Electrophoretic mobility of supercoiled,catenated and knotted DNA molecules

We systematically varied conditions of two-dimensional (2D) agarose gel electrophoresis to optimize separation of DNA topoisomers that differ either by the extent of knotting, the extent of catenation or the extent of supercoiling. To this aim we compared electrophoretic behavior of three different families of DNA topoisomers: (i) supercoiled DNA molecules, where supercoiling covered the range extending from covalently closed relaxed up to naturally supercoiled DNA molecules; (ii) postreplicative catenanes with catenation number increasing from 1 to ∼15, where both catenated rings were nicked; (iii) knotted but nicked DNA molecules with a naturally arising spectrum of knots. For better comparison, we studied topoisomer families where each member had the same total molecular mass. For knotted and supercoiled molecules, we analyzed dimeric plasmids whereas catenanes were composed of monomeric forms of the same plasmid. We observed that catenated, knotted and supercoiled families of topoisomers showed different reactions to changes of agarose concentration and voltage during electrophoresis. These differences permitted us to optimize conditions for their separation and shed light on physical characteristics of these different types of DNA topoisomers during electrophoresis.     

Separation of topological forms of plasmid DNA by anion-exchange HPLC: shifts in elution order of linear DNA

We sought to establish a single anion-exchange HPLC method for the separation of linear, open circular and supercoiled plasmid topoisomers using purified topoisomeric forms of three plasmids (3.0, 5.5 and 7.6 kb). However, finding one condition proved elusive as the topoisomer elution order was determined to depend on salt gradient slope. The observed change in selectivity increased with plasmid size and was most pronounced for the linear form. Indeed, the elution order of the linear 7.6 kb plasmid was reversed relative to the supercoiled form. This observation may have implications for methods used in quality control of plasmid DNA.     

Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae?

The effects of exogenous reducing agents on a number of biological properties of purified Chlamydia trachomatis LGV-434 and Chlamydia psittaci meningopneumonitis elementary bodies (EBs) have been examined in an attempt to identify in vitro correlates of early events in the differentiation of the infectious EB to the replicative cell type, the reticulate body (RB). Treatment of EBs with dithiothreitol elicited a number of changes normally associated with differentiation to the RB. EBs in the presence of 10 mM dithiothreitol displayed enhanced rates of [14C]glutamate oxidation, reduced infectivity, and decreased osmotic stability, and their Machiavello staining properties changed to those characteristic of the RB. A true differentiation of EB to RB did not take place under these conditions, since EBs treated in this manner and examined by transmission electron microscopy did not demonstrate increased size or decreased electron density as do isolated RBs. Additional studies were initiated to identify the macromolecules involved in this process. With polyacrylamide gel electrophoresis and immunoblotting procedures with monoclonal and polyclonal monospecific antibodies, the chlamydial major outer membrane protein was found to be the predominant component that varied under reducing versus nonreducing conditions. Furthermore, the extent of disulfide-mediated cross-linking of the major outer membrane protein varied between the infective and replicative forms of the C. trachomatis LGV-434 life cycle. Implications of disulfide interactions in the life cycle of chlamydiae are discussed.     

Analysis of laser-induced plasmid DNA photolysis by capillary electrophoresis

Capillary electrophoresis (CE) was used to monitor the laser-induced conversion of supercoiled pKOL8UV5 plasmid DNA into nicked conformers. The plasmid samples (0.1 mg/ml) were incubated in the absence or presence of 110 μmol/l ethidium bromide (EB) and then exposed to 110 J of argon laser radiation (488 nm). The nicked, open circular conformers were separated from the supercoiled DNA by a 15% increase in retention time. Approximately 90% of the control DNA was in the supercoiled form. Laser radiation in the presence of EB caused complete conversion of the supercoiled plasmid DNA into nicked conformers. Laser-induced fluorescence CE (LIF-CE) was about 100-fold more sensitive than UV-CE in the detection of these conformers. Agarose gel electrophoresis confirmed these findings and showed the presence of the nicked plasmid conformers. Based on these comparisons, CE is an efficient analytical tool for the identification of laser-induced conformational changes in plasmid DNA.     

Interactions of Drosophila DNA topoisomerase II with left-handed Z-DNA in supercoiled minicircles.

The native form of Drosophila melanogaster DNA topoisomerase II was purified from Schneider's S3 tissue culture cells and studied with two supercoiled minicircle preparations, mini and mini-CG, 354 bp and 370 bp in length, respectively. Mini-CG contains a d(CG)7 insert which assumes a left-handed Z-DNA conformation in negative supercoiled topoisomers with a negative linking number difference - delta Lk greater than or equal to 2. The interactions of topoisomerase II with topoisomer families of mini and mini-CG were studied by band-shift gel electrophoresis in which the individual topoisomers and their discrete or aggregated protein complexes were resolved. A monoclonal anti-Z-DNA IgG antibody (23B6) bound and aggregated only mini-CG, thereby confirming the presence of Z-DNA. Topoisomerase II bound and relaxed mini-CG more readily than mini. In both cases, there was a preference for more highly negatively supercoiled topoisomers. The topoisomerase II inhibitor VM-26 induced the formation of stable covalent DNA-protein intermediates. In addition, the non-hydrolyzable GTP analogue GTP gamma S inhibited the binding and relaxation activities. Experiments to detect topoisomerase cleavage sites failed to elicit specific loci on either minicircle preparation. We conclude that Drosophila topoisomerase II is able to bind and process small minicircles with lengths as short as 360 bp and negative superhelix densities, - sigma, which can exceed 0.1. Furthermore, the enzyme has a preferential affinity for topoisomers containing Z-DNA segments and relaxes these molecules, presumably by cleavage external to the inserts. Thus, a potentially functional relationship between topoisomerase II, an enzyme regulating the topological state of DNA-chromatin in vivo, and left-handed Z-DNA, a conformation stabilized by negative supercoiling, has been established.     

Penicillin Induced Persistence in Chlamydia trachomatis: High Quality Time Lapse Video Analysis of the Developmental Cycle

Background

Chlamydia trachomatis is a major human pathogen with a unique obligate intracellular developmental cycle that takes place inside a modified cytoplasmic structure known as an inclusion. Following entry into a cell, the infectious elementary body (EB) differentiates into a non - infectious replicative form known as a reticulate body (RB). RBs divide by binary fission and at the end of the cycle they redifferentiate into EBs. Treatment of C.trachomatis with penicillin prevents maturation of RBs which survive and enlarge to become aberrant RBs within the inclusion in a non - infective persistent state. Persistently infected individuals may be a reservoir for chlamydial infection. The C.trachomatis genome encodes the enzymes for peptidoglycan (PG) biosynthesis but a PG sacculus has never been detected. This coupled to the action of penicillin is known as the chlamydial anomaly. We have applied video microscopy and quantitative DNA assays to the chlamydial developmental cycle to assess the effects of penicillin treatment and establish a framework for investigating penicillin induced chlamydial persistence.

Principal Findings

Addition of penicillin at the time of cell infection does not prevent uptake and the establishment of an inclusion. EB to RB transition occurs but bacterial cytokinesis is arrested by the second binary fission. RBs continue to enlarge but not divide in the presence of penicillin. The normal developmental cycle can be recovered by the removal of penicillin although the large, aberrant RBs do not revert to the normal smaller size but remain present to the completion of the developmental cycle. Chromosomal and plasmid DNA replication is unaffected by the addition of penicillin but the arrest of bacterial cytokinesis under these conditions results in RBs accumulating multiple copies of the genome.

Conclusions

We have applied video time lapse microscopy to the study of the chlamydial developmental cycle. Linked with accurate measures of genome replication this provides a defined framework to analyse the developmental cycle and to investigate and provide new insights into the effects of antibiotic treatments. Removal of penicillin allows recovery of the normal developmental cycle by 10–20 hrs and the process occurs by budding from aberrant RBs.     

Early melting of supercoiled DNA.

Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.     

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.     

Quantitative proteomics reveals metabolic and pathogenic properties of Chlamydia trachomatis developmental forms

Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography - tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyse the membranes of the pathogen-containing vacuole ('inclusion'). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of > 54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient type III secretion (T3S) in the RB stage at 18 h post infection, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a 'per organism' basis. Our results show that EB and RB proteomes are streamlined to fulfil their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.     

Early melting of supercoiled DNA topoisomers observed by TGGE

We have used temperature gradient gel electrophoresis (TGGE) to measure the progress of local denaturation in closed circular topoisomer DNA as a function of temperature and superhelicity (σ). We describe the versatility of this method as a tool for detecting various conformational modifications of plasmid DNAs. The early melting temperature of a structural transition for any topoisomer is dependent on the value of superhelicity. Supercoiled topoisomers represent a system of molecules that is sensitive to changes in temperature. We show that the topoisomer with the highest absolute value of superhelicity melts earlier than topoisomers with lower values. Thermal sensitivity of highly supercoiled plasmids could play a biologically important role in regulation of replication and expression in cells under thermal stress. The estimated melting temperature for plasmids with σ < –0.05 is very significant because these temperatures for early melting are below physiological temperatures.     

Detection of surface-exposed epitopes on Chlamydia trachomatis by immune electron microscopy

The cell surfaces of two Chlamydia trachomatis serovars were explored by immune electron microscopy with monoclonal antibodies that recognize a number of chlamydial outer-membrane components. Species, subspecies and serovar-reactive epitopes on the major outer-membrane protein (MOMP) of a lymphogranuloma venereum biovar strain, L2/434/Bu, and a trachoma biovar strain, F/UW-6/Cx, were exposed on the surfaces of both elementary bodies (EBs) and reticulate bodies (RBs). Three epitopes on MOMP were inaccessible on EBs and RBs of both strains. These included a genus-reactive, species-reactive, and a subspecies-reactive epitope. In contrast, genus-specific epitopes on lipopolysaccharide (LPS) were not detected on the EB surface, but were clearly expressed on RBs of both L2/434/Bu and F/UW-6/Cx chlamydiae. Antibodies specific for the 60 kDa and 12 kDa 'cysteine-rich' outer-membrane proteins did not react with surface epitopes on either EBs or RBs. These data provide evidence that MOMP is a major surface antigen of both morphological forms, whereas some portions of the LPS molecule are exposed on the RB surface but become inaccessible to antibody after conversion to the infectious EB form.     

Disulfide bonding within components of the Chlamydia type III secretion apparatus correlates with development

Chlamydia spp. exhibit a unique biphasic developmental cycle whereby infectious elementary bodies (EBs) invade host epithelial cells and differentiate into noninfectious, metabolically active reticulate bodies (RBs). EBs posses a unique outer envelope where rigidity is achieved by disulfide bonding among cysteine-rich envelope-associated proteins. Conversely, these disulfide bonds become reduced in RBs to accommodate vegetative growth, thereby linking the redox status of cysteine-rich envelope proteins with progression of the developmental cycle. We investigated the potential role of disulfide bonding within the chlamydial type III secretion system (T3SS), since activity of this system is also closely linked to development. We focused on structural components of the T3S apparatus that contain an unusually high number of cysteine residues compared to orthologs in other secretion systems. Nonreducing SDS-PAGE revealed that EB-localized apparatus proteins such as CdsF, CdsD, and CdsC form higher-order complexes mediated by disulfide bonding. The most dramatic alterations were detected for the needle protein CdsF. Significantly, disulfide bonding patterns shifted during differentiation of developmental forms and were completely reduced in RBs. Furthermore, at later time points during infection following RB to EB conversion, we found that CdsF is reoxidized into higher-order complexes. Overall, we conclude that the redox status of specific T3SS apparatus proteins is intimately linked to the developmental cycle and constitutes a newly appreciated aspect of functionally significant alterations within proteins of the chlamydial envelope.     

Plasmid migration using orthogonal-field-alternation gel electrophoresis.

The migration properties of a series of supercoiled plasmids ranging in size from 4 to 16 kilobases (kb) have been analyzed by orthogonal-field-alternation gel electrophoresis (OFAGE). These circular DNAs enter the gel and are well resolved. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a wide range of pulse times, from 10 to 120 seconds, as well as over a broad range of total running times, from 6 to 24 hours. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of pBR322 shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presented.     

Electron microscopic observations of surface projections on Chlamydia psittaci reticulate bodies.

Electron microscopic observations were carried out to confirm the presence of surface projections on Chlamydia psittaci reticulate bodies (RBs). The morphology of the projections on RBs was identical with that on elementary bodies (EBs); one end of each projection was connected with the cytoplasmic membrane, but the other end of the projection protruded beyond the cell wall through a fine hole or rosette in the cell wall. The results demonstrated that the rosettes seen in RB cell walls were morphological markers indicating the presence of the surface projections. A statistical anaylsis of the number of projections on EBs and the number of rosettes in RB cell walls prepared at 10, 15, and 20 h after infection demonstrated that all RBs had the projections and that the number of projections was maximal by 10 h after infection and then decreased gradually to approximately the same number of projections on EBs.     

Extent of helix perturbation associated with DNA modification by the o-acetyl derivative of the carcinogen 4-hydroxyaminoquinoline-1-oxide

Duplex unwinding associated with DNA modification by 4-acetoxyaminoquinoline-1-oxide, a model ultimate carcinogen of 4-nitroquinoline-1-oxide, has been determined by the agarose gel electrophoresis band-shift method. An average unwinding angle per stable adduct of -15.1 degrees +/- 1.5 degrees for negatively supercoiled topoisomers and -6.5 degrees +/- 1.4 degrees for positively supercoiled topoisomers was obtained. Because of the different proportion of stable adducts (dGuo-N2-AQO, dGuo-C8-AQO, dAdo-N6-AQO) between negatively (8:1.5:0.5) and positively (5:2.5:1) supercoiled topoisomers, the difference in unwinding angles is suggestive of a diverse contribution of the various adducts to the overall conformational change. Since the largest unwinding angle was coupled with the highest proportion of dGuo-N2-AQO adduct, it is likely that this adduct is the most distortive lesion. A contribution of sites of base loss to DNA unwinding was also observed.     

Conformational isomerization of the Holliday junction associated with a cruciform during branch migration in supercoiled plasmid DNA

The variable positions of a branch-migrating cruciform junction in supercoiled plasmid DNA were mapped following cleavage of the DNA with bacteriophage T7 endonuclease I. T7 endonuclease I specifically cleaved, and thereby resolved, the Holliday junction existing at the base of the cruciform in the circular bacterial plasmid pSA1B.56A. Cruciform extrusion of cloned sequences in pSA1B.56A (containing a 322 base-pair inverted repeat insert composed of poxvirus telomeric sequences) topologically relaxed the plasmid substrate in vitro. Thus, numerous crossover positions were identified within the region of cloned sequences, reflecting the range of superhelical densities in the native plasmid preparation. Endonuclease I-sensitive crossover positions, mapped to both strands of the viral insert following the T7 endonuclease I digestion of either plasmid preparations or individual topoisomers, were regularly separated by approximately ten nucleotides. The appearance of sensitive crossovers every ten nucleotides corresponds to a change in linking difference (delta Lk) of +/- 2 in the circular core domain of the plasmid during branch point migration. In contrast, individual topoisomers of a plasmid preparation differ in linking number in increments of +/- 1. Thus, the observed linearization of each individual topoisomer following enzyme treatment, as a result of resolution of the crossovers associated with each topoisomer, showed that branch point migration to sensitive crossover positions must have occurred facilely. T7 endonuclease I randomly resolved across either axis of the cruciform, though some discrimination (related to the sequence specificity of the enzyme) was observed. The ten-nucleotide spacing between sensitive crossover positions is accounted for by an isomerization of the cruciform junction on branch point migration. An hypothesis is that this isomerization was imposed upon the cruciform junction by the change in helix twist (delta Tw) in the two branches that compose the topologically closed, circular domain of the plasmid. T7 endonuclease I may discriminate between the various isomeric forms and cleave a sensitive conformation that appears with every turn of branch migration which leads to the extrusion, or absorption, of two turns of helix from the circular core.