Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme From Thermus Thermophilus

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX ( and γ), and components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions

Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s−1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s−1µM−1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC50 values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC50 of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition

Identification and Characterization of a Chemical Compound that Inhibits Methionyl-tRNA Synthetase from Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is an opportunistic pathogen problematic in causing nosocomial infections and is highly susceptible to development of resistance to multiple antibiotics. The gene encoding methionyl-tRNA synthetase (MetRS) from P. aeruginosa was cloned and the resulting protein characterized. Methods: MetRS was kinetically evaluated and the KM for its three substrates, methionine, ATP and tRNAMet were determined to be 35, 515, and 29 μM, respectively. P. aeruginosaMetRS was used to screen two chemical compound libraries containing 1690 individual compounds. Results: A natural product compound (BM01C11) was identified that inhibited the aminoacylation function. The compound inhibited P. aeruginosa MetRS with an IC50 of 70 μM. The minimum inhibitory concentration (MIC) of BM01C11 was determined against nine clinically relevant bacterial strains, including efflux pump mutants and hypersensitive strains of P. aeruginosa and E. coli. The MIC against the hypersensitive strain of P. aeruginosa was 16 μg/ml. However, the compound was not effective against the wild-type and efflux pump mutant strains, indicating that efflux may not be responsible for the lack of activity against the wild-type strains. When tested in human cell cultures, the cytotoxicity concentration (CC50) was observed to be 30 μg/ml. The compound did not compete with methionine or ATP for binding MetRS, indicating that the mechanism of action of the compound likely occurs outside the active site of aminoacylation. Conclusion: An inhibitor of P. aeruginosa MetRS, BM01C11, was identified as a flavonoid compound named isopomiferin. Isopomiferin inhibited the enzymatic activity of MetRS and displayed broad spectrum antibacterial activity. These studies indicate that isopomiferin may be amenable to development as a therapeutic for bacterial infections

Interaction of Mitochondrial Elongation Factor Tu With Aminoacyl-tRNA and Elongation Factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu·GTP·aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu·GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu·Ts). Mammalian mitochondrial EF-Tu (EF-Tumt) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tumt subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K tRNA) and for the dissociation of the EF-Tu·Tsmt complex (K Ts) were investigated. The equilibrium dissociation constant for the ternary complex was 18 ± 4 nM, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 × 10− 4 s− 1, which is essentially equivalent to that observed for the ternary complex inEscherichia coli. The binding of EF-Tumt to EF-Tsmt is mutually exclusive with the formation of the ternary complex. K Ts was determined by quantifying the effects of increasing concentrations of EF-Tsmt on the amount of ternary complex formed with EF-Tumt. The value obtained for K Ts(5.5 ± 1.3 nM) is comparable to the value ofK tRNA

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes (tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s–1 and 3.1, 3.8, and 1.9 s–1, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition

Reconstitution of a Minimal DNA Replicase From Pseudomonas Aeruginosa and Stimulation by Non-Cognate Auxiliary Factors

DNA polymerase III holoenzyme is responsible for chromosomal replication in bacteria. The components and functions of Escherichia coli DNA polymerase III holoenzyme have been studied extensively. Here, we report the reconstitution of replicase activity by essential components of DNA polymerase holoenzyme from the pathogen Pseudomonas aeruginosa. We have expressed and purified the processivity factor (β), single-stranded DNA-binding protein, a complex containing the polymerase (α) and exonuclease (ϵ) subunits, and the essential components of the DnaX complex (τ3δδ′). Efficient primer elongation requires the presence of αϵ, β, and τ3δδ′. Pseudomonas aeruginosa αϵ can substitute completely for E. coli polymerase III in E. coli holoenzyme reconstitution assays. Pseudomonas β and τ3δδ′ exhibit a 10-fold lower activity relative to their E. coli counterparts in E. coli holoenzyme reconstitution assays. Although the Pseudomonas counterpart to the E. coli ψ subunit was not apparent in sequence similarity searches, addition of purified E. coli χ and ψ (components of the DnaX complex) increases the apparent specific activity of the Pseudomonas τ3δδ′ complex ∼10-fold and enables the reconstituted enzyme to function better under physiological salt conditions

Identification of Chemical Compounds That Inhibit the Function of Histidyl-tRNA Synthetase from Pseudomonas aeruginosa

Pseudomonas aeruginosa histidyl-tRNA synthetase (HisRS) was selected as a target for antibiotic drug development. The HisRS protein was overexpressed in Escherichia coli and kinetically evaluated. The KM values for interaction of HisRS with its three substrates, histidine, ATP, and tRNAHis, were 37.6, 298.5, and 1.5 μM, while the turnover numbers were 8.32, 16.8, and 0.57 s-1, respectively. A robust screening assay was developed, and 800 natural products and 890 synthetic compounds were screened for inhibition of activity. Fifteen compounds with inhibitory activity were identified, and the minimum inhibitory concentration (MIC) was determined for each against a panel of nine pathogenic bacteria. Each compound exhibited broad-spectrum activity. Based on structural similarity and MIC results, four compounds, BT02C02, BT02D04, BT08E04, and BT09C11, were selected for additional analysis. These compounds inhibited the activity of HisRS with IC50 values of 4.4, 9.7, 14.1, and 11.3 µM, respectively. Time-kill studies indicated a bacteriostatic mode of inhibition for each compound. BT02D04 and BT08E04 were noncompetitive with both histidine and ATP, BT02C02 was competitive with histidine but noncompetitive with ATP, and BT09C11 was uncompetitive with histidine and noncompetitive with ATP. These compounds were not observed to be toxic to human cell cultures

Two Homologous EF-G Proteins From Pseudomonas Aeruginosa Exhibit Distinct Functions

Genes encoding two proteins corresponding to elongation factor G (EF-G) were cloned from Pseudomonas aeruginosa. The proteins encoded by these genes are both members of the EFG I subfamily. The gene encoding one of the forms of EF-G is located in the str operon and the resulting protein is referred to as EF-G1A while the gene encoding the other form of EF-G is located in another part of the genome and the resulting protein is referred to as EF-G1B. These proteins were expressed and purified to 98% homogeneity. Sequence analysis indicated the two proteins are 90/84% similar/identical. In other organisms containing multiple forms of EF-G a lower degree of similarity is seen. When assayed in a poly(U)-directed poly-phenylalanine translation system, EF-G1B was 75-fold more active than EF-G1A. EF-G1A pre-incubate with ribosomes in the presence of the ribosome recycling factor (RRF) decreased polymerization of poly-phenylalanine upon addition of EF-G1B in poly(U)-directed translation suggesting a role for EF-G1A in uncoupling of the ribosome into its constituent subunits. Both forms of P. aeruginosa EF-G were active in ribosome dependent GTPase activity. The kinetic parameters (KM) for the interaction of EF-G1A and EF-G1B with GTP were 85 and 70 μM, respectively. However, EF-G1B exhibited a 5-fold greater turnover number (observed kcat) for the hydrolysis of GTP than EF-G1A; 0.2 s-1 vs. 0.04 s-1. These values resulted in specificity constants (kcatobs/KM) for EF-G1A and EF-G1B of 0.5 x 103 s-1 M-1 and 3.0 x 103 s-1 M-1, respectively. The antibiotic fusidic acid (FA) completely inhibited poly(U)-dependent protein synthesis containing P. aeruginosa EF-G1B, but the same protein synthesis system containing EF-G1A was not affected. Likewise, the activity of EF-G1B in ribosome dependent GTPase assays was completely inhibited by FA, while the activity of EF-G1A was not affected