Regulated phase transitions of bacterial chromatin: a non-enzymatic pathway for generic DNA protection.

The enhanced stress resistance exhibited by starved bacteria represents a central facet of virulence, since nutrient depletion is regularly encountered by pathogens in their natural in vivo and ex vivo environments. Here we explore the notion that the regular stress responses, which are mediated by enzymatically catalyzed chemical transactions and promote endurance during the logarithmic growth phase, can no longer be effectively induced during starvation. We show that survival of bacteria in nutrient-depleted habitats is promoted by a novel strategy: finely tuned and fully reversible intracellular phase transitions. These non-enzymatic transactions, detected and studied in bacteria as well as in defined in vitro systems, result in DNA sequestration and generic protection within tightly packed and highly ordered assemblies. Since this physical mode of defense is uniquely independent of enzymatic activity or de novo protein synthesis, and consequently does not require energy consumption, it promotes virulence by enabling long-term bacterial endurance and enhancing antibiotic resistance in adverse habitats.

Ordered intracellular RecA-DNA assemblies: a potential site of in vivo RecA-mediated activities.

The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role. Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli. We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly. The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement. The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur.

Flow of structural information between four DNA conformational levels.

Closed-circular supercoiled DNA molecules have been shown to form a cholesteric assembly within bacteria as well as in vitro under physiological DNA and salt concentrations. Circular dichroism and X-ray scattering studies indicate that the macroscopic structural properties of the chiral mesophase are directly and uniquely dictated by the supercoiling parameters of the constituent molecules. Specifically, we find that the pitch of the DNA cholesteric phase derived from supercoiled DNA is determined by the superhelical density, which, in turn, is modulated by secondary conformational changes. A direct interrelationship among four DNA structural levels, namely, DNA sequence, secondary structural transitions, the tertiary superhelical conformation, and the quaternary, supramolecular organization is accordingly pointed out. Since secondary conformational changes are both sequence and environment dependent, alterations of cellular conditions may effectively modulate the properties of the packed DNA organization, through their effects on secondary structural transitions and hence on the superhelical parameters. On the basis of these results we suggest that liquid crystallinity represents an effectively regulated packaging mode of plectonemic, nucleosome-free DNA molecules in living systems.

Supercoiling-regulated liquid-crystalline packaging of topologically-constrained, nucleosome-free DNA molecules.

Electron microscopy and circular dichroism studies of cholesteric aggregates derived from topologically-constrained DNA molecules indicate that the overall morphology and structural properties of these aggregates are fundamentally different from those characterizing condensed structures of nonconstrained DNA species. Specifically, the cholesteric pitch and twist of all hitherto characterized lyotropic mesophases of biopolymers--including those obtained from linear DNA--depend predominantly upon environmental parameters such as the dielectric constant of the solvent. In contrast, the properties of aggregates derived from closed circular supercoiled DNA are found to be solely and directly dictated by the superhelical density and handedness. On the basis of these results, as well as on the demonstrated ubiquity of liquid-crystalline DNA organizations in vivo, we suggest that supercoiling-regulated liquid crystallinity represents an effective packaging mode of nucleosome-free, topologically-constrained DNA molecules in living systems.

Effects of adenine tracts on the B-Z transition. Fine tuning of DNA conformational transition processes.

The effects exerted by short runs of adenines (A-tracts), alternating (AT)n segments, and single-stranded DNA upon the right- to left-handed DNA transition, as well as upon the energetic and structural parameters of the B/Z junctions, were investigated by using synthetic segments in which these motifs are coupled to a potentially Z-forming core. UV, CD, and 31P NMR studies of the salt-induced B to Z transition occurring in the various segments indicate that the transition is composed of two phases: a slow rate-determining induction of an initial structural deformation followed by a cooperative propagation of this "nucleus" in the form of a left-handed Z-DNA. The first phase is found to be crucially affected by the nature of the sequences coupled to the potentially Z-forming core. Thus, a higher rigidity of the flanking segments, such as that characterizing adenine tracts, is associated with higher energy values required for the induction of the initial conformational deformation, as well as with more defined structural parameters of the ultimate B/Z junctions. The second phase is affected mainly by the composition and sequence of the Z-forming segment. The observations that DNA conformational changes can be finely tuned and modulated by parameters pertaining to both the segment which undergoes the transition and the flanking sequences support the notion that DNA secondary motifs, such as the Z form and A-tracts, might be involved in the regulation of cellular processes.