). PBP5, PBP6 and DacD are primarily expressed in early exponential, stationary and midexponential phases, respectively (Buchanan & Sowell, 1982;Baquero et al., 1996;Santos et al., 2002), which may explain the different roles of these proteins in maintenance of cell shape. The number of PBP5 molecules is also two-to threefold higher than the number of PBP6 molecules in exponentially growing cells (Spratt, 1977;Dougherty et al., 1996). METHODSBacterial strains and antibiotics. Bacterial strains used in this study were derived from E. coli 2443 and are listed in Table 1. CS18-2K was a gift from Professor Kevin D. Young, University of Arkansas Medical School, AR, USA. The strains were grown in Luria-Bertani (LB) broth, agar (Hi-Media), Muller-Hinton (MH) broth (Hi-Media) and M9-Glucose minimal medium, supplemented with the required amino acids (arginine, proline, leucine and threonine) and thiamine. Chloramphenicol (20 mg ml -1 ), kanamycin (50 mg ml -1 ), tetracycline (25 mg ml -1 ) and ampicillin (50 mg ml -1 ) were added where necessary. Unless otherwise specified, chemicals and reagents were purchased from Sigma. (Table 1). Double and triple mutants were constructed from parents from which the res-npt-res cassette...

Of the five dd-carboxypeptidases in Escherichia coli, only PBP5 demonstrates its physiological significance by maintaining cell shape and intrinsic beta-lactam resistance. DacD can partially compensate for the lost beta-lactam resistance in PBP5 mutant, although its biochemical reason is unclear. To understand the mechanism(s) underlying such behaviour, we constructed soluble DacD (sDacD) and compared its biophysical and biochemical properties with those of sPBP5, in vitro. Unlike sPBP6, sDacD can deacylate Bocillin significantly, which is very similar to sPBP5. sDacD shows weak dd-carboxypeptidase activity, although lower than that of sPBP5. Bioinformatics analyses reveal a similar architecture of sPBP5 and sDacD. Therefore, based on the obtained results we can infer that biochemically DacD and PBP5 are more closely related to each other than to PBP6, enabling DacD and PBP5 to play a nearly similar physiological function in terms of recovering the lost beta-lactam resistance.

DD-Carboxypeptidases (DD-CPases) are low-molecular-mass (LMM) penicillin-binding proteins (PBPs) that are mainly involved in peptidoglycan remodelling, but little is known about the DD-CPases of mycobacteria. In this study, a putative DD-CPase of Mycobacterium smegmatis, MSMEG_2433 is characterized. The gene for the membrane-bound form of MSMEG_2433 was cloned and expressed in Escherichia coli in its active form, as revealed by its ability to bind to the Bocillin-FL (fluorescent penicillin). Interestingly, in vivo expression of MSMEG_2433 could restore the cell shape oddities of the septuple PBP mutant of E. coli, which was a prominent physiological characteristic of DD-CPases. Moreover, expression of MSMEG_2433 in trans elevated beta-lactam resistance in PBP deletion mutants (DdacAdacC) of E. coli, strengthening its physiology as a DD-CPase. To confirm the biochemical reason behind such physiological behaviours, a soluble form of MSMEG_2433 (sMSMEG_2433) was created, expressed and purified. In agreement with the observed physiological phenomena, sMSMEG_2433 exhibited DD-CPase activity against artificial and peptidoglycan-mimetic DD-CPase substrates. To our surprise, enzymic analyses of MSMEG_2433 revealed efficient deacylation for beta-lactam substrates at physiological pH, which is a unique characteristic of beta-lactamases. In addition to the MSMEG_2433 active site that favours DD-CPase activity, in silico analyses also predicted the presence of an omega-loop-like region in MSMEG_2433, which is an important determinant of its beta-lactamase activity. Based on the in vitro, in vivo and in silico studies, we conclude that MSMEG_2433 is a dual enzyme, possessing both DD-CPase and beta-lactamase activities.

The combination of antibiotics is one of the strategies to combat drug-resistant bacteria, though only a handful of such combinations are in use, such as the β-lactam combinations. In the present study, the efficacy of a specific sub-inhibitory concentration of cefsulodin with other β-lactams was evaluated against a range of Gram-negative clinical isolates. This approach increased the sensitivity of the isolates, regardless of the β-lactamase production. The preferred target and mechanism of action of cefsulodin were identified in laboratory strains of Escherichia coli, by examining the effects of deleting the penicillin-binding protein (PBP) 1a and 1b encoding genes individually. Deletion of PBP1b was involved in sensitizing the bacteria to β-lactam agents, irrespective of its O-antigen status. Moreover, the use of a sub-inhibitory concentration of cefsulodin in combination with a β-lactam exerted an effect similar to that one obtained for PBP1b gene deletion. We conclude that the identified β-lactam/cefsulodin combination works by inhibiting PBP1b (at least partially) despite the involvement of β-lactamases, and therefore could be extended to a broad range of Gram-negative pathogens.

A single amino acid substitution in the V-like loop of E. coli PBP5 disrupts its ability to maintain cell shape and intrinsic beta-lactam resistance PBP5 has an 'V-loop'-like region similar to that in class A beta-lactamases. It was previously predicted that Leu182 present in the 'V-like' loop of PBP5 corresponds to Glu166 in PER-1 beta-lactamase. Here, we studied the physiological and biochemical effects of the Leu182Glu mutation in PBP5. Upon overexpression in septuple PBP mutants,~75 % of cells were abnormally shaped and intrinsic beta-lactam resistance maintenance was partially lost. Biochemically, the purified soluble mutated PBP5 (smPBP5) retained low acylation ability for penicillin. The turnover number of smPBP5 for artificial and peptidoglycan mimetic substrates was~10-fold less than that of the wild-type. Superimposition of the active-site residues of smPBP5 on PBP5 revealed that perturbation in the orientating key residues may explain the low turnover numbers. Therefore, we establish the involvement of Leu182 in maintaining the physiological and biochemical behaviour of E. coli PBP5. INTRODUCTIONOne of the main bacterial cell-wall components is the peptidoglycan layer, which is indispensable for cellular viability and maintenance of cellular morphology. Penicillin-binding proteins (PBPs) are enzymes required for the cross-linking of the glycan chain (transglycosylase) and peptide chain (transpeptidase). Most of these proteins have three signature motifs, namely SXXK, SXN and KTG, and these motifs are responsible for their respective enzymic activities (Ghuysen, 1991). In Escherichia coli, five PBPs (PBP4, 5, 6, DacD and AmpH) are documented as DD-carboxypeptidase (DDCPase) (Korat et al., 1991;Baquero et al., 1996; Höltje, 1998;Nelson & Young 2000, 2001 González-Leiza et al., 2011). These PBPs are thought to prevent the unwanted cross-link formation by removing the terminal D-alanine residue from the pentapeptide side-chain of N-acetyl muramic acid (Höltje, 1998;Ghosh et al., 2008). PBP5 is the key DD-CPase that helps maintain a uniform cell shape in E. coli (Nelson & Young 2000, 2001Ghosh et al., 2008). E. coli mutants lacking at least three DD-CPases including PBP5 show aberrant morphology. However, the abnormalities are abolished upon ectopic expression of PBP5 (Nelson & Young, 2000;Ghosh & Young, 2003). The ability of PBP5 to repair the cellular abnormalities depends on a stretch of 20 aa (200-219) around the KTG motif of PBP5, known as the 'morphology maintenance domain ' (Ghosh & Young, 2003). Apart from maintaining cell shape, PBP5 also plays an important role in maintaining the intrinsic beta-lactam resistance in E. coli (Sarkar et al., 2010). The absence of PBP5 sensitizes the E. coli cell to beta-lactam antibiotics, while in trans expression of the same helps to reverse the lost resistance (Sarkar et al., 2010). The high copy number of PBP5 at the exponential phase of growth may be responsible for trapping the beta-lactam antibiotics, which prevents them from binding to the essential PBPs (...