The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts.

In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability, although mutants show growth impairment, genetic instability, and sensitivity to DNA damaging agents. In this study, we isolate spontaneous suppressor mutants in a ΔholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, or alleles of sspA, encoding stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (ß), and RpoC (ß') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC, and sspA An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC's absence.

Structure-Activity Relationship of Peptide-Conjugated Chloramphenicol for Inhibiting Escherichia coli.

Intravenous administration of a prodrug, chloramphenicol succinate (CLsu), is ineffective. Recently, we have shown that conjugation of diglycine of CLsu (CLsuGG) not only increases the antibiotic efficacy against Escherichia coli but also reduces adverse drug effects against bone marrow stromal cells. Here, we report the synthesis of structural analogues of CLsuGG and their activities against E. coli. These analogues reveal several trends: (i) except the water-insoluble analogues, the attachment of peptides to CLsu enhances the efficacy of the prodrugs; (ii) negative charges, high steric hindrance in the side chains, or a rigid diester decreases the activities of prodrugs in comparison to CLsuGG; (iii) dipeptides apparently increase the efficacy of the prodrugs most effectively; and so forth. This work suggests that conjugating peptides to CLsu effectively modulates the properties of prodrugs. The structure-activity relationship of these new conjugates may provide useful insights for expanding the pool of antibiotics.

Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram-negative Bacteria.

Antimicrobial drug resistance demands novel approaches for improving the efficacy of antibiotics, especially against Gram-negative bacteria. Herein, we report that conjugating a diglycine (GG) to an antibiotic prodrug drastically accelerates intrabacterial ester-bond hydrolysis required for activating the antibiotic. Specifically, the attachment of GG to chloramphenicol succinate (CLsu) generates CLsuGG, which exhibits about an order of magnitude higher inhibitory efficacy than CLsu against Escherichia coli. Further studies reveal that CLsuGG undergoes rapid hydrolysis, catalyzed by intrabacterial esterases (e.g., BioH and YjfP), to generate chloramphenicol (CL) in E. coli. Importantly, the conjugate exhibits lower cytotoxicity to bone marrow stromal cells than CL. Structural analogues of CLsuGG indicate that the conjugation of GG to an antibiotic prodrug is an effective strategy for accelerating enzymatic prodrug hydrolysis and enhancing the antibacterial efficacy of antibiotics.

A new approach to understanding structure-function relationships in cytochromes P450 by targeting terpene metabolism in the wild.

A strategy for elucidating sequence determinants of function in the class of cytochrome P450 (CYP) enzymes that catalyze the first steps of terpene metabolism in wild microbiomes is described. Wild organisms that can use camphor, terpineol, pinene and limonene were isolated from soils rich in coniferous waste. Cell free extracts and growth beers were analyzed by gas chromatography/mass spectrometry to identify primary oxidative metabolites. For one organism, Pseudomonas nitroreducens TPJM, a cytochrome P450 (CYP108B1) isolated from cell free extracts was demonstrated to catalyze the oxidation of α-terpineol in assays combining the native ferredoxin and putidaredoxin reductase, and the resulting oxidation products identified by gas chromatography/mass spectrometry. Shotgun sequencing of PnTPJM identified four candidate P450 genes, including an apparently fragmentary gene with a high degree of homology with the known enzyme CYP108 (P450terp).

Recombinational branch migration by the RadA/Sms paralog of RecA in Escherichia coli.

RadA (also known as 'Sms') is a highly conserved protein, found in almost all eubacteria and plants, with sequence similarity to the RecA strand exchange protein and a role in homologous recombination. We investigate here the biochemical properties of the E. coli RadA protein and several mutant forms. RadA is a DNA-dependent ATPase, a DNA-binding protein and can stimulate the branch migration phase of RecA-mediated strand transfer reactions. RadA cannot mediate synaptic pairing between homologous DNA molecules but can drive branch migration to extend the region of heteroduplex DNA, even without RecA. Unlike other branch migration factors RecG and RuvAB, RadA stimulates branch migration within the context of the RecA filament, in the direction of RecA-mediated strand exchange. We propose that RadA-mediated branch migration aids recombination by allowing the 3' invading strand to be incorporated into heteroduplex DNA and to be extended by DNA polymerases.

Genetic analysis of Escherichia coli†RadA: functional motifs and genetic interactions.

The RadA/Sms protein is a RecA-related protein found universally in eubacteria and plants, implicated in processing of recombination intermediates. Here we show that the putative Zn finger, Walker A motif, KNRXG motif and Lon protease homology domain of the Escherichia coli RadA protein are required for DNA damage survival. RadA is unlikely to possess protease activity as the putative active site serine is not required. Mutants in RadA have strong synergistic phenotypes with those in the branch migration protein RecG. Sensitivity of radA recG mutants to azidothymidine (AZT) can be rescued by blocking recombination with recA or recF mutations or by overexpression of RuvAB, suggesting that lethal recombination intermediates accumulate in the absence of RadA and RecG. Synthetic genetic interactions for survival to AZT or ciprofloxacin exposure were observed between RadA and known or putative helicases including DinG, Lhr, PriA, Rep, RuvAB, UvrD, YejH and YoaA. These represent the first affected phenotypes reported for Lhr, YejH and YoaA. The specificity of these effects sheds new light on the role of these proteins in DNA damage avoidance and repair and implicates a role in replication gap processing for DinG and YoaA and a role in double-strand break repair for YejH.

Toxicity and tolerance mechanisms for azidothymidine, a replication gap-promoting agent, in Escherichia coli.

Azidothymidine (AZT, zidovudine) is used to treat HIV-AIDS and prevent maternal transmission to newborns. Because the azido group replaces the 3' OH of thymidine, AZT is believed to act as a chain terminator during reverse transcription of viral RNA into DNA, although other mechanisms of viral inhibition have been suggested. There is evidence that AZT is genotoxic, particularly to the mitochondria. In this study, we use the bacterium Escherichia coli to investigate the mechanism of AZT toxicity and the cellular mechanisms that aid survival. We show that that replication arrests quickly after treatment, accompanied by induction of the SOS DNA damage response. AZT appears to produce single-strand DNA gaps, as evident by RecF-dependent induction of the SOS response and visualization of single-strand DNA binding protein foci within the cell. Some of these gaps must be converted to breaks, since mutants in the RecBCD nuclease, required for recombinational double-strand break repair, are highly sensitive to AZT. Blocks in the late recombination functions, the RuvAB branch migration helicase and RuvC Holliday junction endonuclease, caused extreme AZT sensitivity that could be relieved by mutations in the early recombination functions, such as RecF, suggesting gaps engage in recombination reactions. Finally, our data suggest that the proofreading exonucleases of DNA polymerases play little role in AZT tolerance. Rather, Exonuclease III appears to be the enzyme that removes AZT: xthA mutants are highly AZT-sensitive, with a sustained SOS response, and overproduction of the enzyme protects wild-type cells. Our findings suggest that incorporation of AZT into human nuclear and mitochondrial DNA has the potential to promote genetic instability and toxicity through the production of ssDNA gaps and dsDNA breaks, and predicts that the human Exonuclease III ortholog, APE1, will be important for drug tolerance.

The ObgE/CgtA GTPase influences the stringent response to amino acid starvation in Escherichia coli.

The stringent response is important for bacterial survival under stressful conditions, such as amino acid starvation, and is characterized by the accumulation of ppGpp and pppGpp. ObgE (CgtA, YhbZ) is an essential conserved GTPase in Escherichia coli and several observations have implicated the protein in the control of the stringent response. However, consequences of the protein on specific responses to amino acid starvation have not been noted. We show that ObgE binds to ppGpp with biologically relevant affinity in vitro, implicating ppGpp as an in vivo ligand of ObgE. ObgE mutants increase the ratio of pppGpp to ppGpp within the cell during the stringent response. These changes are correlated with a delayed inhibition of DNA replication by the stringent response, delayed resumption of DNA replication after release, as well as a decreased survival after amino acid deprivation. With these data, we place ObgE as an active effector of the response to amino acid starvation in vivo. Our data correlate the pppGpp/ppGpp ratio with DNA replication control under bacterial starvation conditions, suggesting a possible role for the relative balance of these two nucleotides.

Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.

We describe a method for synchronization of the cell cycle in the bacterium Escherichia coli. Treatment of asynchronous cultures with the amino acid analog, dl-serine hydroxamate, induces the stringent response, with concomitant arrest of DNA replication at initiation. Following release of the stringent response, cells initiate DNA replication in synchrony, as determined by flow cytometry for DNA content, Southern blotting and microscopy. This method has the advantage that it can be used in fully wild-type cells, at different growth rates, and may be applicable to other bacterial species with replication control by the stringent response. We also elaborate other methods useful for establishing cell cycle parameters in bacterial populations. We describe flow cytometric methods for analyzing bacterial populations for DNA content using the DNA-specific dye PicoGreen, readily detected by most commercial flow cytometers. We also present an method for incorporation of the nucleotide ethynyl-deoxyuridine, EdU, followed by "click" labeling with fluorescent dyes, which allows us to measure and visualize newly replicated DNA in fixed E. coli K-12 cells under non-denaturing conditions.

RecJ exonuclease: substrates, products and interaction with SSB.

The RecJ exonuclease from Escherichia coli degrades single-stranded DNA (ssDNA) in the 5'-3' direction and participates in homologous recombination and mismatch repair. The experiments described here address RecJ's substrate requirements and reaction products. RecJ complexes on a variety of 5' single-strand tailed substrates were analyzed by electrophoretic mobility shift in the absence of Mg2+ ion required for substrate degradation. RecJ required single-stranded tails of 7 nt or greater for robust binding; addition of Mg2+ confirmed that substrates with 5' tails of 6 nt or less were poor substrates for RecJ exonuclease. RecJ is a processive exonuclease, degrading approximately 1000 nt after a single binding event to single-strand DNA, and releases mononucleotide products. RecJ is capable of degrading a single-stranded tail up to a double-stranded junction, although products in such reactions were heterogeneous and RecJ showed a limited ability to penetrate the duplex region. RecJ exonuclease was equally potent on 5' phosphorylated and unphosphorylated ends. Finally, DNA binding and nuclease activity of RecJ was specifically enhanced by the pre-addition of ssDNA-binding protein and we propose that this specific interaction may aid recruitment of RecJ.