We have developed a FRET-based assay for the fingers-closing conformational transition that occurs when a binary complex of DNA polymerase I (Klenow fragment) with a primer-template binds a complementary dNTP and have used this and other fluorescence assays to place the fingers-closing step within the reaction pathway. Because the rate of fingers-closing was substantially faster than the rate of nucleotide incorporation measured in chemical quench experiments, fingers-closing cannot be the ratelimiting prechemistry step defined by earlier kinetic studies. Experiments using Ca 2+ instead of Mg 2+ as the metal cofactor suggest instead that the prechemistry step may involve a change in metal ion occupancy at the polymerase active site. The use of ribonucleotide substrates shows there is a base discriminating step that precedes fingers-closing. This earlier step, detected by 2-AP fluorescence, is promoted by complementary nucleotides (ribo-as well as deoxyribo-) but is blocked by mismatches. The complementary rNTP blocks the subsequent fingers-closing step. Thus, discrimination against rNTPs occurs during the transition from open to closed conformations, whereas selection against mismatched bases is initiated earlier in the pathway, in the open complex. Mismatched dNTPs accelerate DNA release from the polymerase, suggesting the existence of an early intermediate in which DNA binding is destabilized relative to the binary complex; this could correspond to a conformation that allows an incoming dNTP to preview the template base. The early kinetic checkpoints identified by this study provide an efficient mechanism for the rejection of mismatched bases and ribose sugars and thus enhance polymerase throughput.

DNA polymerases of the A and B families, and reverse transcriptases, share a common mechanism for preventing incorporation of ribonucleotides: a highly conserved active site residue obstructing the position that would be occupied by a 2' hydroxyl group on the incoming nucleotide. In the family Y (lesion bypass) polymerases, the enzyme active site is more open, with fewer contacts to the DNA and nucleotide substrates. Nevertheless, ribonucleotide discrimination by the DinB homolog (Dbh) DNA polymerase of Sulfolobus solfataricus is as stringent as in other polymerases. A highly conserved aromatic residue (Phe12 in Dbh) occupies a position analogous to the residues responsible for excluding ribonucleotides in other DNA polymerases. The F12A mutant of Dbh incorporates ribonucleoside triphosphates almost as efficiently as deoxyribonucleoside triphosphates, and, unlike analogous mutants in other polymerase families, shows no barrier to adding multiple ribonucleotides, suggesting that Dbh can readily accommodate a DNA-RNA duplex product. Like other members of the DinB group of bypass polymerases, Dbh makes single-base deletion errors at high frequency in particular sequence contexts. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis, indicating that the geometry of nucleotide binding is similar in both circumstances.

Testing P. aeruginosa efflux pump mutants showed that the LpxC inhibitor CHIR-090 is a substrate for MexAB-OprM, MexCDOprJ, and MexEF-OprN. Utilizing P. aeruginosa PAO1 with a chromosomal mexC::luxCDABE fusion, luminescent mutants arose on medium containing 4 g/ml CHIR-090, indicating upregulation of MexCD-OprJ. These mutants were less susceptible to CHIR-090 (MIC, 4 g/ml) and had mutations in the mexCD-oprJ repressor gene nfxB. Nonluminescent mutants (MIC, 4 g/ ml) that had mutations in the mexAB-oprM regulator gene mexR were also observed. Plating the clinical isolate K2153 on 4 g/ml CHIR-090 selected mutants with alterations in mexS (immediately upstream of mexT), which upregulates MexEF-OprN. A mutant altered in the putative1ribosomal binding site (RBS) upstream of lpxC and overexpressing LpxC was selected on a related LpxC inhibitor and exhibited reduced susceptibility to CHIR-090. Overexpression of LpxC from a plasmid reduced susceptibility to CHIR-090, and introduction of the altered RBS in this construct further increased expression of LpxC and decreased susceptibility to CHIR-090. Using a mutS (hypermutator) strain, a mutant with an altered lpxC target gene (LpxC L18V) was also selected. Purified LpxC L18V had activity similar to that of wild-type LpxC in an in vitro assay but had reduced inhibition by CHIR-090. Finally, an additional class of mutant, typified by an extreme growth defect, was identified. These mutants had mutations in fabG, indicating that alteration in fatty acid synthesis conferred resistance to LpxC inhibitors. Passaging experiments showed progressive decreases in susceptibility to CHIR-090. Therefore, P. aeruginosa can employ several strategies to reduce susceptibility to CHIR-090 in vitro.

Gram-negative outer membrane (OM) integrity is maintained in part by Mg2+ cross-links between phosphates on lipid A and on core sugars of adjacent lipopolysaccharide (LPS) molecules. In contrast to other Gram-negative bacteria, waaP, encoding an inner-core kinase, could not be inactivated in Pseudomonas aeruginosa. To examine this further, expression of the kinases WaaP or WapP/WapQ/PA5006 was placed under the control of the arabinose-regulated pBAD promoter. Growth of these strains was arabinose dependent, confirming that core phosphorylation is essential in P. aeruginosa. Transmission electron micrographs of kinase-depleted cells revealed marked invaginations of the inner membrane. SDS-PAGE of total LPS from WaaP-depleted cells showed accumulation of a fast-migrating band. Mass spectrometry (MS) analysis revealed that LPS from these cells exhibits a unique truncated core consisting of two 3-deoxy-d-manno-octulosonic acids (Kdo), two l-glycero-d-manno-heptoses (Hep), and one hexose but completely devoid of phosphates, indicating that phosphorylation by WaaP is necessary for subsequent core phosphorylations. MS analysis of lipid A from WaaP-depleted cells revealed extensive 4-amino-4-deoxy-l-arabinose modification. OM prepared from these cells by Sarkosyl extraction of total membranes or by sucrose density gradient centrifugation lacked truncated LPS. Instead, truncated LPS was detected in the inner membrane fractions, consistent with impaired transport/assembly of this species into the OM.

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.